Role In Chromatin Organization Research Articles

LINE-1 activation after fertilization regulates global chromatin accessibility in the early mouse embryo.

After fertilization, to initiate development, gametes are reprogramed to become totipotent. Approximately half of the mammalian genome consists of repetitive elements, including retrotransposons, some of which are transcribed after fertilization. Retrotransposon activation is generally assumed to be a side effect of the extensive chromatin remodeling underlying the epigenetic reprogramming of gametes. Here, we used a targeted epigenomic approach to address whether specific retrotransposon families play a direct role in chromatin organization and developmental progression. We demonstrate that premature silencing of LINE-1 elements decreases chromatin accessibility, whereas prolonged activation prevents the gradual chromatin compaction that occurs naturally in developmental progression. Preventing LINE-1 activation and interfering with its silencing decreases developmental rates independently of the coding nature of the LINE-1 transcript, thus suggesting that LINE-1 functions primarily at the chromatin level. Our data suggest that activation of LINE-1 regulates global chromatin accessibility at the beginning of development and indicate that retrotransposon activation is integral to the developmental program.

Chromatin organization at the nuclear periphery as revealed by image analysis of structured illumination microscopy data

The nuclear periphery (NP) plays a substantial role in chromatin organization. Heterochromatin at the NP is interspersed with active chromatin surrounding nuclear pore complexes (NPCs); however, details of the peripheral chromatin organization are missing. To discern the distribution of epigenetic marks at the NP of HeLa nuclei, we used structured illumination microscopy combined with a new MATLAB software tool for automatic NP and NPC detection, measurements of fluorescent intensity and statistical analysis of measured data. Our results show that marks for both active and non-active chromatin associate differentially with NPCs. The incidence of heterochromatin marks, such as H3K27me2 and H3K9me2, was significantly lower around NPCs. In contrast, the presence of marks of active chromatin such as H3K4me2 was only decreased very slightly around the NPCs or not at all (H3K9Ac). Interestingly, the histone demethylases LSD1 (also known as KDM1A) and KDM2A were enriched within the NPCs, suggesting that there was a chromatin-modifying mechanism at the NPCs. Inhibition of transcription resulted in a larger drop in the distribution of H1, H3K9me2 and H3K23me2, which implies that transcription has a role in the organization of heterochromatin at the NP.

Aged induced pluripotent stem cell (iPSCs) as a new cellular model for studying premature aging.

Nuclear integrity and mechanical stability of the nuclear envelope (NE) are conferred by the nuclear lamina, a meshwork of intermediate filaments composed of A- and B-type lamins, supporting the inner nuclear membrane and playing a pivotal role in chromatin organization and epigenetic regulation. During cell senescence, nuclear alterations also involving NE architecture are widely described. In the present study, we utilized induced pluripotent stem cells (iPSCs) upon prolonged in vitro culture as a model to study aging and investigated the organization and expression pattern of NE major constituents. Confocal and four-dimensional imaging combined with molecular analyses, showed that aged iPSCs are characterized by nuclear dysmorphisms, nucleoskeletal components (lamin A/C-prelamin isoforms, lamin B1, emerin, and nesprin-2) imbalance, leading to impaired nucleo-cytoplasmic MKL1 shuttling, actin polymerization defects, mitochondrial dysfunctions, SIRT7 downregulation and NF-kBp65 hyperactivation. The observed age-related NE features of iPSCs closely resemble those reported for premature aging syndromes (e.g., Hutchinson-Gilford progeria syndrome) and for somatic cell senescence. These findings validate the use of aged iPSCs as a suitable cellular model to study senescence and for investigating therapeutic strategies aimed to treat premature aging.

Impact of the epigenetic modulator BORIS on sensitivity of melanoma cells to UV-induced DNA damage.

e21077 Background: Cutaneous melanomas display considerably higher mutational load compared to other solid tumors, mainly due to DNA damage and absent/defective repair. Chromatin organization plays an important role in these processes, with open chromatin conferring a higher sensitivity to DNA damage. BORIS is an epigenetic modifier that is correlated with open chromatin and is abnormally expressed in primary and metastatic melanoma. Here we set out to obtain a better understanding of BORIS’s role in chromatin organization and DNA damage in melanoma. Methods: A panel of melanoma cell lines was selected and BORIS mRNA expression was determined using qPCR. Cellular localization of BORIS was assessed by immunofluorescence. Ectopic BORIS expression was established exploiting a doxycycline inducible expression vector and proliferation was assessed using crystal violet and Hoechst staining. DNA damage was induced by exposure to 60 J/m2 UVC followed by apoptosis and cell cycle analysis using flow cytometry. Results: We confirmed aberrant BORIS expression in 61% of our melanoma cell line panel and observed nuclear BORIS localization in areas of low chromatin density. Forced expression of BORIS in BORIS low/intermediate cell lines resulted in a significant reduction in cell growth and increased cell death. Interestingly, BORIS overexpressing cells exposed to UV irradiation displayed a higher degree of cell cycle alterations and increased level of apoptosis compared to BORIS low/intermediate cells. Conclusions: Our results demonstrate that BORIS is increased in melanoma, localized in areas of open chromatin, and increased sensitivity to UV-induced DNA damage. Taken together, this indicates a role for BORIS in directing the chromatin state into a more open conformation, allowing increased sensitivity to DNA damage. Currently, we are analyzing DNA damage in BORIS overexpressing cells and are further investigating BORIS’s role in directing chromatin state by exploiting ATAC-seq and ChIP-seq for various epigenetic marks.

Assessing histone demethylase inhibitors in cells: lessons learned

BackgroundHistone lysine demethylases (KDMs) are of interest as drug targets due to their regulatory roles in chromatin organization and their tight associations with diseases including cancer and mental disorders. The first KDM inhibitors for KDM1 have entered clinical trials, and efforts are ongoing to develop potent, selective and cell-active ‘probe’ molecules for this target class. Robust cellular assays to assess the specific engagement of KDM inhibitors in cells as well as their cellular selectivity are a prerequisite for the development of high-quality inhibitors. Here we describe the use of a high-content cellular immunofluorescence assay as a method for demonstrating target engagement in cells.ResultsA panel of assays for the Jumonji C subfamily of KDMs was developed to encompass all major branches of the JmjC phylogenetic tree. These assays compare compound activity against wild-type KDM proteins to a catalytically inactive version of the KDM, in which residues involved in the active-site iron coordination are mutated to inactivate the enzyme activity. These mutants are critical for assessing the specific effect of KDM inhibitors and for revealing indirect effects on histone methylation status. The reported assays make use of ectopically expressed demethylases, and we demonstrate their use to profile several recently identified classes of KDM inhibitors and their structurally matched inactive controls. The generated data correlate well with assay results assessing endogenous KDM inhibition and confirm the selectivity observed in biochemical assays with isolated enzymes. We find that both cellular permeability and competition with 2-oxoglutarate affect the translation of biochemical activity to cellular inhibition.ConclusionsHigh-content-based immunofluorescence assays have been established for eight KDM members of the 2-oxoglutarate-dependent oxygenases covering all major branches of the JmjC-KDM phylogenetic tree. The usage of both full-length, wild-type and catalytically inactive mutant ectopically expressed protein, as well as structure-matched inactive control compounds, allowed for detection of nonspecific effects causing changes in histone methylation as a result of compound toxicity. The developed assays offer a histone lysine demethylase family-wide tool for assessing KDM inhibitors for cell activity and on-target efficacy. In addition, the presented data may inform further studies to assess the cell-based activity of histone lysine methylation inhibitors.

Chromatin organization at the nuclear periphery as revealed by image analysis of structured illumination microscopy data.

The nuclear periphery (NP) plays a substantial role in chromatin organization. Heterochromatin at the NP is interspersed with active chromatin surrounding nuclear pore complexes (NPCs); however, details of the peripheral chromatin organization are missing. To discern the distribution of epigenetic marks at the NP of HeLa nuclei, we used structured illumination microscopy combined with a new MATLAB software tool for automatic NP and NPC detection, measurements of fluorescent intensity and statistical analysis of measured data. Our results show that marks for both active and non-active chromatin associate differentially with NPCs. The incidence of heterochromatin marks, such as H3K27me2 and H3K9me2, was significantly lower around NPCs. In contrast, the presence of marks of active chromatin such as H3K4me2 was only decreased very slightly around the NPCs or not at all (H3K9Ac). Interestingly, the histone demethylases LSD1 (also known as KDM1A) and KDM2A were enriched within the NPCs, suggesting that there was a chromatin-modifying mechanism at the NPCs. Inhibition of transcription resulted in a larger drop in the distribution of H1, H3K9me2 and H3K23me2, which implies that transcription has a role in the organization of heterochromatin at the NP.

SMAR1 binds to T(C/G) repeat and inhibits tumor progression by regulating miR-371-373 cluster

Chromatin architecture and dynamics are regulated by various histone and non-histone proteins. The matrix attachment region binding proteins (MARBPs) play a central role in chromatin organization and function through numerous regulatory proteins. In the present study, we demonstrate that nuclear matrix protein SMAR1 orchestrates global gene regulation as determined by massively parallel ChIP-sequencing. The study revealed that SMAR1 binds to T(C/G) repeat and targets genes involved in diverse biological pathways. We observe that SMAR1 binds and targets distinctly different genes based on the availability of p53. Our data suggest that SMAR1 binds and regulates one of the imperative microRNA clusters in cancer and metastasis, miR-371-373. It negatively regulates miR-371-373 transcription as confirmed by SMAR1 overexpression and knockdown studies. Further, deletion studies indicate that a ~200 bp region in the miR-371-373 promoter is necessary for SMAR1 binding and transcriptional repression. Recruitment of HDAC1/mSin3A complex by SMAR1, concomitant with alteration of histone marks results in downregulation of the miRNA cluster. The regulation of miR-371-373 by SMAR1 inhibits breast cancer tumorigenesis and metastasis as determined by in vivo experiments. Overall, our study highlights the binding of SMAR1 to T(C/G) repeat and its role in cancer through miR-371-373.

Co-ChIP enables genome-wide mapping of histone mark co-occurrence at single-molecule resolution.

Histone modifications play an important role in chromatin organization and transcriptional regulation, but despite the large amount of genome-wide histone modification data collected in different cells and tissues, little is known about co-occurrence of modifications on the same nucleosome. Here we present a genome-wide quantitative method for combinatorial indexed chromatin immunoprecipitation (co-ChIP) to characterize co-occurrence of histone modifications on nucleosomes. Using co-ChIP, we study the genome-wide co-occurrence of 14 chromatin marks (70 pairwise combinations), and find previously undescribed co-occurrence patterns, including the co-occurrence of H3K9me1 and H3K27ac in super-enhancers. Finally, we apply co-ChIP to measure the distribution of the bivalent H3K4me3-H3K27me3 domains in two distinct mouse embryonic stem cell (mESC) states and in four adult tissues. We observe dynamic changes in 5,786 regions and discover both loss and de novo gain of bivalency in key tissue-specific regulatory genes, suggesting a functional role for bivalent domains during different stages of development. These results show that co-ChIP can reveal the complex interactions between histone modifications.

Diverse architectural properties of Sso10a proteins: Evidence for a role in chromatin compaction and organization.

Sso10a proteins are small DNA-binding proteins expressed by the crenarchaeal model organism Sulfolobus solfataricus. Based on the structure of Sso10a1, which contains a winged helix-turn-helix motif, it is believed that Sso10a proteins function as sequence-specific transcription factors. Here we show that Sso10a1 and Sso10a2 exhibit different distinct DNA-binding modes. While the ability to bend DNA is shared between the two proteins, DNA bridging is observed only for Sso10a1 and only Sso10a2 exhibits filament formation along DNA. The architectural properties of Sso10a proteins suggest that these proteins fulfil generic roles in chromatin organization and compaction. As these proteins exhibit different binding behaviour depending on their DNA binding stoichiometry, altered levels of expression in the cell can be exploited to drive changes in local genome folding, which may operate to modulate transcription.

CHANGE POINT ANALYSIS OF HISTONE MODIFICATIONS REVEALS EPIGENETIC BLOCKS LINKING TO PHYSICAL DOMAINS.

Histone modification is a vital epigenetic mechanism for transcriptional control in eukaryotes. High-throughput techniques have enabled whole-genome analysis of histone modifications in recent years. However, most studies assume one combination of histone modification invariantly translates to one transcriptional output regardless of local chromatin environment. In this study we hypothesize that, the genome is organized into local domains that manifest similar enrichment pattern of histone modification, which leads to orchestrated regulation of expression of genes with relevant biological functions. We propose a multivariate Bayesian Change Point (BCP) model to segment the Drosophila melanogaster genome into consecutive blocks on the basis of combinatorial patterns of histone marks. By modeling the sparse distribution of histone marks with a zero-inflated Gaussian mixture, our partitions capture local BLOCKs that manifest relatively homogeneous enrichment pattern of histone marks. We further characterized BLOCKs by their transcription levels, distribution of genes, degree of co-regulation and GO enrichment. Our results demonstrate that these BLOCKs, although inferred merely from histone modifications, reveal strong relevance with physical domains, which suggests their important roles in chromatin organization and coordinated gene regulation.

Distribution and Associations of GATA Repeats in Rice Genome

GATA repeats are associated with sex differentiation in man, buffalo, mouse and even in plants such as papaya. Human X-chromosome region Xp22 that escapes inactivation is ten-fold enriched in GATA repeats suggesting a role in preventing heterochromatinization. The close proximity of GATA repeats to matrix-associated regions (MARs) indicates a role in chromatin organization and function. Chromosome-wise distribution and density of GATA repeats, neighboring genes and Matrix associated regions were analyzed in rice. (GATA)3 and higher repeats were distributed non-randomly with the highest frequency on chromosome 11. About 60% of the repeats were found in intergenic regions flanked by regulatory genes involved in stress response or transposable elements. The GATA associated MAR sequences in rice had at least one or more of the consensus sequence to which GATA factors bind. The genomic milieu around GATA repeats suggests that their genomic contextmay determine their role in chromatin organization and gene regulation.

Specificities and genomic distribution of somatic mammalian histone H1 subtypes

Histone H1 is a structural component of chromatin that may have a role in the regulation of chromatin dynamics. Unlike core histones, the linker histone H1 family is evolutionarily diverse and many organisms have multiple H1 variants or subtypes, distinguishable between germ-line and somatic cells. In mammals, the H1 family includes seven somatic H1 variants with a prevalence that varies between cell types and over the course of differentiation, H1.1 to H1.5 being expressed in a replication-dependent manner, whereas H1.0 and H1X are replication-independent. Until recently, it has not been known whether the different variants had specific roles in the regulation of nuclear processes or were differentially distributed across the genome. To address this, an increasing effort has been made to investigate divergent features among H1 variants, regarding their structure, expression patterns, chromatin dynamics, post-translational modifications and genome-wide distribution. Although H1 subtypes seem to have redundant functions, several reports point to the idea that they are also differently involved in specific cellular processes. Initial studies investigating the genomic distribution of H1 variants have started to suggest that despite a wide overlap, different variants may be enriched or preferentially located at different chromatin types, but this may depend on the cell type, the relative abundance of the variants, the differentiation state of the cell, or whether cells are derived from a neoplastic process. Understanding the heterogeneity of the histone H1 family is crucial to elucidate their role in chromatin organization, gene expression regulation and other cellular processes. This article is part of a Special Issue entitled: Histone H1, edited by Dr. Albert Jordan.

Differential Predictive Roles of A- and B-Type Nuclear Lamins in Prostate Cancer Progression.

BackgroundProstate cancer (PCa) is the most common cancer among men in western countries. While active surveillance is increasingly utilized, the majority of patients are currently treated with radical prostatectomy. In order to avoid over-treatment, there is an indisputable need for reliable biomarkers to identify the potentially aggressive and lethal cases. Nuclear intermediate filament proteins called lamins play a role in chromatin organization, gene expression and cell stiffness. The expression of lamin A is associated with poor outcome in colorectal cancer but to date the prognostic value of the lamins has not been tested in other solid tumors.MethodsWe studied the expression of different lamins with immunohistochemistry in a tissue microarray material of 501 PCa patients undergoing radical prostatectomy and lymph node dissection. Patients were divided into two staining categories (low and high expression). The correlation of lamin expression with clinicopathological variables was tested and the association of lamin status with biochemical recurrence (BCR) and disease specific survival (DSS) was further analyzed.ResultsLow expression of lamin A associated with lymph node positivity (p<0.01) but not with other clinicopathological variables and low expression had a borderline independent significant association with DSS (HR = 0.4; 95% CI 0.2–1.0; p = 0.052). Similarly, low lamin C expression associated with poorer survival (HR = 0.2; 95% CI 0.1–0.6; p = 0.004). Lamin B1 expression did not associate with clinicopathological variables but high expression independently predicted BCR in multivariable Cox regression analysis (HR = 1.8; 95% CI 1.1–2.9; p = 0.023). Low expression of lamin B2 correlated with lymph node positivity (p<0.01) and predicted unfavorable DSS (HR = 0.4; 95% CI 0.2–1.0; p = 0.047).ConclusionsThese results suggest differential roles for lamins in PCa progression. Reduced amounts of lamin A/C and B2 increase risk for lymph node metastasis and disease specific death possibly through increased nuclear deformability while high expression of lamin B1 predicts disease recurrence.

CTCF regulates NELF, DSIF and P-TEFb recruitment during transcription

CTCF is a versatile transcription factor with well-established roles in chromatin organization and insulator function. Recent findings also implicate CTCF in the control of elongation by RNA polymerase (RNAP) II. Here we show that CTCF knockdown abrogates RNAP II pausing at the early elongation checkpoint of c-myc by affecting recruitment of DRB-sensitivity-inducing factor (DSIF). CTCF knockdown also causes a termination defect on the U2 snRNA genes (U2), by affecting recruitment of negative elongation factor (NELF). In addition, CTCF is required for recruitment of positive elongation factor b (P-TEFb), which phosphorylates NELF, DSIF, and Ser2 of the RNAP II CTD to activate elongation of transcription of c-myc and recognition of the snRNA gene-specific 3’ box RNA processing signal. These findings implicate CTCF in a complex network of protein:protein/protein:DNA interactions and assign a key role to CTCF in controlling RNAP II transcription through the elongation checkpoint of the protein-coding c-myc and the termination site of the non-coding U2, by regulating the recruitment and/or activity of key players in these processes.

Role of High‐mobility group box‐1 in diabetic retinal vasculopathy and neuropathy

SummaryHigh‐mobility mobility group box‐1 (HMGB1) is a non‐histone DNA‐binding protein that plays an important role in chromatin organization and transcriptional regulation. Extracellular HMGB1 functions as a proinflammatory cytokine and triggers the inflammatory response through the activation of multiple receptors such as the receptor for advanced glycation end products (RAGE), toll‐like receptor‐2 (TLR2), TLR4 and TLR9. We demonstrated that HMGB1 and RAGE were expressed in fibrovascular epiretinal membranes from patients with proliferative diabetic retinopathy (PDR). In addition, we demonstrated increased levels of HMGB1 in the vitreous samples from patients with PDR and that there were significant positive correlations between the vitreous levels and HMGB1 and the levels of the biomarkers of inflammation and oxidative injury. Furthermore, we demonstrated that diabetes induced significant upregulation of the expression of HMGB1 and RAGE in the retinas of rats and mice and that HMGB1 mediates diabetes‐induced oxidative stress, activation of inflammatory signaling pathways, breakdown of the blood‐retinal barrier and neuropathy in the retina. Therefore, compounds inhibiting HMGB1 may be novel therapeutic agents for diabetic retinopathy.

Chromatin organization in pluripotent cells: emerging approaches to study and disrupt function.

Translating the vast amounts of genomic and epigenomic information accumulated on the linear genome into three-dimensional models of nuclear organization is a current major challenge. In response to this challenge, recent technological innovations based on chromosome conformation capture methods in combination with increasingly powerful functional approaches have revealed exciting insights into key aspects of genome regulation. These findings have led to an emerging model where the genome is folded and compartmentalized into highly conserved topological domains that are further divided into functional subdomains containing physical loops that bring cis-regulatory elements to close proximity. Targeted functional experiments, largely based on designable DNA-binding proteins, have begun to define the major architectural proteins required to establish and maintain appropriate genome regulation. Here, we focus on the accessible and well-characterized system of pluripotent cells to review the functional role of chromatin organization in regulating pluripotency, differentiation and reprogramming.

The Bucentaur (BCNT) protein family: a long-neglected class of essential proteins required for chromatin/chromosome organization and function.

The evolutionarily conserved Bucentaur (BCNT) protein superfamily was identified about two decades ago in bovines, but its biological role has long remained largely unknown. Sparse studies in the literature suggest that BCNT proteins perform important functions during development. Only recently, a functional analysis of the Drosophila BCNT ortholog, called YETI, has provided evidence that it is essential for proper fly development and plays roles in chromatin organization. Here, we introduce the BCNT proteins and comprehensively review data that contribute to clarify their function and mechanistic clues on how they may control development in multicellular organisms.

Protein arginine methyltransferases (PRMTs): role in chromatin organization.

The mammalian genome encodes eleven protein arginine methyltransferases (PRMTs) that are involved in the transfer of a methyl group from S-adenosylmethionine (AdoMet) to the guanidino nitrogen of arginine. The substrates for these enzymes range from histones to several nuclear and cytoplasmic proteins. Methylation of histones by PRMTs can block the docking site for other reader/effector molecules and thus this modification can interfere with histone code orchestration. Several members of the PRMTs have roles in chromatin organization and function. Although PRMT aberrant expression is correlated with several diseases including cancer, the underlying mechanisms are still obscure in most cases.

Ibf1 and Ibf2 are novel CP190-interacting proteins required for insulator function

Insulators are DNA-protein complexes that play a central role in chromatin organization and regulation of gene expression. In Drosophila different proteins, dCTCF, Su(Hw), and BEAF bind to specific subsets of insulators most of them having in common CP190. It has been shown that there are a number of CP190-binding sites that are not shared with any other known insulator protein, suggesting that other proteins could cooperate with CP190 to regulate insulator activity. Here we report on the identification of two previously uncharacterized proteins as CP190-interacting proteins, that we have named Ibf1 and Ibf2. These proteins localize at insulator bodies and associate with chromatin at CP190-binding sites throughout the genome. We also show that Ibf1 and Ibf2 are DNA-binding proteins that form hetero-oligomers that mediate CP190 binding to chromatin. Moreover, Ibf1 and Ibf2 are necessary for insulator activity in enhancer-blocking assays and Ibf2 null mutation cause a homeotic phenotype. Taken together our data reveal a novel pathway of CP190 recruitment to chromatin that is required for insulator activity.

Quantification of the Spatial Organization of the Nuclear Lamina as a Tool for Cell Classification.

The nuclear lamina is the structural scaffold of the nuclear envelope that plays multiple regulatory roles in chromatin organization and gene expression as well as a structural role in nuclear stability. The lamina proteins, also referred to as lamins, determine nuclear lamina organization and define the nuclear shape and the structural integrity of the cell nucleus. In addition, lamins are connected with both nuclear and cytoplasmic structures forming a dynamic cellular structure whose shape changes upon external and internal signals. When bound to the nuclear lamina, the lamins are mobile, have an impact on the nuclear envelop structure, and may induce changes in their regulatory functions. Changes in the nuclear lamina shape cause changes in cellular functions. A quantitative description of these structural changes could provide an unbiased description of changes in cellular function. In this review, we describe how changes in the nuclear lamina can be measured from three-dimensional images of lamins at the nuclear envelope, and we discuss how structural changes of the nuclear lamina can be used for cell classification.