Role In Chromatin Organization Research Articles

The Myc-associated zinc finger protein epigenetically controls expression of interferon-γ-stimulated genes by recruiting STAT1 to chromatin

The MYC-Associated Zinc Finger Protein (MAZ) plays important roles in chromatin organization and gene transcription regulation. Dysregulated expression of MAZ causes diseases, such as glioblastoma, breast cancer, prostate cancer, and liposarcoma. Previously, it has been reported that MAZ controls the proinflammatory response in colitis and colon cancer via STAT3 signaling, suggesting that MAZ is involved in regulating immunity-related pathways. However, the molecular mechanism underlying this regulation remains elusive. Here, we investigate the regulatory effect of MAZ on interferon-gamma (IFN-γ)-stimulated genes via STAT1, a protein that plays an essential role in immune responses to viral, fungal, and mycobacterial pathogens. We demonstrate that about 80% of occupied STAT1-binding sites colocalize with occupied MAZ-binding sites in HAP1/K562 cells after IFN-γ stimulation. MAZ depletion significantly reduces STAT1 binding in the genome. By analyzing genome-wide gene expression profiles in the RNA-Seq data, we show that MAZ depletion significantly suppresses a subset of the immune response genes, which include the IFN-stimulated genes IRF8 and Absent in Melanoma 2. Furthermore, we find that MAZ controls expression of the immunity-related genes by changing the epigenetic landscape in chromatin. Our study reveals an important role for MAZ in regulating immune-related gene expression.

Interplay between charge distribution and DNA in shaping HP1 paralog phase separation and localization

The heterochromatin protein 1 (HP1) family is a crucial component of heterochromatin with diverse functions in gene regulation, cell cycle control, and cell differentiation. In humans, there are three paralogs, HP1α, HP1β, and HP1γ, which exhibit remarkable similarities in their domain architecture and sequence properties. Nevertheless, these paralogs display distinct behaviors in liquid-liquid phase separation (LLPS), a process linked to heterochromatin formation. Here, we employ a coarse-grained simulation framework to uncover the sequence features responsible for the observed differences in LLPS. We highlight the significance of the net charge and charge patterning along the sequence in governing paralog LLPS propensities. We also show that both highly conserved folded and less-conserved disordered domains contribute to the observed differences. Furthermore, we explore the potential co-localization of different HP1 paralogs in multicomponent assemblies and the impact of DNA on this process. Importantly, our study reveals that DNA can significantly reshape the stability of a minimal condensate formed by HP1 paralogs due to competitive interactions of HP1α with HP1β and HP1γ versus DNA. In conclusion, our work highlights the physicochemical nature of interactions that govern the distinct phase-separation behaviors of HP1 paralogs and provides a molecular framework for understanding their role in chromatin organization.

Interplay between charge distribution and DNA in shaping HP1 paralog phase separation and localization.

The heterochromatin protein 1 (HP1) family is a crucial component of heterochromatin with diverse functions in gene regulation, cell cycle control, and cell differentiation. In humans, there are three paralogs, HP1α, HP1β, and HP1γ, which exhibit remarkable similarities in their domain architecture and sequence properties. Nevertheless, these paralogs display distinct behaviors in liquid-liquid phase separation (LLPS), a process linked to heterochromatin formation. Here, we employ a coarse-grained simulation framework to uncover the sequence features responsible for the observed differences in LLPS. We highlight the significance of the net charge and charge patterning along the sequence in governing paralog LLPS propensities. We also show that both highly conserved folded and less-conserved disordered domains contribute to the observed differences. Furthermore, we explore the potential co-localization of different HP1 paralogs in multicomponent assemblies and the impact of DNA on this process. Importantly, our study reveals that DNA can significantly reshape the stability of a minimal condensate formed by HP1 paralogs due to competitive interactions of HP1α with HP1β and HP1γ versus DNA. In conclusion, our work highlights the physicochemical nature of interactions that govern the distinct phase-separation behaviors of HP1 paralogs and provides a molecular framework for understanding their role in chromatin organization.

Abstract 1648: RB represses cohesin-dependent loop formation and activates E2F-independent transcription

Abstract The retinoblastoma protein (RB) suppresses the activity of the E2F transcription factor family, controlling cellular proliferation. Recent studies, however, indicate that RB's role in chromatin organization, which is not yet fully understood, might be distinct from this E2F-dependent regulation. Employing chromosome conformation capture at a single nucleosome resolution, we identified RB as a cell cycle-regulated repressor of cohesin-dependent loop formation at topologically associating domain (TAD) boundaries. RB depletion increased the number and size of cohesin-dependent loops and strengthened topologically associating domains (TADs). This phenomenon was specific to the G1 phase and was not observed in the S phase, indicating that this novel RB function is regulated by the cell cycle. Mechanistically, RB showed extensive colocalization with cohesin in the human genome, and it impacted cohesin’s distribution on the chromatin. Active RB reduced cohesin from RB-bound TAD boundaries and decreased cohesin activity therein, as assessed by the increased K105/106 acetylation of the cohesin subunit SMC3. This led to reduced insulation in chromosome conformation capture assays. Importantly, by weakening the insulation activity of the adjacent insulators, RB non-canonically enhanced the expression of non-E2F target genes salient for cell adhesion and extracellular matrix organization. When RB was lost, cells showed a more rapid cellular detachment rate and an elevated migration rate, indicating that this novel RB function controls specific transcriptional programs rather than arbitrary genes to regulate the process of cell adhesion and migration. Overall, we conclude that RB has a central role in the interplay between cell cycle and chromatin organization, by repressing the cohesin-dependent loop formation at TAD boundaries. This RB function safeguards E2F-independent transcriptional programs driven by active enhancers and helps maintain cellular adhesion to the extracellular matrix. Citation Format: Hanjun Lee, Ioanna-Maria Gkotinakou, Badri Krishnan, Nicholas J. Dyson, Michael S. Lawrence, Ioannis Sanidas. RB represses cohesin-dependent loop formation and activates E2F-independent transcription [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1648.

The Conserved Chromatin Remodeler SMARCAD1 Interacts with TFIIIC and Architectural Proteins in Human and Mouse.

In vertebrates, SMARCAD1 participates in transcriptional regulation, heterochromatin maintenance, DNA repair, and replication. The molecular basis underlying its involvement in these processes is not well understood. We identified the RNA polymerase III general transcription factor TFIIIC as an interaction partner of native SMARCAD1 in mouse and human models using endogenous co-immunoprecipitations. TFIIIC has dual functionality, acting as a general transcription factor and as a genome organizer separating chromatin domains. We found that its partnership with SMARCAD1 is conserved across different mammalian cell types, from somatic to pluripotent cells. Using purified proteins, we confirmed that their interaction is direct. A gene expression analysis suggested that SMARCAD1 is dispensable for TFIIIC function as an RNA polymerase III transcription factor in mouse ESCs. The distribution of TFIIIC and SMARCAD1 in the ESC genome is distinct, and unlike in yeast, SMARCAD1 is not enriched at active tRNA genes. Further analysis of SMARCAD1-binding partners in pluripotent and differentiated mammalian cells reveals that SMARCAD1 associates with several factors that have key regulatory roles in chromatin organization, such as cohesin, laminB, and DDX5. Together, our work suggests for the first time that the SMARCAD1 enzyme participates in genome organization in mammalian nuclei through interactions with architectural proteins.

The dynamic role of cohesin in maintaining human genome architecture.

Recent advances in genomic and imaging techniques have revealed the complex manner of organizing billions of base pairs of DNA necessary for maintaining their functionality and ensuring the proper expression of genetic information. The SMC proteins and cohesin complex primarily contribute to forming higher-order chromatin structures, such as chromosomal territories, compartments, topologically associating domains (TADs) and chromatin loops anchored by CCCTC-binding factor (CTCF) protein or other genome organizers. Cohesin plays a fundamental role in chromatin organization, gene expression and regulation. This review aims to describe the current understanding of the dynamic nature of the cohesin-DNA complex and its dependence on cohesin for genome maintenance. We discuss the current 3C technique and numerous bioinformatics pipelines used to comprehend structural genomics and epigenetics focusing on the analysis of Cohesin-centred interactions. We also incorporate our present comprehension of Loop Extrusion (LE) and insights from stochastic modelling.

Knockdown of DOM/Tip60 Complex Subunits Impairs Male Meiosis of Drosophila melanogaster.

ATP-dependent chromatin remodeling complexes are involved in nucleosome sliding and eviction and/or the incorporation of histone variants into chromatin to facilitate several cellular and biological processes, including DNA transcription, replication and repair. The DOM/TIP60 chromatin remodeling complex of Drosophila melanogaster contains 18 subunits, including the DOMINO (DOM), an ATPase that catalyzes the exchange of the canonical H2A with its variant (H2A.V), and TIP60, a lysine-acetyltransferase that acetylates H4, H2A and H2A.V histones. In recent decades, experimental evidence has shown that ATP-dependent chromatin remodeling factors, in addition to their role in chromatin organization, have a functional relevance in cell division. In particular, emerging studies suggested the direct roles of ATP-dependent chromatin remodeling complex subunits in controlling mitosis and cytokinesis in both humans and D. melanogaster. However, little is known about their possible involvement during meiosis. The results of this work show that the knockdown of 12 of DOM/TIP60 complex subunits generates cell division defects that, in turn, cause total/partial sterility in Drosophila males, providing new insights into the functions of chromatin remodelers in cell division control during gametogenesis.

Cohesin regulates alternative splicing.

Cohesin, a trimeric complex that establishes sister chromatid cohesion, has additional roles in chromatin organization and transcription. We report that among those roles is the regulation of alternative splicing through direct interactions and in situ colocalization with splicing factors. Degradation of cohesin results in marked changes in splicing, independent of its effects on transcription. Introduction of a single cohesin point mutation in embryonic stem cells alters splicing patterns, demonstrating causality. In primary human acute myeloid leukemia, mutations in cohesin are highly correlated with distinct patterns of alternative splicing. Cohesin also directly interacts with BRD4, another splicing regulator, to generate a pattern of splicing that is distinct from either factor alone, documenting their functional interaction. These findings identify a role for cohesin in regulating alternative splicing in both normal and leukemic cells and provide insights into the role of cohesin mutations in human disease.

Distinct Roles of Histone Lysine Demethylases and Methyltransferases in Developmental Eye Disease.

Histone lysine methyltransferase and demethylase enzymes play a central role in chromatin organization and gene expression through the dynamic regulation of histone lysine methylation. Consistent with this, genes encoding for histone lysine methyltransferases (KMTs) and demethylases (KDMs) are involved in complex human syndromes, termed congenital regulopathies. In this report, we present several lines of evidence for the involvement of these genes in developmental ocular phenotypes, suggesting that individuals with structural eye defects, especially when accompanied by craniofacial, neurodevelopmental and growth abnormalities, should be examined for possible variants in these genes. We identified nine heterozygous damaging genetic variants in KMT2D (5) and four other histone lysine methyltransferases/demethylases (KMT2C, SETD1A/KMT2F, KDM6A and KDM5C) in unrelated families affected with developmental eye disease, such as Peters anomaly, sclerocornea, Axenfeld-Rieger spectrum, microphthalmia and coloboma. Two families were clinically diagnosed with Axenfeld-Rieger syndrome and two were diagnosed with Peters plus-like syndrome; others received no specific diagnosis prior to genetic testing. All nine alleles were novel and five of them occurred de novo; five variants resulted in premature truncation, three were missense changes and one was an in-frame deletion/insertion; and seven variants were categorized as pathogenic or likely pathogenic and two were variants of uncertain significance. This study expands the phenotypic spectra associated with KMT and KDM factors and highlights the importance of genetic testing for correct clinical diagnosis.

Lamin A/C-dependent chromatin architecture safeguards naïve pluripotency to prevent aberrant cardiovascular cell fate and function

Tight control of cell fate choices is crucial for normal development. Here we show that lamin A/C plays a key role in chromatin organization in embryonic stem cells (ESCs), which safeguards naïve pluripotency and ensures proper cell fate choices during cardiogenesis. We report changes in chromatin compaction and localization of cardiac genes in Lmna−/− ESCs resulting in precocious activation of a transcriptional program promoting cardiomyocyte versus endothelial cell fate. This is accompanied by premature cardiomyocyte differentiation, cell cycle withdrawal and abnormal contractility. Gata4 is activated by lamin A/C loss and Gata4 silencing or haploinsufficiency rescues the aberrant cardiovascular cell fate choices induced by lamin A/C deficiency. We uncover divergent functions of lamin A/C in naïve pluripotent stem cells and cardiomyocytes, which have distinct contributions to the transcriptional alterations of patients with LMNA-associated cardiomyopathy. We conclude that disruption of lamin A/C-dependent chromatin architecture in ESCs is a primary event in LMNA loss-of-function cardiomyopathy.

Butyrate Induces Modifications of the CTCF-Binding Landscape in Cattle Cells.

Butyrate is produced in the rumen from microbial fermentation and is related to several functions, including cell differentiation and proliferation. Butyrate supplementation in calves can accelerate rumen development. DNA-protein interactions, such as the CCCTC-binding factor (CTCF), play essential roles in chromatin organization and gene expression regulation. Although CTCF-binding sites have been identified recently in cattle, a deeper characterization, including differentially CTCF-binding sites (DCBS), is vital for a better understanding of butyrate’s role in the chromatin landscape. This study aimed to identify CTCF-binding regions and DCBS under a butyrate-induced condition using ChIP-seq in bovine cells; 61,915 CTCF peaks were identified in the butyrate and 51,347 in the control. From these regions, 2265 DCBS were obtained for the butyrate vs. control comparison, comprising ~90% of induced sites. Most of the butyrate DCBS were in distal intergenic regions, showing a potential role as insulators. Gene ontology enrichment showed crucial terms for the induced DCBS, mainly related to cellular proliferation, cell adhesion, and growth regulation. Interestingly, the ECM-receptor interaction pathway was observed for the induced DCBS. Motif enrichment analysis further identified transcription factors, including CTCF, BORIS, TGIF2, and ZIC3. When DCBS was integrated with RNA-seq data, putative genes were identified for the repressed DCBS, including GATA4. Our study revealed promising candidate genes in bovine cells by a butyrate-induced condition that might be related to the regulation of rumen development, such as integrins, keratins, and collagens. These results provide a better understanding of the function of butyrate in cattle rumen development and chromatin landscape regulation.

Pentosan Polysulfate, a Semisynthetic Heparinoid Disease-Modifying Osteoarthritic Drug with Roles in Intervertebral Disc Repair Biology Emulating the Stem Cell Instructive and Tissue Reparative Properties of Heparan Sulfate.

This review highlights the attributes of pentosan polysulfate (PPS) in the promotion of intervertebral disc (IVD) repair processes. PPS has been classified as a disease-modifying osteoarthritic drug (DMOAD) and many studies have demonstrated its positive attributes in the countering of degenerative changes occurring in cartilaginous tissues during the development of osteoarthritis (OA). Degenerative changes in the IVD also involve inflammatory cytokines, degradative proteases, and cell signaling pathways similar to those operative in the development of OA in articular cartilage. PPS acts as a heparan sulfate (HS) mimetic to effect its beneficial effects in cartilage. The IVD contains small cell membrane HS proteoglycans (HSPGs) such as syndecan, and glypican and a large multifunctional HS/chondroitin sulfate (CS) hybrid proteoglycan (HSPG2/perlecan), that have important matrix-stabilizing properties and sequester, control, and present growth factors from the FGF, VEGF, PDGF, and BMP families to cellular receptors to promote cell proliferation, differentiation, and matrix synthesis. HSPG2 also has chondrogenic properties and stimulates the synthesis of extracellular matrix (ECM) components and expansion of cartilaginous rudiments, and has roles in matrix stabilization and repair. Perlecan is a perinuclear and nuclear proteoglycan (PG) in IVD cells with roles in chromatin organization and control of transcription factor activity, immunolocalizes to stem cell niches in cartilage, promotes escape of stem cells from quiescent recycling, differentiation and attainment of pluripotency and migratory properties. These participate in tissue development and morphogenesis, ECM remodeling and repair. PPS also localizes in the nucleus of stromal stem cells, promotes development of chondroprogenitor cell lineages, ECM synthesis and repair and discal repair by resident disc cells. The availability of recombinant perlecan and PPS offers new opportunities in repair biology. These multifunctional agents offer welcome new developments in repair strategies for the IVD.

Superresolution imaging of nucleoplasmic reticulum protrusions in the cell cycle to get more insights about lamin role in chromatin organization

Chromatin regulation and organization are fundamental to ensure proper genome expression, thus the physiological state of the cell. Indeed, variations in its structure and mobility result in different pathologies including aging and cancer. In this context, the Nucleoplasmic Reticulum (NR), and in particular lamin proteins, have a fundamental role in anchoring chromatin and in organizing it within the cell nucleus. NR's role is still under debate, but evidence shows that it is involved in many fundamental cellular processes such as DNA replication, DNA transcription, and DNA Damage Repair.

Nuclear lamins: Structure and function in mechanobiology.

Nuclear lamins are type V intermediate filament proteins that polymerize into complex filamentous meshworks at the nuclear periphery and in less structured forms throughout the nucleoplasm. Lamins interact with a wide range of nuclear proteins and are involved in numerous nuclear and cellular functions. Within the nucleus, they play roles in chromatin organization and gene regulation, nuclear shape, size, and mechanics, and the organization and anchorage of nuclear pore complexes. At the whole cell level, they are involved in the organization of the cytoskeleton, cell motility, and mechanotransduction. The expression of different lamin isoforms has been associated with developmental progression, differentiation, and tissue-specific functions. Mutations in lamins and their binding proteins result in over 15 distinct human diseases, referred to as laminopathies. The laminopathies include muscular (e.g., Emery–Dreifuss muscular dystrophy and dilated cardiomyopathy), neurological (e.g., microcephaly), and metabolic (e.g., familial partial lipodystrophy) disorders as well as premature aging diseases (e.g., Hutchinson–Gilford Progeria and Werner syndromes). How lamins contribute to the etiology of laminopathies is still unknown. In this review article, we summarize major recent findings on the structure, organization, and multiple functions of lamins in nuclear and more global cellular processes.

Effect of 5'-fluoro-2'-deoxycytidine and sodium butyrate on the genes of the intrinsic apoptotic pathway, p21, p53, cell viability, and apoptosis in human hepatocellular carcinoma cell lines

Backgrounds: Epigenetic regulation such as DNA methylation plays a major role in chromatin organization and gene transcription. Additionally, histone modification is an epigenetic regulator of chromatin structure and influences chromatin organization and gene expression. The relationship between DNA methyltransferase (DNMTs) expression and promoter methylation of the tumor suppressor genes (TSGs) has been reported in various cancers. Previously, the effect of 5-aza-2'-deoxycytidine (5-AZA-CdR), trichostatin A (TSA), and valproic acid (VPA) was shown on various cancers. This study aimed to investigate the effect of 5'-fluoro-2'-deoxycytidine (FdCyd) and sodium butyrate on the genes of the intrinsic apoptotic pathway, p21, p53, cell viability, and apoptosis in human hepatocellular carcinoma SNU449, SNU475, and SNU368 cell lines. 
 Materials and Methods: In this lab trial study, the SNU449, SNU475, and SNU368 cells were cultured and treated with 5'-fluoro-2'-deoxycytidine and sodium butyrate. To determine cell viability, cell apoptosis, and the relative gene expression level, MTT assay, flow cytometry assay, and qRT-PCR were done respectively.
 Results: 5'-fluoro-2'-deoxycytidine and sodium butyrate changed the expression level of the BAX, BAK, APAF1, Bcl-2, Bcl-xL, p21, and p53 gene (P<0.0001) by which induced cell apoptosis and inhibit cell growth in all three cell lines, SNU449, SNU475, and SNU368. 
 Conclusion: Both compounds played their roles through the intrinsic apoptotic pathway to induce cell apoptosis.

Interactome of Site-Specifically Acetylated Linker Histone H1.

Linker histone H1 plays a key role in chromatin organization and maintenance, yet our knowledge of the regulation of H1 functions by post-translational modifications is rather limited. In this study, we report on the generation of site-specifically mono- and di-acetylated linker histone H1.2 by genetic code expansion. We used these modified histones to identify and characterize the acetylation-dependent cellular interactome of H1.2 by affinity purification mass spectrometry and show that site-specific acetylation results in overlapping but distinct groups of interacting partners. Among these, we find multiple translational initiation factors and transcriptional regulators such as the NAD+-dependent deacetylase SIRT1, which we demonstrate to act on acetylated H1.2. Taken together, our data suggest that site-specific acetylation of H1.2 plays a role in modulating protein-protein interactions.

Phosphorylation of the HP1β hinge region sequesters KAP1 in heterochromatin and promotes the exit from naïve pluripotency

Heterochromatin binding protein HP1β plays an important role in chromatin organization and cell differentiation, however the underlying mechanisms remain unclear. Here, we generated HP1β−/− embryonic stem cells and observed reduced heterochromatin clustering and impaired differentiation. We found that during stem cell differentiation, HP1β is phosphorylated at serine 89 by CK2, which creates a binding site for the pluripotency regulator KAP1. This phosphorylation dependent sequestration of KAP1 in heterochromatin compartments causes a downregulation of pluripotency factors and triggers pluripotency exit. Accordingly, HP1β−/− and phospho-mutant cells exhibited impaired differentiation, while ubiquitination-deficient KAP1−/− cells had the opposite phenotype with enhanced differentiation. These results suggest that KAP1 regulates pluripotency via its ubiquitination activity. We propose that the formation of subnuclear membraneless heterochromatin compartments may serve as a dynamic reservoir to trap or release cellular factors. The sequestration of essential regulators defines a novel and active role of heterochromatin in gene regulation and represents a dynamic mode of remote control to regulate cellular processes like cell fate decisions.

Hierarchical dinucleotide distribution in genome along evolution and its effect on chromatin packing.

Dinucleotide densities and their distribution patterns vary significantly among species. Previous studies revealed that CpG is susceptible to methylation, enriched at topologically associating domain boundaries and its distribution along the genome correlates with chromatin compartmentalization. However, the multi-scale organizations of CpG in the linear genome, their role in chromatin organization, and how they change along the evolution are only partially understood. By comparing the CpG distribution at different genomic length scales, we quantify the difference between the CpG distributions of different species and evaluate how the hierarchical uneven CpG distribution appears in evolution. The clustering of species based on the CpG distribution is consistent with the phylogenetic tree. Interestingly, we found the CpG distribution and chromatin structure to be correlated in many different length scales, especially for mammals and avians, consistent with the mosaic CpG distribution in the genomes of these species.

Mus musculus Barrier-To-Autointegration Factor 2 (Banf2) is Not Essential for Spermatogenesis or Fertility

The barrier-to-autointegration factor (BAF) is widely expressed in most human tissues and plays a critical role in chromatin organization, nuclear envelope assembly, gonadal development, and embryonic stem cell self-renewal. Complete loss of BAF has been shown to lead to embryonic lethality and gonadal defects. The BAF paralog, namely, barrier-to-autointegration factor 2 (BANF2), exhibits a testis-predominant expression pattern in both humans and mice. Unlike BAF, it may cause isolated male infertility. Therefore, we used the CRISPR/Cas9 system to generate Banf2-knockout mice to further study its function in spermatogenesis. Unexpectedly, knockout mice did not show any detectable abnormalities in histological structure of the testis, epididymis, ovary, and other tissues, and exhibited normal fertility, indicating that Banf2 is not essential for mouse spermatogenesis and fertility.

The Diverse Cellular Functions of Inner Nuclear Membrane Proteins.

The nuclear compartment is delimited by a specialized expanded sheet of the endoplasmic reticulum (ER) known as the nuclear envelope (NE). Compared to the outer nuclear membrane and the contiguous peripheral ER, the inner nuclear membrane (INM) houses a unique set of transmembrane proteins that serve a staggering range of functions. Many of these functions reflect the exceptional position of INM proteins at the membrane-chromatin interface. Recent research revealed that numerous INM proteins perform crucial roles in chromatin organization, regulation of gene expression, genome stability, and mediation of signaling pathways into the nucleus. Other INM proteins establish mechanical links between chromatin and the cytoskeleton, help NE remodeling, or contribute to the surveillance of NE integrity and homeostasis. As INM proteins continue to gain prominence, we review these advancements and give an overview on the functional versatility of the INM proteome.