Karen E. Brown

Association of Transcriptionally Silent Genes with Ikaros Complexes at Centromeric Heterochromatin

Ikaros proteins are required for normal T, B, and NK cell development and are postulated to activate lymphocyte-specific gene expression. Here we examined Ikaros distribution in the nucleus of B lymphocytes using confocal microscopy and a novel immunofluorescence in situ hybridization (immuno-FISH) approach. Unexpectedly, Ikaros localized to discrete heterochromatin-containing foci in interphase nuclei, which comprise clusters of centromeric DNA as defined by gamma-satellite sequences and the abundance of heterochromatin protein-1 (HP-1). Using locus-specific probes for CD2, CD4, CD8alpha, CD19, CD45, and lambda5 genes, we show that transcriptionally inactive but not transcriptionally active genes associate with Ikaros-heterochromatin foci. These findings support a model of organization of the nucleus in which repressed genes are selectively recruited into centromeric domains.

Dynamic Repositioning of Genes in the Nucleus of Lymphocytes Preparing for Cell Division

We show that several transcriptionally inactive genes localize to centromeric heterochromatin in the nucleus of cycling but not quiescent (noncycling) primary B lymphocytes. In quiescent cells, centromeric repositioning of inactive loci was induced after mitogenic stimulation. A dynamic repositioning of selected genes was also observed in developing T cells. Rag and TdT loci were shown to relocate to centromeric domains following heritable gene silencing in primary CD4+8+ thymocytes, but not in a phenotypically similar cell line in which silencing occurred but was not heritable. Collectively, these data indicate that the spatial organization of genes in cycling and noncycling lymphocytes is different and that locus repositioning may be a feature of heritable gene silencing.

Nonequivalent nuclear location of immunoglobulin alleles in B lymphocytes

Individual B lymphocytes normally express immunoglobulin (Ig) proteins derived from single Ig heavy chain (H) and light chain (L) alleles. Allelic exclusion ensures monoallelic expression of Ig genes by each B cell to maintain single receptor specificity. Here we provide evidence that at later stages of B cell development, additional mechanisms may contribute to prioritizing expression of single IgH and IgL alleles. Fluorescent in situ hybridization analysis of primary splenic B cells isolated from normal and genetically manipulated mice showed that endogenous IgH, κ and λ alleles localized to different subnuclear environments after activation and had differential expression patterns. However, this differential recruitment and expression of Ig alleles was not typically seen among transformed B cell lines. These data raise the possibility that epigenetic factors help maintain the monoallelic expression of Ig.

CEP152 is a genome maintenance protein disrupted in Seckel syndrome

Functional impairment of DNA damage response pathways leads to increased genomic instability. Here we describe the centrosomal protein CEP152 as a new regulator of genomic integrity and cellular response to DNA damage. Using homozygosity mapping and exome sequencing, we identified CEP152 mutations in Seckel syndrome and showed that impaired CEP152 function leads to accumulation of genomic defects resulting from replicative stress through enhanced activation of ATM signaling and increased H2AX phosphorylation.

A role for cohesin in T cell receptor rearrangement and thymocyte differentiation

Cohesin enables post-replicative DNA repair and chromosome segregation by holding sister chromatids together from the time of DNA replication in S phase until mitosis. There is growing evidence that cohesin also forms long-range chromosomal cis-interactions and may regulate gene expression in association with CTCF, mediator or tissue-specific transcription factors. Human cohesinopathies such as Cornelia de Lange syndrome are thought to result from impaired non-canonical cohesin functions, but a clear distinction between the cell-division-related and cell-division-independent functions of cohesion—as exemplified in Drosophila—has not been demonstrated in vertebrate systems. To address this, here we deleted the cohesin locus Rad21 in mouse thymocytes at a time in development when these cells stop cycling and rearrange their T-cell receptor (TCR) α locus (Tcra). Rad21-deficient thymocytes had a normal lifespan and retained the ability to differentiate, albeit with reduced efficiency. Loss of Rad21 led to defective chromatin architecture at the Tcra locus, where cohesion-binding sites flank the TEA promoter and the Eα enhancer, and demarcate Tcra from interspersed Tcrd elements and neighbouring housekeeping genes. Cohesin was required for long-range promoter–enhancer interactions, Tcra transcription, H3K4me3 histone modifications that recruit the recombination machinery and Tcra rearrangement. Provision of pre-rearranged TCR transgenes largely rescued thymocyte differentiation, demonstrating that among thousands of potential target genes across the genome, defective Tcra rearrangement was limiting for the differentiation of cohesin-deficient thymocytes. These findings firmly establish a cell-division-independent role for cohesin in Tcra locus rearrangement and provide a comprehensive account of the mechanisms by which cohesin enables cellular differentiation in a well-characterized mammalian system.

Expression of alpha- and beta-globin genes occurs within different nuclear domains in haemopoietic cells

The α- and β-globin gene clusters have been extensively studied. Regulation of these genes ensures that proteins derived from both loci are produced in balanced amounts, and that expression is tissue-restricted and specific to developmental stages. Here we compare the subnuclear location of the endogenous α- and β-globin loci in primary human cells in which the genes are either actively expressed or silent. In erythroblasts, the α- and β-globin genes are localized in areas of the nucleus that are discrete from α-satellite-rich constitutive heterochromatin. However, in cycling lymphocytes, which do not express globin genes, the distribution of α- and β-globin genes was markedly different. β-globin loci, in common with several inactive genes studied here (human c-fms and SOX-1) and previously (mouse λ5, CD4, CD8α, RAGs, TdT and Sox-1), were associated with pericentric heterochromatin in a high proportion of cycling lymphocytes. In contrast, α-globin genes were not associated with centromeric heterochromatin in the nucleus of normal human lymphocytes, in lymphocytes from patients with α-thalassaemia lacking the regulatory HS-40 element or entire upstream region of the α-globin locus, or in mouse erythroblasts and lymphocytes derived from human α-globin transgenic mice. These data show that the normal regulated expression of α- and β-globin gene clusters occurs in different nuclear environments in primary haemopoietic cells.

Dynamic assembly of silent chromatin during thymocyte maturation

Considerable knowledge has been gained from temporal analyses of molecular events culminating in gene activation, but technical hurdles have hindered comparable studies of gene silencing. Here we describe the temporal assembly of silent chromatin at the mouse terminal transferase gene (Dntt), which is silenced and repositioned to pericentromeric heterochromatin during thymocyte maturation. Silencing was nucleated at the Dntt promoter by the ordered deacetylation of histone H3 at Lys9 (H3-Lys9), loss of methylation at H3-Lys4 and acquisition of methylation at H3-Lys9, followed by bidirectional spreading of each event. Deacetylation at H3-Lys9 coincided with pericentromeric repositioning, and neither of these early events required de novo protein synthesis. CpG methylation increased primarily in mature T cells that had left the thymus. A transformed thymocyte line supported reversible inactivation of Dntt without repositioning. In these cells, histone modification changes were nucleated at the promoter but did not spread. These results provide a foundation for elucidating the mechanisms of silent chromatin assembly during development.

Heritable gene silencing in lymphocytes delays chromatid resolution without affecting the timing of DNA replication.

Temporal control of DNA replication has been implicated in epigenetic regulation of gene expression on the basis of observations that certain tissue-specific genes replicate earlier in expressing than non-expressing cells. Here, we show evidence that several leukocyte-specific genes replicate early in lymphocytes regardless of their transcription and also in fibroblasts, where these genes are never normally expressed. Instead, the heritable silencing of some genes (Rag-1, TdT, CD8α and λ5) and their spatial recruitment to heterochromatin domains within the nucleus of lymphocytes resulted in a markedly delayed resolution of sister chromatids into doublet signals discernable by 3D fluorescence in situ hybridization (FISH). Integration of transgenes within heterochromatin (in cis) did, however, confer late replication and this was reversed after variegated transgene expression. These findings emphasise that chromosomal location is important for defining the replication timing of genes and show that retarded sister-chromatid resolution is a novel feature of inactive chromatin.

DNA Synthesis Is Required for Reprogramming Mediated by Stem Cell Fusion

Summary Embryonic stem cells (ESCs) can instruct the conversion of differentiated cells toward pluripotency following cell-to-cell fusion by a mechanism that is rapid but poorly understood. Here, we used centrifugal elutriation to enrich for mouse ESCs at sequential stages of the cell cycle and showed that ESCs in S/G2 phases have an enhanced capacity to dominantly reprogram lymphocytes and fibroblasts in heterokaryon and hybrid assays. Reprogramming success was associated with an ability to induce precocious nucleotide incorporation within the somatic partner nuclei in heterokaryons. BrdU pulse-labeling experiments revealed that virtually all successfully reprogrammed somatic nuclei, identified on the basis of Oct4 re-expression, had undergone DNA synthesis within 24 hr of fusion with ESCs. This was essential for successful reprogramming because drugs that inhibited DNA polymerase activity effectively blocked pluripotent conversion. These data indicate that nucleotide incorporation is an early and critical event in the epigenetic reprogramming of somatic cells in experimental ESC-heterokaryons.

Centromeric Repositioning of Coreceptor Loci Predicts Their Stable Silencing and the CD4/CD8 Lineage Choice

The differentiation of CD4+ CD8+ double positive (DP) thymocytes requires the irreversible choice between two alternative lineages, distinguished by the mutually exclusive expression of either CD4 or CD8. Differentiating DP cells transiently down-regulate both CD4 and CD8, and this has complicated the debate whether the mechanism of CD4/CD8 lineage choice is instructive, stochastic/selective, or more complex in nature. Using fluorescence in situ hybridization, we show that the stable silencing of coreceptor loci, and ultimately lineage choice, is predicted by the spatial repositioning of coreceptor alleles to centromeric heterochromatin domains. These data provide evidence that lineage-specific developmental programs are established early during the transition from the DP to the single positive stage.