The cohesin complex plays an essential role in maintaining genome organization. However, its role in gene regulation remains largely unresolved. Here, we report that the cohesin release factor WAPL creates a pool of free cohesin, in a process known as cohesin turnover, which reloads it to cell-type specific binding sites. Paradoxically, stabilization of cohesin binding, following WAPL ablation, results in depletion of cohesin from these cell-type specific regions, a loss of gene expression and differentiation. Chromosome conformation capture experiments show that cohesin turnover is important for maintaining promoter-enhancer loops. Binding of cohesin to cell-type specific sites is dependent on the pioneer transcription factors OCT4 (POU5F1) and SOX2, but not NANOG. We show the importance of cohesin turnover in controlling transcription and propose that a cycle of cohesin loading and off-loading, instead of static cohesin binding, mediates promoter and enhancer interactions critical for gene regulation.

Conformation capture-approaches like Hi-C can elucidate chromosome structure at a genome-wide scale. Hi-C datasets are large and require specialised software. Here, we present GENOVA: a user-friendly software package to analyse and visualise chromosome conformation capture (3C) data. GENOVA is an R-package that includes the most common Hi-C analyses, such as compartment and insulation score analysis. It can create annotated heatmaps to visualise the contact frequency at a specific locus and aggregate Hi-C signal over user-specified genomic regions such as ChIP-seq data. Finally, our package supports output from the major mapping-pipelines. We demonstrate the capabilities of GENOVA by analysing Hi-C data from HAP1 cell lines in which the cohesin-subunits SA1 and SA2 were knocked out. We find that ΔSA1 cells gain intra-TAD interactions and increase compartmentalisation. ΔSA2 cells have longer loops and a less compartmentalised genome. These results suggest that cohesinSA1 forms longer loops, while cohesinSA2 plays a role in forming and maintaining intra-TAD interactions. Our data supports the model that the genome is provided structure in 3D by the counter-balancing of loop formation on one hand, and compartmentalization on the other hand. By differentially controlling loops, cohesinSA1 and cohesinSA2 therefore also affect nuclear compartmentalization. We show that GENOVA is an easy to use R-package, that allows researchers to explore Hi-C data in great detail.

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Cytotoxic T cell differentiation is guided by epigenome adaptations, but how epigenetic mechanisms control lymphocyte development has not been well defined. Here we show that the histone methyltransferase DOT1L, which marks the nucleosome core on active genes, safeguards normal differentiation of CD8+ T cells. T cell-specific ablation of Dot1L resulted in loss of naïve CD8+ T cells and premature differentiation toward a memory-like state, independent of antigen exposure and in a cell-intrinsic manner. Mechanistically, DOT1L controlled CD8+ T cell differentiation by ensuring normal T cell receptor density and signaling. DOT1L also maintained epigenetic identity, in part by indirectly supporting the repression of developmentally regulated genes. Finally, deletion of Dot1L in T cells resulted in an impaired immune response. Through our study, DOT1L is emerging as a central player in physiology of CD8+ T cells, acting as a barrier to prevent premature differentiation and controlling epigenetic integrity.

Cytotoxic T-cell differentiation is guided by epigenome adaptations but how epigenetic mechanisms control lymphocyte development has not been well defined. Here we show that the histone methyltransferase DOT1L, which marks the nucleosome core on active genes, safeguards normal differentiation of CD8 + T cells. T-cell specific ablation of Dot1L resulted in loss of naïve CD8 + T cells and premature differentiation towards a memory-like state, independent of antigen exposure and in a cellintrinsic manner. Without DOT1L, the memory-like CD8 + cells fail to acquire full effector functions in vitro and in vivo. Mechanistically, DOT1L controlled T-cell differentiation and function by ensuring normal T-cell receptor density and signaling, and by maintaining epigenetic identity, in part by indirectly supporting the repression of developmentally-regulated genes. Through our study DOT1L is emerging as a central player in physiology of CD8 + T cells, acting as a barrier to prevent premature differentiation and supporting the licensing of the full effector potential of cytotoxic T cells.

HIGHLIGHTS 1. The cohesin release factor WAPL is crucial for maintaining a pluripotency-specific phenotype.2. Dynamic cohesin is enriched at lineage specific loci and overlaps with binding sites of pluripotency transcription factors.3. Expression of lineage specific genes is maintained by dynamic cohesin binding through the formation of promoter-enhancer associated self-interaction domains.4. CTCF-independent cohesin binding to chromatin is controlled by the pioneer factor OCT4. SUMMARYThe cohesin complex plays essential roles in sister chromatin cohesin, chromosome organization and gene expression. The role of cohesin in gene regulation is incompletely understood. Here, we report that the cohesin release factor WAPL is crucial for maintaining a pool of dynamic cohesin bound to regions that are associated with lineage specific genes in mouse embryonic stem cells. These regulatory regions are enriched for active enhancer marks and transcription factor binding sites, but largely devoid of CTCF binding sites. Stabilization of cohesin, which leads to a loss of dynamic cohesin from these regions, does not affect transcription factor binding or active enhancer marks, but does result in changes in promoter-enhancer interactions and downregulation of genes.Acute cohesin depletion can phenocopy the effect of WAPL depletion, showing that cohesin plays a crucial role in maintaining expression of lineage specific genes. The binding of dynamic cohesin to chromatin is dependent on the pluripotency transcription factor OCT4, but not NANOG. Finally, dynamic cohesin binding sites are also found in differentiated cells, suggesting that they represent a general regulatory principle. We propose that cohesin dynamically binding to regulatory sites creates a favorable spatial environment in which promoters and enhancers can communicate to ensure proper gene expression.

In this study, we originally aimed to characterize the potential role of Argonaute 2 (AGO2) in the nucleus, a key protein of the miRNA machinery. We combined Chromatin Immunoprecipitation (ChIP) with high throughput sequencing (ChIP-seq) and quantitative mass spectrometry (ChIP-MS) using the broadly used AGO2 11A9 antibody to determine interactions with chromatin and nuclear proteins. We found a previously described interaction between AGO2 and SWI/SNF on chromatin with ChIP-MS and observed enrichment at enhancers and transcription start sites using ChIP-seq. However, antibody specificity issues can produce misleading results for ChIP, RNA-seq and Mass spectrometry. Therefore, we developed a CRISPR/Cas9 engineered AGO2−/− HEK293T cell line to validate our findings. ChIP-qPCR and immunoprecipitation combined with MS (IP-MS) showed that the 11A9 antibody associates with chromatin and SWI/SNF in the absence of AGO2. Furthermore, stoichiometry, IP-MS and co-IP analysis suggests a direct interaction of this antibody with SMARCC1, a component of the SWI/SNF complex. For this reason, particular care should be taken in performing and interpreting experiments in which the 11A9 antibody is used to study a nuclear role of AGO2.

Genome-wide transcriptional activity involves the binding of many transcription factors to thousands of sites in the genome. Determining which sites are directly driving transcription remains a challenge. Here we use acute protein depletion of the pioneer transcription factors OCT4 and SOX2 to establish their functionality in maintaining chromatin accessibility. We show that thousands of accessible sites are lost within an hour of protein depletion, indicating rapid turnover of these sites in the absence of pioneer factors. To understand the relationship with transcription we performed nascent transcription analysis and found that open chromatin sites that are maintained by SOX2 are highly predictive of gene expression, in contrast to SOX2 binding sites that do not maintain accessibility. We use CRISPR-Cas9 genome editing in the Klf2 locus to functionally validate a predicted regulatory element. We conclude that the regulatory activity of SOX2 is exerted largely at sites where it maintains accessibility and that other binding sites are largely dispensable for gene regulation.