Compaction State Of Chromatin Research Articles

Abstract 4734: BRD8-driven EP400 complex hijacks H2AZ to maintain proliferation in glioblastoma

Abstract Glioblastoma (GBM) is the most notorious primary brain tumor. The median survival of GBM patients is only about one year and about 95% patients succumb after five years. This gloomy picture has not been improved for decades, even with extensive treatments including surgery, radiotherapy, and chemotherapy. Hence, a better understanding of the mechanisms underlying GBM development may provide new therapeutic opportunities. Primary GBM generally harbors a low mutational load when compared to other human cancers. Even P53—the guardian of the genome that is essentially disabled in nearly all human cancers, remains unmutated in about 71% of GBM cases. Moreover, these P53 wildtype GBM cases are as aggressive as those with P53 mutations. To explore the mechanism by which P53 wildtype GBM thrive in the presence of intact P53, we performed CRISPR screens in a panel of human cancer cell lines using an sgRNA library specifically targeting the functional domains of ~200 chromatin regulators. We discovered that BRD8’s bromodomain—a druggable domain as shown by the tremendous success in preclinical and clinical trial in diverse cancers—is a vulnerability specifically in P53 wildtype GBM cases. Our mechanistic studies demonstrate that BRD8 functions through the EP400 chromatin remodeling complex and hijacks the histone variant H2AZ at P53 target loci, enforcing a compact chromatin state that blocks P53’s accessibility to its targets. We show that targeting the BRD8 bromodomain releases H2AZ, opens up chromatin, engages P53-mediated transactivation, and triggers growth arrest. Consistent with these findings, BRD8 is highly expressed with H2AZ in patient-derived proliferating GBM cells, and is inversely correlated with the expression of P53 targets. Our work solves a long-standing mystery in P53 wildtype GBM, and presents a promising therapeutic target for the majority of GBM patients. Citation Format: Sherine Xueqin Sun, Olaf Klingbeil, Christopher Vakoc, Alea Mills. BRD8-driven EP400 complex hijacks H2AZ to maintain proliferation in glioblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4734.

Polycomb Paralog Chromodomain Inhibitors Active against Both CBX6 and CBX8*.

Methyllysine reader proteins bind to methylated lysine residues and alter gene transcription by changing either the compaction state of chromatin or by the recruitment of other multiprotein complexes. The polycomb paralog family of methyllysine readers bind to trimethylated lysine on the tail of histone 3 (H3) via a highly conserved aromatic cage located in their chromodomains. Each of the polycomb paralogs are implicated in several disease states. CBX6 and CBX8 are members of the polycomb paralog family with two structurally similar chromodomains. By exploring the structure-activity relationships of a previously reported CBX6 inhibitor we have discovered more potent and cell permeable analogs. Our current report includes potent, dual-selective inhibitors of CBX6 and CBX8. We have shown that the -2 position in our scaffold is an important residue for selectivity amongst the polycomb paralogs. Preliminary cell-based studies show that the new inhibitors impact cell proliferation in a rhabdoid tumor cell line.

Expression and chromatin structures of cellulolytic enzyme gene regulated by heterochromatin protein 1.

BackgroundHeterochromatin protein 1 (HP1, homologue HepA in Penicillium oxalicum) binding is associated with a highly compact chromatin state accompanied by gene silencing or repression. HP1 loss leads to the derepression of gene expression. We investigated HepA roles in regulating cellulolytic enzyme gene expression, as an increasingly number of studies have suggested that cellulolytic enzyme gene expression is not only regulated by transcription factors, but is also affected by the chromatin status.ResultsAmong the genes that exhibited significant differences between the hepA deletion strain (ΔhepA) and the wild type (WT), most (95.0 %) were upregulated in ΔhepA compared with WT. The expression of the key transcription factor for cellulolytic enzyme gene (e.g., repressor CreA and activator ClrB) increased significantly. However, the deletion of hepA led to downregulation of prominent extracellular cellulolytic enzyme genes. Among the top 10 extracellular glycoside hydrolases (Amy15A, Amy13A, Cel7A/CBHI, Cel61A, Chi18A, Cel3A/BGLI, Xyn10A, Cel7B/EGI, Cel5B/EGII, and Cel6A/CBHII), in which secretion amount is from the highest to the tenth in P. oxalicum secretome, eight genes, including two amylase genes (amy15A and amy13A), all five cellulase genes (cel7A/cbh1, cel6A/cbh2, cel7B/eg1, cel5B/eg2, and cel3A/bgl1), and the cellulose-active LPMO gene (cel61A) expression were downregulated. Results of chromatin accessibility real-time PCR (CHART-PCR) showed that the chromatin of all three tested upstream regions opened specifically because of the deletion of hepA in the case of two prominent cellulase genes cel7A/cbh1 and cel7B/eg1. However, the open chromatin status did not occur along with the activation of cellulolytic enzyme gene expression. The overexpression of hepA upregulated the cellulolytic enzyme gene expression without chromatin modification. The overexpression of hepA remarkably activated the cellulolytic enzyme synthesis, not only in WT (~150 % filter paper activity (FPA) increase), but also in the industry strain RE-10 (~20–30 % FPA increase).ConclusionsHepA is required for chromatin condensation of prominent cellulase genes. However, the opening of chromatin mediated by the deletion of hepA was not positively correlated with cellulolytic enzyme gene activation. HepA is actually a positive regulator for cellulolytic enzyme gene expression and could be a promising target for genetic modification to improve cellulolytic enzyme synthesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0624-9) contains supplementary material, which is available to authorized users.

Mo1754 Early Life Environment Interacts With Genetic Risk in Inflammatory Bowel Diseases

CEACAM6 gene promoter in an open chromatin state region characterized by increased H3S10 phosphorylation level. In contrast, Caco-2 cells expressed low levels of CEACAM6 due to a compact chromatin state in CEACAM6 promoter (low level of H3S10p). AIEC infection led to an increase in CEACAM6 expression related to enhanced HIF-1a binding to CEACAM6 promoter. Abnormal H3S10 phosphorylation in CEACAM6 promoter following AIEC infection enhanced HIF-1a binding and subsequent CEACAM6 expression. Moreover, in silico analysis predicted several miRNAs that are potentially able to regulate CEACAM6 expression. Luciferase reporter assays showed that among the predictive miRNAs, miR-765 and miR-29b-2-5p directly bound to the 3'-UTR of CEACAM6 mRNA. After AIEC infection, a down-regulation of miR-765 and an up-regulation of miR-29b-2-5p in T84 cells were observed. This is consistent with the observation that miR-765 decreased CEACAM6 protein expression compared to miR-29b-2-5p after transfection in T84 cells. Conclusion: This study suggests that AIEC bacteria have the ability to modulate gene expression in the host cell for their own benefit by altering different epigenetic marks as miRNA expression profile and histones post-translational modifications enhancing CEACAM6 abnormal expression. Thus, epigenetic pathways could be new targets to prevent AIEC colonization and AIECinduced inflammation in CD patients.

Rapid epigenetic adaptation to uncontrolled heterochromatin spreading.

Heterochromatin, a highly compact chromatin state characterized by histone H3K9 methylation and HP1 protein binding, silences the underlying DNA and influences the expression of neighboring genes. However, the mechanisms that regulate heterochromatin spreading are not well understood. In this study, we show that the conserved Mst2 histone acetyltransferase complex in fission yeast regulates histone turnover at heterochromatin regions to control heterochromatin spreading and prevents ectopic heterochromatin assembly. The combined loss of Mst2 and the JmjC domain protein Epe1 results in uncontrolled heterochromatin spreading and massive ectopic heterochromatin, leading to severe growth defects due to the inactivation of essential genes. Interestingly, these cells quickly recover by accumulating heterochromatin at genes essential for heterochromatin assembly, leading to their reduced expression to restrain heterochromatin spreading. Our studies discover redundant pathways that control heterochromatin spreading and prevent ectopic heterochromatin assembly and reveal a fast epigenetic adaptation response to changes in heterochromatin landscape.

Into the chromatin world: Role of nuclear architecture in epigenome regulation

Epigenome modifications are established early in development and differentiation and generate distinct levels of chromatin complexity. The specific position of chromosomes and the compaction state of chromatin are both typical features that make it possible to distinguish between repressive and permissive environment for gene expression. In this review we describe the distinct levels of epigenome structures, emphasizing the role of nuclear architecture in the control of gene expression. Recent novel insights have increasingly demonstrated that the nuclear environment can influence nuclear processes such as gene expression and DNA repair. These findings have revealed a further important aspect of the chromatin modifications, suggesting that a proper crosstalk between chromatin and nuclear components, such as lamins or nuclear pores, is required to ensure the correct functioning of the nucleus and that this assumes a crucial role in many pathologies and diseases. Knowledge regarding the molecular mechanisms behind most of these developmental and disease-related defects remains incomplete; the influence of the nuclear architecture on chromatin function may provide a new perspective for understanding these phenotypes.

Unique Mechanical Properties of Cell Nuclei Regulated by Chromatin

Nuclear mechanics and structure could affect gene regulation and gene expression. Chromatin, a major component of cell nuclei, could play an important role in maintaining nuclear integrity and their mechanical properties. Previous studies on nuclear mechanical properties have focused largely on the role of the nuclear lamina, using techniques such as AFM and micropipette aspiration. In this work, we explicitly address the contributions of chromatin to nuclear rheology after isolation from the cell using a microfluidic optical stretcher. We find that isolated nuclei swell in volume under uni-axial stress and exhibit significant softening with increased nuclear size, which can be described by a filtration model for the nuclear membrane and a cortical chromatin model, respectively. In addition, changes to the state of chromatin condensation via histone modifications or chromatin remodeling processes (ATP, topoisomerase II) can strongly impact nuclear morphology and compliance. Moreover, isolated nuclear mechanics is also sensitive to ionic conditions: nuclei stiffen with increasing ionic strength of the buffer and exhibit a transition from stretch to contraction in the presence of multivalent ions (only). Finally, we find that in contrast to other studies suggesting a high refractive index of cell nuclei compared to the cytoplasm, the refractive index of isolated cell nuclei of a variety of cell types can be lower than the refractive index of the cells. The presented work establishes a quantitative link between nuclear mechanical properties and the compaction state of chromatin, which can be modulated by a change in nuclear volume, chromatin remodeling or electrochemical environment.

Crowded chromatin is not sufficient for heterochromatin formation and not required for its maintenance

In contrast to cytoplasmic organelles, which are usually separated from the rest of the cell by phospholipid membranes, nuclear compartments are readily accessible to diffusing proteins and must rely on different mechanisms to maintain their integrity. Specific interactions between scaffolding proteins are known to have important roles for the formation and maintenance of nuclear structures. General physical mechanisms such as molecular crowding, phase separation or colloidal behavior have also been suggested, but their physiological significance remains uncertain. For macromolecular crowding, a role in the maintenance of nucleoli and promyelocytic leukemia (PML) nuclear bodies has been shown. Here, we tested whether a modulation of the compaction state of chromatin, which directly influences the local crowding state, has an impact on the formation and maintenance of densely packed heterochromatin. By osmotic perturbations, we could modify the packing state of chromatin in a controlled manner and show that chromatin compaction, which is associated with increased crowding conditions, is not, per se, sufficient to initiate the formation of new bona fide heterochromatin structures nor is it necessary to maintain already established heterochromatin domains. In consequence, if an increase in crowding induced by chromatin compaction maybe an early step in heterochromatin formation, specific protein-protein interactions are nevertheless required to make heterochromatin long lasting and independent of the crowding state.

Modulation of Higher Order Chromatin Conformation in Mammalian Cell Nuclei Can Be Mediated by Polyamines and Divalent Cations

The organisation of the large volume of mammalian genomic DNA within cell nuclei requires mechanisms to regulate chromatin compaction involving the reversible formation of higher order structures. The compaction state of chromatin varies between interphase and mitosis and is also subject to rapid and reversible change upon ATP depletion/repletion. In this study we have investigated mechanisms that may be involved in promoting the hyper-condensation of chromatin when ATP levels are depleted by treating cells with sodium azide and 2-deoxyglucose. Chromatin conformation was analysed in both live and permeabilised HeLa cells using FLIM-FRET, high resolution fluorescence microscopy and by electron spectroscopic imaging microscopy. We show that chromatin compaction following ATP depletion is not caused by loss of transcription activity and that it can occur at a similar level in both interphase and mitotic cells. Analysis of both live and permeabilised HeLa cells shows that chromatin conformation within nuclei is strongly influenced by the levels of divalent cations, including calcium and magnesium. While ATP depletion results in an increase in the level of unbound calcium, chromatin condensation still occurs even in the presence of a calcium chelator. Chromatin compaction is shown to be strongly affected by small changes in the levels of polyamines, including spermine and spermidine. The data are consistent with a model in which the increased intracellular pool of polyamines and divalent cations, resulting from depletion of ATP, bind to DNA and contribute to the large scale hyper-compaction of chromatin by a charge neutralisation mechanism.

The Epigenomics of Embryonic Stem Cell Differentiation

Embryonic stem cells (ESCs) possess an open and highly dynamic chromatin landscape, which underlies their plasticity and ultimately maintains ESC pluripotency. The ESC epigenome must not only maintain the transcription of pluripotency-associated genes but must also, through gene priming, facilitate rapid and cell type-specific activation of developmental genes upon lineage commitment. Trans-generational inheritance ensures that the ESC chromatin state is stably transmitted from one generation to the next; yet at the same time, epigenetic marks are highly dynamic, reversible and responsive to extracellular cues. Once committed to differentiation, the ESC epigenome is remodeled and resolves into a more compact chromatin state. A thorough understanding of the role of chromatin modifiers in ESC fate and differentiation will be important if they are to be used for therapeutic purposes.Recent technical advances, particularly in next-generation sequencing technologies, have provided a genome-scale view of epigenetic marks and chromatin modifiers. More affordable and faster sequencing platforms have led to a comprehensive characterization of the ESC epigenome and epigenomes of differentiated cell types. In this review, we summarize and discuss the recent progress that has highlighted the central role of histone modifications, histone variants, DNA methylation and chromatin modifiers in ESC pluripotency and ESC fate. We provide a detailed and comprehensive discussion of genome-wide studies that are pertinent to our understanding of mammalian development.

Differential Estrogen-Regulation of CXCL12 Chemokine Receptors, CXCR4 and CXCR7, Contributes to the Growth Effect of Estrogens in Breast Cancer Cells

CXCR4 and CXCR7 are the two receptors for the chemokine CXCL12, a key mediator of the growth effect of estrogens (E2) in estrogen receptor (ER)-positive breast cancers. In this study we examined E2-regulation of the CXCL12 axis components and their involvement in the growth of breast cancer cells. CXCR4 and CXCR7 were differentially regulated by E2 which enhanced the expression of both CXCL12 and CXCR4 but repressed the expression of CXCR7. Formaldehyde-associated isolation of regulatory elements (FAIRE) revealed that E2-mediated transcriptional regulation of these genes is linked to the control of the compaction state of chromatin at their promoters. This effect could be accomplished via several distal ER-binding sites in the regions surrounding these genes, all of which are located 20–250 kb from the transcription start site. Furthermore, individual down-regulation of CXCL12, CXCR4 or CXCR7 expression as well as the inhibition of their activity significantly decreases the rate of basal cell growth. In contrast, E2-induced cell growth was differentially affected. Unlike CXCR7, the inhibition of the expression or activity of either CXCL12 or CXCR4 significantly blunted the E2-mediated stimulation of cellular growth. Besides, CXCR7 over-expression increased the basal MCF-7 cell growth rate and decreased the growth effect of E2. These findings indicate that E2 regulation of the CXCL12 signaling axis is important for the E2-mediated growth effect of breast cancer cells. These data also provide support for distinct biological functions of CXCR4 and CXCR7 and suggest that targeting CXCR4 and/or CXCR7 would have distinct molecular effects on ER-positive breast tumors.

Sequence determinants of histone–DNA binding preferences: Comment on “Cracking the chromatin code: Precise rule of nucleosome positioning” by Edward N. Trifonov

DNA in eukaryotic cells is packaged into compact chromatin state. The fundamental unit of chromatin is a nucleosome, a highly conserved protein-DNA complex in which ~ 147 basepairs (bp) of DNA are wrapped around a histone octamer in a left-handed superhelix [1]. Histone-DNA binding affinity depends on the nucleotide sequence of the nucleosomal site: indeed, the ability of the DNA molecule to bend into the superhelix is mostly governed by dinucleotide base-stacking energies. The review by Trifonov [2] emphasizes the role of deformational properties of DNA in nucleosome positioning and energetics, proposing a novel sequence motif CGRAAATTTYCG that favors nucleosome formation. The motif is inferred from a large-scale map of C.elegans nucleosomes [3]. In building up the case for the 10–11 bp-periodic nucleosome positioning motif shown above, the author has chosen to focus mostly on his own work and the work of his colleagues. However, I find myself intrigued by how well the proposed pattern stacks up against some of the other models and datasets (nucleosome positioning determinants and the idea of the “nucleo-some code” have recently garnered a lot of interest in the chromatin field). The author argues that due to steric exclusion between neighboring particles only single-nucleosome conformations can be used to compare experiment with theory [4]. However, techniques similar to dynamic programming in computer science and transfer matrices in physics can be used to convert histone-DNA binding energies into probabilities of nucleosome formation at every bp, without any approximations related to the finite particle size (see e.g. [5]). Furthermore, because the DNA bendability matrix proposed by the author is capable of placing nucleosomes with 1 bp resolution, only seven nucleosomes whose positions are known precisely from experiment have been chosen to test the model, with impressive success [4]. This seems to be too restrictive – certainly a high-resolution algorithm can predict lower-resolution data. Besides, the vast majority of the algorithms proposed in the literature also have 1 bp resolution and have nonetheless been used to predict genome-wide occupancy profiles for nucleosomes mapped with ~ 10–20 bp precision by micrococcal nuclease (MNase). Moreover, C.elegans data from which the model was inferred in the first place employed MNase digestion followed by 454 pyrosequencing [3], and therefore has the usual ~ 10 – 20 bp accuracy. It would be especially interesting to see whether the DNA bendability model proposed by Trifonov and colleagues has greater predictive power against large-scale nucleosome maps than simple models that assign the same scores to mono- and dinucleotides of the same type, regardless of their position with respect to the DNA helical repeat [6, 7]. Finally, nucleosomes on which the analysis by Trifonov and colleagues is based come from in vivo chromatin in a mixture of C.elegans cells. The nucleosome positions in this sample are averaged over cell types and may have been affected, among other things, by chromatin remodeling enzymes and competition with other DNA-binding proteins. It would be reassuring to see the proposed motif also appear in two recent large-scale maps of nucleosomes positioned in vitro on genomic sequences from S.cerevisiae and E.coli [8, 9].

Ring1B Compacts Chromatin Structure and Represses Gene Expression Independent of Histone Ubiquitination

How polycomb group proteins repress gene expression in vivo is not known. While histone-modifying activities of the polycomb repressive complexes (PRCs) have been studied extensively, in vitro data have suggested a direct activity of the PRC1 complex in compacting chromatin. Here, we investigate higher-order chromatin compaction of polycomb targets in vivo. We show that PRCs are required to maintain a compact chromatin state at Hox loci in embryonic stem cells (ESCs). There is specific decompaction in the absence of PRC2 or PRC1. This is due to a PRC1-like complex, since decompaction occurs in Ring1B null cells that still have PRC2-mediated H3K27 methylation. Moreover, we show that the ability of Ring1B to restore a compact chromatin state and to repress Hox gene expression is not dependent on its histone ubiquitination activity. We suggest that Ring1B-mediated chromatin compaction acts to directly limit transcription in vivo.

Histone Acetylation and the Maintenance of Chromatin Compaction by Polycomb Repressive Complexes

Mechanisms controlling higher-order chromatin structure or chromatin compaction and linking this to gene regulation are poorly understood. Previously, we had shown that the PRC1 Polycomb repressive complex is required to maintain a compact chromatin state at Polycomb target loci in embryonic stem cells (ESCs) of the mouse and that this activity, together with the ability to repress target gene expression, is surprisingly independent of the histone ubiquitination activity of the Ring1B component of PRC1. Here we investigate and discuss the role of another histone modification--histone acetylation--in Polycomb function. We show that inhibition of histone deacetylases leads to some decompaction of Hox loci and suggest that histone deacetylation has a role in the pathway of PRC1-mediated chromatin compaction. We discuss whether PRC1 and histone hypoacetylation function together to establish a chromatin template at which stable nucleosomes act to antagonize transcriptional elongation.

Prothymosin α

Prothymosin α (ProTα) is a highly acidic protein widely distributed in mammalian cells. Since its discovery in 1984, the biological role of this protein has been controversial. Initially, ProTα was considered a thymic factor with a hormonal-like role in the maturation of T-lymphocytes. However, molecular and cellular analyses led to conclude that ProTα is a nuclear protein required in proliferation events while failing to show a clear immunological effect. The involvement of ProTα in changes in the compaction state of chromatin has been recently elucidated with the demonstration that this protein induces the unfolding of chromatin fibres in a process that seems to be mediated by the interaction of ProTα with histone H1. This finding opens up new perspectives in the study of the dynamics of the genetic material in mammalian cells. Furthermore, the relationship between ProTα and apoptosis as well as with proliferation makes this protein an attractive target in the search for modulators of cell death and tumour growth.