Disruption Of Chromatin Structure Research Articles

A fluorescent assay for cryptic transcription in Saccharomyces cerevisiae reveals novel insights into factors that stabilize chromatin structure on newly replicated DNA.

The disruption of chromatin structure can result in transcription initiation from cryptic promoters within gene bodies. While the passage of RNA polymerase II is a well-characterized chromatin-disrupting force, numerous factors, including histone chaperones, normally stabilize chromatin on transcribed genes, thereby repressing cryptic transcription. DNA replication, which employs a partially overlapping set of histone chaperones, is also inherently disruptive to chromatin, but a role for DNA replication in cryptic transcription has never been examined. In this study, we tested the hypothesis that, in the absence of chromatin-stabilizing factors, DNA replication can promote cryptic transcription in Saccharomyces cerevisiae. Using a novel fluorescent reporter assay, we show that multiple factors, including Asf1, CAF-1, Rtt106, Spt6, and FACT, block transcription from a cryptic promoter, but are entirely or partially dispensable in G1-arrested cells, suggesting a requirement for DNA replication in chromatin disruption. Collectively, these results demonstrate that transcription fidelity is dependent on numerous factors that function to assemble chromatin on nascent DNA.

Abstract IA002: Structural basis of chromatin transcription

Abstract In eukaryotes, RNA polymerase (Pol) II transcribes chromatin and must move past nucleosomes, often resulting in nucleosome displacement. How Pol II unwraps the DNA from nucleosomes to allow transcription and how DNA rewraps to retain nucleosomes has been unclear. I will present the 3.0 Å cryo–electron microscopy structure of a mammalian Pol II-DSIF-SPT6-PAF1c-TFIIS-nucleosome complex stalled 54 base pairs within the nucleosome. The structure provides a mechanistic basis for nucleosome retention during transcription elongation where upstream DNA emerging from the Pol II cleft has rewrapped the proximal side of the nucleosome. The structure uncovers a direct role for Pol II and transcription elongation factors in nucleosome retention and explains how nucleosomes are retained to prevent the disruption of chromatin structure across actively transcribed genes. Importantly, the structure also provides the mechanistic basis for the survival of histone marks during transcription. Citation Format: Lucas Farnung. Structural basis of chromatin transcription. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA002.

Structural basis of nucleosome retention during transcription elongation.

In eukaryotes, RNA polymerase (Pol) II transcribes chromatin and must move past nucleosomes, often resulting in nucleosome displacement. How Pol II unwraps the DNA from nucleosomes to allow transcription and how DNA rewraps to retain nucleosomes has been unclear. Here, we report the 3.0-angstrom cryo-electron microscopy structure of a mammalian Pol II-DSIF-SPT6-PAF1c-TFIIS-nucleosome complex stalled 54 base pairs within the nucleosome. The structure provides a mechanistic basis for nucleosome retention during transcription elongation where upstream DNA emerging from the Pol II cleft has rewrapped the proximal side of the nucleosome. The structure uncovers a direct role for Pol II and transcription elongation factors in nucleosome retention and explains how nucleosomes are retained to prevent the disruption of chromatin structure across actively transcribed genes.

SAMMY-seq reveals early alteration of heterochromatin and deregulation of bivalent genes in Hutchinson-Gilford Progeria Syndrome

Hutchinson-Gilford progeria syndrome is a genetic disease caused by an aberrant form of Lamin A resulting in chromatin structure disruption, in particular by interfering with lamina associated domains. Early molecular alterations involved in chromatin remodeling have not been identified thus far. Here, we present SAMMY-seq, a high-throughput sequencing-based method for genome-wide characterization of heterochromatin dynamics. Using SAMMY-seq, we detect early stage alterations of heterochromatin structure in progeria primary fibroblasts. These structural changes do not disrupt the distribution of H3K9me3 in early passage cells, thus suggesting that chromatin rearrangements precede H3K9me3 alterations described at later passages. On the other hand, we observe an interplay between changes in chromatin accessibility and Polycomb regulation, with site-specific H3K27me3 variations and transcriptional dysregulation of bivalent genes. We conclude that the correct assembly of lamina associated domains is functionally connected to the Polycomb repression and rapidly lost in early molecular events of progeria pathogenesis.

Extreme disruption of heterochromatin is required for accelerated hematopoietic aging

AbstractLoss of heterochromatin has been proposed as a universal mechanism of aging across different species and cell types. However, a comprehensive analysis of hematopoietic changes caused by heterochromatin loss is lacking. Moreover, there is conflict in the literature around the role of the major heterochromatic histone methyltransferase Suv39h1 in the aging process. Here, we use individual and dual deletion of Suv39h1 and Suv39h2 enzymes to examine the causal role of heterochromatin loss in hematopoietic cell development. Loss of neither Suv39h1 nor Suv39h2 individually had any effect on hematopoietic stem cell function or the development of mature lymphoid or myeloid lineages. However, deletion of both enzymes resulted in characteristic changes associated with aging such as reduced hematopoietic stem cell function, thymic involution and decreased lymphoid output with a skewing toward myeloid development, and increased memory T cells at the expense of naive T cells. These cellular changes were accompanied by molecular changes consistent with aging, including alterations in nuclear shape and increased nucleolar size. Together, our results indicate that the hematopoietic system has a remarkable tolerance for major disruptions in chromatin structure and reveal a role for Suv39h2 in depositing sufficient H3K9me3 to protect the entire hematopoietic system from changes associated with premature aging.

CRYPTIC TRANSCRIPTION IS ASSOCIATED WITH AGE IN MAMMALIAN STEM CELLS

Aging is a multifaceted process that challenges organisms with stresses resulting from the dysregulation of cellular processes. Unsurprisingly, given how tightly regulated it is under normal conditions, transcription is one of the key pathways disrupted during aging. Indeed, dysregulation of transcription contributes to the activation of transposable elements, the loss of cellular identity, and decreased stem cell potency with age. Our previous work identified intragenic cryptic transcription (CT) as a novel type of age-associated transcriptional dysregulation that limits the lifespan of yeast and worms. Continuing this work, we show for the first time that CT increases with age in mammalian stem cells. Increased CT is associated with disrupted chromatin structure, particularly with the reduction of H3K36me3, a histone modification known to inhibit CT throughout eukaryotes. We propose that an age-associated reduction in H3K36me3 in actively transcribed gene bodies drives disruption of chromatin structure in these regions, resulting in an open chromatin state. This open chromatin state is permissive for the entry of RNA Pol II, which can then initiate transcription from within the gene body. These aberrant cryptic transcripts may contribute to the pathological load of mammalian aging.

An epigenetic gateway to brain tumor cell identity.

Precise targeting of genetic lesions alone has been insufficient to extend brain tumor patient survival. Brain cancer cells are diverse in their genetic, metabolic and microenvironmental compositions, accounting for their phenotypic heterogeneity and disparate responses to therapy. These factors converge at the level of the epigenome, representing a unified node that can be disrupted by pharmacologic inhibition. Aberrant epigenomes define many childhood and adult brain cancers, as demonstrated by widespread changes to DNA methylation patterns, redistribution of histone marks and disruption of chromatin structure. In this Review, we describe the convergence of genetic, metabolic and microenvironmental factors on mechanisms of epigenetic deregulation in brain cancer. We discuss how aberrant epigenetic pathways identified in brain tumors affect cell identity, cell state and neoplastic transformation, as well as addressing the potential to exploit these alterations as new therapeutic strategies for the treatment of brain cancer.

Cadmium affects mitotically inherited histone modification pathways in mouse embryonic stem cells

The fetal basis of adult disease (FeBAD) theorizes that embryonic challenges initiate pathologies in adult life through epigenetic modification of gene expression. In addition, inheritance of H3K27 methylation marks, especially in vitro, is still controversial. Metals, such as Cd, are known to affect differentiation, DNA repair and epigenetic status in mES cells. We tested the premise that Cd exerts differential toxicity in mouse embryonic stem (mES) cells by targeting total histone protein (THP) production early in stem cell development, while affecting H3K27-mono-methylation (H3K27me1) in latter stages of differentiation. The inability of mES cells to recover from Cd insult at concentrations greater than IC50 indicates that maximum cytotoxicity occurs during initial hours of exposure. Moreover, as a measure of chromatin stability, low dose acute Cd exposure lowers THP production. The heritable effects of Cd exposure on cell proliferation, chromatin stability and transcription observed through several cell population doublings were detected only during alternate passages on days 3, 7, and 11, presumably due to slower maturation of histone methylation marks. These findings demonstrate a selective disruption of chromatin structure following acute Cd exposure, an effect not seen in developmentally mature cells. Hence, we present that acute Cd toxicity is cumulative and disrupts DNA repair, while concurrently affecting cell cycle progression, chromatin stability and transcriptional state in mES cells.

Abstract 2868: SETDB1 accelerates non-small cell lung cancer (NSCLC) tumorigenesis through WNT signalling pathway

Abstract Histone methylation deregulation leads to disruption of chromatin structure and gene expression pattern in many human cancers. Histone H3 lysine K9 methyltransferase, SETDB1 (SET domain, bifurcated 1) is initially reported to control heterochromatin structure, repress gene expression and be essential in early embryogenesis. Studies have recently shown its involvement in boosting tumorigenesis in different kinds of human cancers, including lung cancer. However, the mechanism underlying its ability to promote tumorigenesis remains largely unknown. In our study, we sought to investigate the oncogenic role of SETDB1 in NSCLC. Our immunohistochemistry found that 72% out of 387-lung cancer cases stained positively for SETDB1, compared to 46% samples of normal bronchial epithelium (p<0.0001). Moreover, the percent positive cells and their intensity of positivity correlated with cancer grades. Ectopic expression of SETDB1 in NSCLC cell lines enhanced clonogenic growth in vitro and significantly enlarges tumor size in vivo; while depleting endogenous SETDB1 expression with shRNAs suppressed NSCLC cells proliferation and tumor formation. Analyzing the expression pattern of cells with forced expression of SETDB1 combined with TOP/FOP luciferase assay and western blotting showed that WNT/β-catenin signaling was activated upon overexpression of SETDB1, as well as upregulation of its downstream targets, c-Myc and Cyclin D1. ChIP assay showed that SETDB1 binds to WNT relevant genes, such as APOE, IGFBP4, FZD1 and LRP8 and upregulates their transcription, which is very distinct from its canonical suppressing function of H3K9 trimethylation. Interestingly, we also noted that SETDB1 was able to repress P53 expression, while diminishing P53 expression reciprocally enhanced SETDB1 expression. In summary, our study demonstrates that SETDB1 enhances NSCLC tumorigenesis through activating WNT/β-catenin signaling and repressing tumor suppressor P53. Our novel findings provide new insight into a non-canonical function of SETDB1 in activating gene expression, and suggest therapeutic benefit of targeting SETDB1 in NSCLC having high expression level of this protein. Citation Format: Qiaoyang Sun, Lingwen Ding, Jinfen Xiao, Lee Goodglick, David Chia, Vei Mah, Mohammad Alavi, Ngan Doan, Jonathan W. Said, Henry Yang, H.Phillip Koeffler. SETDB1 accelerates non-small cell lung cancer (NSCLC) tumorigenesis through WNT signalling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2868. doi:10.1158/1538-7445.AM2015-2868

Thyroid hormone receptor actions on transcription in amphibia: The roles of histone modification and chromatin disruption

Thyroid hormone (T3) plays diverse roles in adult organ function and during vertebrate development. The most important stage of mammalian development affected by T3 is the perinatal period when plasma T3 level peaks. Amphibian metamorphosis resembles this mammalian postembryonic period and is absolutely dependent on T3. The ability to easily manipulate this process makes it an ideal model to study the molecular mechanisms governing T3 action during vertebrate development. T3 functions mostly by regulating gene expression through T3 receptors (TRs). Studies in vitro, in cell cultures and reconstituted frog oocyte transcription system have revealed that TRs can both activate and repress gene transcription in a T3-dependent manner and involve chromatin disruption and histone modifications. These changes are accompanied by the recruitment of diverse cofactor complexes. More recently, genetic studies in mouse and frog have provided strong evidence for a role of cofactor complexes in T3 signaling in vivo. Molecular studies on amphibian metamorphosis have also revealed that developmental gene regulation by T3 involves histone modifications and the disruption of chromatin structure at the target genes as evidenced by the loss of core histones, arguing that chromatin remodeling is an important mechanism for gene activation by liganded TR during vertebrate development.

Epigenetics and B-cell lymphoma

It has only recently become apparent that mutations in epigenetic mechanisms and perturbation of epigenomic patterning are frequent events in B-cell lymphomas. The purpose of this review is to highlight these new findings and provide a conceptual framework for understanding how epigenetic modifications might contribute to lymphomagenesis. Somatic mutations affecting histone methyltransferases such as enhancer of zeste 2 and mixed lineage leukemia 2, histone demethylases including ubiquitously transcribed X chromosome tetratricopeptide repeat and Jumonji domain-containing 2C, and histone acetyltransferases including CBP and p300 are recurrent and common in lymphomas. These mutations result in disruption of chromatin structure and functions of other proteins, ultimately causing aberrant transcriptional programming affecting multiple gene networks. Widespread perturbation of cytosine methylation patterning now appears to be a hallmark of B-cell lymphomas and occurs in specific patterns that can distinguish disease subtypes. Therapeutic targeting strategies can overcome abnormal epigenetic mechanisms and potently kill lymphoma cells. Newly discovered epigenetic lesions may provide critical insights into the genesis of B-cell lymphomas, but further studies are required to understand how they affect biological mechanism. Epigenetic lesions offer tremendous opportunities for the development of improved biomarkers and treatments.

Clothing up DNA for all seasons: Histone chaperones and nucleosome assembly pathways

In eukaryotes, the packaging of DNA into chromatin is essential for cell viability. Several important DNA metabolic events require the transient disruption of chromatin structure, but cells have evolved a number of elaborate pathways that operate throughout the cell cycle to prevent the deleterious effects of chromatin erosion. In this review, we describe a number of distinct nucleosome assembly pathways that function during DNA replication, transcription, cellular senescence and early embryogenesis. In addition, we illustrate some of the physiological consequences associated with defects in nucleosome assembly pathways.

Ectopic histone H3S10 phosphorylation causes chromatin structure remodeling inDrosophila

Histones are subject to numerous post-translational modifications that correlate with the state of higher-order chromatin structure and gene expression. However, it is not clear whether changes in these epigenetic marks are causative regulatory factors in chromatin structure changes or whether they play a mainly reinforcing or maintenance role. In Drosophila phosphorylation of histone H3S10 in euchromatic chromatin regions by the JIL-1 tandem kinase has been implicated in counteracting heterochromatization and gene silencing. Here we show, using a LacI-tethering system, that JIL-1 mediated ectopic histone H3S10 phosphorylation is sufficient to induce a change in higher-order chromatin structure from a condensed heterochromatin-like state to a more open euchromatic state. This effect was absent when a ;kinase dead' LacI-JIL-1 construct without histone H3S10 phosphorylation activity was expressed. Instead, the 'kinase dead' construct had a dominant-negative effect, leading to a disruption of chromatin structure that was associated with a global repression of histone H3S10 phosphorylation levels. These findings provide direct evidence that the epigenetic histone tail modification of H3S10 phosphorylation at interphase can function as a causative regulator of higher-order chromatin structure in Drosophila in vivo.

Deletion of the core region of 5′ HS2 of the mouse β-globin locus control region reveals a distinct effect in comparison with human β-globin transgenes

AbstractThe β-globin locus control region (LCR) is a large DNA element that is required for high-level expression of β-like globin genes from the endogenous mouse locus or in transgenic mice carrying the human β-globin locus. The LCR encompasses 6 DNaseI hypersensitive sites (HSs) that bind transcription factors. These HSs each contain a core of a few hundred base pairs (bp) that has most of the functional activity and exhibits high interspecies sequence homology. Adjoining the cores are 500- to 1000-bp “flanks” with weaker functional activity and lower interspecies homology. Studies of human β-globin transgenes and of the endogenous murine locus show that deletion of an entire HS (core plus flanks) moderately suppresses expression. However, human transgenes in which only individual HS core regions were deleted showed drastic loss of expression accompanied by changes in chromatin structure. To address these disparate results, we have deleted the core region of 5′HS2 from the endogenous murine β-LCR. The phenotype was similar to that of the larger 5′HS2 deletion, with no apparent disruption of chromatin structure. These results demonstrate that the greater severity of HS core deletions in comparison to full HS deletions is not a general property of the β-LCR. (Blood. 2006;107:821-826)

Genome-Scale Analysis of Hematopoietic Regulatory Sequences by Digital Analysis of Chromatin Structure.

In vivo, cis-regulatory elements coincide with focal disruptions in chromatin structure that manifest as DNaseI hypersensitive sites (HSs). We developed a novel stochastic methodology for comprehensive, genome-wide mapping of DNaseI HSs in vivo. We show that 19–20bp genomic DNA tags can be used to effect genome-wide localization of individual DNaseI cutting events in nuclear chromatin, and that such tags can be generated and sequenced in large numbers from limited numbers of starting cells. We analyzed 257,000 tags from a hematopoietic cell line (K562) and applied a quantitative algorithm to discriminate statistically significant tag clustering events. Such tag clusters identified both known and novel functional elements of hematopoietic genes across the genome. A unique feature of this approach is that it permits unbiased evaluation of the chromatin context of regulatory sequences from disperse genomic loci. We observed surprisingly large differences in the chromatin structural configuration of a variety of active erythroid and hematopoietic regulatory sequences, suggesting a discrete hierarchy of nuclear organization that is not apparent with conventional assays. This approach can be applied readily to generate a comprehensive catalogue of cis-regulatory sequences active in hematopoietic stem cells.

Rapid In Vivo Mapping of Hematopoietic Cis-Regulatory Elements.

Cis-acting sequences play defining roles in the control of genes involved in hematopoiesis. However, for the vast majority of such genes, regulatory sequences remain to be defined. We describe a powerful, generic approach to identification of cis-regulatory sequences that may be applied to any gene locus in the context of any cell type. We used densely tiled primers and a novel real-time PCR-based assay to create continuous, high-resolution quantitative profiles of in vivo chromatin structure across entire gene domains. Such profiles can be analyzed using robust statistical algorithms to pinpoint disruptions in chromatin structure that are characteristic of cis-regulatory elements. We analyzed >1Mb of human genomic terrain from diverse gene loci in the context of several hematopoietic cell lines and cleanly delineated a spectrum of classical cis-regulatory activities including enhancers, promoters, insulators, and locus control regions. The approach displayed outstanding sensitivity (100%) and specificity (>99.6%) for known elements and was successful in defining novel elements even in heavily-explored terrain such as the alpha- and beta-globin loci. Since only small quantities of cells are required, the approach can be used readily in the context of hematopoietic progenitors. Systematic application of quantitative chromatin profiling to relevant genes promises to expand dramatically our understanding of the regulation of hematopoiesis.

RNA polymerase II elongation through chromatin.

The machinery that transcribes protein-coding genes in eukaryotic cells must contend with repressive chromatin structures in order to find its target DNA sequences. Diverse arrays of proteins modify the structure of chromatin at gene promoters to help transcriptional regulatory proteins access their DNA recognition sites. The way in which disruption of chromatin structure at a promoter is transmitted through a whole gene has not been defined. Recent breakthroughs suggest that the passage of an RNA polymerase through a gene is coupled to mechanisms that propagate the breakdown of chromatin.

RNA Polymerase-specific Nucleosome Disruption by Transcription in Vivo

The nucleosomal chromatin structure within genes is disrupted upon transcription by RNA polymerase II. To determine whether this disruption is caused by transcription per se as opposed to the RNA polymerase source, we engineered the yeast chromosomal HSP82 gene to be exclusively transcribed by bacteriophage T7 RNA polymerase in vivo. Interestingly, we found that a fraction of the T7-generated transcripts were 3' end processed and polyadenylated at or near the 3' ends of the hsp82 and the immediately downstream CIN2 genes. Surprisingly, the nucleosomal structure of the T7-transcribed hsp82 gene remained intact, in marked contrast to the disrupted structure generated by much weaker, basal level transcription of the wild type gene by RNA polymerase II under non-heat shock conditions. Therefore, disruption of chromatin structure by transcription is dependent on the RNA polymerase source. We propose that the observed RNA polymerase dependence for transcription-induced nucleosome disruption may be related either to the differential recruitment of chromatin remodeling complexes, the rates of histone octamer translocation and nucleosome reformation during polymerase traversal, and/or the degree of transient torsional stress generated by the elongating polymerase.

Identification and analysis of the Arabidopsis thaliana BSH gene, a member of the SNF5 gene family.

The multiprotein complexes involved in active dis-ruption of chromatin structure, homologous to yeast SWI/SNF complex, have been described for human and Drosophila cells. In all SWI/SNF-class complexes characterised so far, one of the key components is the SNF5-type protein. Here we describe the isolation of a plant (Arabidopsis thaliana ) cDNA encoding a 27 kDa protein which we named BSH, with high homology to yeast SNF5p and its human (INI1) and Drosophila (SNR1) counterparts as well as to other putative SNF5-type proteins from Caenorhabditis elegans, fish and yeast. With 240 amino acids, the Arabidopsis BSH is the smallest SNF5-type protein so far identified. When expressed in Saccharomyces cerevisiae, the gene for BSH partially complements the snf5 mutation. BSH is, however, unable to activate transcription in yeast when tethered to DNA. The gene for BSH occurs in single copy in the Arabidopsis genome and is ubiquitously expressed in the plant. Analysis of the whole cell and nuclear protein extracts with antibodies against recombinant BSH indicates that the protein is localised in nuclei. Transgenic Arabidopsis plants with markedly decreased physiological level of the BSH mRNA, resulting from the expression of antisense messenger, are viable but exhibit a distinctive phenotype characterised by bushy growth and flowers that are unable to produce seeds.

Determinants of chromatin disruption and transcriptional regulation instigated by the thyroid hormone receptor: hormone-regulated chromatin disruption is not sufficient for transcriptional activation.

Chromatin disruption and transcriptional activation are both thyroid hormone-dependent processes regulated by the heterodimer of thyroid hormone receptor and 9-cis retinoic acid receptor (TR-RXR). In the absence of hormone, TR-RXR binds to nucleosomal DNA, locally disrupts histone-DNA contacts and generates a DNase I-hypersensitive site. Chromatin-bound unliganded TR-RXR silences transcription of the Xenopus TRbetaA gene within a canonical nucleosomal array. On addition of hormone, the receptor directs the extensive further disruption of chromatin structure over several hundred base pairs of DNA and activates transcription. We define a domain of the TR protein necessary for directing this extensive hormone-dependent chromatin disruption. Particular TR-RXR heterodimers containing mutations in this domain are able to bind both hormone and their thyroid hormone receptor recognition element (TRE) within chromatin, yet are unable to direct the extensive hormone-dependent disruption of chromatin or to activate transcription. We distinguish the hormone-dependent disruption of chromatin and transcriptional activation as independently regulated events through the mutagenesis of basal promoter elements and by altering the position and number of TREs within the TRbetaA promoter. Chromatin disruption alone on a minichromosome is shown to be insufficient for transcriptional activation of the TRbetaA gene.