Active Chromatin Regions Research Articles

LAP2alpha facilitates myogenic gene expression by preventing nucleoplasmic lamin A/C from spreading to active chromatin regions.

A-type lamins form a filamentous meshwork beneath the nuclear membrane that anchors large heterochromatic genomic regions at the nuclear periphery. A-type lamins also exist as a dynamic, non-filamentous pool in the nuclear interior, where they interact with lamin-associated polypeptide 2 alpha (LAP2α). Both proteins associate with largely overlapping euchromatic genomic regions in the nucleoplasm, but the functional significance of this interaction is poorly understood. Here, we report that LAP2α relocates towards regions containing myogenic genes in the early stages of muscle differentiation, possibly facilitating efficient gene regulation, while lamins A and C mostly associate with genomic regions away from these genes. Strikingly, upon depletion of LAP2α, A-type lamins spread across active chromatin and accumulate at regions of active H3K27ac and H3K4me3 histone marks in the vicinity of myogenic genes whose expression is impaired in the absence of LAP2α. Reorganization of A-type lamins on chromatin is accompanied by depletion of the active chromatin mark H3K27ac and a significantly impaired myogenic differentiation. Thus, the interplay of LAP2α and A-type lamins is crucial for proper positioning of intranuclear lamin A/C on chromatin to allow efficient myogenic differentiation.

Identification of a specific APOE transcript and functional elements associated with Alzheimer’s disease

BackgroundThe APOE gene is the strongest genetic risk factor for late-onset Alzheimer’s Disease (LOAD). However, the gene regulatory mechanisms at this locus remain incompletely characterized.MethodsTo identify novel AD-linked functional elements within the APOE locus, we integrated SNP variants with multi-omics data from human postmortem brains including 2,179 RNA-seq samples from 3 brain regions and two ancestries (European and African), 667 DNA methylation samples, and ChIP-seq samples. Additionally, we plotted the expression trajectory of APOE transcripts in human brains during development.ResultsWe identified an AD-linked APOE transcript (jxn1.2.2) particularly observed in the dorsolateral prefrontal cortex (DLPFC). The APOE jxn1.2.2 transcript is associated with brain neuropathological features, cognitive impairment, and the presence of the APOE4 allele in DLPFC. We prioritized two independent functional SNPs (rs157580 and rs439401) significantly associated with jxn1.2.2 transcript abundance and DNA methylation levels. These SNPs are located within active chromatin regions and affect brain-related transcription factor-binding affinities. The two SNPs shared effects on the jxn1.2.2 transcript between European and African ethnic groups.ConclusionThe novel APOE functional elements provide potential therapeutic targets with mechanistic insight into the disease etiology.

P-045 Deciphering meiotic chromatin organization by SCP3

Abstract Study question What’s the pattern of genome-wide chromatin occupancy of SCP3 during meiotic prophase, and how its interaction with chromatin is regulated? Summary answer We document SCP3 enriched at active chromatin regions with largely inherited from leptotene to pachytene stage, which facilitated by transcription and condensate assembly in spermatocytes. What is known already Meiosis is a critical step during the process of gametogenesis to form haploid cells which finally develop into functional gametes. The synaptonemal complex (SC) is a meiosis-specific protein assembly which bridges homologous chromosomes to establish the architecture of meiotic chromosome. Genetic ablation of genes encoding SC components impedes meiosis and gametogenesis in mice and mutation of these genes is frequently found in infertile patients. SCP3 localization changes with chromosome morphology in autosomes and is involved in maintaining the association of sex chromosomes. Male mice with null mutation of SCP3 failed to form SCs, resulting in developmental arrest during meiotic prophase. Study design, size, duration This study included male mice at post-natal day 10 (P10), 4 weeks or adult (8 weeks or older) were maintained on C57BL/6J background. Spermatocytes were performed modified CUT&Tag using antibodies. ChIP-seq data was analyzed together with WGBS and ATAC-seq data. Participants/materials, setting, methods Spermatocytes were obtained by a discontinuous BSA density gradient. Cell pellet was resuspended in culture medium (MEM with 10% FBS) at a concentration of 105 cells/ml and cultured at 37 °C in a 5% CO2 incubator. Spermatocytes were cultured with or without 1,6-hexanediol or α-amanitin. Spermatocytes with or without drug treatment were collected at indicated time points for further examination and then modified CUT&Tag and co-IP using anti-SCP3 and anti-POL II antibodies. Main results and the role of chance Chromatin occupancy of SCP3 in leptotene and pachytene spermatocytes, SCP3-Core sites retained upon treatment of 1,6-hexanediol which dissolves SCP3 condensate, and SCP1 occupancy at meiotic chromatin in pachytene spermatocytes. When we determined inter-peak distances and width of peaks, we observe that: (1) SCP3 occupancy in leptotene stage had greater inter-peak distances and narrower peak width than SCP3 occupancy in pachytene stage, demonstrating gradual accumulated SCP3 at chromatin during meiotic progression; (2) SCP3-Core sites have broader peak width than SCP3-NonCore sites, in agreement with stability of SCP3 occupancy at SCP3-Core sites; (3) SCP1 occupancy had greater inter-peak distances and narrower peak width than SCP3 occupancy in pachytene stage, supporting that SCP1 peaks are a subset of SCP3 peaks and SCP1’s major function in connecting LE and CE respectively. Generally, at pachytene-stage meiosis, SCP3 mainly occupies chromatin regions including transcriptionally active sites, Deu and Alu elements, while SCP1 mainly co-localizes with SCP3 at a subset of regions with open chromatin and high cohesin enrichment to connect meiotic chromosome axes with CE. In this way, a local active chromatin environment is created at meiotic chromosome axes, facilitating homologous chromosome recombination. Limitations, reasons for caution Our test was not successful to examine SCP3 occupancy with as few as less than 1000 spermatocytes using our protocol. It is improvement of efficiency of tagmentation or development of more efficient antibodies against SCP3 will be helpful for clinical sample detection. Wider implications of the findings SCP3 occupancy was largely inherited from leptotene to pachytene stage, facilitated by transcription and condensate assembly, and enriched at specific SINE repeats. Further exploration using knockout mouse models of meiotic axial proteins and epigenetic modifiers will provide valuable information on meiotic chromatin architecture and crosstalk among meiotic chromatin events. Trial registration number N/A

Behaviors of nucleosomes with mutant histone H4s in euchromatic domains of living human cells.

Since Robert Feulgen first stained DNA in the cell, visualizing genome chromatin has been a central issue in cell biology to uncover how chromatin is organized and behaves in the cell. To approach this issue, we have developed single-molecule imaging of nucleosomes, a basic unit of chromatin, to unveil local nucleosome behavior in living cells. In this study, we investigated behaviors of nucleosomes with various histone H4 mutants in living HeLa cells to address the role of H4 tail acetylation, including H4K16Ac and others, which are generally associated with more transcriptionally active chromatin regions. We ectopically expressed wild-type (wt) or mutated H4s (H4K16 point; H4K5,8,12,16 quadruple; and H4 tail deletion) fused with HaloTag in HeLa cells. Cells that expressed wtH4-Halo, H4K16-Halo mutants, and multiple H4-Halo mutants had euchromatin-concentrated distribution. Consistently, the genomic regions of the wtH4-Halo nucleosomes corresponded to Hi-C contact domains (or topologically associating domains, TADs)with active chromatin marks (A-compartment). Utilizing single-nucleosome imaging, we found that none of the H4 deacetylation or acetylation mimicked H4 mutants altered the overall local nucleosome motion. This finding suggests that H4 mutant nucleosomes embedded in the condensed euchromatic domains with excess endogenous H4 nucleosomes cannot cause an observable change in the local motion. Interestingly, H4 with four lysine-to-arginine mutations displayed a substantial freely diffusing fraction in the nucleoplasm, whereas H4 with a truncated N-terminal tail was incorporated in heterochromatic regions as well as euchromatin. Our study indicates the power of single-nucleosome imaging to understand individual histone/nucleosome behavior reflecting chromatin environments in living cells.

Nucleoporin Elys attaches peripheral chromatin to the nuclear pores in interphase nuclei

Transport of macromolecules through the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs) consisting of nucleoporins (Nups). Elys/Mel-28 is the Nup that binds and connects the decondensing chromatin with the reassembled NPCs at the end of mitosis. Whether Elys links chromatin with the NE during interphase is unknown. Here, using DamID-seq, we identified Elys binding sites in Drosophila late embryos and divided them into those associated with nucleoplasmic or with NPC-linked Elys. These Elys binding sites are located within active or inactive chromatin, respectively. Strikingly, Elys knockdown in S2 cells results in peripheral chromatin displacement from the NE, in decondensation of NE-attached chromatin, and in derepression of genes within. It also leads to slightly more compact active chromatin regions. Our findings indicate that NPC-linked Elys, together with the nuclear lamina, anchors peripheral chromatin to the NE, whereas nucleoplasmic Elys decompacts active chromatin.

Epigenetically active chromatin in neonatal iWAT reveals GABPα as a potential regulator of beige adipogenesis.

Thermogenic beige adipocytes, which dissipate energy as heat, are found in neonates and adults. Recent studies show that neonatal beige adipocytes are highly plastic and contribute to >50% of beige adipocytes in adults. Neonatal beige adipocytes are distinct from recruited beige adipocytes in that they develop independently of temperature and sympathetic innervation through poorly defined mechanisms. We characterized the neonatal beige adipocytes in the inguinal white adipose tissue (iWAT) of C57BL6 postnatal day 3 and 20 mice (P3 and P20) by imaging, genome-wide RNA-seq analysis, ChIP-seq analysis, qRT-PCR validation, and biochemical assays. We found an increase in acetylated histone 3 lysine 27 (H3K27ac) on the promoter and enhancer regions of beige-specific gene UCP1 in iWAT of P20 mice. Furthermore, H3K27ac ChIP-seq analysis in the iWAT of P3 and P20 mice revealed strong H3K27ac signals at beige adipocyte-associated genes in the iWAT of P20 mice. The integration of H3K27ac ChIP-seq and RNA-seq analysis in the iWAT of P20 mice reveal epigenetically active signatures of beige adipocytes, including oxidative phosphorylation and mitochondrial metabolism. We identify the enrichment of GA-binding protein alpha (GABPα) binding regions in the epigenetically active chromatin regions of the P20 iWAT, particularly on beige genes, and demonstrate that GABPα is required for beige adipocyte differentiation. Moreover, transcriptomic analysis and glucose oxidation assays revealed increased glycolytic activity in the neonatal iWAT from P20. Our findings demonstrate that epigenetic mechanisms regulate the development of peri-weaning beige adipocytes via GABPα. Further studies to better understand the upstream mechanisms that regulate epigenetic activation of GABPα and characterization of the metabolic identity of neonatal beige adipocytes will help us harness their therapeutic potential in metabolic diseases.

TEAD2 initiates ground-state pluripotency by mediating chromatin looping

The transition of mouse embryonic stem cells (ESCs) between serum/LIF and 2i(MEK and GSK3 kinase inhibitor)/LIF culture conditions serves as a valuable model for exploring the mechanisms underlying ground and confused pluripotent states. Regulatory networks comprising core and ancillary pluripotency factors drive the gene expression programs defining stable naïve pluripotency. In our study, we systematically screened factors essential for ESC pluripotency, identifying TEAD2 as an ancillary factor maintaining ground-state pluripotency in 2i/LIF ESCs and facilitating the transition from serum/LIF to 2i/LIF ESCs. TEAD2 exhibits increased binding to chromatin in 2i/LIF ESCs, targeting active chromatin regions to regulate the expression of 2i-specific genes. In addition, TEAD2 facilitates the expression of 2i-specific genes by mediating enhancer-promoter interactions during the serum/LIF to 2i/LIF transition. Notably, deletion of Tead2 results in reduction of a specific set of enhancer-promoter interactions without significantly affecting binding of chromatin architecture proteins, CCCTC-binding factor (CTCF), and Yin Yang 1 (YY1). In summary, our findings highlight a novel prominent role of TEAD2 in orchestrating higher-order chromatin structures of 2i-specific genes to sustain ground-state pluripotency.

Study on chromatin regulation patterns of expression vectors in the PhiC31 integration site

ABSTRACT The PhiC31 integration system allows for targeted and efficient transgene integration and expression by recognizing pseudo attP sites in mammalian cells and integrating the exogenous genes into the open chromatin regions of active chromatin. In order to investigate the regulatory patterns of efficient gene expression in the open chromatin region of PhiC31 integration, this study utilized Ubiquitous Chromatin Opening Element (UCOE) and activating RNA (saRNA) to modulate the chromatin structure in the promoter region of the PhiC31 integration vector. The study analysed the effects of DNA methylation and nucleosome occupancy changes in the integrated promoter on gene expression levels. The results showed that for the OCT4 promoter with moderate CG density, DNA methylation had a smaller impact on expression compared to changes in nucleosome positioning near the transcription start site, which was crucial for enhancing downstream gene expression. On the other hand, for the SOX2 promoter with high CG density, increased methylation in the CpG island upstream of the transcription start site played a key role in affecting high expression, but the positioning and clustering of nucleosomes also had an important influence. In conclusion, analysing the DNA methylation patterns, nucleosome positioning, and quantity distribution of different promoters can determine whether the PhiC31 integration site possesses the potential to further enhance expression or overcome transgene silencing effects by utilizing chromatin regulatory elements.

Cis-regulatory effect of HPV integration is constrained by host chromatin architecture in cervical cancers.

Human papillomavirus (HPV) infections are the primary drivers of cervical cancers, and often HPV DNA gets integrated into the host genome. Although the oncogenic impact of HPV encoded genes is relatively well known, the cis-regulatory effect of integrated HPV DNA on host chromatin structure and gene regulation remains less understood. We investigated genome-wide patterns of HPV integrations and associated host gene expression changes in the context of host chromatin states and topologically associating domains (TADs). HPV integrations were significantly enriched in active chromatin regions and depleted in inactive ones. Interestingly, regardless of chromatin state, genomic regions flanking HPV integrations showed transcriptional upregulation. Nevertheless, upregulation (both local and long-range) was mostly confined to TADs with integration, but not affecting adjacent TADs. Few TADs showed recurrent integrations associated with overexpression of oncogenes within them (e.g. MYC, PVT1, TP63 and ERBB2) regardless of proximity. Hi-C and 4C-seq analyses in cervical cancer cell line (HeLa) demonstrated chromatin looping interactions between integrated HPV and MYC/PVT1 regions (~ 500 kb apart), leading to allele-specific overexpression. Based on these, we propose HPV integrations can trigger multimodal oncogenic activation to promote cancer progression.

Stage-specific dynamic reorganization of genome topology shapes transcriptional neighborhoods in developing human retinal organoids

We have generated a high-resolution Hi-C map of developing human retinal organoids to elucidate spatiotemporal dynamics of genomic architecture and its relationship with gene expression patterns. We demonstrate progressive stage-specific alterations in DNA topology and correlate these changes with transcription of cell-type-restricted gene markers during retinal differentiation. Temporal Hi-C reveals a shift toward A compartment for protein-coding genes and B compartment for non-coding RNAs, displaying high and low expression, respectively. Notably, retina-enriched genes are clustered near lost boundaries of topologically associated domains (TADs), and higher-order assemblages (i.e., TAD cliques) localize in active chromatin regions with binding sites for eye-field transcription factors. These genes gain chromatin contacts at their transcription start site as organoid differentiation proceeds. Our study provides a global view of chromatin architecture dynamics associated with diversification of cell types during retinal development and serves as a foundational resource for in-depth functional investigations of retinal developmental traits.

IRF2 Regulates De Novo Active Chromatin Regions and Participates in the Development and Pathogenesis of Multiple Myeloma

Multiple Myeloma (MM) is the second most common hematologic neoplasia, and it remains largely incurable due to the genetic heterogeneity of the patients. We have recently characterized the epigenome of plasma cells from patients with MM, describing the de novo activation of the chromatin as a unifying factor underlying the pathobiology of the MM. Precisely, we identified 1556 de novo active regions, showing the H3K27ac chromatin activation mark, in MM in comparison to different subpopulations of normal B cells. Moreover, those de novo active regions where enriched in Transcription Factor Binding Sites (TFBS). In order to defined the potential role of these regions we have performed a CRISPR-Cas9 screening using guides against 56 TF that presented TFBS in de novo active regions and that are expressed in MM. A CRISPR-Cas9 library containing 10 guides for each gene was transduced in two MM cell lines (MM.1R and KMS-11) and the presences of guides were analyzed at days +1, +14 and +21. Out of the 22 TFs shown to be essential for MM survival, there were 3 members of the IRF TF family (IRF1, IRF2 and IRF5). Single knockdowns identified IRF2 and the previously described IRF4 as the best candidates. To characterize the signalling pathways involved in IRF family regulation of MM, the chromatin regions where IRF1, IRF2 and IRF4 are bound were mapped using CUT&RUN in MM.1R and KMS-11 cell lines. Interestingly, the three TF were located in 573 out of the 1556 de novo active regions. Notably, IRF2 was present in the 86.9% of those regions, and in 40% of the cases, the binding occurred independently of IRF1 or IRF4 ( Fig. 1). Additionally, the genomic distribution demonstrated that the regions where IRF2 binds alone were enriched in promoters compared to those regions where IRF1 and IRF4 also bound, being these enriched mostly in the gene body. Gene Ontology studies showed that IRF2, as well as IRF1/4 were involved in regulation of cell death in response to oxidative stress, while only IRF2 autonomously was involved in the regulation of pathways relevant for MM such as the kinase dysregulation, cell migration and osteoblast regulation. Furthermore, using our previous RNA-seq data, we observed that the majority of the genes regulated uniquely by IRF2 presented an overexpression in MM in comparison to different subpopulations of normal B cells. This overexpression was already identified in plasma cells from MGUS (Monoclonal Gammopathy of Undetermined Significance) patients and SMM (Smouldering MM) stages in contrast to normal bone marrow plasma cells. Finally, we examined the impact of IRF2 and IRF4 expression on the outcome of MM using RNA-seq data from 664 patients from MMRF (MM Research Foundation) CoMMpass (IA18) database. We observed that patients with higher expression of IRF2 presented significantly poorer Progression Free Survival (PFS) and Overall Survival (OS) in univariate analysis. Interestingly, in multivariate analyses taking into account the classic high-risk genetic factors for MM, the expression of IRF2 together with del13q and amp1q resulted in a significantly lower PFS (global p < 0.001) and the expression of IRF2 together with amp1q and del17p resulted in statistically significant worse OS (global p < 0.001). On a multivariate model, MM patients were stratified according to the number of events ( IRF2 expression, del13q and amp1q for PFS and IRF2 expression, amp1q and del17p for OS) into four risk groups both for PFS and OS ( Fig. 2). In conclusion, our study demonstrate that IRF2 plays a significant role in the pathogenesis of MM by participating in the regulation of the de novo active chromatin regions, specifically regions related to kinase pathway. Additionally, IRF2 represents a robust biomarker that in combination with classical genetic markers improves stratification of MM patients. These results establish IRF2 as a promising candidate for new therapeutic strategies in patients with MM.

HIV-1 Gag co-localizes with euchromatin histone marks at the nuclear periphery.

The traditional view of retrovirus assembly posits that packaging of gRNA by HIV-1 Gag occurs in the cytoplasm or at the plasma membrane. However, our previous studies showing that HIV-1 Gag enters the nucleus and binds to USvRNA at transcription sites suggest that gRNA selection may occur in the nucleus. In the present study, we observed that HIV-1 Gag trafficked to the nucleus and co-localized with USvRNA within 8 hours of expression. In infected T cells (J-Lat 10.6) reactivated from latency and in a HeLa cell line stably expressing an inducible Rev-dependent HIV-1 construct, we found that Gag preferentially localized with euchromatin histone marks associated with enhancer and promoter regions near the nuclear periphery, which is the favored site HIV-1 integration. These observations support the innovative hypothesis that HIV-1 Gag associates with euchromatin-associated histones to localize to active transcription sites, promoting capture of newly synthesized gRNA for packaging.

Increased histone acetylation is the signature of repressed state on the genes transcribed by RNA polymerase III

Several covalent modifications are found associated with the transcriptionally active chromatin regions constituted by the genes transcribed by RNA polymerase (pol) II. Pol III-transcribed genes code for the small, stable RNA species, which participate in many cellular processes, essential for survival. Pol III transcription is repressed under most of the stress conditions by its negative regulator Maf1. We found that most of the histone acetylations increase with starvation-induced repression on several genes transcribed by the yeast pol III. On one of these genes, SNR6 (coding for the U6snRNA), a strongly positioned nucleosome in the gene upstream region plays regulatory role under repression. On this nucleosome, the changes in H3K9 and H3K14 acetylations show different dynamics. During repression, acetylation levels on H3K9 show steady increase whereas H3K14 acetylation increases with a peak at 40 min after which levels reduce. Both the levels settle by 2 hr to a level higher than the active state, which revert to normal levels with nutrient repletion. The increase in H3 acetylations is seen in the mutants reported to show reduced SNR6 transcription but not in the maf1Δ cells. This increase on a regulatory nucleosome may be part of the signaling mechanisms, which prepare cells for the stress-related quick repression as well as reactivation. The contrasting association of the histone acetylations with pol II and pol III transcription may be an important consideration to make in research studies focused on drug developments targeting histone modifications.

A Multimodal Single Cell Atlas of Adult Murine Haematopoiesis Reveals Nine Differentiation Trajectories and Epigenetic Regulatory Programs

Single cell genomics has revolutionised our understanding of differentiating systems and led to a reinterpretation of haematopoiesis, moving from a step-wise hierarchical tree model towards more continous differentiation landscapes. Recent single cell RNA sequencing (scRNA-seq) studies of Lineage- ckit+ (LK) mouse bone marrow progenitors have defined a highly granular landscape of differentiation from multipotent stem cells to committed progenitors in 8 different lineages and have served as a valuable reference landscape for comparisons with perturbation states. While these landscapes have permitted exploration and interrogation of early haematopoietic progenitors, as yet, no map of mouse total bone marrow haematopoiesis exists at single cell resolution, limiting efforts to fully understand the gene programs which specify and the cell surface phenotypes which identify maturing haematopoietic lineages nor the connection between early bifurcation decisions in progenitor cells and mature cellular outputs. To answer these questions, we performed droplet-based scRNA-seq and cell surface proteomics on whole bone marrow from 4 male and 4 female 12-week old C57Bl6 mice, using a panel of 138 antibodies against cell surface antigens. To increase representation of rare progenitor populations, we complemented total bone marrow mononuclear cells with c-kit enrichment and FACS sorted LK and LK sca1-positive (LSK) populations. After quality control filtering the single cell transcriptomes and proteomes, over 198,000 single cells were analysed separately before multimodal integration into a single UMAP embedding. Addition of cell surface proteome information to the scRNA-seq data through multimodal integration produced a representation with greater resolution of haematopoietic cell types, including improved resolution of T-cell subtypes and separation of a rare population of eosinophil progenitors which were previously indistinguishable from basophil progenitors. This integrated landscape of mouse haematopoiesis was then clustered and annotated though an iterative process of label transfer and manual annotation into 41 cell type states (Figure 1). Using cell fate analysis, which utilises transcriptional similarity and pseudotemporal ordering to estimate the probability of a single cell's commitment to defined terminal states, we were able to resolve cell fate probabilities towards nine haematopoietic lineages (erythroid, neutrophil, megakaryocyte, lymphoid, monocyte, dendritic cell (DC), plasmacytoid dendritic cell (pDC), basophil and eosinophil (Figure 2)). From these probabilities we defined nine trajectories from the earliest uncommitted progenitors to terminally differentiated populations, including a pDC trajectory involving both lymphoid and common DC progenitors. Analysis of trajectory-based gene expression trends provides a framework for discovery of lineage drivers as well as shared gene expression programs across multiple lineages. Leveraging cell surface proteome data permits the use of ‘in-silico-FACS’ to describe phenotypes of cell populations which exist at cell fate branch-points, for further in-vitro characterisation. To demonstrate the utility of our atlas as a reference, we projected perturbation datasets from 7 pre-leukaemic mouse models onto the atlas, allowing quantitation of mutation-associated cellular and tissue-scale alterations. Finally, we have leveraged the transcription-start site enrichment of 5' scRNA-seq to compute enhancer RNA (eRNA) expression in our atlas, allowing the identification of dynamic usage of specific enhancer elements during haematopoietic specification and thus providing a novel framework for correlation of eRNA expression (as a proxy for active distal chromatin regions) with gene expression trends along specific haematopoietic lineages.

Imaging-based study demonstrates how the DEK nanoscale distribution differentially correlates with epigenetic marks in a breast cancer model

Epigenetic dysregulation of chromatin is one of the hallmarks of cancer development and progression, and it is continuously investigated as a potential general bio-marker of this complex disease. One of the nuclear factors involved in gene regulation is the unique DEK protein—a histone chaperon modulating chromatin topology. DEK expression levels increase significantly from normal to cancer cells, hence raising the possibility of using DEK as a tumor marker. Although DEK is known to be implicated in epigenetic and transcriptional regulation, the details of these interactions and their relevance in cancer development remain largely elusive. In this work, we investigated the spatial correlation between the nuclear distribution of DEK and chromatin patterns—alongside breast cancer progression—leveraging image cross-correlation spectroscopy (ICCS) coupled with Proximity Ligation Assay (PLA) analysis. We performed our study on the model based on three well-established human breast cell lines to consider this tumor's heterogeneity (MCF10A, MCF7, and MDA-MB-231 cells). Our results show that overexpression of DEK correlates with the overall higher level of spatial proximity between DEK and histone marks corresponding to gene promoters regions (H3K9ac, H3K4me3), although it does not correlate with spatial proximity between DEK and gene enhancers (H3K27ac). Additionally, we observed that colocalizing fractions of DEK and histone marks are lower for the non-invasive cell subtype than for the highly invasive cell line (MDA-MB-231). Thus, this study suggests that the role of DEK on transcriptionally active chromatin regions varies depending on the subtype of the breast cancer cell line.

Regulation of chromatin microphase separation by binding of protein complexes.

We show evidence of the association of RNA polymerase II (RNAP) with chromatin in a core-shell organization, reminiscent of microphase separation where the cores comprise dense chromatin and the shell, RNAP and chromatin with low density. These observations motivate our physical model for the regulation of core-shell chromatin organization. Here, we model chromatin as a multiblock copolymer, comprising active and inactive regions (blocks) that are both in poor solvent and tend to be condensed in the absence of binding proteins. However, we show that the solvent quality for the active regions of chromatin can be regulated by the binding of protein complexes (e.g., RNAP and transcription factors). Using the theory of polymer brushes, we find that such binding leads to swelling of the active chromatin regions which in turn modifies the spatial organization of the inactive regions. In addition, we use simulations to study spherical chromatin micelles, whose cores comprise inactive regions and shells comprise active regions and bound protein complexes. In spherical micelles the swelling increases the number of inactive cores and controls their size. Thus, genetic modifications affecting the binding strength of chromatin-binding protein complexes may modulate the solvent quality experienced by chromatin and regulate the physical organization of the genome.

Genome architecture and selective signals compensatorily shape plastic response to a new environment

Transcriptional plasticity interacts with natural selection in complex ways and is crucial for the survival of species under rapid climate change. How 3D genome architecture affects transcriptional plasticity and its interaction with genetic adaptation are unclear. We transplanted estuarine oysters to a new environment and found that genes located in active chromatin regions exhibited greater transcriptional plasticity, and changes in these regions were negatively correlated with selective signals. This indicates a trade-off between 3D active regions and selective signals in shaping plastic responses to a new environment. Specifically, a mutation, lincRNA, and changes in the accessibility of a distal enhancer potentially affect its interaction with the ManⅡa gene, which regulates the muscle function and survival of oysters. Our findings reveal that 3D genome architecture compensates for the role of genetic adaptation in environmental response to new environments and provide insights into synergetic genetic and epigenetic interactions critical for fitness-related trait and survival in a model marine species.

MORC proteins regulate transcription factor binding by mediating chromatin compaction in active chromatin regions

BackgroundThe microrchidia (MORC) proteins are a family of evolutionarily conserved GHKL-type ATPases involved in chromatin compaction and gene silencing. Arabidopsis MORC proteins act in the RNA-directed DNA methylation (RdDM) pathway, where they act as molecular tethers to ensure the efficient establishment of RdDM and de novo gene silencing. However, MORC proteins also have RdDM-independent functions although their underlying mechanisms are unknown.ResultsIn this study, we examine MORC binding regions where RdDM does not occur in order to shed light on the RdDM-independent functions of MORC proteins. We find that MORC proteins compact chromatin and reduce DNA accessibility to transcription factors, thereby repressing gene expression. We also find that MORC-mediated repression of gene expression is particularly important under conditions of stress. MORC-regulated transcription factors can in some cases regulate their own transcription, resulting in feedback loops.ConclusionsOur findings provide insights into the molecular mechanisms of MORC-mediated chromatin compaction and transcription regulation.

Condensed but liquid-like domain organization of active chromatin regions in living human cells.

In eukaryotes, higher-order chromatin organization is spatiotemporally regulated as domains, for various cellular functions. However, their physical nature in living cells remains unclear (e.g., condensed domains or extended fiber loops; liquid-like or solid-like). Using novel approaches combining genomics, single-nucleosome imaging, and computational modeling, we investigated the physical organization and behavior of early DNA replicated regions in human cells, which correspond to Hi-C contact domains with active chromatin marks. Motion correlation analysis of two neighbor nucleosomes shows that nucleosomes form physically condensed domains with ~150-nm diameters, even in active chromatin regions. The mean-square displacement analysis between two neighbor nucleosomes demonstrates that nucleosomes behave like a liquid in the condensed domain on the ~150 nm/~0.5 s spatiotemporal scale, which facilitates chromatin accessibility. Beyond the micrometers/minutes scale, chromatin seems solid-like, which may contribute to maintaining genome integrity. Our study reveals the viscoelastic principle of the chromatin polymer; chromatin is locally dynamic and reactive but globally stable.

Development of a Novel Chromatin Activation Score As a Powerful Independent Prognostic Factor in Chronic Lymphocytic Leukemia

Background & Aim: In a previous study, we identified a signature of 498 regulatory regions with de novo increased H3K27ac levels (a mark of chromatin activation) in patients with chronic lymphocytic leukemia (CLL), which seemed to be present in all cases studied (Beekman et al., Nat Med 2018). In this abstract, we show that such de novo chromatin activation is not stable among CLL patients but heterogeneous, and therefore, we aim at exploring whether such heterogeneity has a biological and clinical impact. Results: The signature of 498 de novo active regulatory regions was related to 434 specific H3K27ac peaks based on chromatin immunoprecipitation sequencing (ChIPseq) data from 104 CLLs. Analyzing the signal variability among patients, we observed a high degree of heterogeneity. An unsupervised clustering revealed two different sets of regions whose mean H3K27ac signal was associated with time to first treatment (TTFT) using a univariate Cox model. In detail, a higher mean H3K27ac intensity was associated with a shorter TTFT in one of the sets (P=0.001 and hazard ratio (HR)=3.5) while, intriguingly, showed a trend towards longer TTFT (P=0.09 HR=0.6) in the other set. These findings suggest that not only disease progression but also disease stability seem to be active processes in CLL involving de novo chromatin changes. Then, we argued that the clinical behavior of CLL patients could be better captured by the balance between the activation levels of these two sets of regions. Indeed, the ratio of both means was statistically more significant (P=1.4x10-9 HR=15.4). Next, we further refined these groups by selecting individual regions associated with shorter (HR>1, 77 peaks) or longer (HR<1, 144 peaks) TTFT (FDR 0.2). We subsequently defined the score SPP as the mean signal in the pro-progressive (PP) group (HR>1), the score SPS as the mean signal in the pro-stable (PS) disease group (HR<1), and SB as their ratio. Ultimately, this led to an improvement of the statistical significance of the SB with TTFT (P=1.4x10-14 HR=8.6). From a biological perspective, we explored the validity of the SB performing ChIPseq for H3K27ac in 10 primary CLLs before and after a co-culture system inducing strong proliferation (Mangolini et al., Nat Comms in press). In these experiments, we observed an increased SB (P<10-3) that was mostly associated with a decreased SPS while maintaining the SPP levels. To further validate the biological implications of our findings, we mined RNAseq data in 64 cases with concurrent ChIPseq experimentation. We identified target genes whose expression levels correlated with H3K27ac levels (43 genes associated with the PP regions and 62 target genes associated with the PS regions). Both lists included genes previously described in CLL (e.g. CTLA4, FMOD or KSR2). Analogously to H3K27ac, we defined three new scores based on expression levels: SPP-exp, SPS-exp and SB-exp. These scores highly correlated with the H3K27ac-based scores (Pearson r between 0.82 to 0.88). Interestingly, cases with higher SB-exp were enriched in TNF-α and NF-κB pathways and in RELB, FOS, JUN, JUNB and EGR1 transcription factors regulons. We also identified 21 genetic driver alterations related to a higher SB-exp including trisomy 12, 11q loss and mutations in IGLV3-21R110, ATM, SF3B1, NOTCH1 and U1. Finally, we evaluated the potential of SB and SB-exp as independent prognostic factors in multivariate models. In the ChIPseq series (n=104), SB was significant independently of the IGHV mutational status, Binet stage and number of genetic driver alterations. Similarly, using three independent CLL series of gene expression data (n=110, 174 and 140), we observed that the SB-exp was a highly significant prognostic factor independent from IGHV status, Binet stage and number of drivers. To favour potential clinical translation, we generated a reduced SB-exp with only 12 genes which maintained the clinical impact. Conclusions: This study revealed an unexpected heterogeneity within de novo active chromatin regions in CLL, and that such heterogeneity has a dual clinical impact on CLL. A balance score between regions related to the two tendencies (i.e. more progressive vs more stable disease) identified groups of patients with different biological features. Finally, using both chromatin and gene expression data as a surrogate, the balance score was a highly significant independent prognostic factor in independent CLL series.