Chromatin Structural Proteins Research Articles

BRD4 phosphorylation regulates the structure of chromatin nanodomains.

The interplay between chromatin structure and phase-separating proteins is an emerging topic in cell biology with implications for understanding disease states. Here, we investigate the functional relationship between bromodomain protein 4 (BRD4) and chromatin architecture. By combining molecular dynamics simulations with live-cell imaging, we demonstrate that BRD4, when mutated at specific N-terminus sites, significantly impacts nucleosome nanodomain (NN) organization and dynamics. Our findings reveal that enhanced chromatin binding activity of BRD4 condenses NNs, while both loss or gain of BRD4 chromatin binding reduced diffusion of single nucleosomes, suggesting a role for BRD4 in the regulation of nanoscale chromatin architecture and the chromatin microenvironment. These observations shed light on the nuanced regulation of chromatin structure by BRD4, offering insights into its role in maintaining the nuclear architecture and transcriptional activity.

Generation and analysis of mouse embryonic stem cells with knockout of the Mcph1 (microcephalin) gene.

Chromatin is not randomly distributed within the nucleus, but organized in a three-dimensional structure that plays a critical role in genome functions. Сohesin and condensins are conserved multi-subunit protein complexes that participate in mammalian genome organization by extruding chromatin loops. The fine temporal regulation of these complexes is facilitated by a number of other proteins, one of which is microcephalin (Mcph1). Mcph1 prevents condensin II from associating with chromatin through interphase. Loss of Mcph1 induces chromosome hypercondensation; it is not clear to what extent this reorganization affects gene expression. In this study, we generated several mouse embryonic stem cell (mESC) lines with knockout of the Mcph1 gene and analyzed their gene expression profile. Gene Ontology analyses of differentially expressed genes (DEGs) after Mcph1 knockout revealed gene categories related to general metabolism and olfactory receptor function but not to cell cycle control previously described for Mcph1. We did not find a correlation between the DEGs and their frequency of lamina association. Thus, this evidence questions the hypothesis that Mcph1 knockout-mediated chromatin reorganization governs gene expression in mESCs. Among the negative effects of Mcph1 knockout, we observed numerous chromosomal aberrations, including micronucleus formation and chromosome fusion. This confirms the role of Mcph1 in maintaining genome integrity described previously. In our opinion, dysfunction of Mcph1 may be a kind of "Rosetta stone" for deciphering the function of condensin II in the interphase nucleus. Thus, the cell lines with knocked-out Mcph1 can be used to further study the influence of chromatin structural proteins on gene expression.

Control of Intestinal Stemness and Cell Lineage by Histone Variant H2A.Z Isoforms.

The histone variant H2A.Z plays important functions in the regulation of gene expression. In mammals, it is encoded by two genes, giving rise to two highly related isoforms named H2A.Z.1 and H2A.Z.2, which can have similar or antagonistic functions depending on the promoter. Knowledge of the physiopathological consequences of such functions emerges, but how the balance between these isoforms regulates tissue homeostasis is not fully understood. Here, we investigated the relative role of H2A.Z isoforms in intestinal epithelial homeostasis. Through genome-wide analysis of H2A.Z genomic localization in differentiating Caco-2 cells, we uncovered an enrichment of H2A.Z isoforms on the bodies of genes which are induced during enterocyte differentiation, stressing the potential importance of H2A.Z isoforms dynamics in this process. Through a combination of invitro and invivo experiments, we further demonstrated the two isoforms cooperate for stem and progenitor cells proliferation, as well as for secretory lineage differentiation. However, we found that they antagonistically regulate enterocyte differentiation, with H2A.Z.1 preventing terminal differentiation and H2A.Z.2 favoring it. Altogether, these data indicate that H2A.Z isoforms are critical regulators of intestine homeostasis and may provide a paradigm of how the balance between two isoforms of the same chromatin structural protein can control physiopathological processes.

Multi-omics data-based analysis characterizes molecular alterations of the vesicle genes in human colorectal cancer.

The role of vesicular genes in the development of colorectal cancer (CRC) is crucial. Analyzing alterations in these genes at multi-omics can aid in understanding the molecular pathways behind colorectal carcinogenesis and identifying potential treatment targets. However, studies on the overall alteration of vesicular genes in CRC are still lacking. In this study, we aimed to investigate the relationship between vesicle genetic alterations and CRC progression. To achieve this, we analyzed molecular alterations in CRC vesicle genes at eight levels, including mRNA, protein, and epigenetic levels. Additionally, we examined CRC overall survival-related genes that were obtained from a public database. Our analysis of chromatin structural variants, DNA methylation, chromatin accessibility, and proteins (including phosphorylation, ubiquitination, and malonylation), along with RNA-seq data from the TCGA database, revealed multiple levels of alterations in CRC vesicle genes in the collected tissue samples. We progressively examined the alterations of vesicle genes in mRNA and protein levels in CRC and discovered the hub genes. Further investigation identified the probable essential transcription factors. This study contributes to a thorough knowledge of the connection between vesicle gene alterations at multiple levels and the development of CRC and offers a theoretical framework for the identification of novel treatment targets.

ΔNp63α facilitates proliferation and migration, and modulates the chromatin landscape in intrahepatic cholangiocarcinoma cells

p63 plays a crucial role in epithelia-originating tumours; however, its role in intrahepatic cholangiocarcinoma (iCCA) has not been completely explored. Our study revealed the oncogenic properties of p63 in iCCA and identified the major expressed isoform as ΔNp63α. We collected iCCA clinical data from The Cancer Genome Atlas database and analyzed p63 expression in iCCA tissue samples. We further established genetically modified iCCA cell lines in which p63 was overexpressed or knocked down to study the protein function/function of p63 in iCCA. We found that cells overexpressing p63, but not p63 knockdown counterparts, displayed increased proliferation, migration, and invasion. Transcriptome analysis showed that p63 altered the iCCA transcriptome, particularly by affecting cell adhesion-related genes. Moreover, chromatin accessibility decreased at p63 target sites when p63 binding was lost and increased when p63 binding was gained. The majority of the p63 bound sites were located in the distal intergenic regions and showed strong enhancer marks; however, active histone modifications around the Transcription Start Site changed as p63 expression changed. We also detected an interaction between p63 and the chromatin structural protein YY1. Taken together, our results suggest an oncogenic role for p63 in iCCA.

Regulation of DNA damage and transcriptional output in the vasculature through a cytoglobin-HMGB2 axis

Identifying novel regulators of vascular smooth muscle cell function is necessary to further understand cardiovascular diseases. We previously identified cytoglobin, a hemoglobin homolog, with myogenic and cytoprotective roles in the vasculature. The specific mechanism of action of cytoglobin is unclear but does not seem to be related to oxygen transport or storage like hemoglobin. Herein, transcriptomic profiling of injured carotid arteries in cytoglobin global knockout mice revealed that cytoglobin deletion accelerated the loss of contractile genes and increased DNA damage. Overall, we show that cytoglobin is actively translocated into the nucleus of vascular smooth muscle cells through a redox signal driven by NOX4. We demonstrate that nuclear cytoglobin heterodimerizes with the non-histone chromatin structural protein HMGB2. Our results are consistent with a previously unknown function by which a non-erythrocytic hemoglobin inhibits DNA damage and regulates gene programs in the vasculature by modulating the genome-wide binding of HMGB2.

Light-activated macromolecular phase separation modulates transcription by reconfiguring chromatin interactions.

Biomolecular condensates participate in the regulation of gene transcription, yet the relationship between nuclear condensation and transcriptional activation remains elusive. Here, we devised a biotinylated CRISPR-dCas9-based optogenetic method, light-activated macromolecular phase separation (LAMPS), to enable inducible formation, affinity purification, and multiomic dissection of nuclear condensates at the targeted genomic loci. LAMPS-induced condensation at enhancers and promoters activates endogenous gene transcription by chromatin reconfiguration, causing increased chromatin accessibility and de novo formation of long-range chromosomal loops. Proteomic profiling of light-induced condensates by dCas9-mediated affinity purification uncovers multivalent interaction-dependent remodeling of macromolecular composition, resulting in the selective enrichment of transcriptional coactivators and chromatin structure proteins. Our findings support a model whereby the formation of nuclear condensates at native genomic loci reconfigures chromatin architecture and multiprotein assemblies to modulate gene transcription. Hence, LAMPS facilitates mechanistic interrogation of the relationship between nuclear condensation, genome structure, and gene transcription in living cells.

Differential Regulation of Inflammatory Responses Following 5‐HT2 Receptor Activation in Pulmonary Tissues

IntroductionThe concept of functional selectivity and psychedelics is often regarded as the ability of different 5‐HT2A receptor agonist compounds to exert varying degrees of psychoactivity in the central nervous system. However, the distribution and expression of the 5‐HT2A receptor, the primary pharmacological target for psychedelics, in peripheral and immune‐related tissue suggests selective signaling may occur here as well. Using an ovalbumin (OVA)‐induced model of rodent acute asthma, our lab has previously shown that subtle changes to the 2,5‐dimethoxy‐phenethylamine pharmacophore structure can profoundly impact the ability of these compounds to prevent asthmatic exacerbations. Here, through both in vitroand in vivoassays, we interrogate elements of functional selectivity in peripheral tissues following exposure to the 5‐HT2 receptor selective agonists (R)‐2,5‐dimethoxy‐4‐iodoamphetamine [(R)‐DOI] and 2,5‐dimethoxy‐4‐trifluoromethylamphetamine (DOTFM). (R)‐DOI is a full and potent anti‐inflammatory compound, whereas DOTFM has no measurable anti‐inflammatory activity. We find that elements of selective signaling combined with modulation to overall chromatin structure and downstream protein expression provide mechanistic clues regarding how psychedelics may elicit their long‐lasting, therapeutic effects.ObjectiveTo elucidate mechanistic variations between anti‐inflammatory and non‐anti‐inflammatory 5‐HT2 receptor agonists.MethodsWe utilized fluorescence recovery after photobleaching (FRAP) to probe the kinetics of the linker histone H1c transiently transfected into a HEK293 cell line stably expressing the human 5‐HT2A receptor, and calcium mobilization assays in the same cell line to determine differences in signaling between (R)‐DOI and DOTFM. For in vivoassays we measured enhanced pause (PenH) values for BALB/c mice challenged with OVA and treated with either (R)‐DOI or DOTFM, using PenH values as a proxy measure for pulmonary inflammation. Lungs were removed en bloc,processed, and analyzed by qPCR and quantitative proteomics using tandem tag mass spectrometry (TMT).ResultsIn vitro assays indicate modest differences irrespective of phenethylamine compound, whereas whole‐body plethysmography (WBP), qPRC, and proteomic analysis reveal significant differential regulation of proteins relevant for both asthmatic initiation and chromatin compaction dependent upon 5‐HT2A agonist.ConclusionAs 5‐HT2‐mediated expression of asthma‐relevant proteins are differentially regulated by either (R)‐DOI and DOTFM, the downregulation of inflammatory pathways via activation of the 5‐HT2 receptor likely requires an accessible, non‐sterically hindered subunit at the 4‐position of the standard 2,5‐dimethoxy‐ampetamine structure to maximally induce anti‐inflammatory effects. Changes to both linker histone mobility and the expression of proteins relevant to chromatin architecture suggest that modulation to the overall chromatin structure combined with downstream epigenetic regulation is a potential mechanism by which certain 5‐HT2A agonists can exert their therapeutic effects.

High Mobility Group A1 (HMGA1): Structure, Biological Function, and Therapeutic Potential.

High mobility group A1 (HMGA1) is a nonhistone chromatin structural protein characterized by no transcriptional activity. It mainly plays a regulatory role by modifying the structure of DNA. A large number of studies have confirmed that HMGA1 regulates genes related to tumours in the reproductive system, digestive system, urinary system and haematopoietic system. HMGA1 is rare in adult cells and increases in highly proliferative cells such as embryos. After being stimulated by external factors, it will produce effects through the Wnt/β-catenin, PI3K/Akt, Hippo and MEK/ERK pathways. In addition, HMGA1 also affects the ageing, apoptosis, autophagy and chemotherapy resistance of cancer cells, which are linked to tumorigenesis. In this review, we summarize the mechanisms of HMGA1 in cancer progression and discuss the potential clinical application of targeted HMGA1 therapy, indicating that targeted HMGA1 is of great significance in the diagnosis and treatment of malignancy.

Early adaptive chromatin remodeling events precede pathologic phenotypes and are reinforced in the failing heart

The temporal nature of chromatin structural changes underpinning pathologic transcription are poorly understood. We measured chromatin accessibility and DNA methylation to study the contribution of chromatin remodeling at different stages of cardiac hypertrophy and failure. ATAC-seq and reduced representation bisulfite sequencing were performed in cardiac myocytes after transverse aortic constriction (TAC) or depletion of the chromatin structural protein CTCF. Early compensation to pressure overload showed changes in chromatin accessibility and DNA methylation preferentially localized to intergenic and intronic regions. Most methylation and accessibility changes observed in enhancers and promoters at the late phase (3 weeks after TAC) were established at an earlier time point (3 days after TAC), before heart failure manifests. Enhancers were paired with genes based on chromatin conformation capture data: while enhancer accessibility generally correlated with changes in gene expression, this feature, nor DNA methylation, was alone sufficient to predict transcription of all enhancer interacting genes. Enrichment of transcription factors and active histone marks at these regions suggests that enhancer activity coordinates with other epigenetic factors to determine gene transcription. In support of this hypothesis, ChIP-qPCR demonstrated increased enhancer and promoter occupancy of GATA4 and NKX2.5 at Itga9 and Nppa, respectively, concomitant with increased transcription of these genes in the diseased heart. Lastly, we demonstrate that accessibility and DNA methylation are imperfect predictors of chromatin structure at the scale of A/B compartmentalization—rather, accessibility, DNA methylation, transcription factors and other histone marks work within these domains to determine gene expression. These studies establish that chromatin reorganization during early compensation after pathologic stimuli is maintained into the later decompensatory phases of heart failure. The findings reveal the rules for how local chromatin features govern gene expression in the context of global genomic structure and identify chromatin remodeling events for therapeutic targeting in disease.

MALDI-TOF mass spectrometric protein profiling of THP-1 cells and their microvesicles

Extracellular vesicles that are shed from the plasma membranes take an active part in intercellular communication, transporting a wide range of molecules, including proteins, lipids, nucleic acids and carbohydrates, being of great functional importance. One of the steps to better understanding of distant communications of cells and their regulatory mechanisms is a proteomic study of various extracellular vesicles, including microvesicles and exosomes. Pro-inflammatory cytokines produced by monocytes and individual complement system components play a key role in their specific functioning. The aim of this work was to study proteomic composition of THP-1 monocyte-like cells and their microvesicles. The MALDI-mass spectrometric analysis of electrophoretic protein fractions of cell lysates and microvesicles allowed for identifying 107 proteins that perform various functions. Among 19 determined functional groups, the largest ones comprise transcription regulators and proteins with unknown functions. The smallest functional groups include regulators of cell differentiation and development, proteins participating in immune response and inflammation, cellular receptors and their regulators, transporter and transport regulatory proteins, as well as cell proteins mediating adhesion and matrix structures, processing regulators, proteins of ubiquitin-proteasome system, intracellular signaling, autophagy and exocytosis regulators, chromatin structural proteins, hemostatic regulators, and peptide hormones. An intermediate position is occupied by cytokines and growth factors, enzymes, cytoskeleton and motor proteins, as well as RNA processing and translation regulators. The subsequent DAVID Functional Annotation Clustering analysis allowed for identifying the most common groups distributed by their molecular function, biological processes, and cellular component. Separately, in the microvesicles derived from THP-1 monocyte-like cells, proteins of the immune response and inflammation, cytokines and growth factors, intracellular signaling proteins, cell differentiation regulators and developmental proteins, as well as cell adhesion and matrix proteins were identified among other protein molecules. The data obtained on the partial proteome of THP-1 monocyte-like cells and their microvesicles extend the existing knowledge on distant communications between the cells and suggest new mechanisms of interaction between monocytes/macrophages and their microenvironment.

Poly(ADP-Ribosyl) Code Functions.

Poly(ADP-ribosyl)ation plays a key role in cellular metabolism. Covalent poly(ADP-ribosyl)ation affects the activity of the proteins engaged in DNA repair, chromatin structure regulation, gene expression, RNA processing, ribosome biogenesis, and protein translation. Non-covalent PAR-dependent interactions are involved in the various types of cellular response to stress and viral infection, such as inflammation, hormonal signaling, and the immune response. The review discusses how structurally different poly(ADP-ribose) (PAR) molecules composed of identical monomers can differentially participate in various cellular processes acting as the so-called “PAR code.” The article describes the ability of PAR polymers to form functional biomolecular clusters through a phase-separation in response to various signals. This phase-separation contributes to rapid spatial segregation of biochemical processes and effective recruitment of the necessary components. The cellular PAR level is tightly controlled by a network of regulatory proteins: PAR code writers, readers, and erasers. Impaired PAR metabolism is associated with the development of pathological processes causing oncological, cardiovascular, and neurodegenerative diseases. Pharmacological correction of the PAR level may represent a new approach to the treatment of various diseases.

Relative mRNA and protein stability of epigenetic regulators in musculoskeletal cell culture models

Control of gene expression by epigenetic regulators is fundamental to tissue development and homeostasis. Loss-of-function (LOF) studies using siRNAs for epigenetic regulators require that RNA interference rapidly reduces the cellular levels of the corresponding mRNAs and/or proteins. The most abundant chromatin structural proteins (i.e., the core histones H2A, H2B, H3 and H4) have relatively long half-lives and do not turn over rapidly, although their mRNAs are labile. The question arises whether epigenetic regulatory enzymes (e.g., Ezh2) or proteins that interact with histones via selective modifications (e.g., Cbx1 to Cbx8, Brd4) are stable or unstable. Therefore, we performed classical α-amanitin and cycloheximide inhibition assays that block, respectively, mRNA transcription and protein translation in mouse MC3T3 osteoblasts, ATDC5 chondrocytes and C2C12 myoblasts. We find that mRNA levels of Cbx proteins and Ezh2 were significantly depleted after 24 hrs, while their corresponding proteins remained relatively stable. As positive control, the half-life of the labile cyclin D1 protein was found to be less than 1 hr. Our study suggests that histone code readers and writers are relatively stable chromatin-related proteins, which is consistent with their long-term activities in maintaining chromatin organization and phenotype identity. These findings have conceptual ramifications for the interpretation of RNAi experiments that reduce the mRNA but not protein levels of epiregulatory proteins. We propose that siRNAs for at least some epigenetic regulatory proteins may exert their biological effects by blocking translation and new protein synthesis rather than by decreasing pre-existing protein pools.

Transcriptome profile analysis of two Vicia faba cultivars with contrasting salinity tolerance during seed germination

Faba bean (Vicia faba L.) is an important food legume crop. Salinity soils severely constrain the production of faba bean, however, the seed germination of faba bean, which is a vital plant growth stage, is sensitive to salinity. Planting improved varieties of faba bean, which exhibit salt tolerance in seed germination stage, is an optimal strategy for faba bean product. To investigate the genes dynamics during the seed germination stage under salinity, RNA-seq method was used to investigate genome-wide transcription profiles of two faba bean varieties with contrast salt-tolerance during the seed germination. A total of 4,486 differentially expressed genes (DEGs) were identified among the comparison of salt-tolerant variety Y134 and salt-sensitive variety Y078 treated with salinity or not. Of these, 1,410 candidate DEGs were identified as salt-stress response genes. Furthermore, 623 DEGs were identified as variety-specific response gene during seed germination at 16 h or 24 h with salt treatment. Based on the pathway enrichment according to the Kyoto Encyclopedia of Genes and Genomes database (KEGG), these DEGs involving in cell wall loosening (e.g., xyloglucan endotransglucosylase/hydrolase, chitinase, and expansin), hormone metabolism (e.g., LEA genes, genes associated with ABA or ethylene signal pathway), chromatin remodeling (e.g., chromatin structure proteins, LHP1), small interfering RNA pathway, etc., were significantly up-regulated in salt-tolerance variety with salt treatment, indicating that they play critical roles in regulation of seed germination. The results indicated that a clearer mechanism of gene regulation that regulates the seed germination responding to salinity in faba bean. These findings are helpful to increase the understanding of the salt tolerance mechanism of crops during seed germination, and provide valuable genetic resource for the breeding of salt-tolerant faba bean varieties in future.

SAT-029 Understanding The Functions Of Parp-1 In Embryonic Stem Cells

Poly(ADP-ribose) polymerase 1 (PARP-1) is an abundant nuclear enzyme that plays diverse roles in a variety of nuclear processes, including DNA damage detection and repair, chromatin remodeling, transcriptional regulation, and RNA processing. PARP-1 functions both as an enzyme, through its NAD+-dependent ADP- ribosyltransferase catalytic activity, and a chromatin structural protein, through an NAD+-independent DNA binding activity. We have observed a loss of the maintenance of pluripotency in murine embryonic stem cells (mESCs) lacking PARP-1. Additionally, we have seen a dramatic induction of PARP-1 catalytic activity during the early stages of mESC differentiation. From these observations, we sought to understand the regulation of PARP-1 catalytic activity during differentiation and its effect. We hypothesize that the onset of ADP-ribosylation events by PARP-1, regulated by compartmentalized NAD+, generates site-specific and target-specific modifications (ADP-ribosylation) that assists differentiation. Proteins ADP-ribosylated during this period of enhanced PARP-1 catalytic activity were previously unknown. Using analog NAD+-sensitive PARP-1 technology developed in the Kraus lab, we have identified the PARP-1 ADP-ribosylated proteome of mESCs during the onset of differentiation. These modified proteins appear to be highly associated with specific pathways including - mRNA processing, transcription regulation, and development - shedding light on additional layers of regulation of the differentiation process. We are now using biochemical, molecular, and cellular assays to determine the functional relevance of site-specific ADP-ribosylation events in the differentiation process. This work is supported by theNSF-Graduate Research Fellowship toAJ and agrantfrom the NIH/NIDDK to WLK.

Sporadic DUX4 expression in FSHD myocytes is associated with incomplete repression by the PRC2 complex and gain of H3K9 acetylation on the contracted D4Z4 allele

BackgroundFacioscapulohumeral muscular dystrophy 1 (FSHD1) has an autosomal dominant pattern of inheritance and primarily affects skeletal muscle. The genetic cause of FSHD1 is contraction of the D4Z4 macrosatellite array on chromosome 4 alleles associated with a permissive haplotype causing infrequent sporadic expression of the DUX4 gene. Epigenetically, the contracted D4Z4 array has decreased cytosine methylation and an open chromatin structure. Despite these genetic and epigenetic changes, the majority of FSHD myoblasts are able to repress DUX4 transcription. In this study we hypothesized that histone modifications distinguish DUX4 expressing and non-expressing cells from the same individuals.ResultsFSHD myocytes containing the permissive 4qA haplotype with a long terminal D4Z4 unit were sorted into DUX4 expressing and non-expressing groups. We found similar CpG hypomethylation between the groups of FSHD-affected cells suggesting that CpG hypomethylation is not sufficient to trigger DUX4 expression. A survey of histone modifications present at the D4Z4 region during cell lineage commitment revealed that this region is bivalent in FSHD iPS cells with both H3K4me3 activating and H3K27me3 repressive marks present, making D4Z4 poised for DUX4 activation in pluripotent cells. After lineage commitment, the D4Z4 region becomes univalent with H3K27me3 in FSHD and non-FSHD control myoblasts and a concomitant increase in H3K4me3 in a small fraction of cells. Chromatin immunoprecipitation (ChIP) for histone modifications, chromatin modifier proteins and chromatin structural proteins on sorted FSHD myocytes revealed that activating H3K9Ac modifications were ~ fourfold higher in DUX4 expressing FSHD myocytes, while the repressive H3K27me3 modification was ~ fourfold higher at the permissive allele in DUX4 non-expressing FSHD myocytes from the same cultures. Similarly, we identified EZH2, a member of the polycomb repressive complex involved in H3K27 methylation, to be present more frequently on the permissive allele in DUX4 non-expressing FSHD myocytes.ConclusionsThese results implicate PRC2 as the complex primarily responsible for DUX4 repression in the setting of FSHD and H3K9 acetylation along with reciprocal loss of H3K27me3 as key epigenetic events that result in DUX4 expression. Future studies focused on events that trigger H3K9Ac or augment PRC2 complex activity in a small fraction of nuclei may expose additional drug targets worthy of study.

Abstract 115: Chromatin Microenvironments With Distinct Functionality During Cardiac Stress

Loss of the chromatin structural protein CTCF induces heart failure. Chromatin capture experiments (Hi-C), which detect genomic interactions, revealed global remodeling of chromatin structure after cardiac-specific depletion of CTCF or pressure overload (TAC). In the present study, we investigated epigenomic features (histone marks and DNA methylation) contributing to distinct behaviors of chromatin microenvironments. CTCF-KO or TAC induced gene expression changes mediated by a shift in compartmentalization between active and inactive chromatin (~4% of the genome). At the transcriptome level, genes significantly dysregulated in disease were involved in cardiac function ( e.g. , Mybpc3, Lmna , Hand2 ), chromatin structure ( e.g. , Hmgb2 , Rad21 ), and histone modification ( e.g. , Hdac4 , Mapk14, Foxo1 ). Our Hi-C data allowed us to correlate changes in gene expression with localization within the nucleus, showing efficient segregation of differentially expressed genes into specific neighborhoods with similar expression behavior. Analyses of histone marks in these neighborhoods reveal enrichment of activating and inhibiting marks, H3K9ac and H3K9me3, respectively, in the specialized chromatin environments associated with gene expression or silencing. DNA methylation studies showed that transcriptionally active neighborhoods tended to have unmethylated CpGs, while repressive environments were methylated. Our Hi-C data also showed how active environments positively influence the expression of the genes that physically interact with them. For example, 351 unique genes with interacting partner genes had increased H3K9ac after TAC, and their 3D interacting partners included 200 upregulated genes and 104 downregulated genes, demonstrating similar transcriptional behavior in areas with similar epigenomic features and anatomical distribution in the nucleus. Heart failure involves conserved structural reprogramming of these chromatin microenvironments.

Expression analysis of HMGB1 in histological samples of malignant pleural mesothelioma.

High mobility group box 1 (HMGB1) is a chromatin structural protein, expressed ubiquitously in the nuclei of mammalian cells. When transported extracellularly, it acts as a tumour suppressor and oncogenic protein. In malignant pleural mesothelioma (MPM), high serum levels of HMGB1 have been related to a poor prognosis. Conversely, the significance of HMGB1 expression in MPM tissues is still unclear. Biopsy samples from 170 patients with MPM were assessed by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) to evaluate HMGB1 protein and gene expression. The expression level of HMGB1 protein was scored using a semiquantitative system that sums the intensity (0-3) and the percentage (from 0 to 4) of positively stained cells in nuclei, cytoplasm and in both. The final score was considered as high (>3) or low (<3) expression. Gene expression levels were calculated using the ΔΔCt method. High expression levels of HMGB1 as total (P = 0.0011) and cytoplasmic score (P = 0.0462) were related to a worse disease-specific survival (DSS) in the entire cohort and in the clinicopathological subgroups. No significant correlation was found between HMGB1 gene expression and DSS. These findings indicate that HMGB1 may be a useful prognostic biomarker in MPM when detected by immunohistochemistry. Conversely, as it is also expressed in normal and reactive mesothelial cells, HMGB1 cannot be considered a diagnostic biomarker in histological samples of mesothelioma.

Loss of DEK induces radioresistance of murine restricted hematopoietic progenitors

Self-renewing hematopoietic stem cells and multipotent progenitor cells are responsible for maintaining hematopoiesis throughout an individual's lifetime. For overall health and survival, it is critical that the genome stability of these cells is maintained and that the cell population is not exhausted. Previous reports have indicated that the DEK protein, a chromatin structural protein that functions in numerous nuclear processes, is required for DNA damage repair in vitro and long-term engraftment of hematopoietic stem cells in vivo. Therefore, we investigated the role of DEK in normal hematopoiesis and response to DNA damaging agents in vivo. Here, we report that hematopoiesis is largely unperturbed in DEK knockout mice compared with wild-type (WT) controls. However, DEK knockout mice have fewer radioprotective units, but increased capacity to survive repeated sublethal doses of radiation exposure compared with WT mice. Furthermore, this increased survival correlated with a sustained quiescent state in which DEK knockout restricted hematopoietic progenitor cells (HPC-1) were nearly three times more likely to be quiescent following irradiation compared with WT cells and were significantly more radioresistant during the early phases of myeloid reconstitution. Together, our studies indicate that DEK functions in the normal hematopoietic stress response to recurrent radiation exposure.

High-Resolution Mapping of Chromatin Conformation in Cardiac Myocytes Reveals Structural Remodeling of the Epigenome in Heart Failure.

Cardiovascular disease is associated with epigenomic changes in the heart; however, the endogenous structure of cardiac myocyte chromatin has never been determined. To investigate the mechanisms of epigenomic function in the heart, genome-wide chromatin conformation capture (Hi-C) and DNA sequencing were performed in adult cardiac myocytes following development of pressure overload-induced hypertrophy. Mice with cardiac-specific deletion of CTCF (a ubiquitous chromatin structural protein) were generated to explore the role of this protein in chromatin structure and cardiac phenotype. Transcriptome analyses by RNA-seq were conducted as a functional readout of the epigenomic structural changes. Depletion of CTCF was sufficient to induce heart failure in mice, and human patients with heart failure receiving mechanical unloading via left ventricular assist devices show increased CTCF abundance. Chromatin structural analyses revealed interactions within the cardiac myocyte genome at 5-kb resolution, enabling examination of intra- and interchromosomal events, and providing a resource for future cardiac epigenomic investigations. Pressure overload or CTCF depletion selectively altered boundary strength between topologically associating domains and A/B compartmentalization, measurements of genome accessibility. Heart failure involved decreased stability of chromatin interactions around disease-causing genes. In addition, pressure overload or CTCF depletion remodeled long-range interactions of cardiac enhancers, resulting in a significant decrease in local chromatin interactions around these functional elements. These findings provide a high-resolution chromatin architecture resource for cardiac epigenomic investigations and demonstrate that global structural remodeling of chromatin underpins heart failure. The newly identified principles of endogenous chromatin structure have key implications for epigenetic therapy.