Cameron S. Osborne

REPLICATION AND TRANSCRIPTION: SHAPING THE LANDSCAPE OF THE GENOME

As the relationship between nuclear structure and function begins to unfold, a picture is emerging of a dynamic landscape that is centred on the two main processes that execute the regulated use and propagation of the genome. Rather than being subservient enzymatic activities, the replication and transcriptional machineries provide potent forces that organize the genome in three-dimensional nuclear space. Their activities provide opportunities for epigenetic changes that are required for differentiation and development. In addition, they impose physical constraints on the genome that might help to shape its evolution.

Retrovirus vector silencing is de novo methylase independent and marked by a repressive histone code

Retrovirus vectors are de novo methylated and transcriptionally silent in mammalian stem cells. Here, we identify epigenetic modifications that mark retrovirus‐silenced transgenes. We show that murine stem cell virus (MSCV) and human immunodeficiency virus type 1 (HIV‐1) vectors dominantly silence a linked locus control region (LCR) β‐globin reporter gene in transgenic mice. MSCV silencing blocks LCR hypersensitive site formation, and silent transgene chromatin is marked differentially by a histone code composed of abundant linker histone H1, deacetylated H3 and acetylated H4. Retrovirus‐transduced embryonic stem (ES) cells are silenced predominantly 3 days post‐infection, with a small subset expressing enhanced green fluorescent protein to low levels, and silencing is not relieved in de novo methylase‐null [dnmt3a−/−;dnmt3b−/−] ES cells. MSCV and HIV‐1 sequences also repress reporter transgene expression in Drosophila, demonstrating establishment of silencing in the absence of de novo and maintenance methylases. These findings provide mechanistic insight into a conserved gene silencing mechanism that is de novo methylase independent and that epigenetically marks retrovirus chromatin with a repressive histone code.

The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements

The mammalian genome harbors up to one million regulatory elements often located at great distances from their target genes. Long-range elements control genes through physical contact with promoters and can be recognized by the presence of specific histone modifications and transcription factor binding. Linking regulatory elements to specific promoters genome-wide is currently impeded by the limited resolution of high-throughput chromatin interaction assays. Here we apply a sequence capture approach to enrich Hi-C libraries for >22,000 annotated mouse promoters to identify statistically significant, long-range interactions at restriction fragment resolution, assigning long-range interacting elements to their target genes genome-wide in embryonic stem cells and fetal liver cells. The distal sites contacting active genes are enriched in active histone modifications and transcription factor occupancy, whereas inactive genes contact distal sites with repressive histone marks, demonstrating the regulatory potential of the distal elements identified. Furthermore, we find that coregulated genes cluster nonrandomly in spatial interaction networks correlated with their biological function and expression level. Interestingly, we find the strongest gene clustering in ES cells between transcription factor genes that control key developmental processes in embryogenesis. The results provide the first genome-wide catalog linking gene promoters to their long-range interacting elements and highlight the complex spatial regulatory circuitry controlling mammalian gene expression.

Large Scale Loss of Data in Low-Diversity Illumina Sequencing Libraries Can Be Recovered by Deferred Cluster Calling

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.

The pre‐B‐cell receptor induces silencing of VpreB and λ5 transcription

The pre‐B‐cell receptor (pre‐BCR), composed of Ig heavy and surrogate light chain (SLC), signals pre‐BII‐cell proliferative expansion. We have investigated whether the pre‐BCR also signals downregulation of the SLC genes (VpreB and λ5), thereby limiting this expansion. We demonstrate that, as BM cells progress from the pre‐BI to large pre‐BII‐cell stage, there is a shift from bi‐ to mono‐allelic λ5 transcription, while the second allele is silenced in small pre‐BII cells. A VpreB1‐promoter‐driven transgene shows the same pattern, therefore suggesting that VpreB1 is similarly regulated and thereby defines the promoter as a target for transcriptional silencing. Analyses of pre‐BCR‐deficient mice show a temporal delay in λ5 downregulation, thereby demonstrating that the pre‐BCR is essential for monoallelic silencing at the large pre‐BII‐cell stage. Our data also suggest that SLP‐65 is one of the signaling components important for this process. Furthermore, the VpreB1/λ5 alleles undergo dynamic changes with respect to nuclear positioning and heterochromatin association, thereby providing a possible mechanism for their transcriptional silencing.

Pairing of Homologous Regions in the Mouse Genome Is Associated with Transcription but Not Imprinting Status

Although somatic homologous pairing is common in Drosophila it is not generally observed in mammalian cells. However, a number of regions have recently been shown to come into close proximity with their homologous allele, and it has been proposed that pairing might be involved in the establishment or maintenance of monoallelic expression. Here, we investigate the pairing properties of various imprinted and non-imprinted regions in mouse tissues and ES cells. We find by allele-specific 4C-Seq and DNA FISH that the Kcnq1 imprinted region displays frequent pairing but that this is not dependent on monoallelic expression. We demonstrate that pairing involves larger chromosomal regions and that the two chromosome territories come close together. Frequent pairing is not associated with imprinted status or DNA repair, but is influenced by chromosomal location and transcription. We propose that homologous pairing is not exclusive to specialised regions or specific functional events, and speculate that it provides the cell with the opportunity of trans-allelic effects on gene regulation.

Developmental Regulation of the β-Globin Gene Locus

The β-globin genes have become a classical model for studying regulation of gene expression. Wide-ranging studies have revealed multiple levels of epigenetic regulation that coordinately ensure a highly specialised, tissue- and stage-specific gene transcription pattern. Key players include cis-acting elements involved in establishing and maintaining specific chromatin conformations and histone modification patterns, elements engaged in the transcription process through long-range regulatory interactions, trans-acting general and tissue-specific factors. On a larger scale, molecular events occurring at the locus level take place in the context of a highly dynamic nucleus as part of the cellular epigenetic programme.

Heterogeneity of the ɛγδβ-thalassaemias: characterization of three novel English deletions

We have characterized three novel ɛγδβ‐thalassaemia deletions in three English families. Two of the deletions, 114 and 439 kb, removed the entire β‐globin gene complex, including a variable number of flanking olfactory receptor (HOR) genes. The 98‐kb deletion extended 90‐kb upstream of the ɛ gene to 8 kb upstream of the Gγ‐gene, leaving the γ,δ and β‐genes intact. The 439 kb deletion is the largest deletion reported so far to cause ɛγδβ‐thalassaemia; heterozygotes for this deletion were variably affected by neonatal haemolytic anaemia. Two of the deletions were de novo. Breakpoints of all three deletions occurred within regions of L1 or Alu repeats and contained short regions of direct homology between the flanking sequences, a feature that is likely to have contributed to the illegitimate recombinations.

Novel 112 kb (epsilonGgammaAgamma) deltabeta-thalassaemia deletion in a Dutch family

Summary. An adult autochthonous Dutch patient who had exhibited severe perinatal anaemia, with partial recovery a few months after birth, was studied for the presence of β‐thalassaemia. Southern blotting showed that the patient was heterozygous for a novel deletion in the β‐globin gene cluster, leaving the β‐gene intact. Inverse polymerase chain reaction was used to determine the breakpoint sequence. The deletion removed 112 kb starting upstream of the HOR5′b6 gene to the second intron of the Aγ‐globin gene, including the locus control region. The breakpoint fragment identified a 13‐bp orphan sequence not present at either side of the breakpoint.

Where shall we meet? A role for genome organisation and nuclear sub-compartments in mediating interchromosomal interactions.

A recent spate of examples of specific interactions between loci on separate chromosomes in mammalian nuclei has illuminated another layer of complexity in gene regulation. As the specifics of the cross‐talk between interacting loci are worked out, it is also important to consider exactly how, when and where loci can ever reliably find each other within such an intricate environment. Answers may lie in how the genome is organised in relation to itself and to specialised nuclear sub‐compartments. Here, we discuss how such specialised nuclear bodies may have the potential to specifically sequester loci and provide a context where interchromosomal communications can occur. J. Cell. Biochem. 104: 1553–1561, 2008.