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.

Cis- and trans-regulation in X inactivation.

Female mammalian cells compensate dosage of X-linked gene expression through the inactivation of one of their two X chromosomes. X chromosome inactivation (XCI) in eutherians is dependent on the non-coding RNA Xist that is up-regulated from the future inactive X chromosome, coating it and recruiting factors involved in silencing and altering its chromatin state. Xist lies within the X-inactivation center (Xic), a region on the X that is required for XCI, and is regulated in cis by elements on the X chromosome and in trans by diffusible factors. In this review, we summarize the latest results in cis- and trans-regulation of the Xic. We discuss how the organization of the Xic in topologically associating domains is important for XCI (cis-regulation) and how proteins in the pluripotent state and upon development or differentiation of embryonic stem cells control proper inactivation of one X chromosome (trans-regulation).

Cohesin-mediated interactions organize chromosomal domain architecture.

To ensure proper gene regulation within constrained nuclear space, chromosomes facilitate access to transcribed regions, while compactly packaging all other information. Recent studies revealed that chromosomes are organized into megabase-scale domains that demarcate active and inactive genetic elements, suggesting that compartmentalization is important for genome function. Here, we show that very specific long-range interactions are anchored by cohesin/CTCF sites, but not cohesin-only or CTCF-only sites, to form a hierarchy of chromosomal loops. These loops demarcate topological domains and form intricate internal structures within them. Post-mitotic nuclei deficient for functional cohesin exhibit global architectural changes associated with loss of cohesin/CTCF contacts and relaxation of topological domains. Transcriptional analysis shows that this cohesin-dependent perturbation of domain organization leads to widespread gene deregulation of both cohesin-bound and non-bound genes. Our data thereby support a role for cohesin in the global organization of domain structure and suggest that domains function to stabilize the transcriptional programmes within them.

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.

Orchestrating Asymmetric Expression: Mechanisms behind Xist Regulation.

Compensation for the gene dosage disequilibrium between sex chromosomes in mammals is achieved in female cells by repressing one of its X chromosomes through a process called X chromosome inactivation (XCI), exemplifying the control of gene expression by epigenetic mechanisms. A critical player in this mechanism is Xist, a long, non-coding RNA upregulated from a single X chromosome during early embryonic development in female cells. Over the past few decades, many factors involved at different levels in the regulation of Xist have been discovered. In this review, we hierarchically describe and analyze the different layers of Xist regulation operating concurrently and intricately interacting with each other to achieve asymmetric and monoallelic upregulation of Xist in murine female cells. We categorize these into five different classes: DNA elements, transcription factors, other regulatory proteins, long non-coding RNAs, and the chromatin and topological landscape surrounding Xist.

SPEN is required for Xist upregulation during initiation of X chromosome inactivation.

At initiation of X chromosome inactivation (XCI), Xist is monoallelically upregulated from the future inactive X (Xi) chromosome, overcoming repression by its antisense transcript Tsix. Xist recruits various chromatin remodelers, amongst them SPEN, which are involved in silencing of X-linked genes in cis and establishment of the Xi. Here, we show that SPEN plays an important role in initiation of XCI. Spen null female mouse embryonic stem cells (ESCs) are defective in Xist upregulation upon differentiation. We find that Xist-mediated SPEN recruitment to the Xi chromosome happens very early in XCI, and that SPEN-mediated silencing of the Tsix promoter is required for Xist upregulation. Accordingly, failed Xist upregulation in Spen-/- ESCs can be rescued by concomitant removal of Tsix. These findings indicate that SPEN is not only required for the establishment of the Xi, but is also crucial in initiation of the XCI process.

REX1 is the critical target of RNF12 in imprinted X chromosome inactivation in mice.

In mice, imprinted X chromosome inactivation (iXCI) of the paternal X in the pre-implantation embryo and extraembryonic tissues is followed by X reactivation in the inner cell mass (ICM) of the blastocyst to facilitate initiation of random XCI (rXCI) in all embryonic tissues. RNF12 is an E3 ubiquitin ligase that plays a key role in XCI. RNF12 targets pluripotency protein REX1 for degradation to initiate rXCI in embryonic stem cells (ESCs) and loss of the maternal copy of Rnf12 leads to embryonic lethality due to iXCI failure. Here, we show that loss of Rex1 rescues the rXCI phenotype observed in Rnf12-/- ESCs, and that REX1 is the prime target of RNF12 in ESCs. Genetic ablation of Rex1 in Rnf12-/- mice rescues the Rnf12-/- iXCI phenotype, and results in viable and fertile Rnf12-/-:Rex1-/- female mice displaying normal iXCI and rXCI. Our results show that REX1 is the critical target of RNF12 in XCI.

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.

New Xist-Interacting Proteins in X-Chromosome Inactivation.

To achieve dosage compensation of X-linked gene expression, female mammalian cells inactivate one X chromosome through a process called X-chromosome inactivation (XCI). A central component of this process is the X-encoded long non-coding RNA Xist. Following upregulation from one X chromosome, Xist spreads in cis, kicking off a plethora of events that ultimately results in stable X-linked gene repression, which is then faithfully transmitted to all daughter cells. In the last decades, intensive work has been undertaken to understand each of the steps in XCI, namely Xist transcription control, Xist spreading and localization, and silencing of gene expression. Recently, several groups have spearheaded the research of Xist's interactome and the factors involved in silencing. Several novel proteins have now been shown to be required for the transcriptional silencing of the X chromosome and/or Xist spreading and localization to the inactive X chromosome. Here, we review these new findings in the context of existing knowledge about Xist-interacting factors.

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.