Chromatin Regulatory Mechanisms Research Articles

Overview of chromatin regulatory processes during surface ectodermal development and homeostasis

The ectoderm is the outermost of the three germ layers of the early embryo that arise during gastrulation. Once the germ layers are established, the complex interplay of cellular proliferation, differentiation, and migration results in organogenesis. The ectoderm is the progenitor of both the surface ectoderm and the neural ectoderm. Notably, the surface ectoderm develops into the epidermis and its associated appendages, nails, external exocrine glands, olfactory epithelium, and the anterior pituitary. Specification, development, and homeostasis of these organs demand a tightly orchestrated gene expression program that is often dictated by epigenetic regulation. In this review, we discuss the recent discoveries that have highlighted the importance of chromatin regulatory mechanisms mediated by transcription factors, histone and DNA modifications that aid in the development of surface ectodermal organs and maintain their homeostasis post-development.

Denaturing purifications demonstrate that PRC2 and other widely reported chromatin proteins do not appear to bind directly to RNA in vivo

Polycomb repressive complex 2 (PRC2) is reported to bind to many RNAs and has become a central player in reports of how long non-coding RNAs (lncRNAs) regulate gene expression. Yet, there is a growing discrepancy between the biochemical evidence supporting specific lncRNA-PRC2 interactions and functional evidence demonstrating that PRC2 is often dispensable for lncRNA function. Here, we revisit the evidence supporting RNA binding by PRC2 and show that many reported interactions may not occur invivo. Using denaturing purification of invivo crosslinked RNA-protein complexes in human and mouse cell lines, we observe a loss of detectable RNA binding to PRC2 and chromatin-associated proteins previously reported to bind RNA (CTCF, YY1, and others), despite accurately mapping bona fide RNA-binding sites across others (SPEN, TET2, and others). Taken together, these results argue for a critical re-evaluation of the broad role of RNA binding to orchestrate various chromatin regulatory mechanisms.

Coevolution of the CDCA7-HELLS ICF-related nucleosome remodeling complex and DNA methyltransferases

5-Methylcytosine (5mC) and DNA methyltransferases (DNMTs) are broadly conserved in eukaryotes but are also frequently lost during evolution. The mammalian SNF2 family ATPase HELLS and its plant ortholog DDM1 are critical for maintaining 5mC. Mutations in HELLS, its activator CDCA7, and the de novo DNA methyltransferase DNMT3B, cause immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome, a genetic disorder associated with the loss of DNA methylation. We here examine the coevolution of CDCA7, HELLS and DNMTs. While DNMT3, the maintenance DNA methyltransferase DNMT1, HELLS, and CDCA7 are all highly conserved in vertebrates and green plants, they are frequently co-lost in other evolutionary clades. The presence-absence patterns of these genes are not random; almost all CDCA7 harboring eukaryote species also have HELLS and DNMT1 (or another maintenance methyltransferase, DNMT5). Coevolution of presence-absence patterns (CoPAP) analysis in Ecdysozoa further indicates coevolutionary linkages among CDCA7, HELLS, DNMT1 and its activator UHRF1. We hypothesize that CDCA7 becomes dispensable in species that lost HELLS or DNA methylation, and/or the loss of CDCA7 triggers the replacement of DNA methylation by other chromatin regulation mechanisms. Our study suggests that a unique specialized role of CDCA7 in HELLS-dependent DNA methylation maintenance is broadly inherited from the last eukaryotic common ancestor.

Coevolution of the CDCA7-HELLS ICF-related nucleosome remodeling complex and DNA methyltransferases.

5-Methylcytosine (5mC) and DNA methyltransferases (DNMTs) are broadly conserved in eukaryotes but are also frequently lost during evolution. The mammalian SNF2 family ATPase HELLS and its plant ortholog DDM1 are critical for maintaining 5mC. Mutations in HELLS, its activator CDCA7, and the de novo DNA methyltransferase DNMT3B, cause immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome, a genetic disorder associated with the loss of DNA methylation. We here examine the coevolution of CDCA7, HELLS and DNMTs. While DNMT3, the maintenance DNA methyltransferase DNMT1, HELLS, and CDCA7 are all highly conserved in vertebrates and green plants, they are frequently co-lost in other evolutionary clades. The presence-absence patterns of these genes are not random; almost all CDCA7 harboring eukaryote species also have HELLS and DNMT1 (or another maintenance methyltransferase, DNMT5). Coevolution of presence-absence patterns (CoPAP) analysis in Ecdysozoa further indicates coevolutionary linkages among CDCA7, HELLS, DNMT1 and its activator UHRF1. We hypothesize that CDCA7 becomes dispensable in species that lost HELLS or DNA methylation, and/or the loss of CDCA7 triggers the replacement of DNA methylation by other chromatin regulation mechanisms. Our study suggests that a unique specialized role of CDCA7 in HELLS-dependent DNA methylation maintenance is broadly inherited from the last eukaryotic common ancestor.

Whole genome deconvolution unveils Alzheimer’s resilient epigenetic signature

Assay for Transposase Accessible Chromatin by sequencing (ATAC-seq) accurately depicts the chromatin regulatory state and altered mechanisms guiding gene expression in disease. However, bulk sequencing entangles information from different cell types and obscures cellular heterogeneity. To address this, we developed Cellformer, a deep learning method that deconvolutes bulk ATAC-seq into cell type-specific expression across the whole genome. Cellformer enables cost-effective cell type-specific open chromatin profiling in large cohorts. Applied to 191 bulk samples from 3 brain regions, Cellformer identifies cell type-specific gene regulatory mechanisms involved in resilience to Alzheimer’s disease, an uncommon group of cognitively healthy individuals that harbor a high pathological load of Alzheimer’s disease. Cell type-resolved chromatin profiling unveils cell type-specific pathways and nominates potential epigenetic mediators underlying resilience that may illuminate therapeutic opportunities to limit the cognitive impact of the disease. Cellformer is freely available to facilitate future investigations using high-throughput bulk ATAC-seq data.

Context-defined cancer co-dependency mapping identifies a functional interplay between PRC2 and MLL-MEN1 complex in lymphoma

Interplay between chromatin-associated complexes and modifications critically contribute to the partitioning of epigenome into stable and functionally distinct domains. Yet there is a lack of systematic identification of chromatin crosstalk mechanisms, limiting our understanding of the dynamic transition between chromatin states during development and disease. Here we perform co-dependency mapping of genes using CRISPR-Cas9-mediated fitness screens in pan-cancer cell lines to quantify gene-gene functional relationships. We identify 145 co-dependency modules and further define the molecular context underlying the essentiality of these modules by incorporating mutational, epigenome, gene expression and drug sensitivity profiles of cell lines. These analyses assign new protein complex composition and function, and predict new functional interactions, including an unexpected co-dependency between two transcriptionally counteracting chromatin complexes - polycomb repressive complex 2 (PRC2) and MLL-MEN1 complex. We show that PRC2-mediated H3K27 tri-methylation regulates the genome-wide distribution of MLL1 and MEN1. In lymphoma cells with EZH2 gain-of-function mutations, the re-localization of MLL-MEN1 complex drives oncogenic gene expression and results in a hypersensitivity to pharmacologic inhibition of MEN1. Together, our findings provide a resource for discovery of trans-regulatory interactions as mechanisms of chromatin regulation and potential targets of synthetic lethality.

Epigenetic moonlighting: Catalytic-independent functions of histone modifiers in regulating transcription

The past three decades have yielded a wealth of information regarding the chromatin regulatory mechanisms that control transcription. The "histone code" hypothesis-which posits that distinct combinations of posttranslational histone modifications are "read" by downstream effector proteins to regulate gene expression-has guided chromatin research to uncover fundamental mechanisms relevant to many aspects of biology. However, recent molecular and genetic studies revealed that the function of many histone-modifying enzymes extends independently and beyond their catalytic activities. In this review, we highlight original and recent advances in the understanding of noncatalytic functions of histone modifiers. Many of the histone modifications deposited by these enzymes-previously considered to be required for transcriptional activation-have been demonstrated to be dispensable for gene expression in living organisms. This perspective aims to prompt further examination of these enigmatic chromatin modifications by inspiring studies to define the noncatalytic "epigenetic moonlighting" functions of chromatin-modifying enzymes.

Hippocampus-based behavioral, structural, and molecular dynamics across the estrous cycle.

The activity of neurons in the rodent hippocampus contributes to diverse behaviors, with the activity of ventral hippocampal neurons affecting behaviors related to anxiety and emotion regulation, and the activity of dorsal hippocampal neurons affecting performance in learning- and memory-related tasks. Hippocampal cells also express receptors for ovarian hormones, estrogen and progesterone, and are therefore affected by physiological fluctuations of those hormones that occur over the rodent estrous cycle. In this review, we discuss the effects of cycling ovarian hormones on hippocampal physiology. Starting with behavior, we explore the role of the estrous cycle in regulating hippocampus-dependent behaviors. We go on to detail the cellular mechanisms through which cycling estrogen and progesterone, through changes in the structural and functional properties of hippocampal neurons, may be eliciting these changes in behavior. Then, providing a basis for these cellular changes, we outline the epigenetic, chromatin regulatory mechanisms through which ovarian hormones, by binding to their receptors, can affect the regulation of behavior- and synaptic plasticity-related genes in hippocampal neurons. We also highlight an unconventional role that chromatin dynamics may have in regulating neuronal function across the estrous cycle, including in sex hormone-driven X chromosome plasticity and hormonally-induced epigenetic priming. Finally, we discuss directions for future studies and the translational value of the rodent estrous cycle for understanding the effects of the human menstrual cycle on hippocampal physiology and brain disease risk.

Abstract A015: Cancer co-dependency mapping identifies a functional interplay between PRC2 and MLL-MEN1 complex in lymphoma

Abstract Interplays between chromatin-associated complexes and modifications critically contribute to the partitioning of epigenome into stable and functionally distinct domains. Yet there is a lack of systematic identification of chromatin crosstalk mechanisms, limiting our understanding of the dynamic transition between chromatin states during development and disease. Here we perform co-dependency mapping of genes encoding nuclear proteins using CRISPR-Cas9-mediated fitness screens in pan-cancer cell lines to quantify gene-gene functional relationships. We identify 84 co-dependency modules and further define the molecular context underlying the essentiality of these modules by incorporating mutational, epigenome, gene expression and drug sensitivity profiles of cell lines. These analyses assign new protein complex functions and biochemical interactions, and predict new chromatin crosstalk, including an unexpected co-dependency between two transcriptionally counteracting chromatin complexes - polycomb repressive complex 2 (PRC2) and MLL-MEN1 complex. We show that PRC2-mediated H3K27 tri-methylation regulates the genome-wide distribution of MLL1 and MEN1. In lymphoma cells with EZH2 gain-of-function mutations, the re-localization of MLL-MEN1 complex drives oncogenic gene expression and results in a hypersensitivity to pharmacologic inhibition of MEN1. Together, our findings provide a resource for discovery of trans-regulatory interactions as mechanisms of chromatin regulation and potential targets of synthetic lethality. Citation Format: Xiao Chen, Yinglu Li, Chao Lu. Cancer co-dependency mapping identifies a functional interplay between PRC2 and MLL-MEN1 complex in lymphoma. [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 A015.

Abstract PR004: High-density CRISPR screens reveal mechanisms of chromatin regulation of stemness networks in acute myeloid leukemia

Abstract Several leukemia-associated oncoproteins activate transcriptional circuits resembling a stem-like state in acute myeloid leukemia (AML). This activation of “stemness” genes is achieved by enlisting the activity of specialized components of the epigenetic machinery. We sought to comprehensively map epigenetic regulators critical for perpetuating these stemness networks in AML, as they may represent new therapeutic targets. To this end, we used a GFP-reporter knocked into the endogenous locus of the key leukemia oncogene and self-renewal-associated gene MEIS1 and conducted a pooled domain-focused CRISPR screen targeting >600 epigenetic modifiers. Through this screen, we identified and validated multiple members of eight distinct chromatin-modifying complexes that were required for sustaining MEIS1 expression in diverse AML subtypes. These included several novel MEIS1 regulators such as TAF6, LDB1, KAT2A, AFF2, JADE3, casein kinase 2 (CK2), ENY2 and SGF29, in addition to previously characterized regulators such as DOT1L, AF10, ENL and HBO1. A secondary pooled CRISPR screen, coupled with a single-cell transcriptome readout (CROP-seq) revealed that the deletion of several of these MEIS1 activators not only reversed MEIS1 activation but also reduced expression of stem-cell associated genes, including genes of the HOXA cluster, BMI1, SATB1 and Musashi 2, and concomitantly activated expression of differentiation-associated genes. Of particular interest to us was the TUDOR domain chromatin reader SGF29, a key component of the SAGA (Spt-Ada-Gcn5 acetyltransferase) complex. CRISPR-ko of SGF29 significantly reduced the proliferation of cells with distinct HOX-activating mutations including KMT2A rearranged and AF10-rearranged AML cells. ChIP-seq studies showed that SGF29 occupied the promoters and enhancers of key leukemia-oncogenes in a TUDOR-domain-dependent manner and SGF29 deletion selectively attenuated their transcription. Importantly, chromatin proteomics showed that SGF29 deletion led to the eviction of the SAGA complex subunit KAT2A from chromatin and into the cytoplasm. Further, SGF29 knockout impaired blast colony formation and induced differentiation in the KMT2A-MLLLT3, KMT2A-AF10, and CALM-AF10 mouse AML models but did not affect normal hematopoietic colony formation. Lastly, SGF29 deletion delayed disease latency in two distinct human AML cell line models as well as a patient-derived xenograft model in vivo and resulted in striking antiproliferative effects. Our study revealed several new attractive nodes for therapeutic targeting of leukemia stem cells in AML, including the chromatin reader SGF29, and provides a framework to identify vulnerabilities against recalcitrant oncogenic networks. Citation Format: Karina O. Barbosa Guerra, Anagha Deshpande, Ping Xiang, Anna Minkina, Fiorella Schischlik, Adam Brown, Neil A. Robertson, John Doench, Peter D. Adams, Keith Humphries, Eytan Ruppin, Jay Shendure, Prashant Mali, Aniruddha Deshpande. High-density CRISPR screens reveal mechanisms of chromatin regulation of stemness networks in acute myeloid leukemia. [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 PR004.

Abstract IA003: Catalytic and noncatalytic mechanisms of chromatin modifiers

Abstract Many mechanisms regulate chromatin structure to ensure that genes are active or silent. Some are considered epigenetic, ensuring the heritable transmission of a specific chromatin state. One of the mechanisms of chromatin regulation is based on posttranslational modifications of histones. Chemical groups are deposited, read, and removed by protein complexes that regulate chromatin structure. Histone modifications correlate with and sometimes directly affect critical processes such as transcription, replication, recombination, and repair. They are the subject of many clinical studies, as aberrant histone modifications and deregulation of enzymes responsible for their deposition occur in many cancers. These modifications rarely function in isolation, and there is often crosstalk between them that results in a coordinated transcriptional output. The central question in the field is to understand the regulation of histone modifications and associated molecular complexes, their role in gene expression, and their pathology in cancer. Another critical question is to discover and characterize the intricate relationships between different histone modifications and histone modifying enzymes at the mechanistic level. I will discuss our progress in understanding the roles of histone methylation and how it interfaces with other chromatin modifications that play critical roles in gene regulation. Citation Format: Karim-Jean Armache. Catalytic and noncatalytic mechanisms of chromatin modifiers. [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 IA003.

Light, chromatin, action: nuclear events regulating light signaling in Arabidopsis.

The plant nucleus provides a major hub for environmental signal integration at the chromatin level. Multiple light signaling pathways operate and exchange information by regulating a large repertoire of gene targets that shape plant responses to a changing environment. In addition to the established role of transcription factors in triggering photoregulated changes in gene expression, there are eminent reports on the significance of chromatin regulators and nuclear scaffold dynamics in promoting light-induced plant responses. Here, we report and discuss recent advances in chromatin-regulatory mechanisms modulating plant architecture and development in response to light, including the molecular and physiological roles of key modifications such as DNA, RNA and histone methylation, and/or acetylation. The significance of the formation of biomolecular condensates of key light signaling components is discussed and potential applications to agricultural practices overviewed.

Chromatin basis of the senescence-associated secretory phenotype.

Cellular senescence is a stable cell growth arrest. Senescent cells are metabolically active, as exemplified by the secretion of inflammatory cytokines, chemokines, and growth factors, which is termed senescence-associated secretory phenotype (SASP). The SASP exerts a range of functions in both normal health and pathology, which is possibly best characterized in cancers and physical aging. Recent studies demonstrated that chromatin is instrumental in regulating the SASP both through nuclear transcription and via the innate immune cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway in the cytoplasm. Here, we will review these regulatory mechanisms, with an emphasis on most recent developments in the field. We will highlight the challenges and opportunities in developing intervention approaches, such as targeting chromatin regulatory mechanisms, to alter the SASP as an emerging approach to combat cancers and achieve healthy aging.

Recent Advances in Investigating Functional Dynamics of Chromatin.

Dynamics spanning the picosecond-minute time domain and the atomic-subcellular spatial window have been observed for chromatin in vitro and in vivo. The condensed organization of chromatin in eukaryotic cells prevents regulatory factors from accessing genomic DNA, which requires dynamic stabilization and destabilization of structure to initiate downstream DNA activities. Those processes are achieved through altering conformational and dynamic properties of nucleosomes and nucleosome–protein complexes, of which delineating the atomistic pictures is essential to understand the mechanisms of chromatin regulation. In this review, we summarize recent progress in determining chromatin dynamics and their modulations by a number of factors including post-translational modifications (PTMs), incorporation of histone variants, and binding of effector proteins. We focus on experimental observations obtained using high-resolution techniques, primarily including nuclear magnetic resonance (NMR) spectroscopy, Förster (or fluorescence) resonance energy transfer (FRET) microscopy, and molecular dynamics (MD) simulations, and discuss the elucidated dynamics in the context of functional response and relevance.

RNA matchmaking in chromatin regulation.

Beyond being the product of gene expression, RNA can also influence the regulation of chromatin. The majority of the human genome has the capacity to be transcribed and the majority of the non-protein-coding transcripts made by RNA Polymerase II are enriched in the nucleus. Many chromatin regulators can bind to these ncRNAs in the nucleus; in some cases, there are clear examples of direct RNA-mediated chromatin regulation mechanisms stemming from these interactions, while others have yet to be determined. Recent studies have highlighted examples of chromatin regulation via RNA matchmaking, a term we use broadly here to describe intermolecular base-pairing interactions between one RNA molecule and an RNA or DNA match. This review provides examples of RNA matchmaking that regulates chromatin processes and summarizes the technical approaches used to capture these events.

Determining the Binding Constant of LANA Protein Fragment with Nucleosome

On the surface of the nucleosome, there are many regions for interaction with chromatin proteins, but an acidic patch, a negatively charged region formed by the residues of the histone H2A/H2B dimer, is especially prominent. The acidic patch is a target for anticancer drugs and an interaction locus for various pathogens. For instance, it was shown that this region is the binding site of the LANA protein of human gammaherpesvirus 8 and tethers the virus episomes to mitotic chromosomes. The development of methods for analyzing the binding of various compounds to nucleosomes is necessary both for understanding the mechanisms of chromatin regulation and for therapeutic agents' design. In this work, we propose a technique and measure the binding constant of the LANA protein fragment to the nucleosome. In contrast to previous studies, the measurements were carried out at a physiological salt concentration in the buffer solution. The proposed technique is based on the signal detection from the fluorescently labeled peptides after the separation of complexes of nucleosomes with peptides by gel electrophoresis. The paper also discusses mathematical models for analyzing the interaction between peptides and nucleosome and possible factors that can affect it.

The conserved histone chaperone LIN-53 is required for normal lifespan and maintenance of muscle integrity in Caenorhabditis elegans.

Whether extension of lifespan provides an extended time without health deteriorations is an important issue for human aging. However, to which degree lifespan and aspects of healthspan regulation might be linked is not well understood. Chromatin factors could be involved in linking both aging aspects, as epigenetic mechanisms bridge regulation of different biological processes. The epigenetic factor LIN‐53 (RBBP4/7) associates with different chromatin‐regulating complexes to safeguard cell identities in Caenorhabditis elegans as well as mammals, and has a role in preventing memory loss and premature aging in humans. We show that LIN‐53 interacts with the nucleosome remodeling and deacetylase (NuRD) complex in C. elegans muscles to ensure functional muscles during postembryonic development and in adults. While mutants for other NuRD members show a normal lifespan, animals lacking LIN‐53 die early because LIN‐53 depletion affects also the histone deacetylase complex Sin3, which is required for a normal lifespan. To determine why lin‐53 and sin‐3 mutants die early, we performed transcriptome and metabolomic analysis revealing that levels of the disaccharide trehalose are significantly decreased in both mutants. As trehalose is required for normal lifespan in C. elegans, lin‐53 and sin‐3 mutants could be rescued by either feeding with trehalose or increasing trehalose levels via the insulin/IGF1 signaling pathway. Overall, our findings suggest that LIN‐53 is required for maintaining lifespan and muscle integrity through discrete chromatin regulatory mechanisms. Since both LIN‐53 and its mammalian homologs safeguard cell identities, it is conceivable that its implication in lifespan regulation is also evolutionarily conserved.

The Ulp2 SUMO protease promotes transcription elongation through regulation of histone sumoylation.

Many eukaryotic proteins are regulated by modification with the ubiquitin-like protein small ubiquitin-like modifier (SUMO). This linkage is reversed by SUMO proteases, of which there are two in Saccharomyces cerevisiae, Ulp1 and Ulp2. SUMO-protein conjugation regulates transcription, but the roles of SUMO proteases in transcription remain unclear. We report that Ulp2 is recruited to transcriptionally active genes to control local polysumoylation. Mutant ulp2 cells show impaired association of RNA polymerase II (RNAPII) with, and diminished expression of, constitutively active genes and the inducible CUP1 gene. Ulp2 loss sensitizes cells to 6-azauracil, a hallmark of transcriptional elongation defects. We also describe a novel chromatin regulatory mechanism whereby histone-H2B ubiquitylation stimulates histone sumoylation, which in turn appears to inhibit nucleosome association of the Ctk1 kinase. Ctk1 phosphorylates serine-2 (S2) in the RNAPII C-terminal domain (CTD) and promotes transcript elongation. Removal of both ubiquitin and SUMO from histones is needed to overcome the impediment to S2 phosphorylation. These results suggest sequential ubiquitin-histone and SUMO-histone modifications recruit Ulp2, which removes polySUMO chains and promotes RNAPII transcription elongation.

The Genetics and Epigenetics of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD), a progressive myopathy that afflicts individuals of all ages, provides a powerful model of the complex interplay between genetic and epigenetic mechanisms of chromatin regulation. FSHD is caused by dysregulation of a macrosatellite repeat, either by contraction of the repeat or by mutations in silencing proteins. Both cases lead to chromatin relaxation and, in the context of a permissive allele, aberrant expression of the DUX4 gene in skeletal muscle. DUX4 is a pioneer transcription factor that activates a program of gene expression during early human development, after which its expression is silenced in most somatic cells. When misexpressed in FSHD skeletal muscle, the DUX4 program leads to accumulated muscle pathology. Epigenetic regulators of the disease locus represent particularly attractive therapeutic targets for FSHD, as many are not global modifiers of the genome, and altering their expression or activity should allow correction of the underlying defect.

Chromatin regulatory mechanisms and therapeutic opportunities in cancer.

Research over the past several decades has unmasked a major contribution of disrupted chromatin regulatory processes to human disease, particularly cancer. Advances in genome-wide technologies have highlighted frequent mutations in genes encoding chromatin-associated proteins, identified unexpected synthetic lethal opportunities and enabled increasingly comprehensive structural and functional dissection. Here, we review recent progress in our understanding of oncogenic mechanisms at each level of chromatin organization and regulation, and discuss new strategies towards therapeutic intervention.