Developmental regulation of cohesin positioning on mammalian chromosome arms

Cohesin has a well-­established role in sister chromatid cohesion and postreplicative DNA repair. In addition, previous work in our laboratory suggested a positive correlation between cohesin binding and gene expression (Parelho et al. 2008). We decided to establish the ChIP-­seq technique to address the relationship between cohesin binding and gene expression at the genome-­wide level. However, in the meantime, several other groups reported genome-­wide binding maps of cohesin in embryonic stem (ES) cells, demonstrating cell-­type-­specific cohesin binding and its correlation with gene expression (Schmidt et al. 2010; Kagey et al. 2010; Nitzsche et al. 2011). Kagey and colleagues argue that cohesin is required for the expression of pluripotency-­associated genes, based on cohesin downregulation for an extended time period. We believe this method generates indirect effects such as cell stress, death and enrichment for slowly cycling differentiating cells, biasing the results towards differentiated cells. We have generated ES cells homozygous for conditional Rad21 alleles and have found that, unlike the Kagey approach, rapid 24-­hour cohesin depletion does not induce cell stress responses. We detect a stronger correlation between cohesin-­bound genes and gene expression changes, suggesting our approach is more accurate in understanding the role of cohesin in gene expression. We have expanded our analysis of cohesin binding by generating ES cells expressing epitope-­tagged BORIS, a paralogue of CTCF. We have mapped BORIS binding sites in ES cells and data suggest that BORIS, unlike CTCF, does not recruit cohesin. To study the specific involvement of cohesin in gene expression, two developmentally regulated models, T cell receptor α (Tcrα) rearrangement and X chromosome inactivation (XCI), have been used. Cohesin loss in non-­cycling developing thymocytes leads to impaired Tcrα rearrangement. Finally, we present evidence that cohesin contributes to creating chromatin boundaries that segregate facultative heterochromatin from active chromatin on the inactive X chromosome in differentiating female ES cells

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

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. Chromosomal compartmentalization has been recognized as important for genome function. High-resolution techniques such as Hi-C, ChIP- and 4C-seq offer novel insights into cohesin's dynamic role in shaping the nuclear architecture

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

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.</p

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

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)