Analysis of Cohesin Function in Gene Regulation and Chromatin Organization in Interphase.

Cohesin is essential for the maintenance of chromosomes through the cell cycle. In addition, cohesin contributes to the regulation of gene expression and the organization of chromatin in interphase cells. To study cohesin's role in gene expression and chromatin organization, it is necessary to avoid secondary effects due to disruption of vital cohesin functions in the cell cycle. Here we describe experimental approaches to achieve this and the methods applied to define cohesin's role in interphase.

Cohesin's role in pluripotency and reprogramming.

Cohesin is required for ES cell self-renewal and iPS-mediated reprogramming of somatic cells. This may indicate a special role for cohesin in the regulation of pluripotency genes, perhaps by mediating long-range chromosomal interactions between gene regulatory elements. However, cohesin is also essential for genome integrity, and its depletion from cycling cells induces DNA damage responses. Hence, the failure of cohesin-depleted cells to establish or maintain pluripotency gene expression could be explained by a loss of long-range interactions or by DNA damage responses that undermine pluripotency gene expression. In recent work we began to disentangle these possibilities by analyzing reprogramming in the absence of cell division. These experiments showed that cohesin was not specifically required for reprogramming, and that the expression of most pluripotency genes was maintained when ES cells were acutely depleted of cohesin. Here we take this analysis to its logical conclusion by demonstrating that deliberately inflicted DNA damage - and the DNA damage that results from proliferation in the absence of cohesin - can directly interfere with pluripotency and reprogramming. The role of cohesin in pluripotency and reprogramming may therefore be best explained by essential cohesin functions in the cell cycle.

Initiation and maintenance of pluripotency gene expression in the absence of cohesin.

Cohesin is implicated in establishing and maintaining pluripotency. Whether this is because of essential cohesin functions in the cell cycle or in gene regulation is unknown. Here we tested cohesin's contribution to reprogramming in systems that reactivate the expression of pluripotency genes in the absence of proliferation (embryonic stem [ES] cell heterokaryons) or DNA replication (nuclear transfer). Contrary to expectations, cohesin depletion enhanced the ability of ES cells to initiate somatic cell reprogramming in heterokaryons. This was explained by increased c-Myc (Myc) expression in cohesin-depleted ES cells, which promoted DNA replication-dependent reprogramming of somatic fusion partners. In contrast, cohesin-depleted somatic cells were poorly reprogrammed in heterokaryons, due in part to defective DNA replication. Pluripotency gene induction was rescued by Myc, which restored DNA replication, and by nuclear transfer, where reprogramming does not require DNA replication. These results redefine cohesin's role in pluripotency and reveal a novel function for Myc in promoting the replication-dependent reprogramming of somatic nuclei.

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.

Presumptive germ cells derived from mouse pluripotent somatic cell hybrids.

The fusion of embryonic stem (ES) cells with differentiated somatic cells is an approach that reverses a somatic cell nucleus to a state of pluripotency. The resulting ES-somatic cell hybrids (ES-SCH) retain most of the properties of ES cells: differentiate into multiple cell types and have the ability to produce embryoid bodies (EB) and chimeras. However, it is still unknown whether ES-SCH will be able to complete the differentiation into germ cells (GC) in vitro similar to ES cells. Here, we show that near diploid ES-SCH, obtained by the fusion of mouse ES and spleen cells, were able to differentiate in vitro into presumptive GC. Differentiation of ES-SCH was induced through EB formation and by the addition of retinoic acid. Presumptive GC obtained reacted positively with anti-EMA, Vasa, Fragilis and Dazl antibodies and expressed GC-specific genes, such as Vasa, Stella, Dazl, Piwil 2, Tex14, Bmp8b, Tdrd1 and Rnf17. Fluorescent in situ hybridization analysis indicates chromosome reduction in the GC-like cells. Expression of meiotic and postmeiotic GC-specific genes such as Haprin, Acrosin, Scyp1, Scyp3 and Stra-8 were also detected. Transmission electron microscopy confirmed ES-SCH differentiation into presumptive GC. The presence of several autosomes and the X chromosome originated from the "somatic" partner did not prevent ES-SCH differentiation towards presumptive GC. Overall our study suggests an interesting in vitro model, which allows the study GC differentiation in reprogrammed somatic cells.

In vitro differentiation of male mouse embryonic stem cells into both presumptive sperm cells and oocytes.

Pioneer work in male mouse embryonic stem (ES) cells differentiation into germ cells (GC) showed generations of male or female gametes in separate experiments, using genetically manipulated or preselected ES cells. In an attempt to produce both types of gametes from male mouse ES cells without any genetic manipulation or preselection, we induce the differentiation by retinoic acid (RA) within nonadherent embryoid bodies (EB). It seems that gamete-like cell formation occurs in the correct manner based on the expression of early and late GC-specific genes such as Oct-4, Mvh, Stella, Dazl, Piwil 2, Pdrd 1, Rex 14, Rnf 17, Bmp8b, Acrosin, Stra-8, Haprin, LH-R, Gdf9, Zp3, Zp2, Sycp1, and Sycp3. Immunofluorescence analysis of morphologically well-formed GC and presumptive gametes showed positive labeling for SSEA1, Oct-4, EMA-1, FE-J1, Dazl, Fragilis, Mvh, Acrosin, and acetylated alpha-tubulin. Conventional cytogenetic and FISH analysis indicated a chromosome reduction in ES-derived GC. Our data suggest that ES cells with XY chromosomes can produce under the same experimental conditions both types of presumptive gametes, and this production depends on their positional and temporal information within the EB context.