DNA in higher eukaryotes is packaged with histone proteins to form nucleosomes which are further assembled into higher-order chromatin fibres to protect and regulate access to the genetic ...information. Chromatin folding above the nucleosomal level is controversial with differing views proposing unfolded irregular structures through to highly organised chromatin fibres. Using a combination of techniques including sedimentation studies, electron microscopy and superresolution imaging views are converging to indicate that local chromatin is organised into a fibre, peppered with numerous discontinuities and points of flexibility. Reconciliation of different views suggests that in vitro and in vivo data are generally consistent but questions remain on how chromatin packaging is altered by cellular processes such as transcription, enzymatic chromatin remodelling and elusive DNA supercoiling.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The importance of three-dimensional chromatin organisation in genome regulation has never been clearer. But in spite of the enormous technological advances to probe chromatin organisation in vivo, ...there is still a lack of mechanistic understanding of how such an arrangement is achieved. Here we review emerging evidence pointing to an intriguing role of nuclear RNA in shaping large-scale chromatin structure and regulating genome function. We suggest this role may be achieved through the formation of a dynamic nuclear mesh that can exploit ATP-driven processes and phase separation of RNA-binding proteins to tune its assembly and material properties.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Chromatin folded into 3D macromolecular structures is often analyzed by chromosome conformation capture (3C) and fluorescence in situ hybridization (FISH) techniques, but these frequently provide ...contradictory results. Chromatin can be modeled as a simple polymer composed of a connected chain of units. By embedding data for epigenetic marks (H3K27ac), chromatin accessibility (assay for transposase-accessible chromatin using sequencing ATAC-seq), and structural anchors (CCCTC-binding factor CTCF), we developed a highly predictive heteromorphic polymer (HiP-HoP) model, where the chromatin fiber varied along its length; combined with diffusing protein bridges and loop extrusion, this model predicted the 3D organization of genomic loci at a population and single-cell level. The model was validated at several gene loci, including the complex Pax6 gene, and was able to determine locus conformations across cell types with varying levels of transcriptional activity and explain different mechanisms of enhancer use. Minimal a priori knowledge of epigenetic marks is sufficient to recapitulate complex genomic loci in 3D and enable predictions of chromatin folding paths.
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•HiP-HoP: highly predictive heteromorphic polymer model to analyze chromatin structure•Polymer simulations use widely available epigenetic and protein binding data as input•Validate HiP-HoP model at complex loci using 3D FISH and Capture-C•Simulations uncover striking conformational variability in chromatin fiber folding
Buckle et al. develop a highly predictive heteromorphic polymer (HiP-HoP) model that uses epigenetic and protein binding data to predict 3D chromatin organizations, validated by FISH and Capture-C experiments. These simulations show striking conformation variability in fiber folding within a cell population and can explain different mechanisms of enhancer use.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Centromeres are highly specialized genomic loci that function during mitosis to maintain genome stability. Formed primarily on repetitive α-satellite DNA sequence characterisation of native ...centromeric chromatin structure has remained challenging. Fortuitously, neocentromeres are formed on a unique DNA sequence and represent an excellent model to interrogate centromeric chromatin structure. This review uncovers the specific findings from independent neocentromere studies that have advanced our understanding of canonical centromere chromatin structure.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Centromeres are essential for chromosome segregation and are specified epigenetically by the presence of the histone H3 variant CENP-A. In flies and humans, replenishment of the centromeric mark is ...uncoupled from DNA replication and requires the removal of H3 "placeholder" nucleosomes. Although transcription at centromeres has been previously linked to the loading of new CENP-A, the underlying molecular mechanism remains poorly understood. Here, we used
tissue culture cells to show that centromeric presence of actively transcribing RNA polymerase II temporally coincides with de novo deposition of dCENP-A. Using a newly developed dCENP-A loading system that is independent of acute transcription, we found that short inhibition of transcription impaired dCENP-A incorporation into chromatin. Interestingly, initial targeting of dCENP-A to centromeres was unaffected, revealing two stability states of newly loaded dCENP-A: a salt-sensitive association with the centromere and a salt-resistant chromatin-incorporated form. This suggests that transcription-mediated chromatin remodeling is required for the transition of dCENP-A to fully incorporated nucleosomes at the centromere.
Experimental approaches have been applied to address questions in understanding three-dimensional chromatin organization and function. As datasets increase in size and complexity, it becomes a ...challenge to reach a mechanistic interpretation of experimental results. Polymer simulations and mechanistic modeling have been applied to explain experimental observations and their links to different aspects of genome function. Here we provide a guide for biologists, explaining different simulation approaches and the contexts in which they have been used.
DNA supercoiling is an inherent consequence of twisting DNA and is critical for regulating gene expression and DNA replication. However, DNA supercoiling at a genomic scale in human cells is ...uncharacterized. To map supercoiling, we used biotinylated trimethylpsoralen as a DNA structure probe to show that the human genome is organized into supercoiling domains. Domains are formed and remodeled by RNA polymerase and topoisomerase activities and are flanked by GC-AT boundaries and CTCF insulator protein-binding sites. Underwound domains are transcriptionally active and enriched in topoisomerase I, 'open' chromatin fibers and DNase I sites, but they are depleted of topoisomerase II. Furthermore, DNA supercoiling affects additional levels of chromatin compaction as underwound domains are cytologically decondensed, topologically constrained and decompacted by transcription of short RNAs. We suggest that supercoiling domains create a topological environment that facilitates gene activation, providing an evolutionary purpose for clustering genes along chromosomes.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Focal adhesion kinase (FAK) promotes anti-tumor immune evasion. Specifically, the kinase activity of nuclear-targeted FAK in squamous cell carcinoma (SCC) cells drives exhaustion of CD8+ T cells and ...recruitment of regulatory T cells (Tregs) in the tumor microenvironment by regulating chemokine/cytokine and ligand-receptor networks, including via transcription of Ccl5, which is crucial. These changes inhibit antigen-primed cytotoxic CD8+ T cell activity, permitting growth of FAK-expressing tumors. Mechanistically, nuclear FAK is associated with chromatin and exists in complex with transcription factors and their upstream regulators that control Ccl5 expression. Furthermore, FAK’s immuno-modulatory nuclear activities may be specific to cancerous squamous epithelial cells, as normal keratinocytes do not have nuclear FAK. Finally, we show that a small-molecule FAK kinase inhibitor, VS-4718, which is currently in clinical development, also drives depletion of Tregs and promotes a CD8+ T cell-mediated anti-tumor response. Therefore, FAK inhibitors may trigger immune-mediated tumor regression, providing previously unrecognized therapeutic opportunities.
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•Depletion or kinase inhibition of FAK can cause squamous cell carcinoma regression•FAK promotes tumor evasion by inducing an immuno-suppressive microenvironment•Nuclear FAK promotes transcription of chemokines that drive recruitment of Tregs•FAK-induced Tregs inhibit cytotoxic CD8+ T cells, allowing tumor tolerance and growth
Nuclear focal adhesion kinase (FAK) regulates transcription of chemokines that drive recruitment of tumor-associated regulatory T cells (Tregs), thereby creating a tumor suppressive microenvironment by inhibiting cytotoxic CD8+ T cell activity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
How polycomb group proteins repress gene expression in vivo is not known. While histone-modifying activities of the polycomb repressive complexes (PRCs) have been studied extensively, in vitro data ...have suggested a direct activity of the PRC1 complex in compacting chromatin. Here, we investigate higher-order chromatin compaction of polycomb targets in vivo. We show that PRCs are required to maintain a compact chromatin state at Hox loci in embryonic stem cells (ESCs). There is specific decompaction in the absence of PRC2 or PRC1. This is due to a PRC1-like complex, since decompaction occurs in
Ring1B null cells that still have PRC2-mediated H3K27 methylation. Moreover, we show that the ability of Ring1B to restore a compact chromatin state and to repress Hox gene expression is not dependent on its histone ubiquitination activity. We suggest that Ring1B-mediated chromatin compaction acts to directly limit transcription in vivo.
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► Hox loci are specifically decompacted in the absence of PRC2 or PRC1 components ►
Ring1B null embryonic stem cells (ESCs) still have PRC2-mediated H3K27 methylation ► Ring1B maintains a compact chromatin state and silences Hox loci in ESCs ► Compaction and silencing are independent of Ring1B's histone ubiquitination activity
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Chemical inhibitors of histone deacetylase (HDAC) activity are used as experimental tools to induce histone hyperacetylation and deregulate gene transcription, but it is not known whether the ...inhibition of HDACs plays any part in the normal physiological regulation of transcription. Using both in vitro and in vivo assays, we show that lactate, which accumulates when glycolysis exceeds the cell's aerobic metabolic capacity, is an endogenous HDAC inhibitor, deregulating transcription in an HDAC-dependent manner. Lactate is a relatively weak inhibitor (IC(50) 40 mM) compared to the established inhibitors trichostatin A and butyrate, but the genes deregulated overlap significantly with those affected by low concentrations of the more potent inhibitors. HDAC inhibition causes significant up and downregulation of genes, but genes that are associated with HDAC proteins are more likely to be upregulated and less likely to be downregulated than would be expected. Our results suggest that the primary effect of HDAC inhibition by endogenous short-chain fatty acids like lactate is to promote gene expression at genes associated with HDAC proteins. Therefore, we propose that lactate may be an important transcriptional regulator, linking the metabolic state of the cell to gene transcription.
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