Complex exon-intron marking by histone modifications is not determined solely by nucleosome distribution

It has recently been shown that nucleosome distribution, histone modifications and RNA polymerase II (Pol II) occupancy show preferential association with exons (“exon-intron marking”), linking chromatin structure and function to co-transcriptional splicing in a variety of eukaryotes. Previous ChIP-sequencing studies suggested that these marking patterns reflect the nucleosomal landscape. By analyzing ChIP-chip datasets across the human genome in three cell types, we have found that this marking system is far more complex than previously observed. We show here that a range of histone modifications and Pol II are preferentially associated with exons. However, there is noticeable cell-type specificity in the degree of exon marking by histone modifications and, surprisingly, this is also reflected in some histone modifications patterns showing biases towards introns. Exon-intron marking is laid down in the absence of transcription on silent genes, with some marking biases changing or becoming reversed for genes expressed at different levels. Furthermore, the relationship of this marking system with splicing is not simple, with only some histone modifications reflecting exon usage/inclusion, while others mirror patterns of exon exclusion. By examining nucleosomal distributions in all three cell types, we demonstrate that these histone modification patterns cannot solely be accounted for by differences in nucleosome levels between exons and introns. In addition, because of inherent differences between ChIP-chip array and ChIP-sequencing approaches, these platforms report different nucleosome distribution patterns across the human genome. Our findings confound existing views and point to active cellular mechanisms which dynamically regulate histone modification levels and account for exon-intron marking. We believe that these histone modification patterns provide links between chromatin accessibility, Pol II movement and co-transcriptional splicing

Characterization of the ZFX family of transcription factors that bind downstream of the start site of CpG island promoters

Our study focuses on a family of ubiquitously expressed human C₂H₂ zinc finger proteins comprised of ZFX, ZFY and ZNF711. Although their protein structure suggests that ZFX, ZFY and ZNF711 are transcriptional regulators, the mechanisms by which they influence transcription have not yet been elucidated. We used CRISPR-mediated deletion to create bi-allelic knockouts of ZFX and/or ZNF711 in female HEK293T cells (which naturally lack ZFY). We found that loss of either ZFX or ZNF711 reduced cell growth and that the double knockout cells have major defects in proliferation. RNA-seq analysis revealed that thousands of genes showed altered expression in the double knockout clones, suggesting that these TFs are critical regulators of the transcriptome. To gain insight into how these TFs regulate transcription, we created mutant ZFX proteins and analyzed them for DNA binding and transactivation capability. We found that zinc fingers 11–13 are necessary and sufficient for DNA binding and, in combination with the N terminal region, constitute a functional transactivator. Our functional analyses of the ZFX family provides important new insights into transcriptional regulation in human cells by members of the large, but under-studied family of C₂H₂ zinc finger proteins

Hyperosmotic priming of arabidopsis seedlings establishes a long-term somatic memory accompanied by specific changes of the epigenome

<p>Background: In arid and semi-arid environments, drought and soil salinity usually occur at the beginning and end of a plant's life cycle, offering a natural opportunity for the priming of young plants to enhance stress tolerance in mature plants. Chromatin marks, such as histone modifications, provide a potential molecular mechanism for priming plants to environmental stresses, but whether transient exposure of seedlings to hyperosmotic stress leads to chromatin changes that are maintained throughout vegetative growth remains unclear.</p> <p>Results: We have established an effective protocol for hyperosmotic priming in the model plant Arabidopsis, which includes a transient mild salt treatment of seedlings followed by an extensive period of growth in control conditions. Primed plants are identical to non-primed plants in growth and development, yet they display reduced salt uptake and enhanced drought tolerance after a second stress exposure. ChIP-seq analysis of four histone modifications revealed that the priming treatment altered the epigenomic landscape; the changes were small but they were specific for the treated tissue, varied in number and direction depending on the modification, and preferentially targeted transcription factors. Notably, priming leads to shortening and fractionation of H3K27me3 islands. This effect fades over time, but is still apparent after a ten day growth period in control conditions. Several genes with priming-induced differences in H3K27me3 showed altered transcriptional responsiveness to the second stress treatment.</p> <p>Conclusion: Experience of transient hyperosmotic stress by young plants is stored in a long-term somatic memory comprising differences of chromatin status, transcriptional responsiveness and whole plant physiology.</p&gt

Control of VEGF-A transcriptional programs by pausing and genomic compartmentalization.

Vascular endothelial growth factor A (VEGF-A) is a master regulator of angiogenesis, vascular development and function. In this study we investigated the transcriptional regulation of VEGF-A-responsive genes in primary human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) using genome-wide global run-on sequencing (GRO-Seq). We demonstrate that half of VEGF-A-regulated gene promoters are characterized by a transcriptionally competent paused RNA polymerase II (Pol II). We show that transition into productive elongation is a major mechanism of gene activation of virtually all VEGF-regulated genes, whereas only ∼40% of the genes are induced at the level of initiation. In addition, we report a comprehensive chromatin interaction map generated in HUVECs using tethered conformation capture (TCC) and characterize chromatin interactions in relation to transcriptional activity. We demonstrate that sites of active transcription are more likely to engage in chromatin looping and cell type-specific transcriptional activity reflects the boundaries of chromatin interactions. Furthermore, we identify large chromatin compartments with a tendency to be coordinately transcribed upon VEGF-A stimulation. We provide evidence that these compartments are enriched for clusters of regulatory regions such as super-enhancers and for disease-associated single nucleotide polymorphisms (SNPs). Collectively, these findings provide new insights into mechanisms behind VEGF-A-regulated transcriptional programs in endothelial cells

Nascent RNA sequencing reveals mechanisms of gene regulation in the human malaria parasite Plasmodium falciparum.

Gene expression in Plasmodium falciparum is tightly regulated to ensure successful propagation of the parasite throughout its complex life cycle. The earliest transcriptomics studies in P. falciparum suggested a cascade of transcriptional activity over the course of the 48-hour intraerythrocytic developmental cycle (IDC); however, the just-in-time transcriptional model has recently been challenged by findings that show the importance of post-transcriptional regulation. To further explore the role of transcriptional regulation, we performed the first genome-wide nascent RNA profiling in P. falciparum. Our findings indicate that the majority of genes are transcribed simultaneously during the trophozoite stage of the IDC and that only a small subset of genes is subject to differential transcriptional timing. RNA polymerase II is engaged with promoter regions prior to this transcriptional burst, suggesting that Pol II pausing plays a dominant role in gene regulation. In addition, we found that the overall transcriptional program during gametocyte differentiation is surprisingly similar to the IDC, with the exception of relatively small subsets of genes. Results from this study suggest that further characterization of the molecular players that regulate stage-specific gene expression and Pol II pausing will contribute to our continuous search for novel antimalarial drug targets

Identification of H3K4me1-associated proteins at mammalian enhancers.

Enhancers act to regulate cell-type-specific gene expression by facilitating the transcription of target genes. In mammalian cells, active or primed enhancers are commonly marked by monomethylation of histone H3 at lysine 4 (H3K4me1) in a cell-type-specific manner. Whether and how this histone modification regulates enhancer-dependent transcription programs in mammals is unclear. In this study, we conducted SILAC mass spectrometry experiments with mononucleosomes and identified multiple H3K4me1-associated proteins, including many involved in chromatin remodeling. We demonstrate that H3K4me1 augments association of the chromatin-remodeling complex BAF to enhancers in vivo and that, in vitro, H3K4me1-marked nucleosomes are more efficiently remodeled by the BAF complex. Crystal structures of the BAF component BAF45C indicate that monomethylation, but not trimethylation, is accommodated by BAF45C's H3K4-binding site. Our results suggest that H3K4me1 has an active role at enhancers by facilitating binding of the BAF complex and possibly other chromatin regulators