Mammalian demethylation studies have recently benefited from a flurry of papers detailing 5hmC and its potential role in demethylation. This base is produced from the further modification of 5mC by TET enzymes (TET1-3), and appears to allow demethylation to occur through a series of intermediate conversions (He et al., 2011; Ito et al., 2011; Kriaucionis & Heintz, 2009; Tahiliani et al., 2009). It has been suggested that 5hmC is converted to 5-formylcytosine (5fC), then 5-carboxylcytosine (5caC); which is subsequently substituted for cytosine by the action of TDG (Ito et al., 2011; He et al., 2011). This pathway is yet to be studied in zebrafish, though at least one unpublished report exists claiming the presence of 5hmC in zebrafish (Yen & Jia, 2010). All 3 mammalian TET proteins have homologous sequences in zebrafish (see Table 1). Mammalian 5hmC could be suggested to have a dual functional role, as it does not appear to be immediately converted, and is present at considerably greater levels than 5fC and 5caC. Such roles could include repression of transcription SIN3A recruitment (Williams et al., 2011) and hypomethylation of target regions allowing PRC2 recruitment (Wu et al., 2011). It will be interesting to explore the distribution of 5hmC and TET binding/expression profiles in zebrafish. Immunostaining of mitotic chromosome spreads of mouse pre-implantation embryos demonstrated that paternal 5hmC is gradually lost during pre-implantation development (Inoue & Zhang, 2011). Here it is suggested that although the conversion of 5mC to 5hmC in zygotes is an enzyme-catalysed process, loss of 5hmC during pre-implantation may be a

DNA methylation has long been studied in the chicken, with the globin genes being the primary model of study; and is complimented by structural studies (Heitmann et al., 2003;

The first evidence for functionally relevant DNA methylation in chicken came when methylation sensitive restriction enzymes were used to study the methylation profile around the chicken beta globin gene (McGhee & Ginder, 1979). Lack of methylation was observed in tissues expressing the gene; while tissues not expressing it showed high

intermediate, explaining the RNA dependency reported.

DNA replication dependent but passive process.

**3. Chicken –** *Gallus gallus*

Scarsdale et al., 2011).

Especially interesting is the increase in non-CpG methylation seen in zebrafish embryos compared to mouse embryos, equivalent to a 3-4 fold increase; but similar to mouse ES cells. BS-seq showed that 5mC is modestly enriched at repetitive elements and within gene bodies, but depleted at CpG islands covering TSSs. Overall, the organisation of the 5mC methylome is similar in all vertebrates, which have methylation throughout the genome except at CpG islands (Feng et al., 2010a). Gene body methylation is conserved with clear preference for exons in most organisms.

Mammalian development is known to be highly dependent upon methylation and there are many studies which also highlight the importance of methylation in zebrafish. First, the apparent presence of maternal Dnmt1 in zebrafish oocytes points towards an essential role of 5mC in zebrafish development (Goll et al., 2009). Treatment of embryos with 5 azacytidine resulted in abnormal tail development, and incorrect patterning of somites (Martin et al., 1999). Morpholino knockout (zdnmt1MO) of Dnmt1 in zebrafish embryos has been shown to interfere with terminal differentiation of the intestine, exocrine pancreas and retina; but not the liver or endocrine pancreas (Rai et al., 2006). This is interesting as many embryos survive past gastrulation, unlike in mice. Surviving zdnmt1MO embryos exhibit developmental defects including curled tails, pericardial oedema, and jaw defects; with eyes appearing normal. This may suggest altered dependence on DNA methylation or Dnmts in zebrafish, or alternative mechanisms of DNA methylation maintenance in non-mammalian animals. It chimes with the situation in *Xenopus*, where certain key roles of DNMT1 do not depend on it methylating DNA (Dunican et al., 2008; Stancheva et al., 2001). Genetic analysis reinforces this difference, as zebrafish mutants in uhrf1 and dnmt1 also have defects in lens development and maintenance and they are not embryonic lethal, despite being hypomethylated (Anderson et al., 2009; Tittle et al., 2011). This suggests that attributing developmental defects in these mutants to hypomethylation may be premature, as the biology may be more complex. Here it is worth noting that Lsh mutants in mice are as globally hypomethylated as Dnmt1 mutants, yet certain Lsh -/- mutant mice exhibit growth retardation and a premature aging phenotype (Sun et al., 2004). This may be partly due to the different classes of gene misexpression compared to Dnmt1 mutants in mice (Myant et al., 2011). This implies that the role of DNA methylation and its associated components is still unclear, although DNA methylation patterns at promoters are highly correlated with transcription state.

Demethylation of DNA may occur either actively or passively, with passive demethylation occurring via the absence of methylation maintenance upon cell division and active methylation via direct enzymatic action resulting in the reversion of 5mC to C (Wu & Zhang, 2010). Active demethylation in the mouse is most obvious during early development, where detection of 5mC is rapidly lost in the male pronucleus (Santos et al., 2002), and also appears to occur during PGC development (Monk et al., 1987). Whether an equivalent process occurs within zebrafish is still unclear. Early experiments looking for 5mC changes during early zebrafish development did not detect any (Macleod et al., 1999), though this investigation was not comprehensive; while a more recent report did (Mhanni & McGowan, 2004). Promising results were obtained using immunological methods in zebrafish, and it was seen that methylation is lost around 1.5-2 hpf (cleavage and early blastula stages), and increases by 4 hpf (MacKay et al., 2007). This result is particularly encouraging, as the study made use of a similar technique to that used to obtain positive results in mouse (Santos et al., 2002). The authors suggest possible explanations for negative

Especially interesting is the increase in non-CpG methylation seen in zebrafish embryos compared to mouse embryos, equivalent to a 3-4 fold increase; but similar to mouse ES cells. BS-seq showed that 5mC is modestly enriched at repetitive elements and within gene bodies, but depleted at CpG islands covering TSSs. Overall, the organisation of the 5mC methylome is similar in all vertebrates, which have methylation throughout the genome except at CpG islands (Feng et al., 2010a). Gene body methylation is conserved with clear

Mammalian development is known to be highly dependent upon methylation and there are many studies which also highlight the importance of methylation in zebrafish. First, the apparent presence of maternal Dnmt1 in zebrafish oocytes points towards an essential role of 5mC in zebrafish development (Goll et al., 2009). Treatment of embryos with 5 azacytidine resulted in abnormal tail development, and incorrect patterning of somites (Martin et al., 1999). Morpholino knockout (zdnmt1MO) of Dnmt1 in zebrafish embryos has been shown to interfere with terminal differentiation of the intestine, exocrine pancreas and retina; but not the liver or endocrine pancreas (Rai et al., 2006). This is interesting as many embryos survive past gastrulation, unlike in mice. Surviving zdnmt1MO embryos exhibit developmental defects including curled tails, pericardial oedema, and jaw defects; with eyes appearing normal. This may suggest altered dependence on DNA methylation or Dnmts in zebrafish, or alternative mechanisms of DNA methylation maintenance in non-mammalian animals. It chimes with the situation in *Xenopus*, where certain key roles of DNMT1 do not depend on it methylating DNA (Dunican et al., 2008; Stancheva et al., 2001). Genetic analysis reinforces this difference, as zebrafish mutants in uhrf1 and dnmt1 also have defects in lens development and maintenance and they are not embryonic lethal, despite being hypomethylated (Anderson et al., 2009; Tittle et al., 2011). This suggests that attributing developmental defects in these mutants to hypomethylation may be premature, as the biology may be more complex. Here it is worth noting that Lsh mutants in mice are as globally hypomethylated as Dnmt1 mutants, yet certain Lsh -/- mutant mice exhibit growth retardation and a premature aging phenotype (Sun et al., 2004). This may be partly due to the different classes of gene misexpression compared to Dnmt1 mutants in mice (Myant et al., 2011). This implies that the role of DNA methylation and its associated components is still unclear, although DNA methylation patterns at promoters are highly correlated with

Demethylation of DNA may occur either actively or passively, with passive demethylation occurring via the absence of methylation maintenance upon cell division and active methylation via direct enzymatic action resulting in the reversion of 5mC to C (Wu & Zhang, 2010). Active demethylation in the mouse is most obvious during early development, where detection of 5mC is rapidly lost in the male pronucleus (Santos et al., 2002), and also appears to occur during PGC development (Monk et al., 1987). Whether an equivalent process occurs within zebrafish is still unclear. Early experiments looking for 5mC changes during early zebrafish development did not detect any (Macleod et al., 1999), though this investigation was not comprehensive; while a more recent report did (Mhanni & McGowan, 2004). Promising results were obtained using immunological methods in zebrafish, and it was seen that methylation is lost around 1.5-2 hpf (cleavage and early blastula stages), and increases by 4 hpf (MacKay et al., 2007). This result is particularly encouraging, as the study made use of a similar technique to that used to obtain positive results in mouse (Santos et al., 2002). The authors suggest possible explanations for negative

preference for exons in most organisms.

transcription state.

results reported by other groups; including the relevant time point being missed, and potential contamination of samples with hypermethylated mitochondrial DNA (MacKay et al., 2007). In any case, the lack of large scale demethylation events would not prevent the existence of active demethylation, and there is evidence that such non-global processes occur in zebrafish. It has been shown that a methylated plasmid is demethylated when inserted into a one-cell zebrafish embryo, independently of DNA replication (Collas, 1998). It was also reported that the demethylase activity was dependent upon RNA, due to its inhibition by RNaseH. Upregulation of both MBD4 and AID results in global DNA demethylation, and both are found to be recruited to methylated DNA by Gadd45a (Rai et al., 2008). AID is a 5mC-deaminase which converts 5mC to T; and MBD4 has G:T mismatch thymine glycosylase. These enzymes are thus expected to promote demethylation via the conversion of 5mC to T, and the subsequent substitution of T to C. Interestingly, Gadd45a has been shown to bind neither single or double stranded DNA whether methylated or unmethylated (Sytnikova et al., 2011). However, RNA binding activity was observed, indicating that recruitment of the demethylation couplet may proceed via an RNA intermediate, explaining the RNA dependency reported.

Mammalian demethylation studies have recently benefited from a flurry of papers detailing 5hmC and its potential role in demethylation. This base is produced from the further modification of 5mC by TET enzymes (TET1-3), and appears to allow demethylation to occur through a series of intermediate conversions (He et al., 2011; Ito et al., 2011; Kriaucionis & Heintz, 2009; Tahiliani et al., 2009). It has been suggested that 5hmC is converted to 5-formylcytosine (5fC), then 5-carboxylcytosine (5caC); which is subsequently substituted for cytosine by the action of TDG (Ito et al., 2011; He et al., 2011). This pathway is yet to be studied in zebrafish, though at least one unpublished report exists claiming the presence of 5hmC in zebrafish (Yen & Jia, 2010). All 3 mammalian TET proteins have homologous sequences in zebrafish (see Table 1). Mammalian 5hmC could be suggested to have a dual functional role, as it does not appear to be immediately converted, and is present at considerably greater levels than 5fC and 5caC. Such roles could include repression of transcription SIN3A recruitment (Williams et al., 2011) and hypomethylation of target regions allowing PRC2 recruitment (Wu et al., 2011). It will be interesting to explore the distribution of 5hmC and TET binding/expression profiles in zebrafish. Immunostaining of mitotic chromosome spreads of mouse pre-implantation embryos demonstrated that paternal 5hmC is gradually lost during pre-implantation development (Inoue & Zhang, 2011). Here it is suggested that although the conversion of 5mC to 5hmC in zygotes is an enzyme-catalysed process, loss of 5hmC during pre-implantation may be a DNA replication dependent but passive process.

#### **3. Chicken –** *Gallus gallus*

DNA methylation has long been studied in the chicken, with the globin genes being the primary model of study; and is complimented by structural studies (Heitmann et al., 2003; Scarsdale et al., 2011).

The first evidence for functionally relevant DNA methylation in chicken came when methylation sensitive restriction enzymes were used to study the methylation profile around the chicken beta globin gene (McGhee & Ginder, 1979). Lack of methylation was observed in tissues expressing the gene; while tissues not expressing it showed high

DNA Methylation in Mammalian and Non-Mammalian Organisms 133

A potential active demethylation system in chicken was first seen when chicken embryonic nuclear extracts were shown to be capable of demethylation, and that the extent of demethylation was different across developmental time points (Jost, 1993). This action was subsequently suggested to involve the combined action of glycosylases, (Jost et al., 1995) and repair pathways (Jost et al., 1995; Zhu et al., 2000) in an RNA dependant manner (Fremont et al., 1997; Jost et al., 1997). There is potential for 5hmC to be involved in this pathway, as the chicken genome contains potential homologues for all three TET proteins, which share all

A particularly interesting region found in the chicken is the chicken male hypermethylation region (cMHM). This region is hypermethylated in males but hypomethylated in females where the region is transcribed into ncRNAs which accumulate at DMRT1 – a gene required for chicken testis differentiation; and in a region enriched for dosage compensated genes. A recent study investigated this region in chickens subject to sex-reversal (Yang et al., 2011).  The cMHM in gonad cells was seen to be highly methylated in males and hypomethylated in standard females, as expected. Methylation was slightly increased in the slightly sex-reversed group of females, and there was no significant difference in cMHM methylation between highly sex-reversed chickens and males. However, in liver the cMHM was hypermethylated in males, and low in females but remained low in each of the sex-reversed groups. DMRT1 expression was seen to increase towards male levels upon sex-reversal, but the expression of other sex specific genes changed less dramatically. A link between chicken DNA methylation and development was further seen by the interesting regulation of DNMTs by miRNAs during PGC development (Rengaraj et al., 2011), a finding similar to that seen in mouse PGCs (Takada et al., 2009).

Due to the extensive use of chick in agriculture, study of disease in the organism is relevant not only as models for human diseases; but also due to potential economic benefits. A recent study sought to find links between DNA methylation and neoplastic diseases in chickens using two lines, one susceptible to tumours and one resistant (Yu et al, 2008a, 2008b). Possible links between methylation of viral DNA (Yu et al., 2008a) and DNMT genes (Yu et al., 2008b) were reported. The general importance of methylation to chicken health was seen when *in vivo* treatment with 5-azacytidine negatively affected lymphatic organs, and increased the prevalence of autoimmune diseases in chicks (Schauenstein et al., 1991). Findings particularly relevant to humans came from experiments which used betaine as a dietary supplement (Xing et al., 2011) - a methyl donor known to reduce fat deposition. A modest reduction and change in pattern of promoter methylation was noted for the LPL gene, involved in lipoprotein catabolism; which coincided with a change in LPL levels.

*Xenopus laevis* has a solid history in methylation research, and was used in the experiments which pioneered the use of methylation-sensitive restriction enzymes to analyse gene expression (Bird & Southern, 1978). This method allowed extensive discoveries to be made

domains with their mouse equivalents (Table 1 and Figure 3).

**4. Frog –** *Xenopus tropicalis / Xenopus laevis*

and permitted functional DNA methylation studies to begin.

methylation in the region. Soon after this initial study, it was found that around the genes encoding ovalbumin, conalbumin, and ovomucoid; DNA methylation was reduced in the oviduct when they are expressed (Mandel & Chambon, 1979). It was also noted that the highest levels of methylation were found in sperm. The observation that methylation is particularly high in sperm was replicated in a study reporting a negative correlation between methylation and expression of the adult and embryonic alpha globin genes during chicken development (Haigh et al., 1982). Despite these results linking methylation to expression for globin genes, *in vivo* demethylation by 5-azacytidine treatment in adult chickens did not result in reactivation of embryonic alpha globin (Ginder et al., 1983); it was also subsequently shown that a difference in methylation profile does not necessarily correspond to a difference in expression level (Cooper et al., 1983). The link between methylation and expression was nevertheless enhanced by experiments showing by HPLC and restriction analysis that chicken methylation profiles appear to change in an age- and tissue-dependent manner (Harasawa & Mitsuoka, 1984). This idea is strengthened by a more recent study showing significant differences in global methylation levels between different chicken tissues (Xu et al., 2007), but this may only be true for a subset of genes, as no correlation was observed between methylation of the lysozyme promoter and its transcriptional activity (Wolfl et al., 1991).

Structural evidence for a link between methylation and expression of target genes in chicken came when it was shown that the presence of methylation at just 3 CpGs in the beta globin promoter was sufficient to exclude binding of histone proteins (Davey et al., 1997). The finding that a small number of CpGs could be the major determinant in gene expression changes was also seen when chickens were transfected with GFP under the control of the RSV promoter (Park et al., 2010). It was found that GFP expression varied between tissues, but didn't appear to have inserted into a tissue specific gene cluster. To test if methylation was involved in this, the methylation status of the promoter was tested, and was seen to be slightly lower in tissues with higher GFP expression, but mainly at the set of CpGs at the very start of the promoter, where the majority of CpGs were unmethylated.

The methylation of the GFP reporter construct demonstrates *de novo* methylation in chicken. The chicken genome contains homologues of each mammalian DNMT (Table 1) with the exception of DNMT3L (Yokomine et al., 2006) each having high conservation with mammalian proteins; with DNMT1 having 94% identical amino acid sequence (Tajima et al., 1995).

The chicken genome consists of 39 chromosomes, 33 of which are classed as microchromosomes (McQueen et al., 1998). These microchromosomes are gene rich, early replicating and enriched for CpG islands (McQueen et al., 1996, 1998); but remain incompletely sequenced due technical difficulties (Dodgson et al., 2011). The sequencing of the chicken genome (International Chicken Genome Sequencing Consortium, 2004) allowed a genomewide methylation profile to be obtained. Liver and muscle tissue was analysed from two breeds of chicken, and found to be enriched within gene bodies and at repetitive sequences but depleted at TSS and TTSs. The majority of CpG islands were in an unmethylated state, and promoter methylation correlated with gene expression. No differentially methylated regions were found, consistent with the previous lack of evidence for imprinting in birds and the lack of a DNMT3L homologue in the chicken genome sequence. UHRF1 appears in the chicken genome, as do several MBDs (Table 1 and Figure 2).

methylation in the region. Soon after this initial study, it was found that around the genes encoding ovalbumin, conalbumin, and ovomucoid; DNA methylation was reduced in the oviduct when they are expressed (Mandel & Chambon, 1979). It was also noted that the highest levels of methylation were found in sperm. The observation that methylation is particularly high in sperm was replicated in a study reporting a negative correlation between methylation and expression of the adult and embryonic alpha globin genes during chicken development (Haigh et al., 1982). Despite these results linking methylation to expression for globin genes, *in vivo* demethylation by 5-azacytidine treatment in adult chickens did not result in reactivation of embryonic alpha globin (Ginder et al., 1983); it was also subsequently shown that a difference in methylation profile does not necessarily correspond to a difference in expression level (Cooper et al., 1983). The link between methylation and expression was nevertheless enhanced by experiments showing by HPLC and restriction analysis that chicken methylation profiles appear to change in an age- and tissue-dependent manner (Harasawa & Mitsuoka, 1984). This idea is strengthened by a more recent study showing significant differences in global methylation levels between different chicken tissues (Xu et al., 2007), but this may only be true for a subset of genes, as no correlation was observed between methylation of the lysozyme promoter and its

Structural evidence for a link between methylation and expression of target genes in chicken came when it was shown that the presence of methylation at just 3 CpGs in the beta globin promoter was sufficient to exclude binding of histone proteins (Davey et al., 1997). The finding that a small number of CpGs could be the major determinant in gene expression changes was also seen when chickens were transfected with GFP under the control of the RSV promoter (Park et al., 2010). It was found that GFP expression varied between tissues, but didn't appear to have inserted into a tissue specific gene cluster. To test if methylation was involved in this, the methylation status of the promoter was tested, and was seen to be slightly lower in tissues with higher GFP expression, but mainly at the set of CpGs at the

The methylation of the GFP reporter construct demonstrates *de novo* methylation in chicken. The chicken genome contains homologues of each mammalian DNMT (Table 1) with the exception of DNMT3L (Yokomine et al., 2006) each having high conservation with mammalian

The chicken genome consists of 39 chromosomes, 33 of which are classed as microchromosomes (McQueen et al., 1998). These microchromosomes are gene rich, early replicating and enriched for CpG islands (McQueen et al., 1996, 1998); but remain incompletely sequenced due technical difficulties (Dodgson et al., 2011). The sequencing of the chicken genome (International Chicken Genome Sequencing Consortium, 2004) allowed a genomewide methylation profile to be obtained. Liver and muscle tissue was analysed from two breeds of chicken, and found to be enriched within gene bodies and at repetitive sequences but depleted at TSS and TTSs. The majority of CpG islands were in an unmethylated state, and promoter methylation correlated with gene expression. No differentially methylated regions were found, consistent with the previous lack of evidence for imprinting in birds and the lack of a DNMT3L homologue in the chicken genome sequence. UHRF1 appears in the chicken

proteins; with DNMT1 having 94% identical amino acid sequence (Tajima et al., 1995).

very start of the promoter, where the majority of CpGs were unmethylated.

transcriptional activity (Wolfl et al., 1991).

genome, as do several MBDs (Table 1 and Figure 2).

A potential active demethylation system in chicken was first seen when chicken embryonic nuclear extracts were shown to be capable of demethylation, and that the extent of demethylation was different across developmental time points (Jost, 1993). This action was subsequently suggested to involve the combined action of glycosylases, (Jost et al., 1995) and repair pathways (Jost et al., 1995; Zhu et al., 2000) in an RNA dependant manner (Fremont et al., 1997; Jost et al., 1997). There is potential for 5hmC to be involved in this pathway, as the chicken genome contains potential homologues for all three TET proteins, which share all domains with their mouse equivalents (Table 1 and Figure 3).

A particularly interesting region found in the chicken is the chicken male hypermethylation region (cMHM). This region is hypermethylated in males but hypomethylated in females where the region is transcribed into ncRNAs which accumulate at DMRT1 – a gene required for chicken testis differentiation; and in a region enriched for dosage compensated genes. A recent study investigated this region in chickens subject to sex-reversal (Yang et al., 2011).  The cMHM in gonad cells was seen to be highly methylated in males and hypomethylated in standard females, as expected. Methylation was slightly increased in the slightly sex-reversed group of females, and there was no significant difference in cMHM methylation between highly sex-reversed chickens and males. However, in liver the cMHM was hypermethylated in males, and low in females but remained low in each of the sex-reversed groups. DMRT1 expression was seen to increase towards male levels upon sex-reversal, but the expression of other sex specific genes changed less dramatically. A link between chicken DNA methylation and development was further seen by the interesting regulation of DNMTs by miRNAs during PGC development (Rengaraj et al., 2011), a finding similar to that seen in mouse PGCs (Takada et al., 2009).

Due to the extensive use of chick in agriculture, study of disease in the organism is relevant not only as models for human diseases; but also due to potential economic benefits. A recent study sought to find links between DNA methylation and neoplastic diseases in chickens using two lines, one susceptible to tumours and one resistant (Yu et al, 2008a, 2008b). Possible links between methylation of viral DNA (Yu et al., 2008a) and DNMT genes (Yu et al., 2008b) were reported. The general importance of methylation to chicken health was seen when *in vivo* treatment with 5-azacytidine negatively affected lymphatic organs, and increased the prevalence of autoimmune diseases in chicks (Schauenstein et al., 1991). Findings particularly relevant to humans came from experiments which used betaine as a dietary supplement (Xing et al., 2011) - a methyl donor known to reduce fat deposition. A modest reduction and change in pattern of promoter methylation was noted for the LPL gene, involved in lipoprotein catabolism; which coincided with a change in LPL levels.

#### **4. Frog –** *Xenopus tropicalis / Xenopus laevis*

*Xenopus laevis* has a solid history in methylation research, and was used in the experiments which pioneered the use of methylation-sensitive restriction enzymes to analyse gene expression (Bird & Southern, 1978). This method allowed extensive discoveries to be made and permitted functional DNA methylation studies to begin.

DNA Methylation in Mammalian and Non-Mammalian Organisms 135

xMBD3 is highly expressed in *X. laevis* embryos, in a spatially distinctive pattern which overlaps significantly with DNMT1 expression (Iwano et al., 2004). Knockdown of xMBD3 resulted in the eye developmental transcription factor Pax6, consistent with the eye being one of the regions with particularly high xMBD3 expression. This is in contrast with Mbd3 being indispensible for mouse development (Hendrich et al., 2001) The *X. tropicalis* genome

The xDnmt1 sequence has all the hallmarks of a maintenance methyltransferase which can propagate pre-existing patterns of methylation that are present in the early *Xenopus* embryos (Stancheva & Meehan, 2000). Transient anti-sense RNA depletion of xDnmt1 levels by 90% results in DNA hypomethylation and premature activation of gene expression before the mid-blastula transition (MBT), a developmental landmark that coincides with general zygotic gene activation (Dunican et al., 2008; Newport & Kirschner, 1982; Stancheva & Meehan, 2000). Loss of xDnmt1 results in embryonic lethality due to activation of a programmed cell death pathway (Jackson-Grusby et al., 2001; Stancheva & Meehan, 2000). DNA methylation levels recover after anti-sense RNA depletion of xDnmt1 suggesting that a functional de novo methylation pathway is present ion *Xenopus laevis* (Stancheva & Meehan, 2000). Morphant knockdown of xDNMT1 (xDMO) matched the anti-sense RNA phenotype by exhibiting p53 dependant apoptotic embryo lethality and premature activation of zygotic transcription before the MBT. Very surprisingly, this occurred without global changes in DNA methylation levels. The underlying explanation was that a moderate reduction in xDnmt1p levels was sufficient to prematurely activate gene expression in *X. laevis* embryos independently of changes in DNA methylation levels or histone modifications (Dunican et al., 2008). Crucially, repression of target genes was re-imposed in xDMO morphants by co-injection of mRNA encoding a catalytically inactive form of DNMT1. In addition, it was observed that histone modifications (H3K4me3, H3K9me2 and H4K20me3) accumulate after the MBT and are not prematurely accrued when xDnmt1 levels were reduced and transcription occurs *before* the MBT (Akkers et al., 2009; Dunican et al., 2008). Here there is separation between the transcriptional outcome and specification by the histone code. A model was proposed in which xDnmt1 has a major silencer role in early *Xenopus* development as a chromatin bound non-enzymatic protein that regulated the timing of zygotic gene activation (Dunican et al., 2008). xDnmt1p may serve as a titratable repressor component that has been previously invoked for *Xenopus* embryos (Almouzni &

appears to contain multiple MBD proteins (Table 1).

Wolffe, 1995; Newport & Kirschner, 1982; Prioleau et al., 1994)

amphibians (Imokawa & Yoshizato, 1997; Torok et al., 1999).

The study of DNA methylation in amphibians is particularly interesting due to the possibility of regenerative mechanisms being elucidated. Methylation of the enhancer of *Shh -* the ZRS (Lettice et al., 2003), was found to be low in tadpoles, which have full limb regeneration ability, but high in adult frogs capable of only limited regeneration (Yakushiji et al., 2007). Re-expression of *Shh* has been shown to occur during limb regeneration in other

Although the majority of frog DNA methylation studies have been performed in *Xenopus laevis*, the recent sequencing of the *Xenopus tropicalis* genome (Hellsten et al., 2010) has allowed the 5mC profile of this organism to be investigated. Genome wide 5mC profiling in embryos revealed that 5mC is mainly present in the CpG context, 45% of which are methylated, but also as CpA methylation. It appeared particularly enriched within repetitive

Although both *X. laevis* and *X. tropicalis* have a DNMT3 homologue, *de novo* methylation in *Xenopus* is not well studied, and it is notable that they do not appear to have a DNMT3B homologue (Table 1). When *Xenopus* eggs were injected with *Xenopus* globin transgenes, no *de novo* methylation was observed (Bendig & Williams, 1983; Harland, 1982). *In vitro* methylation of the transgenes did, however, result in the maintenance of transgene methylation, and importantly, low transgene expression was observed regardless of methylation status (Bendig & Williams, 1983).

It has been shown that when *in vitro* methylated plasmids are injected into *Xenopus* embryos, the methylation is maintained (Harland, 1982) and that adenoviral genes injected into *Xenopus* were repressed by methylation (Vardimon et al., 1982b). Further studies went on to show that methylation is only repressive at certain CpG sites (Langner et al., 1984; Vardimon et al., 1982a, 1983). An attempt to link gene induction by estrogen to methylation was unsuccessful, as no methylation change was detected (Folger et al., 1983).

Dnmt1 was confirmed to be expressed in *Xenopus* (Kimura et al., 1996) and is accumulated in oocytes (Kimura et al., 1999), suggesting an *in vivo* importance during development. Functional evidence for such a role came when dnmt1 depletion in embryos resulted in temporal misexpression of genes, with various developmental markers being expressed early (Stancheva & Meehan, 2000); and misexpression of dnmts was linked to activation of apoptosis by various studies (Kaito et al., 2001;Kimura et al., 2002; Stancheva et al., 2001). However, it has recently been shown that xDnmt1 can alter gene expression in a methylation independent manner (Dunican et al., 2008), making it possible that the apoptotic phenotypes were also methylation independent. Further, when the function of dnmt1 oocyte accumulation was probed using monoclonal antibodies for xDnmt1, the resulting inhibition of cell division was also found to be unrelated to methylation (Hashimoto et al., 2003). Nevertheless, several studies link methylation to expression in *Xenopus*, such as the finding that methylation directly inhibits transcription (Harvey & Newport, 2003; Lopes et al., 2008); 5mC promotes HDAC mediated transcriptional repression (Jones et al., 1998; Wade et al., 1999); and the finding that the methyl CpG binding activity of the transcription factor Kaiso is specifically required during development; although some of the Kaiso targets do not appear to be directly regulated by promoter methylation (Ruzov et al., 2009a, 2009b). Kaiso can also interact directly with components of the Wnt signalling pathways in development and cancer, which may expand its functional repertoire (Ruzov et al., 2009a). It was demonstrated that the zinc finger regions (ZF1-3) of xKaiso (*Xenopus)*, dKaiso (*Drosophila*) and gKaiso (chicken) are sufficient for direct interaction with a terminal component of the canonical Wnt pathway, xTcf3, via its HMG domain *in vitro* (Ruzov et al., 2009a); suggesting that the interaction between Kaiso and Tcf3/4 is mutually exclusive of their DNA binding. Over-expression of xKaiso in developing *Xenopus* embryos actually mimics certain aspects of xTcf3 depletion, such as ectopic Siamois expression. A potential intersection of Kaiso with Wnt signalling pathways may occur in cancer, where over-expression of Kaiso could attenuate constitutive Wnt signalling, while at the same time promoting cancer progression through silencing of *de novo* methylated tumour suppressor genes (Lopes et al., 2008). Recently published work shows that disruption of the Tcf4:Kaiso interaction in human colon cancer cell lines releases Tcf4, enabling its mutual association with β-catenin and the formation of a transcriptional complex (Del Valle-Perez et al., 2011). This also permits Kaiso binding to the methylated CDKN2A promoter in cells, leading to its decreased expression.

Although both *X. laevis* and *X. tropicalis* have a DNMT3 homologue, *de novo* methylation in *Xenopus* is not well studied, and it is notable that they do not appear to have a DNMT3B homologue (Table 1). When *Xenopus* eggs were injected with *Xenopus* globin transgenes, no *de novo* methylation was observed (Bendig & Williams, 1983; Harland, 1982). *In vitro* methylation of the transgenes did, however, result in the maintenance of transgene methylation, and importantly, low transgene expression was observed regardless of

It has been shown that when *in vitro* methylated plasmids are injected into *Xenopus* embryos, the methylation is maintained (Harland, 1982) and that adenoviral genes injected into *Xenopus* were repressed by methylation (Vardimon et al., 1982b). Further studies went on to show that methylation is only repressive at certain CpG sites (Langner et al., 1984; Vardimon et al., 1982a, 1983). An attempt to link gene induction by estrogen to methylation

Dnmt1 was confirmed to be expressed in *Xenopus* (Kimura et al., 1996) and is accumulated in oocytes (Kimura et al., 1999), suggesting an *in vivo* importance during development. Functional evidence for such a role came when dnmt1 depletion in embryos resulted in temporal misexpression of genes, with various developmental markers being expressed early (Stancheva & Meehan, 2000); and misexpression of dnmts was linked to activation of apoptosis by various studies (Kaito et al., 2001;Kimura et al., 2002; Stancheva et al., 2001). However, it has recently been shown that xDnmt1 can alter gene expression in a methylation independent manner (Dunican et al., 2008), making it possible that the apoptotic phenotypes were also methylation independent. Further, when the function of dnmt1 oocyte accumulation was probed using monoclonal antibodies for xDnmt1, the resulting inhibition of cell division was also found to be unrelated to methylation (Hashimoto et al., 2003). Nevertheless, several studies link methylation to expression in *Xenopus*, such as the finding that methylation directly inhibits transcription (Harvey & Newport, 2003; Lopes et al., 2008); 5mC promotes HDAC mediated transcriptional repression (Jones et al., 1998; Wade et al., 1999); and the finding that the methyl CpG binding activity of the transcription factor Kaiso is specifically required during development; although some of the Kaiso targets do not appear to be directly regulated by promoter methylation (Ruzov et al., 2009a, 2009b). Kaiso can also interact directly with components of the Wnt signalling pathways in development and cancer, which may expand its functional repertoire (Ruzov et al., 2009a). It was demonstrated that the zinc finger regions (ZF1-3) of xKaiso (*Xenopus)*, dKaiso (*Drosophila*) and gKaiso (chicken) are sufficient for direct interaction with a terminal component of the canonical Wnt pathway, xTcf3, via its HMG domain *in vitro* (Ruzov et al., 2009a); suggesting that the interaction between Kaiso and Tcf3/4 is mutually exclusive of their DNA binding. Over-expression of xKaiso in developing *Xenopus* embryos actually mimics certain aspects of xTcf3 depletion, such as ectopic Siamois expression. A potential intersection of Kaiso with Wnt signalling pathways may occur in cancer, where over-expression of Kaiso could attenuate constitutive Wnt signalling, while at the same time promoting cancer progression through silencing of *de novo* methylated tumour suppressor genes (Lopes et al., 2008). Recently published work shows that disruption of the Tcf4:Kaiso interaction in human colon cancer cell lines releases Tcf4, enabling its mutual association with β-catenin and the formation of a transcriptional complex (Del Valle-Perez et al., 2011). This also permits Kaiso binding to the methylated

was unsuccessful, as no methylation change was detected (Folger et al., 1983).

CDKN2A promoter in cells, leading to its decreased expression.

methylation status (Bendig & Williams, 1983).

xMBD3 is highly expressed in *X. laevis* embryos, in a spatially distinctive pattern which overlaps significantly with DNMT1 expression (Iwano et al., 2004). Knockdown of xMBD3 resulted in the eye developmental transcription factor Pax6, consistent with the eye being one of the regions with particularly high xMBD3 expression. This is in contrast with Mbd3 being indispensible for mouse development (Hendrich et al., 2001) The *X. tropicalis* genome appears to contain multiple MBD proteins (Table 1).

The xDnmt1 sequence has all the hallmarks of a maintenance methyltransferase which can propagate pre-existing patterns of methylation that are present in the early *Xenopus* embryos (Stancheva & Meehan, 2000). Transient anti-sense RNA depletion of xDnmt1 levels by 90% results in DNA hypomethylation and premature activation of gene expression before the mid-blastula transition (MBT), a developmental landmark that coincides with general zygotic gene activation (Dunican et al., 2008; Newport & Kirschner, 1982; Stancheva & Meehan, 2000). Loss of xDnmt1 results in embryonic lethality due to activation of a programmed cell death pathway (Jackson-Grusby et al., 2001; Stancheva & Meehan, 2000). DNA methylation levels recover after anti-sense RNA depletion of xDnmt1 suggesting that a functional de novo methylation pathway is present ion *Xenopus laevis* (Stancheva & Meehan, 2000). Morphant knockdown of xDNMT1 (xDMO) matched the anti-sense RNA phenotype by exhibiting p53 dependant apoptotic embryo lethality and premature activation of zygotic transcription before the MBT. Very surprisingly, this occurred without global changes in DNA methylation levels. The underlying explanation was that a moderate reduction in xDnmt1p levels was sufficient to prematurely activate gene expression in *X. laevis* embryos independently of changes in DNA methylation levels or histone modifications (Dunican et al., 2008). Crucially, repression of target genes was re-imposed in xDMO morphants by co-injection of mRNA encoding a catalytically inactive form of DNMT1. In addition, it was observed that histone modifications (H3K4me3, H3K9me2 and H4K20me3) accumulate after the MBT and are not prematurely accrued when xDnmt1 levels were reduced and transcription occurs *before* the MBT (Akkers et al., 2009; Dunican et al., 2008). Here there is separation between the transcriptional outcome and specification by the histone code. A model was proposed in which xDnmt1 has a major silencer role in early *Xenopus* development as a chromatin bound non-enzymatic protein that regulated the timing of zygotic gene activation (Dunican et al., 2008). xDnmt1p may serve as a titratable repressor component that has been previously invoked for *Xenopus* embryos (Almouzni & Wolffe, 1995; Newport & Kirschner, 1982; Prioleau et al., 1994)

The study of DNA methylation in amphibians is particularly interesting due to the possibility of regenerative mechanisms being elucidated. Methylation of the enhancer of *Shh -* the ZRS (Lettice et al., 2003), was found to be low in tadpoles, which have full limb regeneration ability, but high in adult frogs capable of only limited regeneration (Yakushiji et al., 2007). Re-expression of *Shh* has been shown to occur during limb regeneration in other amphibians (Imokawa & Yoshizato, 1997; Torok et al., 1999).

Although the majority of frog DNA methylation studies have been performed in *Xenopus laevis*, the recent sequencing of the *Xenopus tropicalis* genome (Hellsten et al., 2010) has allowed the 5mC profile of this organism to be investigated. Genome wide 5mC profiling in embryos revealed that 5mC is mainly present in the CpG context, 45% of which are methylated, but also as CpA methylation. It appeared particularly enriched within repetitive

DNA Methylation in Mammalian and Non-Mammalian Organisms 137

al., 2006). In contrast with mammals, the honeybee genome contains two homologues of the DNMT1 maintenance methylase, and one for the DNMT3 *de novo* methylases (Table 1); with each having high sequence conservation with the mammalian genes (Wang et al., 2006).  Inevitably, once the genome sequence was available, a methylome was obtained (Zemach et al., 2010). This initial analysis was performed on DNA obtained from whole bodies of worker bees, and revealed that overall methylation levels in honeybee are low, with CpG methylation being 0.51%; CHG methylation 0.11%; and CHH methylation 0.16%. Hypomethylation of transposable elements was observed, with methylation mainly being observed within gene bodies, and no enrichment seen at promoters. Interestingly, when transcriptional activity was compared to CpG methylation it was seen that the methylation profile peaked at around the 50th percentile of transcriptional activity, with lowly expressed genes and highly expressed genes sharing a relatively low amount of CpG methylation. Due to this study being from whole bodies of honeybees, it is possible that the results are not biologically relevant, and are an artefact of the amalgamation of all cell types and tissues being analysed together. More informative honeybee methylomes were obtained when BS-seq was used to map the 5mC profile of *Apis mellifera* in brains from both worker and queen bees (Lyko et al., 2010). This allowed both the study of 5mC within a distinct tissue; and the comparison of 5mC profiles between two genomically identical castes with massive behavioural and physiological differences. This study replicated the finding that global levels of DNA methylation are low in honeybees, with approximately 70,000 cytosines methylated from a total of 60,000,000 in the genome (although it remains possible that methylation levels are high in other tissues), and showed that honeybee cytosine methylation is almost exclusive to CpG dinucleotides. Further, the vast majority of 5mC (over 85% in both workers and queens), was within gene bodies; most abundant in exons (over 75% in both castes) and found significantly at splice sites. The lack of methylation of repeats and transposons was also confirmed by this study, with the authors suggesting that it may represent that these elements do not confer genome instability in honeybees. Interestingly, the genes found to be methylated showed higher sequence conservation across a wide range of species than non-methylated genes. Finally, the authors found a significant group of genes to be differentially methylated between worker and queen bees, pointing towards DNA methylation being one mechanism of genetic control contributing to the differences between these castes. Interestingly, when Dnmt3 is knocked down in honeybees using siRNA, they are born with queen-like characteristics such as developed ovaries, mimicking the effects of a royal jelly diet (Kucharski et al., 2008). This study was complimented by the finding that DNMT3 activity reduced with each day that larvae were fed royal jelly (tested at days 3,4 and 5) (Shi et al., 2011). The existence of MBDs in the honeybee is unclear, with Mbd3 being the only classical-type MBD apparent in the genome (Table 1). However, MBD domains are seen in other contexts (Figure 1C). The hypothetical honeybee protein LOC725950 contains an MBD domain and shares most amino acid similarity in mouse with the PHF20L1 protein, which lacks any MBD. Another hypothetical protein in Apis, LOC412607, shares most amino acid similarity in mouse with the FBXL7 protein, with which it shares an F-BOX domain but no MBD. In fact, the MBD:F-BOX architecture appears to be unique to insects, based on the Sanger pfam

regions (except for CpG depleted microsatellites), gene bodies and promoters; with TSSs being hypomethylated (Bogdanovic et al., 2011).

H3K4/27 trimethylation and RNA polymerase II (RNAPII) maps identify promoters and transcribed regions in *Xenopus tropicalis* (Akkers et al., 2009). Spatial differences in H3K27me3 deposition are predictive of localised gene expression. In agreement with a previous study, the appearance of K3K4me3 coincides with zygotic gene activation, whereas H3K27me3 is predominantly deposited upon subsequent spatial restriction or repression of transcriptional regulators (Akkers et al., 2009; Dunican et al., 2008).

Deep sequencing of purified methylated DNA obtained from early *X. tropicalis* embryos demonstrates that its genome is heavily methylated during blastula and gastrula stages (Bogdanovic et al., 2011). DNA methylation is absent in large H3K27me3 domains, indicating that these two repression pathways may be mutually antagonistic. Strikingly, genes that are highly expressed in *X. tropicalis* embryos but not in differentiated cells exhibit relatively high DNA methylation. Direct testing with reporter template demonstrates that methylated promoters are robustly transcribed in blastula- and gastrula-stage embryos, but not in oocytes or late embryos. This complements the situation in *X. laevis*, in which depletion of xDnmt1 leads to premature zygotic activation on a background of global methylation and a *tabula rasa* of histone modifications (Dunican et al., 2008). These findings have implications for epigenetic regulation of gene expression in early embryos and subsequent differentiation. It is noteworthy that mouse ES cells that lack *Dnmt1*, *Dnmt3a* and *Dnmt3b* survive in culture very well, but cannot differentiate properly (Tsumura et al., 2006).

It has been shown that global loss of 5mC does not occur during early *Xenopus* development (Stancheva et al., 2002; Veenstra & Wolffe, 2001). However, evidence exists for active demethylation; for example, demethylation of exogenous mouse *Oct4* is required for its transcription when micro injected into *X. laevis* oocytes (Simonsson & Gurdon, 2004), and gadd45a recruitment occurs at the demethylated region, with demethylation proceeding via action of the repair enzyme XPG (Barreto et al., 2007). It has subsequently been shown that *Xenopus* demethylation makes use of the NER system rather than BER (Schafer et al., 2010). Interestingly, gadd45a has been implicated in the switch from pluripotency to differentiation in the early *Xenopus* embryo (Kaufmann & Niehrs, 2011). *X. tropicalis* appears to possess homologues for at least TET2 and TET3 (Table 1 and Fig 3), but again the 5hmC system has not yet been investigated in *Xenopus*; though the results of such investigation would certainly be of interest. The apparent absence of a TET1 homologue (the only TET with a definite CXXC domain), is particularly interesting and could represent a demethylation mechanism absent in this species; although the function of the TET1 CXXC domain is still unclear (Frauer et al., 2011). Also notable is the presence of a potential lipid attachment site in Tet2, which is normally found in prokaryotes (Figure 1B).

#### **5. Honeybee –** *Apis mellifera*

The sequencing of the genome of the honeybee *Apis mellifera* (Honeybee Genome Sequencing Consortium, 2006) allowed potential DNA methylation genes to be identified. This was a significant turning point in the study of insect DNA methylation, with honeybee being the first insect shown to contain at least one homologue of DNMT1, 2 and 3 (Wang et

regions (except for CpG depleted microsatellites), gene bodies and promoters; with TSSs

H3K4/27 trimethylation and RNA polymerase II (RNAPII) maps identify promoters and transcribed regions in *Xenopus tropicalis* (Akkers et al., 2009). Spatial differences in H3K27me3 deposition are predictive of localised gene expression. In agreement with a previous study, the appearance of K3K4me3 coincides with zygotic gene activation, whereas H3K27me3 is predominantly deposited upon subsequent spatial restriction or repression of

Deep sequencing of purified methylated DNA obtained from early *X. tropicalis* embryos demonstrates that its genome is heavily methylated during blastula and gastrula stages (Bogdanovic et al., 2011). DNA methylation is absent in large H3K27me3 domains, indicating that these two repression pathways may be mutually antagonistic. Strikingly, genes that are highly expressed in *X. tropicalis* embryos but not in differentiated cells exhibit relatively high DNA methylation. Direct testing with reporter template demonstrates that methylated promoters are robustly transcribed in blastula- and gastrula-stage embryos, but not in oocytes or late embryos. This complements the situation in *X. laevis*, in which depletion of xDnmt1 leads to premature zygotic activation on a background of global methylation and a *tabula rasa* of histone modifications (Dunican et al., 2008). These findings have implications for epigenetic regulation of gene expression in early embryos and subsequent differentiation. It is noteworthy that mouse ES cells that lack *Dnmt1*, *Dnmt3a* and *Dnmt3b* survive in culture very

It has been shown that global loss of 5mC does not occur during early *Xenopus* development (Stancheva et al., 2002; Veenstra & Wolffe, 2001). However, evidence exists for active demethylation; for example, demethylation of exogenous mouse *Oct4* is required for its transcription when micro injected into *X. laevis* oocytes (Simonsson & Gurdon, 2004), and gadd45a recruitment occurs at the demethylated region, with demethylation proceeding via action of the repair enzyme XPG (Barreto et al., 2007). It has subsequently been shown that *Xenopus* demethylation makes use of the NER system rather than BER (Schafer et al., 2010). Interestingly, gadd45a has been implicated in the switch from pluripotency to differentiation in the early *Xenopus* embryo (Kaufmann & Niehrs, 2011). *X. tropicalis* appears to possess homologues for at least TET2 and TET3 (Table 1 and Fig 3), but again the 5hmC system has not yet been investigated in *Xenopus*; though the results of such investigation would certainly be of interest. The apparent absence of a TET1 homologue (the only TET with a definite CXXC domain), is particularly interesting and could represent a demethylation mechanism absent in this species; although the function of the TET1 CXXC domain is still unclear (Frauer et al., 2011). Also notable is the presence of a potential lipid

attachment site in Tet2, which is normally found in prokaryotes (Figure 1B).

The sequencing of the genome of the honeybee *Apis mellifera* (Honeybee Genome Sequencing Consortium, 2006) allowed potential DNA methylation genes to be identified. This was a significant turning point in the study of insect DNA methylation, with honeybee being the first insect shown to contain at least one homologue of DNMT1, 2 and 3 (Wang et

**5. Honeybee –** *Apis mellifera*

being hypomethylated (Bogdanovic et al., 2011).

transcriptional regulators (Akkers et al., 2009; Dunican et al., 2008).

well, but cannot differentiate properly (Tsumura et al., 2006).

al., 2006). In contrast with mammals, the honeybee genome contains two homologues of the DNMT1 maintenance methylase, and one for the DNMT3 *de novo* methylases (Table 1); with each having high sequence conservation with the mammalian genes (Wang et al., 2006).  Inevitably, once the genome sequence was available, a methylome was obtained (Zemach et al., 2010). This initial analysis was performed on DNA obtained from whole bodies of worker bees, and revealed that overall methylation levels in honeybee are low, with CpG methylation being 0.51%; CHG methylation 0.11%; and CHH methylation 0.16%. Hypomethylation of transposable elements was observed, with methylation mainly being observed within gene bodies, and no enrichment seen at promoters. Interestingly, when transcriptional activity was compared to CpG methylation it was seen that the methylation profile peaked at around the 50th percentile of transcriptional activity, with lowly expressed genes and highly expressed genes sharing a relatively low amount of CpG methylation. Due to this study being from whole bodies of honeybees, it is possible that the results are not biologically relevant, and are an artefact of the amalgamation of all cell types and tissues being analysed together. More informative honeybee methylomes were obtained when BS-seq was used to map the 5mC profile of *Apis mellifera* in brains from both worker and queen bees (Lyko et al., 2010). This allowed both the study of 5mC within a distinct tissue; and the comparison of 5mC profiles between two genomically identical castes with massive behavioural and physiological differences. This study replicated the finding that global levels of DNA methylation are low in honeybees, with approximately 70,000 cytosines methylated from a total of 60,000,000 in the genome (although it remains possible that methylation levels are high in other tissues), and showed that honeybee cytosine methylation is almost exclusive to CpG dinucleotides. Further, the vast majority of 5mC (over 85% in both workers and queens), was within gene bodies; most abundant in exons (over 75% in both castes) and found significantly at splice sites. The lack of methylation of repeats and transposons was also confirmed by this study, with the authors suggesting that it may represent that these elements do not confer genome instability in honeybees. Interestingly, the genes found to be methylated showed higher sequence conservation across a wide range of species than non-methylated genes. Finally, the authors found a significant group of genes to be differentially methylated between worker and queen bees, pointing towards DNA methylation being one mechanism of genetic control contributing to the differences between these castes. Interestingly, when Dnmt3 is knocked down in honeybees using siRNA, they are born with queen-like characteristics such as developed ovaries, mimicking the effects of a royal jelly diet (Kucharski et al., 2008). This study was complimented by the finding that DNMT3 activity reduced with each day that larvae were fed royal jelly (tested at days 3,4 and 5) (Shi et al., 2011). The existence of MBDs in the honeybee is unclear, with Mbd3 being the only classical-type MBD apparent in the genome (Table 1). However, MBD domains are seen in other contexts (Figure 1C). The hypothetical honeybee protein LOC725950 contains an MBD domain and shares most amino acid similarity in mouse with the PHF20L1 protein, which lacks any MBD. Another hypothetical protein in Apis, LOC412607, shares most amino acid similarity in mouse with the FBXL7 protein, with which it shares an F-BOX domain but no MBD. In fact, the MBD:F-BOX architecture appears to be unique to insects, based on the Sanger pfam

DNA Methylation in Mammalian and Non-Mammalian Organisms 139

mismatch repair defects and increased mutation rates at a CA17 microsatellite (Loughery et al., 2011). This is associated with decreases in mismatch repair protein levels, including MBD4 following activation of the DNA damage response (DDR). Blocking the DDR, and in

Thanks to the study of DNA modification patterns, their generation and the consequences of inactivating components of the epigenetic pathways in different model organisms we are gaining a fuller understanding of their function in mammalian and especially human

Work in the SP and RM labs are supported by the MRC, BBSRC and Breakthrough Breast

Akkers, R.C., S.J.van Heeringen, U.G.Jacobi, E.M.Janssen-Megens, K.J.Francoijs,

An, W., H.K.van, & J.Zlatanova. 1998. The non-histone chromatin protein HMG1 protects

Anderson, R.M., J.A.Bosch, M.G.Goll, D.Hesselson, P.D.Dong, D.Shin, N.C.Chi, C.H.Shin,

Andrews, A.J. & K.Luger. 2011. Nucleosome structure(s) and stability: variations on a

Baillie, J.K., M.W.Barnett, K.R.Upton, D.J.Gerhardt, T.A.Richmond, S.F.De, P.Brennan,

retrotransposition alters the genetic landscape of the human brain. *Nature*. Barreto, G., A.Schafer, J.Marhold, D.Stach, S.K.Swaminathan, V.Handa, G.Doderlein,

activation by repair-mediated DNA demethylation. *Nature* 445: 671-675. Barski, A., S.Cuddapah, K.Cui, T.Y.Roh, D.E.Schones, Z.Wang, G.Wei, I.Chepelev, &

Bendig, M.M. & J.G.Williams. 1983. Replication and expression of Xenopus laevis globin

H.G.Stunnenberg, & G.J.Veenstra. 2009. A hierarchy of H3K4me3 and H3K27me3 acquisition in spatial gene regulation in Xenopus embryos. *Dev. Cell* 17: 425-434. Almouzni, G. & A.P.Wolffe. 1995. Constraints on transcriptional activator function

contribute to transcriptional quiescence during early Xenopus embryogenesis.

linker DNA on the side opposite to that protected by linker histones. *J. Biol. Chem.*

A.Schlegel, M.Halpern, & D.Y.Stainier. 2009. Loss of Dnmt1 catalytic activity reveals multiple roles for DNA methylation during pancreas development and

P.Rizzu, S.Smith, M.Fell, R.T.Talbot, S.Gustincich, T.C.Freeman, J.S.Mattick, D.A.Hume, P.Heutink, P.Carninci, J.A.Jeddeloh, & G.J.Faulkner. 2011. Somatic

N.Maltry, W.Wu, F.Lyko, & C.Niehrs. 2007. Gadd45a promotes epigenetic gene

K.Zhao. 2007. High-resolution profiling of histone methylations in the human

genes injected into fertilized Xenopus eggs. *Proc. Natl. Acad. Sci. U. S. A* 80: 6197-

particular PARP over-activation, also increases survival of the DNMT1 knockdowns.

disease models.

**8. References**

Cancer.

**7. Acknowlegements**

*EMBO J.* 14: 1752-1765.

regeneration. *Dev. Biol.* 334: 213-223.

theme. *Annu. Rev. Biophys.* 40: 99-117.

genome. *Cell* 129: 823-837.

Bird, A. 2007. Perceptions of epigenetics. *Nature* 447: 396-398.

6201.

273: 26289-26291.

database. In both of these examples, the compared mouse proteins are probably not homologues, with similarity only being due to the shared domain. It is therefore likely that DNA methylation in honeybees is acted on via mechanisms unseen in non-insects. Further evidence for novel epigenetic systems in honeybee include the finding that hypermethylated genes tend to be significantly longer than hypomethylated genes (Zeng & Yi, 2010). Other potential MBD-containing proteins in honeybee include BAZ2B-like, and SETDB1, both of which appear to have legitimate homologues in mouse, which they share MBD and other domains with. Interestingly, honeybee appears to have a TET1 homologue complete with CXXC domain, and may therefore may have an active demethylation system making use of this protein, but not TET2/3 homologues (Table 1). Alternatively, TET1 may function non-catalytically to recruit/block other proteins.

#### **6. Conclusions**

Although interspecific 5mC distributions can vary, methylation of cytosine offers a selective potential as a mechanism for regulating gene expression across a wide range of species. The fundamental mechanism of DNA methylation mediated repression seems to be conserved, with active DNMT enzymes appearing in each of the species discussed, and each having homologues of UHRF1 and MBDs. Additionally, there is genomic evidence for active demethylation mechanisms via 5hmC in each of the species discussed, with each having at least one TET homologue present; although it is unclear whether large-scale active demethylation events such as those seen in mammalian development also occur in nonmammalian animals. It will be particularly interesting to find roles of active demethylation in non-mammalian animals; and to see whether TETs/5hmC are involved. Additionally, it will be interesting to investigate potential roles for domains apparently unique to non-mammalian methylation enzymes; such as the CH domain of zebrafish dnmt3/7; and the potential lipid attachment of *Xenopus* Tet2. However, attribution of the molecular pathology of mutants in components of DNA methylation machinery is not always so clear, especially as non-catalytic versions (e.g. in xDnmt1) can rescue phenotypes. For example, a model for the activation of xDnmt1-mediated apoptosis in *X. laevis* embryos has been proposed in which a chromatin associated complex of xDnmt11, xMbd4 and xMlh1 in embryos responds to DNA damage or replication stress by either repairing the lesion or to activate an apoptotic response through the activation/release of Mbd4/Mlh1 from chromatin-bound xDnmt1 (Ruzov et al., 2009c). The Mbd4/Mlh1 complex signals, perhaps via the DNA-damage kinases ATM and ATR, to activate the p53-dependent programmed cell death pathway.

A recent report has validated this model in human cells by demonstrating that DNMT1 is rapidly but transiently recruited to double stranded breaks (DSBs) (Ha et al., 2011), dependent on its ability to interact with both PCNA and CHK1, but independent of its catalytic activity. What is the potential importance of the Dnmt1 signalling mechanism? Cancers arise from the sequential acquisition of genetic alterations in specific genes leading to cellular transformation in step with epigenetic alterations (Fang et al., 2011; Figueroa et al., 2010). One possibility is that Dnmt1 is part of a signalling cascade that activates a barrier against tumour progression. Cellular sensitivity to changes in DNMT1 levels are lost when components of the signalling cascade are either absent or mutated, contributing to the generation of an altered cancer epigenome in tumours and cell lines. In a recent paper it was shown that depleting DNMT1 in proliferating human fibroblasts is sufficient to cause mismatch repair defects and increased mutation rates at a CA17 microsatellite (Loughery et al., 2011). This is associated with decreases in mismatch repair protein levels, including MBD4 following activation of the DNA damage response (DDR). Blocking the DDR, and in particular PARP over-activation, also increases survival of the DNMT1 knockdowns.

Thanks to the study of DNA modification patterns, their generation and the consequences of inactivating components of the epigenetic pathways in different model organisms we are gaining a fuller understanding of their function in mammalian and especially human disease models.

#### **7. Acknowlegements**

Work in the SP and RM labs are supported by the MRC, BBSRC and Breakthrough Breast Cancer.

#### **8. References**

138 DNA Methylation – From Genomics to Technology

database. In both of these examples, the compared mouse proteins are probably not homologues, with similarity only being due to the shared domain. It is therefore likely that DNA methylation in honeybees is acted on via mechanisms unseen in non-insects. Further evidence for novel epigenetic systems in honeybee include the finding that hypermethylated genes tend to be significantly longer than hypomethylated genes (Zeng & Yi, 2010). Other potential MBD-containing proteins in honeybee include BAZ2B-like, and SETDB1, both of which appear to have legitimate homologues in mouse, which they share MBD and other domains with. Interestingly, honeybee appears to have a TET1 homologue complete with CXXC domain, and may therefore may have an active demethylation system making use of this protein, but not TET2/3 homologues (Table 1).

Alternatively, TET1 may function non-catalytically to recruit/block other proteins.

activate the p53-dependent programmed cell death pathway.

Although interspecific 5mC distributions can vary, methylation of cytosine offers a selective potential as a mechanism for regulating gene expression across a wide range of species. The fundamental mechanism of DNA methylation mediated repression seems to be conserved, with active DNMT enzymes appearing in each of the species discussed, and each having homologues of UHRF1 and MBDs. Additionally, there is genomic evidence for active demethylation mechanisms via 5hmC in each of the species discussed, with each having at least one TET homologue present; although it is unclear whether large-scale active demethylation events such as those seen in mammalian development also occur in nonmammalian animals. It will be particularly interesting to find roles of active demethylation in non-mammalian animals; and to see whether TETs/5hmC are involved. Additionally, it will be interesting to investigate potential roles for domains apparently unique to non-mammalian methylation enzymes; such as the CH domain of zebrafish dnmt3/7; and the potential lipid attachment of *Xenopus* Tet2. However, attribution of the molecular pathology of mutants in components of DNA methylation machinery is not always so clear, especially as non-catalytic versions (e.g. in xDnmt1) can rescue phenotypes. For example, a model for the activation of xDnmt1-mediated apoptosis in *X. laevis* embryos has been proposed in which a chromatin associated complex of xDnmt11, xMbd4 and xMlh1 in embryos responds to DNA damage or replication stress by either repairing the lesion or to activate an apoptotic response through the activation/release of Mbd4/Mlh1 from chromatin-bound xDnmt1 (Ruzov et al., 2009c). The Mbd4/Mlh1 complex signals, perhaps via the DNA-damage kinases ATM and ATR, to

A recent report has validated this model in human cells by demonstrating that DNMT1 is rapidly but transiently recruited to double stranded breaks (DSBs) (Ha et al., 2011), dependent on its ability to interact with both PCNA and CHK1, but independent of its catalytic activity. What is the potential importance of the Dnmt1 signalling mechanism? Cancers arise from the sequential acquisition of genetic alterations in specific genes leading to cellular transformation in step with epigenetic alterations (Fang et al., 2011; Figueroa et al., 2010). One possibility is that Dnmt1 is part of a signalling cascade that activates a barrier against tumour progression. Cellular sensitivity to changes in DNMT1 levels are lost when components of the signalling cascade are either absent or mutated, contributing to the generation of an altered cancer epigenome in tumours and cell lines. In a recent paper it was shown that depleting DNMT1 in proliferating human fibroblasts is sufficient to cause

**6. Conclusions**

DNA Methylation in Mammalian and Non-Mammalian Organisms 141

Esteve, P.O., H.G.Chin, J.Benner, G.R.Feehery, M.Samaranayake, G.A.Horwitz, S.E.Jacobsen,

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**7**

**Could Tissue-Specific Genes**

**Be Silenced in Cattle Carrying the**

Alicia Postiglioni1, Rody Artigas1, Andrés Iriarte1, Wanda Iriarte1, Nicolás Grasso1 and Gonzalo Rincón2 *1Depto. de Genética y Mejora Animal, Facultad de Veterinaria,*

 *Universidad de la República, Montevideo,*

*2Department of Animal Science, University of California, Davis,*

> *1Uruguay 2USA*

**Rob(1;29) Robertsonian Translocation?**

Robertsonian translocations (rob) are frequent chromosome rearrangements observed in human (Nielsen & Wohlert, 1991), plants (Friebe et al., 2005), mice (Saferali et al., 2010) and domestic animals (Fries & Popescu, 1999), that are considered in arthropods classical mechanisms of chromosome evolution (Robertson, 1916). In cattle, this type of translocation is the most widespread chromosomal abnormality in European and American Creole cattle breeds. In Uruguayan Creole cattle the monocentric Robertsonian translocation rob(1;29) presents a frequency of 4%, whereas a frequency between 4 to 10% is described in beef and dairy cattle breeds (King, 1991; Postiglioni et al., 1996). In these populations there is a high incidence of heterozygous carriers and a very small number or even absence of homozygous individuals (Kastelic & Mapletoft, 2003). This situation has also been observed in the majority of cattle populations in which rob(1;29) has been

**1.1 The finding of rob(1;29) when trying to solve a sub-fertility problem in an Artificial**

In 1969, I. Gustavsson described for the first time the Robertsonian translocation rob(1;29) in cattle. This finding, together with the discoveries of pioneers in the field such as Ohno et al., (1962) and Herzog & Hohn (1968), have been important milestones in the development of domestic animal cytogenetics. Particularly, rob(1;29) was found in the Swedish Artificial Insemination Center of Black and White cattle, associated with decreased fertility. This aneuploid alteration causes a reduction in reproductive efficiency, increasing calving intervals, non-return to service and culling rates; thus leading to important economic losses

in commercial cattle herds (Bonnet-Garnier et al., 2006, 2008).

**1. Introduction**

detected (Ducos et al., 2008).

**Insemination Center**

## **Could Tissue-Specific Genes Be Silenced in Cattle Carrying the Rob(1;29) Robertsonian Translocation?**

Alicia Postiglioni1, Rody Artigas1, Andrés Iriarte1, Wanda Iriarte1, Nicolás Grasso1 and Gonzalo Rincón2 *1Depto. de Genética y Mejora Animal, Facultad de Veterinaria, Universidad de la República, Montevideo, 2Department of Animal Science, University of California, Davis, 1Uruguay 2USA*

#### **1. Introduction**

150 DNA Methylation – From Genomics to Technology

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http://www.zymoresearch.com/zrc/pdf/10xgen-poster1.pdf

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expression, functional enrichment covary with the evolutionary signature of DNA

methylcytosine-DNA glycosylase activity is present in a cloned G/T mismatch DNA glycosylase associated with the chicken embryo DNA demethylation

Robertsonian translocations (rob) are frequent chromosome rearrangements observed in human (Nielsen & Wohlert, 1991), plants (Friebe et al., 2005), mice (Saferali et al., 2010) and domestic animals (Fries & Popescu, 1999), that are considered in arthropods classical mechanisms of chromosome evolution (Robertson, 1916). In cattle, this type of translocation is the most widespread chromosomal abnormality in European and American Creole cattle breeds. In Uruguayan Creole cattle the monocentric Robertsonian translocation rob(1;29) presents a frequency of 4%, whereas a frequency between 4 to 10% is described in beef and dairy cattle breeds (King, 1991; Postiglioni et al., 1996). In these populations there is a high incidence of heterozygous carriers and a very small number or even absence of homozygous individuals (Kastelic & Mapletoft, 2003). This situation has also been observed in the majority of cattle populations in which rob(1;29) has been detected (Ducos et al., 2008).

#### **1.1 The finding of rob(1;29) when trying to solve a sub-fertility problem in an Artificial Insemination Center**

In 1969, I. Gustavsson described for the first time the Robertsonian translocation rob(1;29) in cattle. This finding, together with the discoveries of pioneers in the field such as Ohno et al., (1962) and Herzog & Hohn (1968), have been important milestones in the development of domestic animal cytogenetics. Particularly, rob(1;29) was found in the Swedish Artificial Insemination Center of Black and White cattle, associated with decreased fertility. This aneuploid alteration causes a reduction in reproductive efficiency, increasing calving intervals, non-return to service and culling rates; thus leading to important economic losses in commercial cattle herds (Bonnet-Garnier et al., 2006, 2008).

Could Tissue-Specific Genes Be Silenced

Iriarte,W., 2008).

(Postiglioni et al., 2002).

in Cattle Carrying the Rob(1;29) Robertsonian Translocation? 153

Fig. 2. Lymphocyte mephases of male and females cattle carriers of rob(1;29) (courtesy:

Treatments on metaphase chromosomes obtained from lymphocyte culture have been done (C-banding, G banding, Restriction-banding, R-banding, FISH), where the chromatin showed differential expression in the trivalent: chromosome 1, chromosome 29 and the submetacentric rob(1;29). G-banding is obtained with Giemsa stain following digestion of chromosomes with trypsin. It shows clearly dark banding along the rob(1;29) (Fig. 3A).

When metaphases of cattle carriers of rob(1;29) were treated with restriction enzymes (MspI) and C-banding, and counter-staining with propidium yodide, all the autosome centromeres were brilliant in contrast to pale regions in rob(1;29) and X chromosomes (Fig. 3B)

Fig. 3. Lymphocyte metaphases of cattle carriers of rob(1;29). A) treatment to G-banding. B) treatment to restriction-enzyme (MspI) and C-banding, counter staining with propidium yodide. Arrow signed the pale pericentromeric region in rob(1;29) and X chromosomes, in

Changes in chromatin structure may occur during the establishment of rob(1;29), altering nucleotide sequences that could be involved in embryo development and enhancing epigenetic changes (Postiglioni et al., 2011). Furthermore, King (1991) suggested an

contrast all the autosome centromeres (courtesy: Postiglioni, A., 2002).

#### **1.2 Chromosome rearrangements in the bovine genome – The origin of rob(1;29)**

Bovine presents a complex genome due to the presence of high number of repetitive sequences involved in macro and micro-rearrangements that are essential in the evolution of bovids. Schibler et al., (1998) presented a high resolution integrated bovine comparative map where the analysis of break point regions revealed specific repeated density patterns, suggesting that transposons (TEs) may have played a significant role in chromosome evolution and genome plasticity. However, bovines have a morphologically simple karyotype, corresponding to 2n=60, XX; XY.  In females the sex chromosomes correspond two large submetacentric X´s.

Fig. 1. Karyotype of cattle female, treated to G-banding. Observe all the acrocentric autosome pairs and the large submetacentric X ´s. Observe light G-banding in BTA1 and X`s chromosomes (courtesy: Postiglioni,A., 1987).

Theorically, all the acrocentric autosomes have the same probability to be involved in centric fusions, but the only known monocentric Robertsonian translocation corresponds to rob(1;29), being considered as a chromosome polymorphism (Di Meo et al., 2006). Lymphocyte culture in cattle carriers of rob(1;29) clearly present all the metaphases with this chromosome rearrangement.

Today, it is known that this chromosome translocation may have originated both from a centric fusion of chromosomes 1 and 29 and from chromosome rearrangements like pericentric inversions, in which a DNA probe (INRA143) normally mapping to BTA29, appeared proximally to rob(1;29)q-arm. Recently, Di Meo et al.,(2006) suggested a transposition as a complex rearrangement that could also be involved in the origin of this chromosome alteration.

Bovine presents a complex genome due to the presence of high number of repetitive sequences involved in macro and micro-rearrangements that are essential in the evolution of bovids. Schibler et al., (1998) presented a high resolution integrated bovine comparative map where the analysis of break point regions revealed specific repeated density patterns, suggesting that transposons (TEs) may have played a significant role in chromosome evolution and genome plasticity. However, bovines have a morphologically simple karyotype, corresponding to 2n=60, XX; XY.  In females the

**1.2 Chromosome rearrangements in the bovine genome – The origin of rob(1;29)**

Fig. 1. Karyotype of cattle female, treated to G-banding. Observe all the acrocentric

chromosomes (courtesy: Postiglioni,A., 1987).

chromosome rearrangement.

chromosome alteration.

autosome pairs and the large submetacentric X ´s. Observe light G-banding in BTA1 and X`s

Theorically, all the acrocentric autosomes have the same probability to be involved in centric fusions, but the only known monocentric Robertsonian translocation corresponds to rob(1;29), being considered as a chromosome polymorphism (Di Meo et al., 2006). Lymphocyte culture in cattle carriers of rob(1;29) clearly present all the metaphases with this

Today, it is known that this chromosome translocation may have originated both from a centric fusion of chromosomes 1 and 29 and from chromosome rearrangements like pericentric inversions, in which a DNA probe (INRA143) normally mapping to BTA29, appeared proximally to rob(1;29)q-arm. Recently, Di Meo et al.,(2006) suggested a transposition as a complex rearrangement that could also be involved in the origin of this

sex chromosomes correspond two large submetacentric X´s.

Fig. 2. Lymphocyte mephases of male and females cattle carriers of rob(1;29) (courtesy: Iriarte,W., 2008).

Treatments on metaphase chromosomes obtained from lymphocyte culture have been done (C-banding, G banding, Restriction-banding, R-banding, FISH), where the chromatin showed differential expression in the trivalent: chromosome 1, chromosome 29 and the submetacentric rob(1;29). G-banding is obtained with Giemsa stain following digestion of chromosomes with trypsin. It shows clearly dark banding along the rob(1;29) (Fig. 3A).

When metaphases of cattle carriers of rob(1;29) were treated with restriction enzymes (MspI) and C-banding, and counter-staining with propidium yodide, all the autosome centromeres were brilliant in contrast to pale regions in rob(1;29) and X chromosomes (Fig. 3B) (Postiglioni et al., 2002).

Fig. 3. Lymphocyte metaphases of cattle carriers of rob(1;29). A) treatment to G-banding. B) treatment to restriction-enzyme (MspI) and C-banding, counter staining with propidium yodide. Arrow signed the pale pericentromeric region in rob(1;29) and X chromosomes, in contrast all the autosome centromeres (courtesy: Postiglioni, A., 2002).

Changes in chromatin structure may occur during the establishment of rob(1;29), altering nucleotide sequences that could be involved in embryo development and enhancing epigenetic changes (Postiglioni et al., 2011). Furthermore, King (1991) suggested an

Could Tissue-Specific Genes Be Silenced

**carriers**

arm of rob(1;29).

et al., 2009)

proportion of pachytene spermatocytes due to lack of synapsis.

similar mechanism could take place in carriers of rob(1;29).

in Cattle Carrying the Rob(1;29) Robertsonian Translocation? 155

So, the hypothesis tested in mice was that the carriers of the translocation had lost expression of tissue specific genes from the pericentromeric region of chromosome 12 in a

Considering that DNA methylation is a potent transcriptional repressor, these authors demonstrated that lack of synapsis of chromosomes 8 and 12 during the early pachytene stage of meiosis interfere with the proper establishment of gene methylation imprinting and suggests transient silencing of genes in the unsynapsed regions. These findings support the notion that imprinting establishment extends into the prophase I of meiosis and that a

**2. An approach to understand the causes of genetic sub-fertility in rob(1;29)**

Cattle chromosomes BTA1 and BTA29 have genes that could be involved in early embryonic mortality, the main reproductive cause of sub-fertility of rob(1;29) carriers. The tissuespecific gene referred to as collagen type VIII alpha 1 (Col8A1) is related to extracellular matrix proteins that could be associated with placental mammal-specific gene groups. This gen is located in rob(1q13/21;29), next to the microsatellite BMS4015. Chromosome 1 has also another member of the collagen family, Col6A1 gene located in rob(1q4.3;29). Besides, the well-known and imprinted gene insulin -like growth factor 2 (IGF2) is located in the short

As this is a gene that has an important role in embryonic and fetal gestation, as it promotes growth of early embryos, it could also be involved in early embryonic mortality (Postiglioni

Fig. 4. The sub-metacentric chromosome rob(1;29), with the location of possible gene

In a recent study we analysed the pericentromeric region where chromosomes 1 and 29 might have suffered chromatin rearrangements due to the Robertsonian translocation polymorphism constitution. After applying different banding techniques, such as C, G and

involved in genetic sub-fertility (courtesy: Postiglioni,A., 2009).

**2.1 Chromosomal regions and clastogenic agents**

important role of gene mutation occurring on the early embryonic development of rob(1;29) carriers, probably involving collagen genes.

#### **1.3 What happens in the pachytene synapsis of cattle carrier of rob(1;29)?**

Synaptonemal complex analysis in bull heterozygous for rob(1;29) translocation revealed an absence of pairing (unsynapsing) between the proximal regions of chromosome 1 and 29 and the rob(1;29) at early pachytene indicating a lack of homology in these regions of the trivalent (Switonski et al., 1987). These authors also observed that the end-to-end association of segments of the X and Y chromosomes at early pachytene apparently resulted in a high incidence of dissociation. Recently, Raudsepp et al., (2011) demonstrated that the pseudoautosomic region (PAR) in cattle has moved to the end of the long arm (Xq) due to X chromosome rearrangements. The location of the PAR on the long or short arm of the sex chromosomes does not affect X-Y pairing.

However, these kind of transpositions might have genetic implications in the case that these structural chromosomal rearrangements were associated with the gene content of the involved regions, that may critically implicate embryonic survival. A similar situation could be involved in the chromosome rearrangements that generated the rob(1;29), where a gene of the collagen multifamily (COL8A1) is proximal to this affected region (Postiglioni et al., 2011).

Genetic causes of these effects are not yet well understood, but there are indications that the PAR and the proximal region of rob(1;29) have tissue specific genes that might be critically involved in placental and trophoblastic embryo membranes formation, and that could have a genomic imprinting effect during early embryo development (Postiglioni et al., 2009, 2011; Raudsepp et al., 2011). This statement implies that these functional regions are not only limited to morphological segregation in male meiosis, but encourared silenced of expression of tissue specific genes involved in different stages of development in carriers of rob(1;29).

Similar events have also been shown in heterozygous individuals for Robertsonian translocations in human and mice. Noreover, these actions can cause infertility if unpaired autosomes or autosomal segments are paired with the X chromosome. A clear case was shown in a cross-breed, female Limousin-Jersey studied by Basrur et al., (2001) in which chromosome 1 was associated with the inactive X chromosome.

Recently a meiotic silencing of unsynapsed chromatin (MSUC) occurs in mice germ cells of rob(8;12) translocation carriers causing meiotic arrest and infertility. In normal mice spermatogenesis, an accumulation of DNMT3A protein was shown during the midpachytene stage and also a distinct association with the XY body. But in the carriers of a translocation, this protein was proportionally less abundant in unsynapsed pericentromeric regions of chromosomes 8 and 12. This event was associated to silencing of Dnmt3a gene, with the consequence of incomplete methylation of impronted genes (Saferali et al., 2010). If *Dnmt3a* is silenced, it will lead to abnormal epigenetic marking of the spermatocyte genome, including those imprinted regions that gain methylation during spermatogenesis. In mice, heterozygous carriers of the rob(8;12) translocation are fertile but fail to completely establish methylation imprints in a proportion of their sperm. Saferali et al. (2010), proposed that this imprinting defect is due to meiotic silencing of a gene or genes located in the pericentric regions of chromosome 8 and 12 during pachytene.

important role of gene mutation occurring on the early embryonic development of rob(1;29)

Synaptonemal complex analysis in bull heterozygous for rob(1;29) translocation revealed an absence of pairing (unsynapsing) between the proximal regions of chromosome 1 and 29 and the rob(1;29) at early pachytene indicating a lack of homology in these regions of the trivalent (Switonski et al., 1987). These authors also observed that the end-to-end association of segments of the X and Y chromosomes at early pachytene apparently resulted in a high incidence of dissociation. Recently, Raudsepp et al., (2011) demonstrated that the pseudoautosomic region (PAR) in cattle has moved to the end of the long arm (Xq) due to X chromosome rearrangements. The location of the PAR on the long or short arm of the sex

However, these kind of transpositions might have genetic implications in the case that these structural chromosomal rearrangements were associated with the gene content of the involved regions, that may critically implicate embryonic survival. A similar situation could be involved in the chromosome rearrangements that generated the rob(1;29), where a gene of the collagen multifamily (COL8A1) is proximal to this affected region (Postiglioni et al.,

Genetic causes of these effects are not yet well understood, but there are indications that the PAR and the proximal region of rob(1;29) have tissue specific genes that might be critically involved in placental and trophoblastic embryo membranes formation, and that could have a genomic imprinting effect during early embryo development (Postiglioni et al., 2009, 2011; Raudsepp et al., 2011). This statement implies that these functional regions are not only limited to morphological segregation in male meiosis, but encourared silenced of expression of tissue specific genes involved in different stages of development in carriers of rob(1;29). Similar events have also been shown in heterozygous individuals for Robertsonian translocations in human and mice. Noreover, these actions can cause infertility if unpaired autosomes or autosomal segments are paired with the X chromosome. A clear case was shown in a cross-breed, female Limousin-Jersey studied by Basrur et al., (2001) in which

Recently a meiotic silencing of unsynapsed chromatin (MSUC) occurs in mice germ cells of rob(8;12) translocation carriers causing meiotic arrest and infertility. In normal mice spermatogenesis, an accumulation of DNMT3A protein was shown during the midpachytene stage and also a distinct association with the XY body. But in the carriers of a translocation, this protein was proportionally less abundant in unsynapsed pericentromeric regions of chromosomes 8 and 12. This event was associated to silencing of Dnmt3a gene, with the consequence of incomplete methylation of impronted genes (Saferali et al., 2010). If *Dnmt3a* is silenced, it will lead to abnormal epigenetic marking of the spermatocyte genome, including those imprinted regions that gain methylation during spermatogenesis. In mice, heterozygous carriers of the rob(8;12) translocation are fertile but fail to completely establish methylation imprints in a proportion of their sperm. Saferali et al. (2010), proposed that this imprinting defect is due to meiotic silencing of a gene or genes located in the pericentric

chromosome 1 was associated with the inactive X chromosome.

regions of chromosome 8 and 12 during pachytene.

**1.3 What happens in the pachytene synapsis of cattle carrier of rob(1;29)?**

carriers, probably involving collagen genes.

chromosomes does not affect X-Y pairing.

2011).

So, the hypothesis tested in mice was that the carriers of the translocation had lost expression of tissue specific genes from the pericentromeric region of chromosome 12 in a proportion of pachytene spermatocytes due to lack of synapsis.

Considering that DNA methylation is a potent transcriptional repressor, these authors demonstrated that lack of synapsis of chromosomes 8 and 12 during the early pachytene stage of meiosis interfere with the proper establishment of gene methylation imprinting and suggests transient silencing of genes in the unsynapsed regions. These findings support the notion that imprinting establishment extends into the prophase I of meiosis and that a similar mechanism could take place in carriers of rob(1;29).

#### **2. An approach to understand the causes of genetic sub-fertility in rob(1;29) carriers**

Cattle chromosomes BTA1 and BTA29 have genes that could be involved in early embryonic mortality, the main reproductive cause of sub-fertility of rob(1;29) carriers. The tissuespecific gene referred to as collagen type VIII alpha 1 (Col8A1) is related to extracellular matrix proteins that could be associated with placental mammal-specific gene groups. This gen is located in rob(1q13/21;29), next to the microsatellite BMS4015. Chromosome 1 has also another member of the collagen family, Col6A1 gene located in rob(1q4.3;29). Besides, the well-known and imprinted gene insulin -like growth factor 2 (IGF2) is located in the short arm of rob(1;29).

As this is a gene that has an important role in embryonic and fetal gestation, as it promotes growth of early embryos, it could also be involved in early embryonic mortality (Postiglioni et al., 2009)

Fig. 4. The sub-metacentric chromosome rob(1;29), with the location of possible gene involved in genetic sub-fertility (courtesy: Postiglioni,A., 2009).

#### **2.1 Chromosomal regions and clastogenic agents**

In a recent study we analysed the pericentromeric region where chromosomes 1 and 29 might have suffered chromatin rearrangements due to the Robertsonian translocation polymorphism constitution. After applying different banding techniques, such as C, G and

Could Tissue-Specific Genes Be Silenced

in Cattle Carrying the Rob(1;29) Robertsonian Translocation? 157

Chromosomes of Creole cows carrying rob(1;29) revealed heterozygosity in the expression of the fragile site rob(1q13/21;29), when processed with the clastogenic agent aphidicolin.

Comparing APC effects on BTA1q13/21 and rob(1q13/21 ;29), the high damage found in this specific region of the chromosome rearrangements, revealed an heterozygous expression

Table 1. Comparing APC effects on BTA1q13/21 and rob(1q13/21 ;29). Simultaneous expresion

It is important to connect this results to those that were discussed in: *"What happens in the pachytene synapsis of cattle carrier of rob(1;29)?"* (1.1.3). Two important facts, an unsynapsed region of the trivalent and the heterozygous expression of APC, indirectly suggest the incorporation of a nucleotide sequence rich in CTP in this region of rob(1;29), as the action of APC was higher than its "homologous" BTA1 chromosome. This particular APC effect could be related to epigenetic mechanisms (Wang, 2006). So, this particular region of late replication has been transformed to a heterozygous segregation region (Sutherland & Hecht, 1985).

In this particular region, we identified the microsatellite BMS4015 and the tissue-specific gene collagen type VIII alpha 1 (Col8A1) related to extracellular matrix proteins that could be associated with placental mammal-specific gene groups (Artigas et al., 2008a; Di Meo et

To prove the existence of a foreign nucleotide sequence in this particular chromatin region we could incorporate the research of Joerg et al., (2001). The analysis of the microsatellite BMS4015 allowed them to find a specific allele in all rob(1;29) carriers, that clearly

A pronounced chromatin despiralization of rob(1;29) similar to the inactive X chromosome of female mammals, was demonstrated when lymphocyte cultures were exposed to a DNA demethylating agent (5-azacytidine-C) (Artigas et al., 2008b; Artigas et al., 2010). This demethylating agent is considered a useful tool to study chromatin decondensation (Verma