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1.  Pairing of Homologous Regions in the Mouse Genome Is Associated with Transcription but Not Imprinting Status 
PLoS ONE  2012;7(7):e38983.
PMCID: PMC3389011  PMID: 22802932
2.  Meet the neighbours: tools to dissect nuclear structure and function 
The eukaryotic cell nucleus displays a high degree of spatial organization, with discrete functional subcompartments that provide microenvironments where specialized processes take place. Concordantly, the genome also adopts defined conformations that, in part, enable specific genomic regions to interface with these functional centers. Yet the roles of many subcompartments and the genomic regions that contact them have not been explored fully. More fundamentally, it is not entirely clear how genome organization impacts function, and vice versa. The past decade has witnessed the development of a new breed of methods that are capable of assessing the spatial organization of the genome. These stand to further our understanding of the relationship between genome structure and function, and potentially assign function to various nuclear subcompartments. Here, we review the principal techniques used for analyzing genomic interactions, the functional insights they have afforded and discuss the outlook for future advances in nuclear structure and function dynamics.
PMCID: PMC3080762  PMID: 21258046
genome organization; nuclear structure and function; chromosome conformation capture; next generation sequencing
3.  Large Scale Loss of Data in Low-Diversity Illumina Sequencing Libraries Can Be Recovered by Deferred Cluster Calling 
PLoS ONE  2011;6(1):e16607.
Massively parallel DNA sequencing is capable of sequencing tens of millions of DNA fragments at the same time. However, sequence bias in the initial cycles, which are used to determine the coordinates of individual clusters, causes a loss of fidelity in cluster identification on Illumina Genome Analysers. This can result in a significant reduction in the numbers of clusters that can be analysed. Such low sample diversity is an intrinsic problem of sequencing libraries that are generated by restriction enzyme digestion, such as e4C-seq or reduced-representation libraries. Similarly, this problem can also arise through the combined sequencing of barcoded, multiplexed libraries. We describe a procedure to defer the mapping of cluster coordinates until low-diversity sequences have been passed. This simple procedure can recover substantial amounts of next generation sequencing data that would otherwise be lost.
PMCID: PMC3030592  PMID: 21305042
4.  Myc Dynamically and Preferentially Relocates to a Transcription Factory Occupied by Igh 
PLoS Biology  2007;5(8):e192.
Transcription in mammalian nuclei is highly compartmentalized in RNA polymerase II-enriched nuclear foci known as transcription factories. Genes in cis and trans can share the same factory, suggesting that genes migrate to preassembled transcription sites. We used fluorescent in situ hybridization to investigate the dynamics of gene association with transcription factories during immediate early (IE) gene induction in mouse B lymphocytes. Here, we show that induction involves rapid gene relocation to transcription factories. Importantly, we find that the Myc proto-oncogene on Chromosome 15 is preferentially recruited to the same transcription factory as the highly transcribed Igh gene located on Chromosome 12. Myc and Igh are the most frequent translocation partners in plasmacytoma and Burkitt lymphoma. Our results show that transcriptional activation of IE genes involves rapid relocation to preassembled transcription factories. Furthermore, the data imply a direct link between the nonrandom interchromosomal organization of transcribed genes at transcription factories and the incidence of specific chromosomal translocations.
Author Summary
Many different types of cancer result from gene translocations. Specifically, two different chromosomes can be joined that fuse growth control genes with powerful regulatory elements, leading to unrestricted control of cell growth. Translocation partner genes must physically encounter each other in the nucleus to undergo a translocation; how they find each other in the crowded nucleus is unknown. We showed previously that gene transcription occurs at a few hundred discrete nuclear sites called transcription factories. In the current study we investigated the effects of activation of the Myc proto-oncogene and examined its location with respect to transcription factories and its common translocation partner, the immunoglobulin heavy chain (Igh) gene. We found that switching on the Myc gene leads to its rapid relocation to a transcription factory. Surprisingly, we found that the activated Myc frequently chooses the same transcription factory as the highly transcribing Igh gene. This close juxtaposition of translocation partner genes at a shared transcription factory may provide the opportunity for a chromosomal translocation, and thus may be the first step in the genesis of several types of cancers.
Transcription in mammalian nuclei occurs in nuclear foci known as transcription factories. Genes from different chromosomes can share the same factory, suggesting that factories are preassembled and genes migrate to them.
PMCID: PMC1945077  PMID: 17622196
5.  Intergenic Transcription, Cell-Cycle and the Developmentally Regulated Epigenetic Profile of the Human Beta-Globin Locus 
PLoS ONE  2007;2(7):e630.
Several lines of evidence have established strong links between transcriptional activity and specific post-translation modifications of histones. Here we show using RNA FISH that in erythroid cells, intergenic transcription in the human β-globin locus occurs over a region of greater than 250 kb including several genes in the nearby olfactory receptor gene cluster. This entire region is transcribed during S phase of the cell cycle. However, within this region there are ∼20 kb sub-domains of high intergenic transcription that occurs outside of S phase. These sub-domains are developmentally regulated and enriched with high levels of active modifications primarily to histone H3. The sub-domains correspond to the β-globin locus control region, which is active at all developmental stages in erythroid cells, and the region flanking the developmentally regulated, active globin genes. These results correlate high levels of non-S phase intergenic transcription with domain-wide active histone modifications to histone H3.
PMCID: PMC1910613  PMID: 17637845
6.  Molecular Determinants of NOTCH4 Transcription in Vascular Endothelium 
Molecular and Cellular Biology  2005;25(4):1458-1474.
The process whereby the primitive vascular network develops into the mature vasculature, known as angiogenic vascular remodeling, is controlled by the Notch signaling pathway. Of the two mammalian Notch receptors expressed in vascular endothelium, Notch1 is broadly expressed in diverse cell types, whereas Notch4 is preferentially expressed in endothelial cells. As mechanisms that confer Notch4 expression were unknown, we investigated how NOTCH4 transcription is regulated in human endothelial cells and in transgenic mice. The NOTCH4 promoter and the 5′ portion of NOTCH4 assembled into an endothelial cell-specific histone modification pattern. Analysis of NOTCH4 primary transcripts in human umbilical vein endothelial cells by RNA fluorescence in situ hybridization revealed that 36% of the cells transcribed one or both NOTCH4 alleles. The NOTCH4 promoter was sufficient to confer endothelial cell-specific transcription in transfection assays, but intron 1 or upstream sequences were required for expression in the vasculature of transgenic mouse embryos. Cell-type-specific activator protein 1 (AP-1) complexes occupied NOTCH4 chromatin and conferred endothelial cell-specific transcription. Vascular angiogenic factors activated AP-1 and reprogrammed the endogenous NOTCH4 gene in HeLa cells from a repressed to a transcriptionally active state. These results reveal an AP-1-Notch4 pathway, which we propose to be crucial for transducing angiogenic signals and to be deregulated upon aberrant signal transduction in cancer.
PMCID: PMC548019  PMID: 15684396
7.  Retrovirus silencer blocking by the cHS4 insulator is CTCF independent 
Nucleic Acids Research  2003;31(18):5317-5323.
Silencing of retrovirus vectors poses a significant obstacle to genetic manipulation of stem cells and their use in gene therapy. We describe a mammalian silencer blocking assay using insulator elements positioned between retrovirus silencer elements and an LCRβ-globin reporter transgene. In transgenic mice, we show that retrovirus silencers are blocked by the cHS4 insulator. Silencer blocking is independent of the CTCF binding site and is most effective when flanking the internal reporter transgene. These data distinguish silencer blocking activity by cHS4 from its enhancer blocking activity. Retrovirus vectors can be created at high titer with one but not two internal dimer cHS4 cores. cHS4 in the LTRs has no effect on expression in transduced F9 cells, suggesting that position effect blocking is not sufficient to escape silencing. The Drosophila insulators gypsy and Scs fail to block silencing in transgenic mice, but gypsy stimulates vector expression 2-fold when located in the LTRs of an infectious retrovirus. The silencer blocking assay complements existing insulator assays in mammalian cells, provides new insight into mechanisms of insulation and is a valuable tool to identify additional silencer blocking insulators that cooperate with cHS4 to improve stem cell retrovirus vector design.
PMCID: PMC203325  PMID: 12954767
8.  Amelioration of Retroviral Vector Silencing in Locus Control Region β-Globin-Transgenic Mice and Transduced F9 Embryonic Cells 
Journal of Virology  1999;73(7):5490-5496.
Retroviral vectors are transcriptionally silenced in hematopoietic stem cells, and this phenomenon must be overcome for effective gene therapy of blood diseases. The murine stem cell virus (MSCV) vector completely silences β-globin reporter genes regulated by locus control region (LCR) elements 5′HS2 to 5′HS4 in seven of eight transgenic mice. Here, we show that no single known MSCV silencer element is sufficient for complete LCR β-globin transgene silencing. However, partial silencing of high-copy transgenes is conveyed by the MSCV direct repeat and promoter elements. The CpG methylation pattern of silenced and expressed MSCV promoter transgenes is virtually identical, demonstrating that silencing does not absolutely correlate with methylation status. Combined mutations in all four MSCV silencer elements leads to expression of β-globin in 6 of 10 transgenic mice. The same mutations incorporated into the HSC1 retrovirus vector direct neo gene expression in 71% of transduced F9 embryonic carcinoma cells. These studies demonstrate that combined mutation of four retroviral silencer elements relieves complete silencing in most transgenic mice and transduced F9 cells and suggests that novel silencer elements remain. Enhanced expression of the HSC1 vector in primitive stem cells is well suited for blood gene therapy applications.
PMCID: PMC112606  PMID: 10364297

Results 1-8 (8)