Table 2. A list of the genes induced in radioresistant cells by irradiation at 6 Gy. We listed the genes that showed more than a 2-fold increase in almost all of the

radioresistant cells. The numbers indicate the gene expression value in comparison with unirradiated Saos-2 cells. The colored columns indicate more than a 2-fold increase.

expression of many genes that were induced in cells harboring wild-type *p53*, but showed induction of some genes that were not induced in the latter. The 245S mutant cell line showed a particularly marked increase in the expression of many TNF-associated genes upon irradiation. As the expression of TNF-associated genes inhibits apoptosis, this would explain the radioresistance of 245S cells. We also noticed that the expression of TNF-associated apoptosis-inducing genes, such as TNFSF9 (0.15), TNFSF10 (0.18) and TNFSF21 (0.22), was decreased by more than half, in the 245S mutant cell lines. We were unable to explain the radiosensitivity of other mutant cell lines upon induction of apoptosis-related genes.

Table 1. Induction of gene expression in the apoptosis pathway by irradiation at 6 Gy. We listed the apoptosis genes whose expression was increased more than 2-fold in mutant cells. The numbers indicate the gene expression value in comparison with unirradiated Saos-2 cells. The colored columns indicate more than a 2-fold increase.

We speculated that certain genes might play an important role in making some cells radioresistant. In this connection, we listed those genes whose expression was increased more than 2-fold in radioresistant cells. Table 2 shows a list of genes whose expression was increased in more than 4 of the mutant strain of radioresistant cells. We paid attention to the level of gene expression in the radiosensitive mutant cells, because genes that play an important role in conferring radioresistance would be show lower levels of expression in radiosensitive cells. Expression of the genes CADPS2, DNPEP, NKTR, OVOS2, PSENEN, RASSF4, RBM14 and WTAP was not increased more than 2-fold in almost all of the

expression of many genes that were induced in cells harboring wild-type *p53*, but showed induction of some genes that were not induced in the latter. The 245S mutant cell line showed a particularly marked increase in the expression of many TNF-associated genes upon irradiation. As the expression of TNF-associated genes inhibits apoptosis, this would explain the radioresistance of 245S cells. We also noticed that the expression of TNF-associated apoptosis-inducing genes, such as TNFSF9 (0.15), TNFSF10 (0.18) and TNFSF21 (0.22), was decreased by more than half, in the 245S mutant cell lines. We were unable to explain the

radiosensitivity of other mutant cell lines upon induction of apoptosis-related genes.

Table 1. Induction of gene expression in the apoptosis pathway by irradiation at 6 Gy. We listed the apoptosis genes whose expression was increased more than 2-fold in mutant cells. The numbers indicate the gene expression value in comparison with unirradiated Saos-2

We speculated that certain genes might play an important role in making some cells radioresistant. In this connection, we listed those genes whose expression was increased more than 2-fold in radioresistant cells. Table 2 shows a list of genes whose expression was increased in more than 4 of the mutant strain of radioresistant cells. We paid attention to the level of gene expression in the radiosensitive mutant cells, because genes that play an important role in conferring radioresistance would be show lower levels of expression in radiosensitive cells. Expression of the genes CADPS2, DNPEP, NKTR, OVOS2, PSENEN, RASSF4, RBM14 and WTAP was not increased more than 2-fold in almost all of the

cells. The colored columns indicate more than a 2-fold increase.

radiosensitive cells. CADPS2 acts as a calcium sensor in constitutive vesicle trafficking and secretion (Cisternas et al. 2003), and DNPEP is an aspartyl aminopeptidase that catalyzes the sequential removal of amino acids from the unblocked N termini of peptides and proteins (Nakamura et al. 2011). NKTR plays an important role in NK-cell cytotoxicity (Anderson et al. 1993). OVOS2 is a member of the ovostatin family and possesses trypsin-inhibitory activity (Saxxena and Tayyab, 1997). PSENEN (Presenilin enhancer-2) is a component of the -secretase complex which catalyzes the final cleavage of amyloid precursor protein to generate the toxic amiloid protein, the major component of plaques in the brain of Alzheimer disease patients, and protects embryos from apoptosis (Zetterberg et al. 2006). RASSF4 binds directly to activated K-Ras in a GTP-dependent manner via the effector domain, thus exhibits the basic nature of a Ras effector and plays an important role in Rasdependent apoptosis (Eckfeld et al. 2004). RBM14 (CoAA) is a nuclear receptor coactivator protein at the interface of transcriptional coactivation and RNA splicing (Auboeuf et al. 2004). WTAP (Wilms' tumor 1-associating protein) is essential for embryonic development, and appears to exert an antiproliferative effect, inhibiting G1-to-S phase cell cycle transition and also promoting apoptosis (Small et al. 2007).

Table 2. A list of the genes induced in radioresistant cells by irradiation at 6 Gy. We listed the genes that showed more than a 2-fold increase in almost all of the radioresistant cells. The numbers indicate the gene expression value in comparison with unirradiated Saos-2 cells. The colored columns indicate more than a 2-fold increase.

Radiation-Sensitivity and Transcription Profiles in Various Mutant p53 Cells 125

(karyopherin-alpha) proteins are responsible for the transport of proteins into and out of the nucleus through the nuclear pore complex, and KPUNA3 contributes genetically to schizophrenia (Wei and Hemmings 2005). MFAP5 (microfibrillar associated protein 5), also known as a microfibril-associated protein (MAGP2), is a highly significant indicator of survival and chemosensitivity of the cells (Spivey and Banyard, 2010). NEK3 is a serine/threonine kinase that contributes to PRL-mediated breast cell cancer motility through mechanisms involving Rac1 activation and paxillin phosphorylation (Miller et al. 2007). TRIM (tripartite motif-containing) proteins are a family comprising more than 70 members in humans and contain conserved RING, G-box, coiled-coil, and SPRY domains, most of which are involved in protein ubiquitination, but only a few of them have been well studied. TRIM2 mediates the p42/p44 MAPK-dependent ubiquitination of Bim (Bcl-2-interacting mediator of cell death) in rapid ischemic tolerance, and suppression of TRIM2 expression stabilizes the level of Bim protein and blocks neuroprotection (Thompson et al. 2011). TRIM38 has E3 ubiquitin ligase activity and can be degraded during virus infection (Liu

As CBR4 is involved in the induction of apoptosis, this gene may play an important role in radiosensitivity. However, the precise role of these genes in radiosensitivity remains

We have attempted to perform hierarchical clustering analysis of RNA expression in these mutant *p53* cell lines using Gene Tree software, but were unable to find any clear

Ionizing radiation is used extensively in medical diagnostic and treatment protocols. With a better understanding of radiation induced molecular processes, it might become possible to identify the radiosensitivity of individuals before the start of radiation therapy, leading to individualization of radiation treatment. Radiation-induced transcriptional responses have been studied using DNA microarray (Kis et al. 2006; Jen and Cheung, 2006). Some previous studies have also examined cells harboring mutant *p53* using DNA microarray (Amandson

In the present study, we prepared 15 mutant *p53* cell lines, cells harboring wild-type *p53* and Saos-2 cells (null for *p53*). We examined the radiosensitivity of the mutant cell lines and classified them as R (resistant), M (medium) or S (sensitive). We then studied the radiationinduced transcriptional responses in these cell lines, and examined the relationship between their radiation-induced gene expression and radiosensitivity. We found some genes that appeared to have some correlation with radiosensitivity, for example PSENEN and CBR4. However, none of the genes directly determined the radiosensitivity of the cells. Further study will be needed to determine which of these genes is the main determinant of

Radiosensitivity may be determined by several genes working in collaboration. Mutation of *p53* leads not only to loss of function, but also gain of function. If such functions are related to growth arrest or DNA repair, then loss of function would confer radiosensitivity, and gain of function to radioresistance. On the other hand, if such functions are related to apoptosis, then loss of function would confer radioresistance and gain of function to radiosensitivity. Each mutation of *p53* may thus lead to loss of function and gain of some other function at the same time. This makes it very difficult to determine whether a certain

et al. 2003; Scian et al. 2004), but they did not examine each type of mutation.

relationship between radiosensitivity and gene expression.

et al. 2011).

unknown.

**5. Conclusions**

radiosensitivity.

Among these genes, RASSF4 and WTAP are related to apoptosis, but exert a negative effect on radioresistant. As PSENEN blocks apoptosis, this gene may play an important role in radioresistance.

We approached this issue from the opposite perspective, and searched for genes that played an important role in conferring radiosensitivity. We listed genes showing more than a 2-fold increase in expression in radiosensitive cells. Table 3 shows a list of genes whose expression was increased in more than 4 of the radiosensitive mutant cell lines. Expression of CBR4, FOXP1, KPNA3, MFAP5, NEK3, TRIM2 and TRIM38 was not increased more than 2-fold in almost of all radioresistant cell lines. CBR4 (carbonyl reductase 4) is a mitochondrial NADPH-dependent quinine reductase that may be involved in the induction of apoptosis by cytotoxic 9, 10-phenanthrenequinone (Endo et al. 2008). FOXP1 is a forkhead transcription factor with functions in tissue and cell-type specific gene expression, and its gene is a direct target of p53-induced microRNA miR-34a (Rao et al. 2010). KPNA

Table 3. A list of the genes induced in radiosensitive cells by irradiation at 6 Gy. We listed the genes that showed more than a 2-fold increase of expression in almost all of the radiosensitive cells. The numbers indicate the gene expression value in comparison with unirradiated Saos-2 cells. The colored columns indicate more than a 2-fold increase.

(karyopherin-alpha) proteins are responsible for the transport of proteins into and out of the nucleus through the nuclear pore complex, and KPUNA3 contributes genetically to schizophrenia (Wei and Hemmings 2005). MFAP5 (microfibrillar associated protein 5), also known as a microfibril-associated protein (MAGP2), is a highly significant indicator of survival and chemosensitivity of the cells (Spivey and Banyard, 2010). NEK3 is a serine/threonine kinase that contributes to PRL-mediated breast cell cancer motility through mechanisms involving Rac1 activation and paxillin phosphorylation (Miller et al. 2007). TRIM (tripartite motif-containing) proteins are a family comprising more than 70 members in humans and contain conserved RING, G-box, coiled-coil, and SPRY domains, most of which are involved in protein ubiquitination, but only a few of them have been well studied. TRIM2 mediates the p42/p44 MAPK-dependent ubiquitination of Bim (Bcl-2-interacting mediator of cell death) in rapid ischemic tolerance, and suppression of TRIM2 expression stabilizes the level of Bim protein and blocks neuroprotection (Thompson et al. 2011). TRIM38 has E3 ubiquitin ligase activity and can be degraded during virus infection (Liu et al. 2011).

As CBR4 is involved in the induction of apoptosis, this gene may play an important role in radiosensitivity. However, the precise role of these genes in radiosensitivity remains unknown.

We have attempted to perform hierarchical clustering analysis of RNA expression in these mutant *p53* cell lines using Gene Tree software, but were unable to find any clear relationship between radiosensitivity and gene expression.

## **5. Conclusions**

124 Current Topics in Ionizing Radiation Research

Among these genes, RASSF4 and WTAP are related to apoptosis, but exert a negative effect on radioresistant. As PSENEN blocks apoptosis, this gene may play an important role in

We approached this issue from the opposite perspective, and searched for genes that played an important role in conferring radiosensitivity. We listed genes showing more than a 2-fold increase in expression in radiosensitive cells. Table 3 shows a list of genes whose expression was increased in more than 4 of the radiosensitive mutant cell lines. Expression of CBR4, FOXP1, KPNA3, MFAP5, NEK3, TRIM2 and TRIM38 was not increased more than 2-fold in almost of all radioresistant cell lines. CBR4 (carbonyl reductase 4) is a mitochondrial NADPH-dependent quinine reductase that may be involved in the induction of apoptosis by cytotoxic 9, 10-phenanthrenequinone (Endo et al. 2008). FOXP1 is a forkhead transcription factor with functions in tissue and cell-type specific gene expression, and its gene is a direct target of p53-induced microRNA miR-34a (Rao et al. 2010). KPNA

Table 3. A list of the genes induced in radiosensitive cells by irradiation at 6 Gy.

unirradiated Saos-2 cells. The colored columns indicate more than a 2-fold increase.

We listed the genes that showed more than a 2-fold increase of expression in almost all of the radiosensitive cells. The numbers indicate the gene expression value in comparison with

radioresistance.

Ionizing radiation is used extensively in medical diagnostic and treatment protocols. With a better understanding of radiation induced molecular processes, it might become possible to identify the radiosensitivity of individuals before the start of radiation therapy, leading to individualization of radiation treatment. Radiation-induced transcriptional responses have been studied using DNA microarray (Kis et al. 2006; Jen and Cheung, 2006). Some previous studies have also examined cells harboring mutant *p53* using DNA microarray (Amandson et al. 2003; Scian et al. 2004), but they did not examine each type of mutation.

In the present study, we prepared 15 mutant *p53* cell lines, cells harboring wild-type *p53* and Saos-2 cells (null for *p53*). We examined the radiosensitivity of the mutant cell lines and classified them as R (resistant), M (medium) or S (sensitive). We then studied the radiationinduced transcriptional responses in these cell lines, and examined the relationship between their radiation-induced gene expression and radiosensitivity. We found some genes that appeared to have some correlation with radiosensitivity, for example PSENEN and CBR4. However, none of the genes directly determined the radiosensitivity of the cells. Further study will be needed to determine which of these genes is the main determinant of radiosensitivity.

Radiosensitivity may be determined by several genes working in collaboration. Mutation of *p53* leads not only to loss of function, but also gain of function. If such functions are related to growth arrest or DNA repair, then loss of function would confer radiosensitivity, and gain of function to radioresistance. On the other hand, if such functions are related to apoptosis, then loss of function would confer radioresistance and gain of function to radiosensitivity. Each mutation of *p53* may thus lead to loss of function and gain of some other function at the same time. This makes it very difficult to determine whether a certain

Radiation-Sensitivity and Transcription Profiles in Various Mutant p53 Cells 127

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## **6. Acknowledgments**

This work was partly supported by the Global Center of Excellence (GCOE) of the Ministry of Education, Culture, Sports, Science and Technology of Japan.

## **7. References**

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**8**

*Japan*

**Modulation of Gene Expression**

Mitsuru Nenoi and Vares Guillaume *National Institute of Radiological Sciences,*

**After Exposure to Ionizing Radiation**

Regulation of gene expression is one of the fundamental mechanisms by which cells utilize the information in their DNA to obtain diverse characteristics, such as ability of differentiated cells to play specific roles and ability to respond to extracellular stresses including ionizing radiation (IR). The research on transcriptional regulation of genes after exposure to IR has a long history tracing back to early studies in late 1980's. In those studies, expression of individual genes was separately measured by a classical hybridization method. Recently, however, functional genomics approaches, such as microarray profiling, enable us to simultaneously monitor the expression of thousands of genes, and are now

The porpose of investigation of transcriptional gene regulation by IR is primarily to gain an insight into how the human bodies respond to IR and eventually how radiation hazards develop. IR-induced hematopoietic death is an example where proapoptotic gene expression is enhanced in radiosensitive hematopoietic stem cells after exposure to high doses of IR, which causes untolerable loss of peripheral blood cells, ultimately resulting in the individual death. Secondary, the purpose of the reserach on transcriptional gene regulation is to search for biomarkers which indicate the quantitative IR-exposure records. Such biomarkers may help us estimate the exposed dose of public peolpe in the emergency cases. For these purposes, studies have been extensively carried out by lots of researchers, and plenty of

In this review article, some of these studies, including those carried out in our laboratory, will be overviewed with an emphasis on the important roles of a tumor supressor p53 in transcriptional regulation after IR exposure and organ-dependence of transcriptional regulation *in vivo*. Methodology for the research on transcriptional regulation is also briefly touched. And the future perspectives of the research on transcriptional responses to IR will

The SOS response is a well known bacterial response to DNA damage (d'Ari, 1985). There is a group of bacterial genes called SOS genes which are induced by severe DNA damages causing stoppage of DNA synthesis. The SOS genes play roles in DNA repair, cell cycle arrest and mutagenesis. Under normal growth conditions, the SOS genes are negatively regulated by

recognized as a firmly established methodology in radiation biosciences.

**1. Introduction**

insights were obtained.

**2. Studies with bacteria and yeasts**

be discussed.

## **Modulation of Gene Expression After Exposure to Ionizing Radiation**

Mitsuru Nenoi and Vares Guillaume *National Institute of Radiological Sciences, Japan*

## **1. Introduction**

128 Current Topics in Ionizing Radiation Research

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and Fenaux P (1994) *p53* mutations are associated with resistance to chemotherapy

secretase component Presenilin enhancer 2 protects Zebrafish embryos from

Regulation of gene expression is one of the fundamental mechanisms by which cells utilize the information in their DNA to obtain diverse characteristics, such as ability of differentiated cells to play specific roles and ability to respond to extracellular stresses including ionizing radiation (IR). The research on transcriptional regulation of genes after exposure to IR has a long history tracing back to early studies in late 1980's. In those studies, expression of individual genes was separately measured by a classical hybridization method. Recently, however, functional genomics approaches, such as microarray profiling, enable us to simultaneously monitor the expression of thousands of genes, and are now recognized as a firmly established methodology in radiation biosciences.

The porpose of investigation of transcriptional gene regulation by IR is primarily to gain an insight into how the human bodies respond to IR and eventually how radiation hazards develop. IR-induced hematopoietic death is an example where proapoptotic gene expression is enhanced in radiosensitive hematopoietic stem cells after exposure to high doses of IR, which causes untolerable loss of peripheral blood cells, ultimately resulting in the individual death. Secondary, the purpose of the reserach on transcriptional gene regulation is to search for biomarkers which indicate the quantitative IR-exposure records. Such biomarkers may help us estimate the exposed dose of public peolpe in the emergency cases. For these purposes, studies have been extensively carried out by lots of researchers, and plenty of insights were obtained.

In this review article, some of these studies, including those carried out in our laboratory, will be overviewed with an emphasis on the important roles of a tumor supressor p53 in transcriptional regulation after IR exposure and organ-dependence of transcriptional regulation *in vivo*. Methodology for the research on transcriptional regulation is also briefly touched. And the future perspectives of the research on transcriptional responses to IR will be discussed.

## **2. Studies with bacteria and yeasts**

The SOS response is a well known bacterial response to DNA damage (d'Ari, 1985). There is a group of bacterial genes called SOS genes which are induced by severe DNA damages causing stoppage of DNA synthesis. The SOS genes play roles in DNA repair, cell cycle arrest and mutagenesis. Under normal growth conditions, the SOS genes are negatively regulated by

Modulation of Gene Expression After Exposure to Ionizing Radiation 131

how the cells respond to IR. First, the methodologies in research on transcriptional

Transcriptional activity of a gene is usually measured, as an amount transcription products (mRNA), by Northern blot analysis. However, mRNA level does not necessarily represent the transcription rate accurately. Instead, a nuclear run-on assay is performed to measure the transcription rate at a certain time point, which often differ from steady state mRNA levels revealed by Northern blot analysis. It is believed that the nuclear run-on assay is the

As illustrated in Figure 2, the cell nuclei are isolated rapidly, and incubated in a short period of time in the presence of radio-labelled nucleotides, and the transcribed products are

Reporter genes are the genes that confer the cells characteristics easily identified and measured. In order to investigate the promoter activity, the researchers often attach reporter genes to a DNA fragment containing regulatory elements, and introduce them into cultured cells. The promoter activity can be measured by signal intensity produced by reporter genes

regulation is briefly introduced below.

most reliable method to measure transcription rates directly.

Fig. 2. Schematic illustration of the nuclear run-on assay.

hybridized to a slot-blotted cDNA of interested genes.

Fig. 3. Schematic illustration of reporter gene analysis.

**3.1 Nuclear run-on analysis**

**3.2 Reporter gene analysis**

as shown in Figure 3.

LexA repressor protein by binding to a 20 bp consensus sequence (the SOS box) in the operator region for the SOS genes. RecA, one of the SOS gene products, is expressed at certain levels even in the repressed state, because of the low affinity of LexA to its SOS box. When DNA damages are produced, DNA polymerase is blocked at the damages, generating single stranded (ssDNA) regions at replication forks. RecA forms a filament around the ssDNA regions, and becomes activated. The activated RecA facilitates dissociation of the LexA repressor from the operator. Then the SOS genes are transcriptionarily activated (Figure 1). First, the genes having SOS box with a lower affinity to LexA repressor protein (such as lexA, recA, uvrA, uvrB, and uvrD) are fully induced in response to mild DNA damages. Thus the first SOS repair mechanism to be induced is nucleotide excision repair (NER). However, if NER is not capable of fixing the damage, the LexA is further inactivated, so the transcription of genes containing SOS box with a stronger affinity to LexA (such as sulA, umuD, umuC - these are expressed late) is induced. SulA binds to FtsZ causing aborted cell division, which results in filamentation and the induction of UmuDC-dependent mutagenic repair.

Regulation of the SOS genes is utilized to detect the environmental mutagen and carcinogens (Oda et al., 1985). The umu-test is based on the ability of the DNA-damaging agents, most of which are potential carcinogens, to induce the umu operon. A bacterial strain containing a fusion gene of umuC and lacZ can be used to monitor the levels of umu operon expression by measuring the β-galactosidase activity. Using this strain, a wide range of environmental mutagens and carcinogens were shown to be inexpensively and sensitively screened.

Fig. 1. Illustration of SOS response in bacterial cells.

#### **3. Methodology in research on transcriptional regulation in mammalian cells**

Transcriptional regulation of eukaryotic cells is usually mediated by binding of transcription factors (TF) to a DNA region (promoter) neighboring to the protein coding region of a gene. The activity of transcription is determined by a combination of basal TFs, such as RNA polymerase II, TATA binding protein (TBP), TBP-associated factor (TAF), and inducible TFs which act to either enhance or repress initiation of transcription by RNA polymerase II. Regulation of specific gene transcription is attributable to the inducible TFs. Therefore, identification of the inducible TF for IR-responsive genes provides important insight into how the cells respond to IR. First, the methodologies in research on transcriptional regulation is briefly introduced below.

## **3.1 Nuclear run-on analysis**

130 Current Topics in Ionizing Radiation Research

LexA repressor protein by binding to a 20 bp consensus sequence (the SOS box) in the operator region for the SOS genes. RecA, one of the SOS gene products, is expressed at certain levels even in the repressed state, because of the low affinity of LexA to its SOS box. When DNA damages are produced, DNA polymerase is blocked at the damages, generating single stranded (ssDNA) regions at replication forks. RecA forms a filament around the ssDNA regions, and becomes activated. The activated RecA facilitates dissociation of the LexA repressor from the operator. Then the SOS genes are transcriptionarily activated (Figure 1). First, the genes having SOS box with a lower affinity to LexA repressor protein (such as lexA, recA, uvrA, uvrB, and uvrD) are fully induced in response to mild DNA damages. Thus the first SOS repair mechanism to be induced is nucleotide excision repair (NER). However, if NER is not capable of fixing the damage, the LexA is further inactivated, so the transcription of genes containing SOS box with a stronger affinity to LexA (such as sulA, umuD, umuC - these are expressed late) is induced. SulA binds to FtsZ causing aborted cell division, which results

Regulation of the SOS genes is utilized to detect the environmental mutagen and carcinogens (Oda et al., 1985). The umu-test is based on the ability of the DNA-damaging agents, most of which are potential carcinogens, to induce the umu operon. A bacterial strain containing a fusion gene of umuC and lacZ can be used to monitor the levels of umu operon expression by measuring the β-galactosidase activity. Using this strain, a wide range of environmental mutagens and carcinogens were shown to be inexpensively and

DNA damage

dissociation of LexA

**3. Methodology in research on transcriptional regulation in mammalian cells**  Transcriptional regulation of eukaryotic cells is usually mediated by binding of transcription factors (TF) to a DNA region (promoter) neighboring to the protein coding region of a gene. The activity of transcription is determined by a combination of basal TFs, such as RNA polymerase II, TATA binding protein (TBP), TBP-associated factor (TAF), and inducible TFs which act to either enhance or repress initiation of transcription by RNA polymerase II. Regulation of specific gene transcription is attributable to the inducible TFs. Therefore, identification of the inducible TF for IR-responsive genes provides important insight into

in filamentation and the induction of UmuDC-dependent mutagenic repair.

SOS-box uvrA SOS-box umuC

DNA repair (NER)

Fig. 1. Illustration of SOS response in bacterial cells.

Lex A

Strongly repressed

activated RecA

replication fork

abortion of cell division mutagenic repair

Lex A

RecA

sensitively screened.

SOS-box recA

Lex A

> Lex A

weakly repressed Transcriptional activity of a gene is usually measured, as an amount transcription products (mRNA), by Northern blot analysis. However, mRNA level does not necessarily represent the transcription rate accurately. Instead, a nuclear run-on assay is performed to measure the transcription rate at a certain time point, which often differ from steady state mRNA levels revealed by Northern blot analysis. It is believed that the nuclear run-on assay is the most reliable method to measure transcription rates directly.

Fig. 2. Schematic illustration of the nuclear run-on assay.

As illustrated in Figure 2, the cell nuclei are isolated rapidly, and incubated in a short period of time in the presence of radio-labelled nucleotides, and the transcribed products are hybridized to a slot-blotted cDNA of interested genes.

### **3.2 Reporter gene analysis**

Reporter genes are the genes that confer the cells characteristics easily identified and measured. In order to investigate the promoter activity, the researchers often attach reporter genes to a DNA fragment containing regulatory elements, and introduce them into cultured cells. The promoter activity can be measured by signal intensity produced by reporter genes as shown in Figure 3.

Fig. 3. Schematic illustration of reporter gene analysis.

Modulation of Gene Expression After Exposure to Ionizing Radiation 133

Chromatin Immunoprecipitation (ChIP) is a technique used to investigate the interaction between proteins and DNA in the cell, especially to determine whether specific proteins are interacted with specific genomic regions. It is applicable to investigate interaction of TFs with promoters *in vivo*. Protein and genomic DNA are temporarily crosslinked *in vivo*, and the DNA-protein complexes are fragmented by shear stresses as illustrated in Figure 6. Then the DNA fragments associated with the proteins of interest are selectively immunoprecipitated by using antibodies specific to those proteins. The associated DNA

**3.5 Chromatin immunoprecipitation assay**

fragments are purified and their sequence is determined.

Fig. 6. Schematic illustration of Chromatin Immunoprecipitation assay

**4. p53-mediated regulation of gene transcription after IR in cultured cells**

Systematic studies on transcriptional regulation of mammalian genes have started in late 1980's. The representative studies were performed by Fornace and his colleagues (Fornace et al., 1988, Fornace et al., 1989). They screened a cDNA library for UV-induced genes by use of a hybridization subtraction method to obtain 20 differentially expressed genes including unidentified at that time. Some of these genes were co-ordinately regulated by DNA damaging agents and growth arrest signals, and were therefore called as gadd (**g**rowth **a**rrest and **D**NA **d**amage responsive) genes. The most widely characterized of these genes is *GADD45A*. The increase in *GADD45A* protein levels is correlated with a block in S phase progression as well as a stimulation of DNA repair (*Smith, et al., 1994, Chan, et al., 1995, Zhan, et al., 1994*). *GADD45A* gene was soon shown to respond to IR through a tumor supressor p53. The function of p53 in DNA damage responses was vigorously investigated at that time, and its pivotal roles as the guardian of the genome was established (Fei & El-Deiry, 2003). P53 functions primarily as a TF, and has been shown to be involved in multiple cellular pathways including cell cycle arrest, apoptosis, DNA repair, and so on, by controling many other genes. *CDKN1A* (p21CIP1/WAF1) gene is another member of gadd genes which was identified among 20 clones originally isolated by cDNA subtraction. *CDKN1A* gene is induced after exposure to IR also through p53, and inhibits the cycln dependent kinase activity resulting in arrest of cell cycle progression at G1/S border (Hartwell & Weinert, 1989). Although both of *GADD45A* and *CDKN1A* were clearly demonstrated to be regulated through p53, involvement of other TFs has not been investigated. Elucidation of a set of TFs involved in gene regulation after IR should be pivotal for understanding of delicate mechanisms for biological responses to IR which include variety of cellular

## **3.3 Electrophoretic mobility shift assay**

An electrophoretic mobility shift assay (EMSA) is an affinity electrophoresis technique used to determine if a protein is capable of binding to a given DNA sequence, as well as to identify the protein molecule. It is often performed to study binding of TFs to promoter region of interested genes. Electrophoresis of a DNA-protein mixture on a polyacrylamide gel can separate the protein-bound DNA from unbound DNA fragments, producing an additional band on the gel that represents the less mobile complex of DNA bound to protein (Figure 4). The DNA fragment is usually radio-labeled to be visible. An antibody that recognizes the protein is often added to the DNA-protein mixture to form an even larger complex with a greater shift. This method, called as a supershift assay, is used to unambiguously identify a protein present in the DNA-protein complex.

Fig. 4. Scematic illustration of electrophoretic mobility shift assay (EMSA)

## **3.4 DNase I footprinting assay**

A DNase I footprinting assay is a molecular biological technique by which DNA-protein interaction can be detected and located using the fact that a DNA region bound to proteins is often protected from enzymatic cleavage (Figure 5). The method uses an enzyme, deoxyribonuclease I(DNnase I, for short) to cut the radioactively end-labeled DNA, followed by gel electrophoresis to detect the resulting cleavage pattern.

Fig. 5. Schematic illustration of DNase I footprinting assay

## **3.5 Chromatin immunoprecipitation assay**

132 Current Topics in Ionizing Radiation Research

An electrophoretic mobility shift assay (EMSA) is an affinity electrophoresis technique used to determine if a protein is capable of binding to a given DNA sequence, as well as to identify the protein molecule. It is often performed to study binding of TFs to promoter region of interested genes. Electrophoresis of a DNA-protein mixture on a polyacrylamide gel can separate the protein-bound DNA from unbound DNA fragments, producing an additional band on the gel that represents the less mobile complex of DNA bound to protein (Figure 4). The DNA fragment is usually radio-labeled to be visible. An antibody that recognizes the protein is often added to the DNA-protein mixture to form an even larger complex with a greater shift. This method, called as a supershift assay, is used to

Unbound DNA

Radio-label

Antibody-protein-DNA complex (supershifted band)

Protein-DNA complex (shifted band)

unambiguously identify a protein present in the DNA-protein complex.

Polyacrylamide gel

followed by gel electrophoresis to detect the resulting cleavage pattern.

Fig. 5. Schematic illustration of DNase I footprinting assay

Fig. 4. Scematic illustration of electrophoretic mobility shift assay (EMSA)

A DNase I footprinting assay is a molecular biological technique by which DNA-protein interaction can be detected and located using the fact that a DNA region bound to proteins is often protected from enzymatic cleavage (Figure 5). The method uses an enzyme, deoxyribonuclease I(DNnase I, for short) to cut the radioactively end-labeled DNA,

**3.3 Electrophoretic mobility shift assay**

electrophoresis

**3.4 DNase I footprinting assay**

Chromatin Immunoprecipitation (ChIP) is a technique used to investigate the interaction between proteins and DNA in the cell, especially to determine whether specific proteins are interacted with specific genomic regions. It is applicable to investigate interaction of TFs with promoters *in vivo*. Protein and genomic DNA are temporarily crosslinked *in vivo*, and the DNA-protein complexes are fragmented by shear stresses as illustrated in Figure 6. Then the DNA fragments associated with the proteins of interest are selectively immunoprecipitated by using antibodies specific to those proteins. The associated DNA fragments are purified and their sequence is determined.

Fig. 6. Schematic illustration of Chromatin Immunoprecipitation assay

## **4. p53-mediated regulation of gene transcription after IR in cultured cells**

Systematic studies on transcriptional regulation of mammalian genes have started in late 1980's. The representative studies were performed by Fornace and his colleagues (Fornace et al., 1988, Fornace et al., 1989). They screened a cDNA library for UV-induced genes by use of a hybridization subtraction method to obtain 20 differentially expressed genes including unidentified at that time. Some of these genes were co-ordinately regulated by DNA damaging agents and growth arrest signals, and were therefore called as gadd (**g**rowth **a**rrest and **D**NA **d**amage responsive) genes. The most widely characterized of these genes is *GADD45A*. The increase in *GADD45A* protein levels is correlated with a block in S phase progression as well as a stimulation of DNA repair (*Smith, et al., 1994, Chan, et al., 1995, Zhan, et al., 1994*). *GADD45A* gene was soon shown to respond to IR through a tumor supressor p53. The function of p53 in DNA damage responses was vigorously investigated at that time, and its pivotal roles as the guardian of the genome was established (Fei & El-Deiry, 2003). P53 functions primarily as a TF, and has been shown to be involved in multiple cellular pathways including cell cycle arrest, apoptosis, DNA repair, and so on, by controling many other genes. *CDKN1A* (p21CIP1/WAF1) gene is another member of gadd genes which was identified among 20 clones originally isolated by cDNA subtraction. *CDKN1A* gene is induced after exposure to IR also through p53, and inhibits the cycln dependent kinase activity resulting in arrest of cell cycle progression at G1/S border (Hartwell & Weinert, 1989). Although both of *GADD45A* and *CDKN1A* were clearly demonstrated to be regulated through p53, involvement of other TFs has not been investigated. Elucidation of a set of TFs involved in gene regulation after IR should be pivotal for understanding of delicate mechanisms for biological responses to IR which include variety of cellular

Modulation of Gene Expression After Exposure to Ionizing Radiation 135

Fig. 8. Reporter gene analysis for the promoter activity of the *GADD45A* gene. (A) Various *GADD45A*-luciferase constructs are schematically illustrated. The light gray bars and the dark gray bars represent the 5'-flanking region and the third intron region of the *GADD45A* gene, respectively. The transcription start site is indicated by an arrow. The nucleotide sequence of the region, which reduced the basal promoter activity significantly as shown in (B), is presented at the bottom. Two OCT sites and a CCAAT box are underlined. (B) MCF-7 cells transiently transfected with reporter constructs were either irradiated with 10 Gy of Xrays (closed bars) or mock-treated as a control (open bars), and the luciferase activity in the cell lysates prepared 5 h after irradiation was measured. The relative light unit (RLU) per protein concentration is plotted. (C)*,* Fold-induction of the luciferase activity after irradiation with 10 Gy of X-rays is plotted. Results are the mean of at least three

independent experiments. Error bars represent the S.D. (\*\*\* p < 0.001 versus P/I, student's t-

test). (Reproduced with permission of Elsevier B. V. from Ref (Daino et al., 2006))

pathways. In the following sections, researches performed in our laboratory on regulation mechanisms for the *GADD45A* and *CDKN1A* genes are reviewed.

## **4.1 Regulation** *GADD45A* **gene**

In human cells, *GADD45A* is up-regulated in response to IR. Figure 7 is a Northern blot analysis for induction of the *GADD45A* gene expression in human breast adenocarcinoma cell line MCF7 after exposure to IR. The induction was transient with the maximum induction at 2 h after irradiation.

Fig. 7. Northern analysis for induction of the *GADD45A* gene expression after exposure to IR. (A) Total RNA isolated from the MCF-7 cells at various time intervals after irradiation with 10 Gy of X-rays was hybridized with a *GADD45A* gene-specific probe. The *GAPDH* gene was used as a control. (B) Induction of the *GADD45A* gene after irradiation with various doses of X-rays was analyzed. (Reproduced with permission of Elsevier B. V. from Ref (Daino et al., 2006))

To search for the DNA element functioning in transcriptional regulation of the *GADD45A* gene, a series of reporter gene constructs were designed. As the p53 recognition sequence was known to reside in the third intron, the third intron was fused to the 3' end of the luciferase gene (*luc* +) (Figure 8A). MCF-7 cells were transiently transfected with these constructs, and luciferase activity was measured after treatment with or without X-rays. As shown in Figure 8B, constructs P/I through P6/I gave rise to a similar level of the basal luciferase activity, suggesting that there was no major DNA element functioning in transcriptional activity in the -2244 bp/-107 bp region. However, when the 5' flanking region was truncated to -62 bp in P7/I, the luciferase activity was drastically decreased, suggesting that a regulatory element required for the basal expression of the *GADD45A*

pathways. In the following sections, researches performed in our laboratory on regulation

In human cells, *GADD45A* is up-regulated in response to IR. Figure 7 is a Northern blot analysis for induction of the *GADD45A* gene expression in human breast adenocarcinoma cell line MCF7 after exposure to IR. The induction was transient with the maximum

Fig. 7. Northern analysis for induction of the *GADD45A* gene expression after exposure to IR. (A) Total RNA isolated from the MCF-7 cells at various time intervals after irradiation with 10 Gy of X-rays was hybridized with a *GADD45A* gene-specific probe. The *GAPDH* gene was used as a control. (B) Induction of the *GADD45A* gene after irradiation with various doses of X-rays was analyzed. (Reproduced with permission of Elsevier B. V. from

To search for the DNA element functioning in transcriptional regulation of the *GADD45A* gene, a series of reporter gene constructs were designed. As the p53 recognition sequence was known to reside in the third intron, the third intron was fused to the 3' end of the luciferase gene (*luc* +) (Figure 8A). MCF-7 cells were transiently transfected with these constructs, and luciferase activity was measured after treatment with or without X-rays. As shown in Figure 8B, constructs P/I through P6/I gave rise to a similar level of the basal luciferase activity, suggesting that there was no major DNA element functioning in transcriptional activity in the -2244 bp/-107 bp region. However, when the 5' flanking region was truncated to -62 bp in P7/I, the luciferase activity was drastically decreased, suggesting that a regulatory element required for the basal expression of the *GADD45A*

mechanisms for the *GADD45A* and *CDKN1A* genes are reviewed.

**4.1 Regulation** *GADD45A* **gene**

induction at 2 h after irradiation.

Ref (Daino et al., 2006))

Fig. 8. Reporter gene analysis for the promoter activity of the *GADD45A* gene. (A) Various *GADD45A*-luciferase constructs are schematically illustrated. The light gray bars and the dark gray bars represent the 5'-flanking region and the third intron region of the *GADD45A* gene, respectively. The transcription start site is indicated by an arrow. The nucleotide sequence of the region, which reduced the basal promoter activity significantly as shown in (B), is presented at the bottom. Two OCT sites and a CCAAT box are underlined. (B) MCF-7 cells transiently transfected with reporter constructs were either irradiated with 10 Gy of Xrays (closed bars) or mock-treated as a control (open bars), and the luciferase activity in the cell lysates prepared 5 h after irradiation was measured. The relative light unit (RLU) per protein concentration is plotted. (C)*,* Fold-induction of the luciferase activity after irradiation with 10 Gy of X-rays is plotted. Results are the mean of at least three independent experiments. Error bars represent the S.D. (\*\*\* p < 0.001 versus P/I, student's ttest). (Reproduced with permission of Elsevier B. V. from Ref (Daino et al., 2006))

Modulation of Gene Expression After Exposure to Ionizing Radiation 137

Fig. 9. Requirement of p53 and AP-1 recognition sites for both basal expression and transcriptional enhancement of the *GADD45A* gene after exposure to IR. (A) Mutant *GADD45A*-luciferase constructs are schematically illustrated. The light gray bars and the dark gray bars represent the 5'-flanking region and the third intron region of the *GADD45A* gene, respectively. The nucleotide sequences at the p53-deletion in P6/I p53∆ and the AP-1 mutation in P6/I AP-1m are shown on the right of the panel. The small letters represent the substituted nucleotides. The closed box and open box represent the p53 site and the AP-1 site, respectively. The arrowhead represents the site of p53-deletion, and the hatched box represents the mutated AP-1 site. (B) MCF-7 cells transiently transfected with reporter constructs were either irradiated with 10 Gy of X-rays (closed bars) or mock-treated as a control (open bars), and luciferase activity in the cell lysates prepared 5 h after irradiation was measured. The relative light unit (RLU) per protein concentration is plotted. (C) Foldinduction of the luciferase activity after irradiation with 10 Gy of X-rays is plotted. Results are the mean of at least three independent experiments. Error bars represent the S.D. (\*\* p < 0.01, \*\*\* p < 0.001 versus P6/I, student's t-test). (D) and (E) MCF-7 cells transduced with reporter constructs by use of rAAV were either irradiated with 10 Gy of X-rays or mocktreated, and luciferase activity in the cell lysates prepared 5 h after irradiation was measured. Fold-induction of luciferase activity after irradiation with 10 Gy of X-rays is plotted. Results are the mean of at least three independent experiments. Error bars represent the S.D. (\*\* p < 0.01 versus P6/I, student's t-test). (Reproduced with permission of Elsevier

B. V. from Ref (Daino et al., 2006))

gene was present in the region between -107 bp and -62 bp. There were two OCT sites and one CCAAT box in this region (Figure 8A). It was thought that these OCT and CCAAT elements functioned as the core promoter because a canonical TATA sequences could not be found. It can be seen, in Figure 8C, that fold-induction of luciferase expression after irradiation with 10 Gy of X-rays was not reduced even when the 5'-flanking region was deleted down to -39 bp. This result indicated the absence of major regulatory elements functioning in response to IR in the upstream region of the *GADD45A* gene.

When the region +1747 bp/+2488 bp in the third intron was deleted, the luciferase activity was not so much reduced. However, when the region +1625 bp/+2488 bp was deleted, the luciferase activity was decreased dramatically (data not shown), suggesting the presence of a DNA element, in +1625/+1746, that plays a role in the basal expression of the *GADD45A* gene. As AP-1 recognition element existed at the locus +1625 bp/+1631 bp, we constructed another reporter gene experiment with a plasmid (P6/I AP-1m) in which the AP-1 recognition sequence was mutated (Figure 9A). As shown in Figure 9B, the basal luciferase expression from P6/I AP-1m was drastically reduced compared with P6/I which contains wild type AP1 sequence. This result demonstrate the important role of AP-1. Deletion of the p53 site (P6/I p53∆) also resulted in reduction of basal luciferase activity compared to that of the P6/I construct. Then it was suggested that both the AP-1 and p53 sites in the third intron of the *GADD45A* gene are critical for the basal expression. Concerning IR responsiveness, Figure 9C shows that deletion of the p53 site (P6/I p53∆) diminished IR-response, while fold induction for the construct containing the mutated AP-1 site (P6/I AP-1m) was reduced, suggesting that the AP-1 site contributes to a part of p53-dependent transcriptional activation of the *GADD45A* gene after exposure to IR.

It was speculated that transiently transfected plasmid DNAs may not form tight chromatin complexes. Then, we used the recombinant adeno-associated virus (rAAV) vectors, which can stably integrate reporter genes into the genome of host cells. As a result, a remarkably higher fold-induction of luciferase expression (~3.4-fold, rAAV-P6/I) could be observed after IR irradiation as shown in Figure 9D. With this vector system, rAAV-P6/I AP-1m containing mutation at the AP-1 site gave rise to significantly decreased fold induction compared to the rAAV-P6/I construct (Figure 9E). Taken together, these results indicate that the AP-1 recognition site plays a significant role in the p53-dependent transcriptional activation of the *GADD45A* gene by IR.

When EMSA was performed with a DNA probe containing the AP-1 recognition sequence and nuclear extracts from MCF-7 cells, DNA-protein complexes were created. And these DNA-protein complexes disappeared when an anti-JunD antibody was added in the DNAprotein mixture. This result indicated the presence of JunD in the DNA-protein complexes.

When ChIP assay was carried out (Figure 10A), the binding of p53 and JunD to the third intron of the *GADD45A* gene was confirmed. However they were detected even in unirradiated cells. While the affinity of p53 to its site in the intron 3 was enhanced in X-rayirradiated cells (Figure 10C), the bining of JunD to its recognition site was not increased in the X-ray irradiated cells (Figures. 10B and 10C). The binding of phospho-p53 (Ser20) to the p53 site was detected only in X-ray-irradiated cells (Figure 10C). These results indicated that JunD constitutively binds to the third intron of the *GADD45A* gene even in unstressed cells *in vivo*. JunD is considered to play an important role both in basal expression of the *GADD45A* gene and in transcriptional enhancement after IR irradiation via binding to the AP-1 site in the third intron.

gene was present in the region between -107 bp and -62 bp. There were two OCT sites and one CCAAT box in this region (Figure 8A). It was thought that these OCT and CCAAT elements functioned as the core promoter because a canonical TATA sequences could not be found. It can be seen, in Figure 8C, that fold-induction of luciferase expression after irradiation with 10 Gy of X-rays was not reduced even when the 5'-flanking region was deleted down to -39 bp. This result indicated the absence of major regulatory elements

When the region +1747 bp/+2488 bp in the third intron was deleted, the luciferase activity was not so much reduced. However, when the region +1625 bp/+2488 bp was deleted, the luciferase activity was decreased dramatically (data not shown), suggesting the presence of a DNA element, in +1625/+1746, that plays a role in the basal expression of the *GADD45A* gene. As AP-1 recognition element existed at the locus +1625 bp/+1631 bp, we constructed another reporter gene experiment with a plasmid (P6/I AP-1m) in which the AP-1 recognition sequence was mutated (Figure 9A). As shown in Figure 9B, the basal luciferase expression from P6/I AP-1m was drastically reduced compared with P6/I which contains wild type AP1 sequence. This result demonstrate the important role of AP-1. Deletion of the p53 site (P6/I p53∆) also resulted in reduction of basal luciferase activity compared to that of the P6/I construct. Then it was suggested that both the AP-1 and p53 sites in the third intron of the *GADD45A* gene are critical for the basal expression. Concerning IR responsiveness, Figure 9C shows that deletion of the p53 site (P6/I p53∆) diminished IR-response, while fold induction for the construct containing the mutated AP-1 site (P6/I AP-1m) was reduced, suggesting that the AP-1 site contributes to a part of p53-dependent transcriptional

It was speculated that transiently transfected plasmid DNAs may not form tight chromatin complexes. Then, we used the recombinant adeno-associated virus (rAAV) vectors, which can stably integrate reporter genes into the genome of host cells. As a result, a remarkably higher fold-induction of luciferase expression (~3.4-fold, rAAV-P6/I) could be observed after IR irradiation as shown in Figure 9D. With this vector system, rAAV-P6/I AP-1m containing mutation at the AP-1 site gave rise to significantly decreased fold induction compared to the rAAV-P6/I construct (Figure 9E). Taken together, these results indicate that the AP-1 recognition site plays a significant role in the p53-dependent transcriptional

When EMSA was performed with a DNA probe containing the AP-1 recognition sequence and nuclear extracts from MCF-7 cells, DNA-protein complexes were created. And these DNA-protein complexes disappeared when an anti-JunD antibody was added in the DNAprotein mixture. This result indicated the presence of JunD in the DNA-protein complexes. When ChIP assay was carried out (Figure 10A), the binding of p53 and JunD to the third intron of the *GADD45A* gene was confirmed. However they were detected even in unirradiated cells. While the affinity of p53 to its site in the intron 3 was enhanced in X-rayirradiated cells (Figure 10C), the bining of JunD to its recognition site was not increased in the X-ray irradiated cells (Figures. 10B and 10C). The binding of phospho-p53 (Ser20) to the p53 site was detected only in X-ray-irradiated cells (Figure 10C). These results indicated that JunD constitutively binds to the third intron of the *GADD45A* gene even in unstressed cells *in vivo*. JunD is considered to play an important role both in basal expression of the *GADD45A* gene and in transcriptional enhancement after IR irradiation via binding to the

functioning in response to IR in the upstream region of the *GADD45A* gene.

activation of the *GADD45A* gene after exposure to IR.

activation of the *GADD45A* gene by IR.

AP-1 site in the third intron.

Fig. 9. Requirement of p53 and AP-1 recognition sites for both basal expression and transcriptional enhancement of the *GADD45A* gene after exposure to IR. (A) Mutant *GADD45A*-luciferase constructs are schematically illustrated. The light gray bars and the dark gray bars represent the 5'-flanking region and the third intron region of the *GADD45A* gene, respectively. The nucleotide sequences at the p53-deletion in P6/I p53∆ and the AP-1 mutation in P6/I AP-1m are shown on the right of the panel. The small letters represent the substituted nucleotides. The closed box and open box represent the p53 site and the AP-1 site, respectively. The arrowhead represents the site of p53-deletion, and the hatched box represents the mutated AP-1 site. (B) MCF-7 cells transiently transfected with reporter constructs were either irradiated with 10 Gy of X-rays (closed bars) or mock-treated as a control (open bars), and luciferase activity in the cell lysates prepared 5 h after irradiation was measured. The relative light unit (RLU) per protein concentration is plotted. (C) Foldinduction of the luciferase activity after irradiation with 10 Gy of X-rays is plotted. Results are the mean of at least three independent experiments. Error bars represent the S.D. (\*\* p < 0.01, \*\*\* p < 0.001 versus P6/I, student's t-test). (D) and (E) MCF-7 cells transduced with reporter constructs by use of rAAV were either irradiated with 10 Gy of X-rays or mocktreated, and luciferase activity in the cell lysates prepared 5 h after irradiation was measured. Fold-induction of luciferase activity after irradiation with 10 Gy of X-rays is plotted. Results are the mean of at least three independent experiments. Error bars represent the S.D. (\*\* p < 0.01 versus P6/I, student's t-test). (Reproduced with permission of Elsevier B. V. from Ref (Daino et al., 2006))

Modulation of Gene Expression After Exposure to Ionizing Radiation 139

C 0.5h 1h 5h 3h 8h

A

B

3

0

Radiation Research Society of the USA from Ref (Daino et al., 2002))

(rAAV-del5).

012345678 Time (h)

Fig. 11. Nuclear run-on analysis for transcription rates of the *CDKN1A* and *GADD45* genes in human myeloblastic leukemia cell line ML-1 after irradiation with 0.5 Gy of X-rays. (A) The resulting hybridization images. cDNA probes were blotted as shown in the right side panel. (B) The signal intensity was analyzed and plotted. (Reproduced with permission of

The functional DNA region involved in the p53-mediated IR response was investigated by reporter gene analysis using AAV vectors, which can give rise to remarkably high responsiveness to IR as shown in Figure 12A. A series of reporter gene constructs with or without deletions in the 5 flanking region of the *CDKN1A* gene was prepared as shown in Figure 12B, and were transduced into MCF-7 cells. The p53 recognition site at –2.2 kb was preserved in every construct. It can be seen, in Figure 12C, that the fold induction of luciferase activity increased linearly with radiation dose for every constructs. However, IR responsiveness of the constructs rAAV-del2, rAAV-del4 and rAAV-del5 was much reduced compared to that of rAAV-PLS which contains an intact 5 flanking region of the *CDKN1A* gene, suggesting the presence of pivotal DNA elements for IR responsiveness in the regions –1962 bp/–1679 bp (rAAV-del2), –1398 bp/–1119 bp (rAAV-del4), and –1118 bp/–839 bp

1

2

GAPDH GADD5A CDKN1A

Fig. 10. Chromatin immunoprecipitation (ChIP) analysis for the in vivo binding of JunD at the third intron. (A) Schematic illustration of genomic structure of the *GADD45A* gene. PCR primers were designed to amplify the region containing both the p53 site and the AP-1 site, as represented by arrows. (B) ChIP analysis was performed with MCF-7 cells, unirradiated (B) or exposed to 10 Gy of X-rays with 1.5 h postincubated (C). Sheared chromatin was incubated without antibodies (no Ab) or with the IgG, anti-p53 antibody, anti-JunD antibody, and anti-phospho-p53 (Ser20) antibody. Immunoprecipitants were analyzed by PCR. The *GAPDH* promoter containing neither an AP-1 nor p53 site was analyzed as a negative control. PCR was performed for 32 cycles, which was in the linear range of amplification, except for the case using the sample prepared from X-ray-treated cells with the anti-p53 antibody. (Reproduced with permission of Elsevier B. V. from Ref (Daino et al., 2006))

#### **4.2 Regulation** *CDKN1A* **gene**

Regulation of *CDKN1A* gene after exposure to IR occurs in transcription level as shown by a nuclear run-on analysis (Figure 11)*.* While the induction kinetics of *CDKN1A* is similar to that of *GADD45A*, the fold-increase of *CDKN1A* is much higher, and is thought to be one of the most responsive genes to IR.

Fig. 10. Chromatin immunoprecipitation (ChIP) analysis for the in vivo binding of JunD at the third intron. (A) Schematic illustration of genomic structure of the *GADD45A* gene. PCR primers were designed to amplify the region containing both the p53 site and the AP-1 site, as represented by arrows. (B) ChIP analysis was performed with MCF-7 cells, unirradiated (B) or exposed to 10 Gy of X-rays with 1.5 h postincubated (C). Sheared chromatin was incubated without antibodies (no Ab) or with the IgG, anti-p53 antibody, anti-JunD antibody, and anti-phospho-p53 (Ser20) antibody. Immunoprecipitants were analyzed by PCR. The *GAPDH* promoter containing neither an AP-1 nor p53 site was analyzed as a negative control. PCR was performed for 32 cycles, which was in the linear range of amplification, except for the case using the sample prepared from X-ray-treated cells with the anti-p53 antibody. (Reproduced with permission of Elsevier B. V. from Ref (Daino et al.,

Regulation of *CDKN1A* gene after exposure to IR occurs in transcription level as shown by a nuclear run-on analysis (Figure 11)*.* While the induction kinetics of *CDKN1A* is similar to that of *GADD45A*, the fold-increase of *CDKN1A* is much higher, and is thought to be one of

2006))

**4.2 Regulation** *CDKN1A* **gene**

the most responsive genes to IR.

Fig. 11. Nuclear run-on analysis for transcription rates of the *CDKN1A* and *GADD45* genes in human myeloblastic leukemia cell line ML-1 after irradiation with 0.5 Gy of X-rays. (A) The resulting hybridization images. cDNA probes were blotted as shown in the right side panel. (B) The signal intensity was analyzed and plotted. (Reproduced with permission of Radiation Research Society of the USA from Ref (Daino et al., 2002))

The functional DNA region involved in the p53-mediated IR response was investigated by reporter gene analysis using AAV vectors, which can give rise to remarkably high responsiveness to IR as shown in Figure 12A. A series of reporter gene constructs with or without deletions in the 5 flanking region of the *CDKN1A* gene was prepared as shown in Figure 12B, and were transduced into MCF-7 cells. The p53 recognition site at –2.2 kb was preserved in every construct. It can be seen, in Figure 12C, that the fold induction of luciferase activity increased linearly with radiation dose for every constructs. However, IR responsiveness of the constructs rAAV-del2, rAAV-del4 and rAAV-del5 was much reduced compared to that of rAAV-PLS which contains an intact 5 flanking region of the *CDKN1A* gene, suggesting the presence of pivotal DNA elements for IR responsiveness in the regions –1962 bp/–1679 bp (rAAV-del2), –1398 bp/–1119 bp (rAAV-del4), and –1118 bp/–839 bp (rAAV-del5).

Modulation of Gene Expression After Exposure to Ionizing Radiation 141

Fig. 13. Reporter vectors were constructed in which the Oct-1 site at –1.8 kb, TP53 site at –1.4 kb, and Oct-1 site at –1.1 kb were specifically deleted, resulting in rAAV-del2/Oct1, rAAVdel4/TP53, and rAAV-del5/Oct1, respectively (D). X-ray response of each construct was assayed at a dose range of 0.2-2.0 Gy (E). (Reproduced with permission of Elsevier B. V.

The potential recognition sequences for TFs were searched in these regions, and Oct-1 recognition sequences were found at –1.8 kb and –1.1 kb. It was interesting to note that Oct-1 is the TF involved in regulation of IR-responsive genes *GADD45A*, *Prx1*, *Notch1*, and endothelial lipase, suggesting that Oct-1 may play a role in regulation of variety of IRresponsive genes by cooperating with p53. Then, mutant reporter vectors rAAV-del2/Oct1, rAAV-del4/TP53, and rAAV-del5/Oct1 were constructed in which the Oct-1 site at –1.8 kb, the p53 site at –1.4 kb, and the Oct-1 site at –1.1 kb, respectively, were specifically deleted (Figure 13A). Figure 13B shows that the IR responsiveness of these reporter constructs was mostly diminished, indicating that the recognition sequences for these TFs play pivotal roles

EMSA analysis revealed that the DNA probes Oct-1/–1.8kb (Figure 14A) and Oct-1/–1.1kb (Figure 14B) could form complexes with nuclear proteins as indicated by arrows. These bands were supershifted by addition of anti-Oct-1 antibodies and competed away by addition of an excess amount of unlabeled oligonucleotides containing the Oct-1 consensus sequence. These results demonstrate Oct-1 could bind to its recognition sequences at -1.8 kb and -1.1 kb. However, binding of Oct-1 to these sites was not X ray-dependent. In accordance with the EMSA analysis, constitutive binding of Oct-1 to the sites at –1.8 kb and –1.1 kb was observed by ChIP analysis as shown in Figures 14C and 14E. In contrast, binding of p53 to the site at –1.4 kb was not constitutive but inducible after irradiation

Overall, it was considered that Oct-1 is constitutively bound to the sites at –1.8 kb and –1.1 kb and plays a cooperative role with p53 in induction of the *CDKN1A* gene promoter in

from Ref (Nenoi et al., 2009))

(Figure 14D).

response to IR.

in the IR response of the *CDKN1A* gene promoter.

Fig. 12. DNA regions functioning in the IR response of the *CDKN1A* gene. A: Reporter gene constructs containing the luciferase gene under the control of the *CDKN1A* gene promoter were either transfected by electroporation (PLS) or transduced by rAAV vectors (rAAV-PLS) into MCF-7 cells. The cells were irradiated with X-rays and assayed for luciferase activity 5 h after irradiation or mock treatment. Results are the means of three independent experiments. Error bars represent the S.D. B: The reporter gene constructs with or without deletions in the 5 flanking region are schematically represented. The TP53 site at –2.2 kb is indicated by a heavy line. These constructs were transduced into MCF-7 cells using rAAV vectors. C: X-ray response of each construct was assayed in the dose range of 0.2-2.0 Gy. Since the fold induction increased linearly with dose, the slope was used as an index of IR responsiveness. Results are the mean of three independent experiments. Error bars represent the S.D. (Reproduced with permission of Elsevier B. V. from Ref (Nenoi et al., 2009))

Fig. 12. DNA regions functioning in the IR response of the *CDKN1A* gene. A: Reporter gene constructs containing the luciferase gene under the control of the *CDKN1A* gene promoter were either transfected by electroporation (PLS) or transduced by rAAV vectors (rAAV-PLS) into MCF-7 cells. The cells were irradiated with X-rays and assayed for luciferase activity 5 h

experiments. Error bars represent the S.D. B: The reporter gene constructs with or without deletions in the 5 flanking region are schematically represented. The TP53 site at –2.2 kb is indicated by a heavy line. These constructs were transduced into MCF-7 cells using rAAV vectors. C: X-ray response of each construct was assayed in the dose range of 0.2-2.0 Gy. Since the fold induction increased linearly with dose, the slope was used as an index of IR responsiveness. Results are the mean of three independent experiments. Error bars represent the S.D. (Reproduced with permission of Elsevier B. V. from Ref (Nenoi et al.,

after irradiation or mock treatment. Results are the means of three independent

2009))

Fig. 13. Reporter vectors were constructed in which the Oct-1 site at –1.8 kb, TP53 site at –1.4 kb, and Oct-1 site at –1.1 kb were specifically deleted, resulting in rAAV-del2/Oct1, rAAVdel4/TP53, and rAAV-del5/Oct1, respectively (D). X-ray response of each construct was assayed at a dose range of 0.2-2.0 Gy (E). (Reproduced with permission of Elsevier B. V. from Ref (Nenoi et al., 2009))

The potential recognition sequences for TFs were searched in these regions, and Oct-1 recognition sequences were found at –1.8 kb and –1.1 kb. It was interesting to note that Oct-1 is the TF involved in regulation of IR-responsive genes *GADD45A*, *Prx1*, *Notch1*, and endothelial lipase, suggesting that Oct-1 may play a role in regulation of variety of IRresponsive genes by cooperating with p53. Then, mutant reporter vectors rAAV-del2/Oct1, rAAV-del4/TP53, and rAAV-del5/Oct1 were constructed in which the Oct-1 site at –1.8 kb, the p53 site at –1.4 kb, and the Oct-1 site at –1.1 kb, respectively, were specifically deleted (Figure 13A). Figure 13B shows that the IR responsiveness of these reporter constructs was mostly diminished, indicating that the recognition sequences for these TFs play pivotal roles in the IR response of the *CDKN1A* gene promoter.

EMSA analysis revealed that the DNA probes Oct-1/–1.8kb (Figure 14A) and Oct-1/–1.1kb (Figure 14B) could form complexes with nuclear proteins as indicated by arrows. These bands were supershifted by addition of anti-Oct-1 antibodies and competed away by addition of an excess amount of unlabeled oligonucleotides containing the Oct-1 consensus sequence. These results demonstrate Oct-1 could bind to its recognition sequences at -1.8 kb and -1.1 kb. However, binding of Oct-1 to these sites was not X ray-dependent. In accordance with the EMSA analysis, constitutive binding of Oct-1 to the sites at –1.8 kb and –1.1 kb was observed by ChIP analysis as shown in Figures 14C and 14E. In contrast, binding of p53 to the site at –1.4 kb was not constitutive but inducible after irradiation (Figure 14D).

Overall, it was considered that Oct-1 is constitutively bound to the sites at –1.8 kb and –1.1 kb and plays a cooperative role with p53 in induction of the *CDKN1A* gene promoter in response to IR.

Modulation of Gene Expression After Exposure to Ionizing Radiation 143

and the pivotal role played by p53 have been elucidated. However, few studies investigated the *in vivo* modulation of gene expression profiles after exposure to IR and its underlying mechanisms. Especially importance of studies with low dose and low dose-rate IR which

C57BL/6J mice were continuously irradiated with γ-rays for 485 days at the dose-rates in the range of 0.032 – 13 μGy/min. The lowest dose rates were only about 10 times higher than the normal background level. Gene expression profiles in the kidney and testis from irradiated and unirradiated mice were analyzed, and differentially expressed genes were identified after the standard procedure of quality control of the data. By hierarchical cluster analysis of differentially expressed genes (Figure 15), it was suggested that expression of the

**5.1 Modulation of gene expression in the kidneys and testes of mice after**

Fig. 15. Hierarchical cluster analysis of genes whose expression was modulated after irradiation with a statistical significance in the kidney. A: Expression levels of 621 of significantly modulated genes are indicated by color. Green, yellow, and red colors represent low, medium, and high expression, respectively. On the basis of the expression pattern, genes were classified into 16 clusters. B: Expression profiles of genes in each of the 16 clusters are collectively shown. C: Gene Ontology categories significantly overlapped with the gene clusters are shown. No Gene Ontology categories significantly similar to the clusters other than C1, C10 and C13 were found. (Reproduced with permission of the

Japanese Radiation Research Society from Ref (Taki et al., 2009))

people may encounter in social life should be emphasized.

**exposure to IR**

Fig. 14. DNA/chromatin binding of Oct-1. *In vitro* DNA binding of Oct-1 to the sites at –1.8 kb (A) and –1.1 kb (B) was analyzed by EMSA. Sequences of the probe and competitor oligonucleotides for Oct-1 are described in the text. Arrows indicate the DNA-protein complex containing Oct-1. Arrowheads indicate the supershifts caused by addition of anti-Oct-1 antibodies. *In vivo* chromatin binding of Oct-1 and TP53 was analyzed by ChIP. MCF-7 cells were either not irradiated or irradiated with 2.0 Gy of X-rays. After a period of 1.5 h, the chromatin was immunoprecipitated with anti-Oct-1 antibodies (C and E) or anti-p53 antibodies (D). Normal rabbit IgG was used as a control. Binding of the factors to chromatin was assessed using site-specific PCR. (Reproduced with permission of Elsevier B. V. from Ref (Nenoi et al., 2009))

## **5.** *In vivo* **studies**

Regulated initiation of DNA replication (entry of S-phase) is one of the important cellular responses to IR. When DNA damages are produced by IR, multiple factors involved in cell cycle regulation such as CDKN1A which causes cell cycle arrest at G1/S are modulated. The so-called checkpoint regulation is thought to be necessary for sufficient repair of damaged DNA before DNA replication. However, the checkpoint regulation is absent in some of fully differentiated cells such as those *in vivo* organs. Thus, the cellular response to IR *in vivo* is much different from that in cultured cells, and it should have wide variety. In order to understand the difference in transcriptional responses to IR between tissues of mammals and also to reveal the underlying mechanisms for those differences, a microarray analysis of the global gene expression profile is a promising approach. There have been lots of *in vitro* studies investigating gene expression modulation after exposure to IR, where evident modulation of genes associated with DNA repair, stress response, cell cycle and apoptosis

Oct1

TP53

Oct1

PCR X-ray dose

antibody

Fig. 14. DNA/chromatin binding of Oct-1. *In vitro* DNA binding of Oct-1 to the sites at –1.8 kb (A) and –1.1 kb (B) was analyzed by EMSA. Sequences of the probe and competitor oligonucleotides for Oct-1 are described in the text. Arrows indicate the DNA-protein complex containing Oct-1. Arrowheads indicate the supershifts caused by addition of anti-Oct-1 antibodies. *In vivo* chromatin binding of Oct-1 and TP53 was analyzed by ChIP. MCF-7 cells were either not irradiated or irradiated with 2.0 Gy of X-rays. After a period of 1.5 h, the chromatin was immunoprecipitated with anti-Oct-1 antibodies (C and E) or anti-p53 antibodies (D). Normal rabbit IgG was used as a control. Binding of the factors to chromatin was assessed using site-specific PCR. (Reproduced with permission of Elsevier B. V. from

Regulated initiation of DNA replication (entry of S-phase) is one of the important cellular responses to IR. When DNA damages are produced by IR, multiple factors involved in cell cycle regulation such as CDKN1A which causes cell cycle arrest at G1/S are modulated. The so-called checkpoint regulation is thought to be necessary for sufficient repair of damaged DNA before DNA replication. However, the checkpoint regulation is absent in some of fully differentiated cells such as those *in vivo* organs. Thus, the cellular response to IR *in vivo* is much different from that in cultured cells, and it should have wide variety. In order to understand the difference in transcriptional responses to IR between tissues of mammals and also to reveal the underlying mechanisms for those differences, a microarray analysis of the global gene expression profile is a promising approach. There have been lots of *in vitro* studies investigating gene expression modulation after exposure to IR, where evident modulation of genes associated with DNA repair, stress response, cell cycle and apoptosis

N.R.IgG

 TP53/-1.4kb 0 Gy 2.0Gy

TP53

TP53 N.R.IgG

probe X-ray dose

competitor

PCR X-ray dose

antibody

Ref (Nenoi et al., 2009))

**5.** *In vivo* **studies**

antibody

TP53

Oct1

C D

 Oct-1/-1.8kb 0 Gy 2.0Gy

Oct1

N.R.IgG

Oct1 N.R.IgG

Oct-1/-1.8kb 0 Gy 0.2Gy 1.0Gy 2.0Gy

TP53

TP53

A B

Oct1

Oct1

Oct1

probe X-ray dose

competitor

antibody

TP53

Oct1

Oct-1/-1.1kb 0 Gy 0.2Gy 1.0Gy 2.0Gy

TP53

TP53

PCR X-ray dose

antibody

 Oct-1/-1.1kb 0 Gy 2.0Gy

Oct1

N.R.IgG

Oct1 N.R.IgG

Oct1

E

Oct1

Oct1

Oct1

TP53

Oct1

and the pivotal role played by p53 have been elucidated. However, few studies investigated the *in vivo* modulation of gene expression profiles after exposure to IR and its underlying mechanisms. Especially importance of studies with low dose and low dose-rate IR which people may encounter in social life should be emphasized.

#### **5.1 Modulation of gene expression in the kidneys and testes of mice after exposure to IR**

C57BL/6J mice were continuously irradiated with γ-rays for 485 days at the dose-rates in the range of 0.032 – 13 μGy/min. The lowest dose rates were only about 10 times higher than the normal background level. Gene expression profiles in the kidney and testis from irradiated and unirradiated mice were analyzed, and differentially expressed genes were identified after the standard procedure of quality control of the data. By hierarchical cluster analysis of differentially expressed genes (Figure 15), it was suggested that expression of the

Fig. 15. Hierarchical cluster analysis of genes whose expression was modulated after irradiation with a statistical significance in the kidney. A: Expression levels of 621 of significantly modulated genes are indicated by color. Green, yellow, and red colors represent low, medium, and high expression, respectively. On the basis of the expression pattern, genes were classified into 16 clusters. B: Expression profiles of genes in each of the 16 clusters are collectively shown. C: Gene Ontology categories significantly overlapped with the gene clusters are shown. No Gene Ontology categories significantly similar to the clusters other than C1, C10 and C13 were found. (Reproduced with permission of the Japanese Radiation Research Society from Ref (Taki et al., 2009))

Modulation of Gene Expression After Exposure to Ionizing Radiation 145

By use of microarray data, it is possible to pick up TFs whose recognition sequences are preferentially identified in the promoter of genes modulated by IR.  If Z>1.960 then p<0.05, and if Z>2.576 then p<0.01.

Table 1. Statistical analysis of difference in the proportion of genes containing recognition sequences for TFs between unchanged, up-regulated and down-regulated gene groups. Boxes of yellow and red indicate that the difference in populationproportion is significant with p < 0.05 and p < 0.01, respectively. (Reproduced with permission of the Japanese

Po is the population proportion defined by Po=(Noxx•Proxx+Noyy•Proyy)/(Noxx+Noyy), where xx and yy is replaced either by "unaff.", "down" or "up".

"No unaff.", "No down" and "No up" are the number of genes contained in the unaffected, down-regulated and up-regulated gene groups, respectively.

"Pro unaff.", "Pro down" and "Pro up" are the proportion of genes containing recognition sequence for the TF.

Radiation Research Society from Ref (Vares et al., 2011))

(NNNN · ·) (GGTAT · ·)

(p65) (AGAY · ·) (NGAT · ·) unchanged 0.538 0.032 0.591 0.430 0.505 0.280 0.086 0.935 0.624 0.656 0.742 down-regulated 0.595 0.119 0.571 0.643 0.595 0.119 0.000 0.976 0.571 0.500 0.738 na 93.000 93.000 93.000 93.000 93.000 93.000 93.000 93.000 93.000 93.000 93.000 nb 42.000 42.000 42.000 42.000 42.000 42.000 42.000 42.000 42.000 42.000 42.000 P 0.556 0.059 0.585 0.496 0.533 0.230 0.059 0.948 0.607 0.607 0.741 Z 0.624 1.977 0.218 2.289 0.969 2.053 1.960 0.988 0.575 1.717 0.047

E47 AP-4 Elk-1 NF-kappaB Evi-1 Evi-1 CDP S8 HFH-1 Brn-2 HSF1

**5.2 Transcription factors**

p=0.01: Z=2.576 p=0.05: Z=1.960

 Evi-1 recognition sequence, NGATANGANWAGATA <sup>g</sup> GR recognition sequence, NNNNNNCNNTNTGTNCTNN <sup>h</sup> GR recognition sequence, GGTACAANNTGTYCTK

Evi-1 recognition sequence, AGAYAAGATAA

e

e

d

c

b

a

genes involved in mitochondrial oxidative phosphorylation was elevated in the kidney after irradiation at the dose-rates of 0.65 μGy/min (the level of dose rate comparable to that in the spaceshuttle) and 13 μGy/min. A particular mitochondrial response may have occurred after low dose rate irradiation. It was reported that several cell cycle-related genes are upregulated in the kidney after acute whole-body irradiation with 10 Gy of γ-rays (Zhao et al., 2006). However, we did not observe such gene modulation after irradiation at 13μGy/min We did not either extract clusters of significantly modulated genes that overlapped with the Gene Ontology category "cell cycle" (Figure 15C). These results suggested that modulation of the cell cycle-related genes *in vivo* following exposure to radiation may have a dose-rate threshold.

It was also demonstrated that alteration of the gene expression profile in the testis was largely different from that in the kidney. Gene Ontology categories "DNA metabolism", "response to DNA damage" and "DNA replication" were significantly overlapped to upregulated genes in the testis in a dose rate-dependent manner (Figure 16). These results provide fundamental insight into the organ-specific responses to IR.

Fig. 16. Hierarchical cluster analysis of genes whose expression was significantly modulated after irradiation in the testis. A: Expression levels of 2056 of significantly modulated genes are indicated by color. On the basis of the expression pattern, genes were classified into 16 clusters. The cluster 12 contained no gene. B: Expression profiles of genes in each of the 16 clusters are collectively shown. C: Gene Ontology categories significantly overlapped with the gene clusters C1, C2, C9 and C10 are shown. (Reproduced with permission of the Japanese Radiation Research Society from Ref (Taki et al., 2009))

#### **5.2 Transcription factors**

144 Current Topics in Ionizing Radiation Research

genes involved in mitochondrial oxidative phosphorylation was elevated in the kidney after irradiation at the dose-rates of 0.65 μGy/min (the level of dose rate comparable to that in the spaceshuttle) and 13 μGy/min. A particular mitochondrial response may have occurred after low dose rate irradiation. It was reported that several cell cycle-related genes are upregulated in the kidney after acute whole-body irradiation with 10 Gy of γ-rays (Zhao et al., 2006). However, we did not observe such gene modulation after irradiation at 13μGy/min We did not either extract clusters of significantly modulated genes that overlapped with the Gene Ontology category "cell cycle" (Figure 15C). These results suggested that modulation of the cell cycle-related genes *in vivo* following exposure to radiation may have a dose-rate

It was also demonstrated that alteration of the gene expression profile in the testis was largely different from that in the kidney. Gene Ontology categories "DNA metabolism", "response to DNA damage" and "DNA replication" were significantly overlapped to upregulated genes in the testis in a dose rate-dependent manner (Figure 16). These results

provide fundamental insight into the organ-specific responses to IR.

Control 0.032μGy/min

Japanese Radiation Research Society from Ref (Taki et al., 2009))

13μGy/min 0.65μGy/min Control 0.032μGy/min

13μGy/min 0.65μGy/min

Fig. 16. Hierarchical cluster analysis of genes whose expression was significantly modulated after irradiation in the testis. A: Expression levels of 2056 of significantly modulated genes are indicated by color. On the basis of the expression pattern, genes were classified into 16 clusters. The cluster 12 contained no gene. B: Expression profiles of genes in each of the 16 clusters are collectively shown. C: Gene Ontology categories significantly overlapped with the gene clusters C1, C2, C9 and C10 are shown. (Reproduced with permission of the

threshold.

2056 genes

> Control 0.032μGy/min 13μGy/min 0.65μGy/min

By use of microarray data, it is possible to pick up TFs whose recognition sequences are preferentially identified in the promoter of genes modulated by IR. These TFs are suggested

a "Pro unaff.", "Pro down" and "Pro up" are the proportion of genes containing recognition sequence for the TF.

b "No unaff.", "No down" and "No up" are the number of genes contained in the unaffected, down-regulated and up-regulated gene groups, respectively.

c Po is the population proportion defined by Po=(Noxx•Proxx+Noyy•Proyy)/(Noxx+Noyy), where xx and yy is replaced either by "unaff.", "down" or "up".

d Z is the random variable defined by Z=|Proxx-Proyy|/sqrt(Po•(1-Po)•(1/Noxx+1/Noyy)), where xx and yy is replaced either by "unaff.", "down" or "up". If Z>1.960 then p<0.05, and if Z>2.576 then p<0.01. e Evi-1 recognition sequence, AGAYAAGATAA

e Evi-1 recognition sequence, NGATANGANWAGATA

<sup>g</sup> GR recognition sequence, NNNNNNCNNTNTGTNCTNN <sup>h</sup> GR recognition sequence, GGTACAANNTGTYCTK

p=0.01: Z=2.576 p=0.05: Z=1.960

Table 1. Statistical analysis of difference in the proportion of genes containing recognition sequences for TFs between unchanged, up-regulated and down-regulated gene groups. Boxes of yellow and red indicate that the difference in populationproportion is significant with p < 0.05 and p < 0.01, respectively. (Reproduced with permission of the Japanese Radiation Research Society from Ref (Vares et al., 2011))

Modulation of Gene Expression After Exposure to Ionizing Radiation 147

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Fei, P. & El-Deiry, W. S. (2003). P53 and radiation responses. *Oncogene*. Vol.22, No.37,

Fornace, A. J. Jr, Alamo, I. Jr & Hollander, M. C. (1988). DNA damage-inducible transcripts

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Hartwell, L. H. & Weinert, T. A. (1989). Checkpoints: controls that ensure the order of cell

Nenoi, M., Daino, K., Nakajima, T., Wang, B., Taki, K. & Kakimoto, A. (2009). Involvement

Oda, Y., Nakamura, S., Oki, I., Kato, T. & Shinagawa, H. (1985). Evaluation of the new

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ionizing radiation. *Biochim Biophys Acta*. Vol.1789, No.3, pp.225-223.

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p21Waf1/Cip1 with PCNA. *Oncogene*. Vo.11, No.10, pp.1931-1937.

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d'Ari, R. (1985). The SOS system. *Biochimie.* Vol.67, No.3-4, pp.343-347.

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*Journal of Radiation Research*. Vol.50, No.3, pp.241-252.

*Radiation Research*. Vol.52, No.2, pp.249-256.

Gadd45 with PCNA and evidence for competitive interaction of Gadd45 and

mRNA by a low dose of ionizing radiation is due to their dose-dependent post-

GADD45A gene and its enhancement after ionizing irradiation are mediated by

in mammalian cells. *Proceedings of the National Academy of Sciences.* 8Vol.5, No.23,

J. & Holbrook, N.J. (1989). Mammalian genes coordinately regulated by growth arrest signals and DNA-damaging agents. *Molecular and Cellular Biology*. Vo.9,

of Oct-1 in the regulation of CDKN1A in response to clinically relevant doses of

system (umu-test) for the detection of environmental mutagens and carcinogens.

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Tanaka, K., Ichinohe, K., Nakamura, S., Tanaka, S., Magae, J., Kakimoto, A. & Nenoi, M. (2009), Microarray analysis of differentially expressed genes in the kidneys and testes of mice after long-term irradiation with low-dose-rate γ-rays.

Tanaka, K., Ichinohe, K., Nakamura, S., Tanaka, S. & Nenoi M. (2011). Transcription Factor-recognition Sequences Potentially Involved in Modulation of Gene Expression after Exposure to Low-dose-rate γ-rays in the Mouse Liver. *Journal of*

**8. References**

pp.5774-5783.

pp.8800-8804.

1380.

No.10, pp.4196-4203.

to be functionally involved in gene modulations. Then, nucleotide sequences in the neighboring region of the up-regulated, down-regulated, and unaffected genes were retrieved from the Entrez Gene database, and recognition sequences for TFs were searched on the database using the TFSEARCH. As a result, 21 potential TF-binding sites with significantly different incidence between the three gene groups (up-regulated, downregulated and unaffected gene groups) could be identified (Table 1).

The binding sites for sterol regulatory element-binding protein 1 (SREBP-1), aryl hydrocarbon receptor (AhR/Ar) and olfactory 1 (Olf-1) were suggested to be involved in up-regulation, while the binding sites for glucocorticoid receptor (GR) and hepatocyte nuclear factor 1 (HNF-1) were suggested to be involved in down-regulation of the genes. In addition, the binding sites for activating enhancer-binding protein 4 (AP-4), nuclear factorkappaB (NFκB), and early growth response 3 (Egr-3) were correlated with modulation of gene expression regardless of the direction of modulation. Among them, GR and/or SREBP-1 are interesting TFs in association with the recent findings of a trend of elevated body weight in irradiated mice. GR is known to be associated to the metabolic syndrome. Similarly, SREBP-1 is also involved in metabolic process by mediating the final regulatory step in LDL metabolism. Alteration of TF activity of GR and/or SREBP-1 as well as the related signaling pathways after exposure to IR may be the underlying mechanisms for this metabolic process.

## **6. Conclusion**

Studies of modulated gene expression after exposure to IR have historically played an important role in elucidating the molecular mechanisms underlying cellular IR response. Especially finding of transcriptional network derived from p53 made a deep impact on understanding of biological defence system as well as development of radiation hazards such as radiation carcinogenesis. Recent technologies enable us simultaneously monitor modulation of gene expression across essentially the entire genome. It was revealed that so many TFs are involved in gene regulation *in vivo* that gene expression is finely modulated depending on tissues. However no single consistent picture of IR response has not been obtained because variety of experimental models, experimental conditions and data analyses have been employed. As a future perspective in this research area, carefully worked out methods for data analysis, data mining and construction of signalling network are strongly need. In addition, protein researches, especially a comprehensive analysis of protein expression, modification, sub-cellular localization is required, because these data cannot be obtained from the researches on transcriptional regulation.

## **7. Acknowledgment**

This article was prepared with a support by the Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (21510060), the Budget for Nuclear Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan based on screening and counseling by the Atomic Energy Commission, and under contract with the Aomori Prefectural Government, Japan.

## **8. References**

146 Current Topics in Ionizing Radiation Research

to be functionally involved in gene modulations. Then, nucleotide sequences in the neighboring region of the up-regulated, down-regulated, and unaffected genes were retrieved from the Entrez Gene database, and recognition sequences for TFs were searched on the database using the TFSEARCH. As a result, 21 potential TF-binding sites with significantly different incidence between the three gene groups (up-regulated, down-

The binding sites for sterol regulatory element-binding protein 1 (SREBP-1), aryl hydrocarbon receptor (AhR/Ar) and olfactory 1 (Olf-1) were suggested to be involved in up-regulation, while the binding sites for glucocorticoid receptor (GR) and hepatocyte nuclear factor 1 (HNF-1) were suggested to be involved in down-regulation of the genes. In addition, the binding sites for activating enhancer-binding protein 4 (AP-4), nuclear factorkappaB (NFκB), and early growth response 3 (Egr-3) were correlated with modulation of gene expression regardless of the direction of modulation. Among them, GR and/or SREBP-1 are interesting TFs in association with the recent findings of a trend of elevated body weight in irradiated mice. GR is known to be associated to the metabolic syndrome. Similarly, SREBP-1 is also involved in metabolic process by mediating the final regulatory step in LDL metabolism. Alteration of TF activity of GR and/or SREBP-1 as well as the related signaling pathways after exposure to IR may be the underlying mechanisms for this

Studies of modulated gene expression after exposure to IR have historically played an important role in elucidating the molecular mechanisms underlying cellular IR response. Especially finding of transcriptional network derived from p53 made a deep impact on understanding of biological defence system as well as development of radiation hazards such as radiation carcinogenesis. Recent technologies enable us simultaneously monitor modulation of gene expression across essentially the entire genome. It was revealed that so many TFs are involved in gene regulation *in vivo* that gene expression is finely modulated depending on tissues. However no single consistent picture of IR response has not been obtained because variety of experimental models, experimental conditions and data analyses have been employed. As a future perspective in this research area, carefully worked out methods for data analysis, data mining and construction of signalling network are strongly need. In addition, protein researches, especially a comprehensive analysis of protein expression, modification, sub-cellular localization is required, because these data

This article was prepared with a support by the Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (21510060), the Budget for Nuclear Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan based on screening and counseling by the Atomic Energy Commission, and under

regulated and unaffected gene groups) could be identified (Table 1).

cannot be obtained from the researches on transcriptional regulation.

contract with the Aomori Prefectural Government, Japan.

metabolic process.

**6. Conclusion**

**7. Acknowledgment**

**The Role of MicroRNAs in the Cellular**

**Response to Ionizing Radiations**

*Center of Genetics and Molecular and Cellular Physiology, CNRS, University Claude Bernard Lyon I, University of Lyon, Villeurbanne,*

Ionising irradiation is a major issue in our society. Although public attention mainly focuses on it when industrial disasters or medical accidents occur, or in military contexts, it is also a permanent natural phenomenon and a powerful tool for diagnosis and therapy, notably in oncology. Understanding the action mechanisms and consequences of these radiations in living organisms is a major aim for public health and environmental conservation. Conceptions have begun to change over the past three decades. Previously, the central theory focused on the irradiated cells themselves, postulating that the biological effects are direct consequences of DNA damage when repair processes fail. More recently, however, there has been accumulating evidence that important biological consequences arise in cells with no direct radiation exposure (Kennedy et al., 1980; Nagasawa & Little, 1992; review in Wright, 2010). On the one hand, progeny cells over many generations present radiationinduced genomic instability, increasing mutation rates and chromosomal abnormality implicating reactive oxygen species. On the other hand, both neighbouring and remote cells are subject to a radiation-induced bystander effect, induced by signals from directly exposed cells *via* a variety of communication pathways. Both cell response phenomena point to an implication of epigenetic mechanisms, such DNA methylation, chromatin remodelling or

MicroRNA was discovered nearly 20 years ago (Lee et al., 1993; Wightman et al., 1993). The growing family of small RNAs, including small interfering RNA (siRNA), piwi-interacting RNA (piRNA), repeat-associated-siRNA (rasiRNA) and heterochromatic small RNA (hcRNA), forms the most abundant class of endogenous RNA in metazoans, but has also been characterised in plants, unicellular algae (Zhao et al., 2007), DNA viruses (Pfeffer et al., 2004) and, controversially, in retroviruses (Klase et al., 2007). MicroRNAs are involved in numerous cellular processes such as development, viral infection, apoptosis and stress response, by regulating protein-coding gene expression at post-transcriptional and translational levels (review in Bushati & Cohen, 2007). They are also differentially expressed between tumour cells and normal counterparts in both benign proliferation and cancer (Lu et al., 2005). Due to this accumulating evidence, it is worthwhile studying microRNA response to ionising radiation, and this was started few years ago. This chapter will review microRNA studies relating to ionising radiation and discuss the perspectives in this field.

**1. Introduction**

small RNA regulation (Aypar et al., 2011).

Nicolas Joly-Tonetti and Jérôme Lamartine

*France*

Zhan, Q., Bae, I., Kastan, M. B. & Fornace, A. J. Jr. (1994). The p53-dependent gamma-ray response of GADD45. *Cancer Research*. Vol.54, No.10, pp.2755-2760. **9**

## **The Role of MicroRNAs in the Cellular Response to Ionizing Radiations**

Nicolas Joly-Tonetti and Jérôme Lamartine *Center of Genetics and Molecular and Cellular Physiology, CNRS, University Claude Bernard Lyon I, University of Lyon, Villeurbanne, France*

#### **1. Introduction**

148 Current Topics in Ionizing Radiation Research

Zhan, Q., Bae, I., Kastan, M. B. & Fornace, A. J. Jr. (1994). The p53-dependent gamma-ray

Ionising irradiation is a major issue in our society. Although public attention mainly focuses on it when industrial disasters or medical accidents occur, or in military contexts, it is also a permanent natural phenomenon and a powerful tool for diagnosis and therapy, notably in oncology. Understanding the action mechanisms and consequences of these radiations in living organisms is a major aim for public health and environmental conservation. Conceptions have begun to change over the past three decades. Previously, the central theory focused on the irradiated cells themselves, postulating that the biological effects are direct consequences of DNA damage when repair processes fail. More recently, however, there has been accumulating evidence that important biological consequences arise in cells with no direct radiation exposure (Kennedy et al., 1980; Nagasawa & Little, 1992; review in Wright, 2010). On the one hand, progeny cells over many generations present radiationinduced genomic instability, increasing mutation rates and chromosomal abnormality implicating reactive oxygen species. On the other hand, both neighbouring and remote cells are subject to a radiation-induced bystander effect, induced by signals from directly exposed cells *via* a variety of communication pathways. Both cell response phenomena point to an implication of epigenetic mechanisms, such DNA methylation, chromatin remodelling or small RNA regulation (Aypar et al., 2011).

MicroRNA was discovered nearly 20 years ago (Lee et al., 1993; Wightman et al., 1993). The growing family of small RNAs, including small interfering RNA (siRNA), piwi-interacting RNA (piRNA), repeat-associated-siRNA (rasiRNA) and heterochromatic small RNA (hcRNA), forms the most abundant class of endogenous RNA in metazoans, but has also been characterised in plants, unicellular algae (Zhao et al., 2007), DNA viruses (Pfeffer et al., 2004) and, controversially, in retroviruses (Klase et al., 2007). MicroRNAs are involved in numerous cellular processes such as development, viral infection, apoptosis and stress response, by regulating protein-coding gene expression at post-transcriptional and translational levels (review in Bushati & Cohen, 2007). They are also differentially expressed between tumour cells and normal counterparts in both benign proliferation and cancer (Lu et al., 2005). Due to this accumulating evidence, it is worthwhile studying microRNA response to ionising radiation, and this was started few years ago. This chapter will review microRNA studies relating to ionising radiation and discuss the perspectives in this field.

The Role of MicroRNAs in the Cellular Response to Ionizing Radiations 151

presenting mismatches or wobbles, which is well conserved in all animal species. Like messenger RNA (mRNA) genes, pri-miR is capped and polyadenylated (Bracht et al., 2004; Cai et al., 2004). MicroRNAs are clustered on the genome and many are transcribed in polycistron RNA. One of the most frequently studied is the miR-17-92 cluster, which encompasses 6 stem-loops and plays an important role in non-solid and solid cancer (He et al., 2005; review in Mendell, 2008). Although few data are available about their transcription regulation and promoters, some microRNA are known to respond to conventional transcription factors, such as p53 (Raver-Shapira et al., 2007; Corney et al., 2007; Xiao et al., 2011) or MYC (O'Donnell et al., 2005), and are subject to chromatin remodelling (review in

The sequence transcribed in the pri-miR does not always correspond to the mature microRNA because of adenosine-to-inosine editing by the dsRNA adenosine desaminase ADAR1 or ADAR2 (review in Nishikura, 2010). Both enzymes catalyse adenine hydrolytic desamination in hypoxanthine (Bass & Weintraub, 1988) which pairs cytosine, creating a non-Watson-Crick pairing I:U. This wobble affects the secondary structure of pri-miR, with consequences for microRNA maturation and action. Depending on the site of edition on primiR, different consequences are possible, for instance impairment of microRNA maturation (Yang et al., 2006; Kawahara et al., 2007b) or modification of the complete target mRNA list

ADAR1 and ADAR2 tend to edit distinct sites and are subjected to regulation since ADAR2 inhibits ADAR1 activity (Kawahara et al., 2007a) and inflammation can increase ADAR1 isoform p150 expression (Yang et al., 2003). Moreover, ADAR1 can occur on pre-miR, in nucleus or cytoplasm. This regulated ADAR-drive edition may explain edition tissuespecificity: depending on the organ, only a few or all microRNA precursors will be desaminated at various sites (Kawahara et al., 2007a; Kawahara et al., 2007b). Although a pioneering general survey of microRNA precursors found editing in 6% of the pri-miRs examined (Blow et al., 2006), *in vitro* studies showed half of pri-miRs to be editable (Yang et al., 2006). This implies that inflammation may impact microRNA biogenesis and mRNA

Canonical maturation of animal pri-miR is then a sequential two-step process that take place in the nucleus and cytoplasm, performed by two bacterial homologue type-III RNase enzymes in partnership with other proteins, with a strong intermediate check-point,

Most microRNA genes are located in intronic regions of protein-coding genes or in noncoding regions of the genome (Rodriguez et al., 2004; Baskerville & Bartel, 2005). Based on pre-miR end characteristics, the endonuclease DROSHA has been identified as a pri-miR trimming enzyme: it identifies the pri-miR hairpin shape and produces what is called microRNA precursor (pre-miR), approximately 70 nucleotides in length (Lee et al., 2003). DROSHA acts in association with many proteins, leading to the formation of microprocessor complexes, including in particular DiGeorge syndrome critical region gene 8 (DGCR8), the homologue of *Drosophila melanogaster* pasha protein (Han et al., 2004; Gregory et al., 2004). The DROSHA-DGCR8 multimeric complex displays the most active *in vitro* pri-miR maturation activity. DGCR8 is therefore thought to be a molecular anchor (Han et al., 2006), positioning DROSHA at 10 bp from the hairpin base for trimming. In an alternative,

respectively producing a microRNA precursor (pre-miR) and the mature microRNA.

**2.1.2 DROSHA – Dependent and independent pathways to pre-miR**

Sato et al., 2011).

(Kawahara et al., 2007a).

targeting.

Before reviewing miRNA function in radiation response, we will begin with an account of the biogenesis of microRNAs, their mode of action in association with Argonaute protein, their function and the methods currently used to study them.

## **2. General information on microRNA: Biosynthesis, function and methods of investigation**

## **2.1 MicroRNA biogenesis and microRNA ribonucleoprotein (miRNP) assembly**

MicroRNA biogenesis is the cellular process producing active 22-nt long microRNA from large transcripts coded by the cell genome (review in Bartel, 2004; cf. fig. 1). Given differing microRNA profiles according to tissue and cell type and in response to environmental change, microRNA biogenesis can be seen to be a regulated process. Moreover, the bad correlation between the primary transcript synthesis rate, called pri-miR, and the mature microRNA reveals significant posttranscriptional regulation (Thomson et al., 2006).

Fig. 1. MicroRNA biogenesis pathway

## **2.1.1 Pri-miR transcription and edition**

Although a few microRNAs are transcribed by non-coding (nc) RNA associated with RNA polymerase III (Borchert et al., 2006), almost all are transcribed by RNA polymerase II (Lee et al., 2004). This primary transcript displays at least one stem-loop hairpin structure,

Before reviewing miRNA function in radiation response, we will begin with an account of the biogenesis of microRNAs, their mode of action in association with Argonaute protein,

**2. General information on microRNA: Biosynthesis, function and methods of**

MicroRNA biogenesis is the cellular process producing active 22-nt long microRNA from large transcripts coded by the cell genome (review in Bartel, 2004; cf. fig. 1). Given differing microRNA profiles according to tissue and cell type and in response to environmental change, microRNA biogenesis can be seen to be a regulated process. Moreover, the bad correlation between the primary transcript synthesis rate, called pri-miR, and the mature

Although a few microRNAs are transcribed by non-coding (nc) RNA associated with RNA polymerase III (Borchert et al., 2006), almost all are transcribed by RNA polymerase II (Lee et al., 2004). This primary transcript displays at least one stem-loop hairpin structure,

**2.1 MicroRNA biogenesis and microRNA ribonucleoprotein (miRNP) assembly**

microRNA reveals significant posttranscriptional regulation (Thomson et al., 2006).

their function and the methods currently used to study them.

**investigation**

Fig. 1. MicroRNA biogenesis pathway

**2.1.1 Pri-miR transcription and edition**

presenting mismatches or wobbles, which is well conserved in all animal species. Like messenger RNA (mRNA) genes, pri-miR is capped and polyadenylated (Bracht et al., 2004; Cai et al., 2004). MicroRNAs are clustered on the genome and many are transcribed in polycistron RNA. One of the most frequently studied is the miR-17-92 cluster, which encompasses 6 stem-loops and plays an important role in non-solid and solid cancer (He et al., 2005; review in Mendell, 2008). Although few data are available about their transcription regulation and promoters, some microRNA are known to respond to conventional transcription factors, such as p53 (Raver-Shapira et al., 2007; Corney et al., 2007; Xiao et al., 2011) or MYC (O'Donnell et al., 2005), and are subject to chromatin remodelling (review in Sato et al., 2011).

The sequence transcribed in the pri-miR does not always correspond to the mature microRNA because of adenosine-to-inosine editing by the dsRNA adenosine desaminase ADAR1 or ADAR2 (review in Nishikura, 2010). Both enzymes catalyse adenine hydrolytic desamination in hypoxanthine (Bass & Weintraub, 1988) which pairs cytosine, creating a non-Watson-Crick pairing I:U. This wobble affects the secondary structure of pri-miR, with consequences for microRNA maturation and action. Depending on the site of edition on primiR, different consequences are possible, for instance impairment of microRNA maturation (Yang et al., 2006; Kawahara et al., 2007b) or modification of the complete target mRNA list (Kawahara et al., 2007a).

ADAR1 and ADAR2 tend to edit distinct sites and are subjected to regulation since ADAR2 inhibits ADAR1 activity (Kawahara et al., 2007a) and inflammation can increase ADAR1 isoform p150 expression (Yang et al., 2003). Moreover, ADAR1 can occur on pre-miR, in nucleus or cytoplasm. This regulated ADAR-drive edition may explain edition tissuespecificity: depending on the organ, only a few or all microRNA precursors will be desaminated at various sites (Kawahara et al., 2007a; Kawahara et al., 2007b). Although a pioneering general survey of microRNA precursors found editing in 6% of the pri-miRs examined (Blow et al., 2006), *in vitro* studies showed half of pri-miRs to be editable (Yang et al., 2006). This implies that inflammation may impact microRNA biogenesis and mRNA targeting.

Canonical maturation of animal pri-miR is then a sequential two-step process that take place in the nucleus and cytoplasm, performed by two bacterial homologue type-III RNase enzymes in partnership with other proteins, with a strong intermediate check-point, respectively producing a microRNA precursor (pre-miR) and the mature microRNA.

### **2.1.2 DROSHA – Dependent and independent pathways to pre-miR**

Most microRNA genes are located in intronic regions of protein-coding genes or in noncoding regions of the genome (Rodriguez et al., 2004; Baskerville & Bartel, 2005). Based on pre-miR end characteristics, the endonuclease DROSHA has been identified as a pri-miR trimming enzyme: it identifies the pri-miR hairpin shape and produces what is called microRNA precursor (pre-miR), approximately 70 nucleotides in length (Lee et al., 2003). DROSHA acts in association with many proteins, leading to the formation of microprocessor complexes, including in particular DiGeorge syndrome critical region gene 8 (DGCR8), the homologue of *Drosophila melanogaster* pasha protein (Han et al., 2004; Gregory et al., 2004). The DROSHA-DGCR8 multimeric complex displays the most active *in vitro* pri-miR maturation activity. DGCR8 is therefore thought to be a molecular anchor (Han et al., 2006), positioning DROSHA at 10 bp from the hairpin base for trimming. In an alternative,

The Role of MicroRNAs in the Cellular Response to Ionizing Radiations 153

There are thermodynamic differences in base-pairing stability on either side of the duplex, and the strand with the greater 5' stability will be preferentially sorted in the effecter complex, called miRNP or RISC. Mismatches in the seed region (nt 2-8) and 3' mid region (nt 12-15) favour strand separation (Kawamata et al., 2009). Loading is highly sensitive to the presence of phosphate at the 5' end, which completely prevents loading if lacking. The loaded strand is called the "guide" strand, and the other, "passenger", strand is discarded. However, the loaded strand is not always the same. Evolutionary pressure on both strand seed regions and their complementary sequences on target mRNA reveals that microRNA originally from opposite strands can regulate different target genes in a coordinated fashion (Okamura et al., 2008). In a former nomenclature, when there is a predominant loaded strand in terms of relative abundance, the mature sequence is named according to the miR form (e.g., miR-17 or let-7b) whereas the opposite arm, if loaded, is called star microRNA and named according to the miR\* form (e.g., miR-17\* or let-7b\*) (Ambros et al., 2003a). However, miR/miR\* nomenclature is phased out in favour of the -5p/-3p nomenclature, the suffixes 5p and 3p referring to the 5' and 3' sides of pre-miR, respectively (e.g., miR-28-5p or miR-28-3p). Argonaute proteins associate with several factors in the RISC, such as GW182 family proteins TNRC6 (Landthaler et al., 2008) or PABPC1 (Huntzinger et al., 2010). TNRC6

MicroRNAs are fundamental posttranscriptional regulators of gene expression (reviewed in Fabian et al., 2010). Although some studies reported that, in particular situations, microRNAs can activate protein translation (Vasudevan et al., 2007; Ørom et al., 2008; Henke et al., 2008) or can be reimported into the nucleus (Hwang et al., 2006), cytoplasm microRNA-mediated gene repression, by mRNA decay or translation repression, is the general mechanism of action. How one of these two mechanisms or both is brought into play, however, is not well understood. In addition, they have been described as reducing stochastic gene expression noise (Cui et al., 2007) and as possible modifiers of gene regulation paradigms, acting *in cis* and *trans* to regulate of MRE-containing RNA (Salmena

In animals, microRNA-mediated gene repression involves two mechanisms: translation inhibition of target mRNA and decay of mRNA after deadenylation. Although the initial dichotomy between animals and plants highlighted translation inhibition by imperfect matching in the former and mRNA decay by perfect matching in the latter, it is now clear that animal microRNAs mainly act on mRNA stability (Bagga et al., 2005; Guo et al., 2010) whereas plants exhibit translation inhibition mechanisms without notable effect on mRNA

Recent debate has focused on three possible mechanisms of target mRNA translational inhibition: inhibition of translation initiation by disturbing mRNA circularisation or ribosome assembly, or ribosome drop-off during translation elongation. MicroRNA-

Several studies revealed that mRNA that lack a functional m7Gppp or present a nonfunctional ApppG cap structure or whose translation is cap-independent are less sensitive to

**2.1.5 Strand selection and miRNP assembly**

lead miRNP localisation to cytoplasmic P-bodies (Liu et al., 2005).

**2.2.1 Mechanistic aspects of microRNA repression**

mediated mRNA degradation is also still in debate.

stability (Brodersen et al., 2008).

**2.2 MicroRNA function**

et al., 2011).

DROSHA-independent process, pre-miRs are generated during mRNA maturation by the formation of a short stem-loop intron, called a mirtron, then linearised by RNA lariat debranching enzyme (DBR1) (Okamura et al., 2007; Ruby et al., 2007). The resulting pre-miR structure is similar to that released by DROSHA. Recent studies have discussed coupling between microprocessor and spliceosome, trimming pre-miR into a larger intron before splicing and enhancing intron degradation (Morlando et al., 2008; Kataoka et al., 2009). In addition, other complex partners appear to be crucial for microRNA subset expression. DROSHA associates with DEAD-box helicase p68 (DDX5) and p70 (DDX17), which are involved in processing one subclass of pri-miR (Fukuda et al., 2007). Pri-miR maturation can also be influenced by various nuclear factors in response to stimuli, such as pri-miR-21 enhancement proceeding by R-SMAD in response to TGF-β and BMP in vascular smooth muscle cells (Davis et al., 2008) or pri-let-7 family excision inhibition by LIN28 in stem cells and tumours (Viswanathan et al., 2008). P53 also associates with DROSHA and helicase p68, enhancing maturation of growth-suppressive microRNA (Suzuki et al., 2009). For half of the microRNA family, DROSHA also cuts a microRNA-offset RNA (moRNA or moR) created from sequences immediately adjacent to pre-miR (Shi et al., 2009). These are systematically generated with RNA of early evolutionary origin (Langenberger et al., 2009), massively enriched in the nucleus (Taft et al., 2010), and sometimes more expressed than hairpinsharing microRNA (Umbach et al., 2010); their function remains unknown.

#### **2.1.3 Nuclear export checkpoint**

Like smaller shuttling RNA (Izaurralde et al., 1997), pre-miR is actively exported by nucleocytoplasmic transport in a RAN-GTP dependent manner (Yi et al., 2003). The karyopherin family protein exportin-5 (XPO5) cargoes pre-miR sequence-independently (Bohnsack et al., 2004; Lund et al., 2004) and controls the quality of pre-miR: at one end, pre-miR needs at least a 18-nt double-stranded stem for effective nuclear export (Zeng & Cullen, 2004); and at the other end, the 5' protruding end and 3' long end inhibit shuttle (Lund et al., 2004; Zeng & Cullen, 2004). The terminal loop structure and the distance to the hairpin base are the only parts unchecked. Moreover, exportin-5 binding protects premiR against degradation (Zeng & Cullen, 2004; Lee et al., 2011). Pre-miR can be subject to edition by ADAR, before exportation by its two isoforms and then by p150 (Kawahara et al., 2007b).

#### **2.1.4 Cytoplasmic generation and loading of microRNA duplex**

In the cytoplasm, a class III RNAse III enzyme, DICER1, recognises pre-miR and cuts both strands of the imperfect duplex at about two helical turns (~22 nucleotides) from the base of the hairpin, creating a short RNA duplex with 3' 2-nucleotide overhang ends (Hutvágner et al., 2001; MacRae et al., 2006). This cleavage, less reliable than with DROSHA, creates a 3' polymorphism for 5'-strand-generated microRNA. DICER1 associates several proteins, like TRBP (Chendrimada et al., 2005), which contribute to his function. The imperfect duplex associates ATP-dependently (Kawamata et al., 2009; Yoda et al., 2010) with Argonaute protein to form an immature complex called microRNA ribonucleoprotein precursor (premiRNP) or RNA-induced silencing complex precursor (pre-RISC or pre-miRISC, with reference to mRNA cleavage siRNA). Central mismatch at position 10-11 (Tomari et al., 2007) and Hsc70/Hsp90 chaperone machinery (Iki et al., 2010; Iwasaki et al., 2010) contribute to duplex loading.

## **2.1.5 Strand selection and miRNP assembly**

152 Current Topics in Ionizing Radiation Research

DROSHA-independent process, pre-miRs are generated during mRNA maturation by the formation of a short stem-loop intron, called a mirtron, then linearised by RNA lariat debranching enzyme (DBR1) (Okamura et al., 2007; Ruby et al., 2007). The resulting pre-miR structure is similar to that released by DROSHA. Recent studies have discussed coupling between microprocessor and spliceosome, trimming pre-miR into a larger intron before splicing and enhancing intron degradation (Morlando et al., 2008; Kataoka et al., 2009). In addition, other complex partners appear to be crucial for microRNA subset expression. DROSHA associates with DEAD-box helicase p68 (DDX5) and p70 (DDX17), which are involved in processing one subclass of pri-miR (Fukuda et al., 2007). Pri-miR maturation can also be influenced by various nuclear factors in response to stimuli, such as pri-miR-21 enhancement proceeding by R-SMAD in response to TGF-β and BMP in vascular smooth muscle cells (Davis et al., 2008) or pri-let-7 family excision inhibition by LIN28 in stem cells and tumours (Viswanathan et al., 2008). P53 also associates with DROSHA and helicase p68, enhancing maturation of growth-suppressive microRNA (Suzuki et al., 2009). For half of the microRNA family, DROSHA also cuts a microRNA-offset RNA (moRNA or moR) created from sequences immediately adjacent to pre-miR (Shi et al., 2009). These are systematically generated with RNA of early evolutionary origin (Langenberger et al., 2009), massively enriched in the nucleus (Taft et al., 2010), and sometimes more expressed than hairpin-