Chromatin Regulatory Proteins Research Articles

Genomic DNA Methylation Signatures Enable Concurrent Diagnosis and Clinical Genetic Variant Classification in Neurodevelopmental Syndromes

Pediatric developmental syndromes present with systemic, complex, and often overlapping clinical features that are not infrequently a consequence of Mendelian inheritance of mutations in genes involved in DNA methylation, establishment of histone modifications, and chromatin remodeling (the "epigenetic machinery"). The mechanistic cross-talk between histone modification and DNA methylation suggests that these syndromes might be expected to display specific DNA methylation signatures that are a reflection of those primary errors associated with chromatin dysregulation. Given the interrelated functions of these chromatin regulatory proteins, we sought to identify DNA methylation epi-signatures that could provide syndrome-specific biomarkers to complement standard clinical diagnostics. In the present study, we examined peripheral blood samples from a large cohort of individuals encompassing 14 Mendelian disorders displaying mutations in the genes encoding proteins of the epigenetic machinery. We demonstrated that specific but partially overlapping DNA methylation signatures are associated with many of these conditions. The degree of overlap among these epi-signatures is minimal, further suggesting that, consistent with the initial event, the downstream changes are unique to every syndrome. In addition, by combining these epi-signatures, we have demonstrated that a machine learning tool can be built to concurrently screen for multiple syndromes with high sensitivity and specificity, and we highlight the utility of this tool in solving ambiguous case subjects presenting with variants of unknown significance, along with its ability to generate accurate predictions for subjects presenting with the overlapping clinical and molecular features associated with the disruption of the epigenetic machinery.

Abstract 4994: Understanding the MYC and WDR5 interaction at chromatin

Abstract MYC is an oncoprotein that is overexpressed in the majority of malignancies and contributes to an estimated 70,000-100,000 cancer deaths in the United States every year. The broad pro-tumorigenic functions of MYC stem from its role as a sequence-specific transcriptional regulator, controlling the expression of thousands of genes linked to cell cycle control, growth, and metabolism. Key to understanding how MYC causes cancer, therefore, is understanding the mechanisms through which it selects its target genes. Histone modifications, DNA sequence, and interactions with other transcription factors have all been suggested to influence where MYC binds chromatin, but a consistent signature has not been defined. Our laboratory recently discovered that the chromatin regulatory protein WDR5—a core component of histone modifying complexes—interacts directly with MYC and co-localizes with MYC at a majority of its target genes in human cells (Thomas et al., Mol. Cell, 58: 440-452, 2015). Point mutations in MYC that disable interaction with WDR5 do not impact the ability of MYC to bind naked DNA, but they do prevent MYC from recognizing target genes in the context of chromatin and from driving tumorigenesis in mice. These studies led us to propose that the MYC-WDR5 interaction is a critical determinant in MYC target gene recognition, in a process we refer to as “facilitated recruitment.” In the facilitated recruitment model, the presence of WDR5 at chromatin promotes MYC binding at certain genomic loci over others. The role of WDR5 in target gene selection by MYC can explain much of the plasticity in genome-wide binding patterns of MYC that have been reported, and may provide a new avenue for therapeutically targeting MYC in cancer. Two important questions are raised by these studies, however. How does WDR5 recognize and select its target genes? And what other functions, if any, does WDR5 play in regulating MYC target genes? To answer these questions, we are employing traditional and quantitative proteomics, together with biochemical approaches, to characterize the composition and stoichiometry of the WDR5-containing complex that associates with MYC. These studies, still ongoing, have demonstrated that this complex is devoid of canonical WDR5-interaction partners such as RBBP5 and HCF-1, revealing that the function of WDR5 in this setting is distinct from its well-characterized roles in histone modifications. We propose that the WDR5 complex that associates with MYC on chromatin is either entirely novel, or is a ‘ghost’ complex in which select protein components have been excluded by the direct interaction of MYC with WDR5. We continue to define the biochemical constituents of the MYC-WDR5 complex, and will interrogate their role in facilitated recruitment using a combination of genetic, biochemical, and genomic, approaches. Citation Format: Alissa D. Guarnaccia, April M. Weissmiller, Lance R. Thomas, William P. Tansey. Understanding the MYC and WDR5 interaction at chromatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4994. doi:10.1158/1538-7445.AM2017-4994

Transcribing the connectome: roles for transcription factors and chromatin regulators in activity-dependent synapse development.

The wiring of synaptic connections in the developing mammalian brain is shaped by both intrinsic and extrinsic signals. One point where these regulatory pathways converge is via the sensory experience-dependent regulation of new gene transcription. Recent studies have elucidated a number of molecular mechanisms that allow nuclear transcription factors and chromatin regulatory proteins to encode aspects of specificity in experience-dependent synapse development. Here we review the evidence for the transcriptional mechanisms that sculpt activity-dependent aspects of synaptic connectivity during postnatal development and discuss how disruption of these processes is associated with aberrant brain development in autism and intellectual disability.

Demethylated HSATII DNA and HSATII RNA Foci Sequester PRC1 and MeCP2 into Cancer-Specific Nuclear Bodies

This study reveals that high-copy satellite II (HSATII) sequences in the human genome can bind and impact distribution of chromatin regulatory proteins and that this goes awry in cancer. In many cancers, master regulatory proteins form two types of cancer-specific nuclear bodies, caused by locus-specific deregulation of HSATII. DNA demethylation at the 1q12 mega-satellite, common in cancer, causes PRC1 aggregation into prominent Cancer-Associated Polycomb (CAP) bodies. These loci remain silent, whereas HSATII loci with reduced PRC1 become derepressed, reflecting imbalanced distribution of UbH2A on these and other PcG-regulated loci. Large nuclear foci of HSATII RNA form and sequester copious MeCP2 into Cancer-Associated Satellite Transcript (CAST) bodies. Hence, HSATII DNA and RNA have an exceptional capacity to act as molecular sponges and sequester chromatin regulatory proteins into abnormal nuclear bodies in cancer. The compartmentalization of regulatory proteins within nuclear structure, triggered by demethylation of "junk" repeats, raises the possibility that this contributes to further compromise of the epigenome and neoplastic progression.

How a lncRNA Shapes Chromatin Structure to Control Gene Expression

Mammalian genomes encode many thousands of long non-coding RNAs (lncRNAs) that play important roles in diverse biological processes. As a class, lncRNAs are generally enriched in the nucleus and specifically within the chromatin-associated fraction. Here, we will discuss the evidence that many nuclear-retained lncRNAs can interact with various chromatin regulatory proteins and recruit them to specific sites on DNA to regulate gene expression and shape three-dimensional nuclear organization. Specifically, we will discuss emerging mechanistic insights derived from the Xist lncRNA, a paradigm for lncRNA-mediated gene regulation, into how lncRNAs regulate gene expression by localizing to genomic target sites, recruiting regulatory complexes, and reshaping nuclear organization.

Driver gene classification reveals a substantial overrepresentation of tumor suppressors among very large chromatin-regulating proteins.

Compiling a comprehensive list of cancer driver genes is imperative for oncology diagnostics and drug development. While driver genes are typically discovered by analysis of tumor genomes, infrequently mutated driver genes often evade detection due to limited sample sizes. Here, we address sample size limitations by integrating tumor genomics data with a wide spectrum of gene-specific properties to search for rare drivers, functionally classify them, and detect features characteristic of driver genes. We show that our approach, CAnceR geNe similarity-based Annotator and Finder (CARNAF), enables detection of potentially novel drivers that eluded over a dozen pan-cancer/multi-tumor type studies. In particular, feature analysis reveals a highly concentrated pool of known and putative tumor suppressors among the <1% of genes that encode very large, chromatin-regulating proteins. Thus, our study highlights the need for deeper characterization of very large, epigenetic regulators in the context of cancer causality.

A Chromatin-Focused siRNA Screen for Regulators of p53-Dependent Transcription.

The protein product of the Homo sapiens TP53 gene is a transcription factor (p53) that regulates the expression of genes critical for the response to DNA damage and tumor suppression, including genes involved in cell cycle arrest, apoptosis, DNA repair, metabolism, and a number of other tumorigenesis-related pathways. Differential transcriptional regulation of these genes is believed to alter the balance between two p53-dependent cell fates: cell cycle arrest or apoptosis. A number of previously identified p53 cofactors covalently modify and alter the function of both the p53 protein and histone proteins. Both gain- and loss-of-function mutations in chromatin modifiers have been strongly implicated in cancer development; thus, we sought to identify novel chromatin regulatory proteins that affect p53-dependent transcription and the balance between the expression of pro-cell cycle arrest and proapoptotic genes. We utilized an siRNA library designed against predicted chromatin regulatory proteins, and identified known and novel chromatin-related factors that affect both global p53-dependent transcription and gene-specific regulators of p53 transcriptional activation. The results from this screen will serve as a comprehensive resource for those interested in further characterizing chromatin and epigenetic factors that regulate p53 transcription.

Small Molecular Inhibitors Targeting Chromatin Regulating Proteins for Cancer.

Chromatin-regulating proteins modulate nucleosome structure by either modifying histones covalently or disrupting DNA-protein interaction directly with ATP hydrolysis. Evidence has shown that chromatin-regulating proteins play critical roles in regulation of molecular processes using DNA as template, including gene expression, DNA replication, DNA damage repair, and chromosome integrity. In most of human malignancies, chromatin-regulating proteins have been shown as functional oncogenes. In some scenarios, chromatin-regulating proteins also could have tumor suppressive functions. Thereby, small molecular inhibitors targeting chromatin-regulating proteins could be used for cancer therapies. Numerous small molecular inhibitors against chromatin-regulating proteins are recently developed by academic and industrial groups. These compounds are evaluated for antitumor effects in vitro and in vivo. Some of them have shown great potential to become a therapeutic drug for cancer, and is currently evaluated in clinical trials. A few compounds have been approved for clinical use in cancer treatment. In this review, we will focus on the recent progress on the development of small inhibitors of chromatin-regulating proteins for cancer therapy.

An Overview of Chromatin-Regulating Proteins in Cells.

In eukaryotic cells, gene expressions on chromosome DNA are orchestrated by a dynamic chromosome structure state that is largely controlled by chromatin-regulating proteins, which regulate chromatin structures, release DNA from the nucleosome, and activate or suppress gene expression by modifying nucleosome histones or mobilizing DNA-histone structure. The two classes of chromatinregulating proteins are 1) enzymes that modify histones through methylation, acetylation, phosphorylation, adenosine diphosphate-ribosylation, glycosylation, sumoylation, or ubiquitylation and 2) enzymes that remodel DNA-histone structure with energy from ATP hydrolysis. Chromatin-regulating proteins, which modulate DNA-histone interaction, change chromatin conformation, and increase or decrease the binding of functional DNA-regulating protein complexes, have major functions in nuclear processes, including gene transcription and DNA replication, repair, and recombination. This review provides a general overview of chromatin-regulating proteins, including their classification, molecular functions, and interactions with the nucleosome in eukaryotic cells.

Bromodomain and extra-terminal (BET) family proteins: New therapeutic targets in major diseases.

The bromodomains and extra-terminal domain (BET) family proteins recognize acetylated chromatin through their bromodomains (BDs) and help in regulating gene expression. BDs are chromatin 'readers': by interacting with acetylated lysines on the histone tails, they recruit chromatin-regulating proteins on the promoter region to regulate gene expression and repression. Extensive efforts have been employed by scientific communities worldwide to identify and develop potential inhibitors of BET family BDs to regulate protein expression by inhibiting acetylated histone (H3/H4) interactions. Several small molecule inhibitors have been reported, which not only have high affinity but also have high specificity to BET BDs. These developments make BET family proteins an important therapeutic targets for major diseases such as cancer, neurological disorders, obesity and inflammation. Here, we review and discuss the structural biology of BET family BDs and their applications in major diseases.

Targeting epigenetic regulations in cancer.

Epigenetic regulation of gene expression is a dynamic and reversible process with DNA methylation, histone modifications, and chromatin remodeling. Recently, groundbreaking studies have demonstrated the importance of DNA and chromatin regulatory proteins from different aspects, including stem cell, development, and tumor genesis. Abnormal epigenetic regulation is frequently associated with diseases and drugs targeting DNA methylation and histone acetylation have been approved for cancer therapy. Although the network of epigenetic regulation is more complex than people expect, new potential druggable chromatin-associated proteins are being discovered and tested for clinical application. Here we review the key proteins that mediate epigenetic regulations through DNA methylation, the acetylation and methylation of histones, and the reader proteins that bind to modified histones. We also discuss cancer associations and recent progress of pharmacological development of these proteins.

Emerging roles of lysine methylation on non-histone proteins.

Lysine methylation is a common posttranslational modification (PTM) of histones that is important for the epigenetic regulation of transcription and chromatin in eukaryotes. Increasing evidence demonstrates that in addition to histones, lysine methylation also occurs on various non-histone proteins, especially transcription- and chromatin-regulating proteins. In this review, we will briefly describe the histone lysine methyltransferases (KMTs) that have a broad spectrum of non-histone substrates. We will use p53 and nuclear receptors, especially estrogen receptor alpha, as examples to discuss the dynamic nature of non-histone protein lysine methylation, the writers, erasers, and readers of these modifications, and the crosstalk between lysine methylation and other PTMs in regulating the functions of the modified proteins. Understanding the roles of lysine methylation in normal cells and during development will shed light on the complex biology of diseases associated with the dysregulation of lysine methylation on both histones and non-histone proteins.

The epigenetic landscape of clear-cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of all kidney tumors. During the last few years, epigenetics has emerged as an important mechanism in ccRCC pathogenesis. Recent reports, involving large-scale methylation and sequencing analyses, have identified genes frequently inactivated by promoter methylation and recurrent mutations in genes encoding chromatin regulatory proteins. Interestingly, three of detected genes (PBRM1, SETD2 and BAP1) are located on chromosome 3p, near the VHL gene, inactivated in over 80% ccRCC cases. This suggests that 3p alterations are an essential part of ccRCC pathogenesis. Moreover, most of the proteins encoded by these genes cooperate in histone H3 modifications. The aim of this review is to summarize the latest discoveries shedding light on deregulation of chromatin machinery in ccRCC. Newly described ccRCC-specific epigenetic alterations could potentially serve as novel diagnostic and prognostic biomarkers and become an object of novel therapeutic strategies.

PSMC5, a 19S Proteasomal ATPase, Regulates Cocaine Action in the Nucleus Accumbens.

ΔFosB is a stable transcription factor which accumulates in the nucleus accumbens (NAc), a key part of the brain’s reward circuitry, in response to chronic exposure to cocaine or other drugs of abuse. While ΔFosB is known to heterodimerize with a Jun family member to form an active transcription factor complex, there has not to date been an open-ended exploration of other possible binding partners for ΔFosB in the brain. Here, by use of yeast two-hybrid assays, we identify PSMC5—also known as SUG1, an ATPase-containing subunit of the 19S proteasomal complex—as a novel interacting protein with ΔFosB. We verify such interactions between endogenous ΔFosB and PSMC5 in the NAc and demonstrate that both proteins also form complexes with other chromatin regulatory proteins associated with gene activation. We go on to show that chronic cocaine increases nuclear, but not cytoplasmic, levels of PSMC5 in the NAc and that overexpression of PSMC5 in this brain region promotes the locomotor responses to cocaine. Together, these findings describe a novel mechanism that contributes to the actions of ΔFosB and, for the first time, implicates PSMC5 in cocaine-induced molecular and behavioral plasticity.

Individual epigenetic status of the pathogenic D4Z4 macrosatellite correlates with disease in facioscapulohumeral muscular dystrophy.

BackgroundBoth forms of facioscapulohumeral muscular dystrophy (FSHD) are associated with aberrant epigenetic regulation of the chromosome 4q35 D4Z4 macrosatellite. Chromatin changes due to large deletions of heterochromatin (FSHD1) or mutations in chromatin regulatory proteins (FSHD2) lead to relaxation of epigenetic repression and increased expression of the deleterious double homeobox 4 (DUX4) gene encoded within the distal D4Z4 repeat. However, many individuals with the genetic requirements for FSHD remain asymptomatic throughout their lives. Here we investigated family cohorts of FSHD1 individuals who were either affected (manifesting) or without any discernible weakness (nonmanifesting/asymptomatic) and their unaffected family members to determine if individual epigenetic status and stability of repression at the contracted 4q35 D4Z4 array in myocytes correlates with FSHD disease.ResultsFamily cohorts were analyzed for DNA methylation on the distal pathogenic 4q35 D4Z4 repeat on permissive A-type subtelomeres. We found DNA hypomethylation in FSHD1-affected subjects, hypermethylation in healthy controls, and distinctly intermediate levels of methylation in nonmanifesting subjects. We next tested if these differences in DNA methylation had functional relevance by assaying DUX4-fl expression and the stability of epigenetic repression of DUX4-fl in myogenic cells. Treatment with drugs that alter epigenetic status revealed that healthy cells were refractory to treatment, maintaining stable repression of DUX4, while FSHD1-affected cells were highly responsive to treatment and thus epigenetically poised to express DUX4. Myocytes from nonmanifesting subjects had significantly higher levels of DNA methylation and were more resistant to DUX4 activation in response to epigenetic drug treatment than cells from FSHD1-affected first-degree relatives containing the same contraction, indicating that the epigenetic status of the contracted D4Z4 array is reflective of disease.ConclusionsThe epigenetic status of the distal 4qA D4Z4 repeat correlates with FSHD disease; FSHD-affected subjects have hypomethylation, healthy unaffected subjects have hypermethylation, and nonmanifesting subjects have characteristically intermediate methylation. Thus, analysis of DNA methylation at the distal D4Z4 repeat could be used as a diagnostic indicator of developing clinical FSHD. In addition, the stability of epigenetic repression upstream of DUX4 expression is a key regulator of disease and a viable therapeutic target.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0072-6) contains supplementary material, which is available to authorized users.

Parallel action of AtDRB2 and RdDM in the control of transposable element expression

BackgroundIn plants and animals, a large number of double-stranded RNA binding proteins (DRBs) have been shown to act as non-catalytic cofactors of DICERs and to participate in the biogenesis of small RNAs involved in RNA silencing. We have previously shown that the loss of Arabidopsis thaliana’s DRB2 protein results in a significant increase in the population of RNA polymerase IV (p4) dependent siRNAs, which are involved in the RNA-directed DNA methylation (RdDM) process.ResultsSurprisingly, despite this observation, we show in this work that DRB2 is part of a high molecular weight complex that does not involve RdDM actors but several chromatin regulator proteins, such as MSI4, PRMT4B and HDA19. We show that DRB2 can bind transposable element (TE) transcripts in vivo but that drb2 mutants do not have a significant variation in TE DNA methylation.ConclusionWe propose that DRB2 is part of a repressive epigenetic regulator complex involved in a negative feedback loop, adjusting epigenetic state to transcription level at TE loci, in parallel of the RdDM pathway. Loss of DRB2 would mainly result in an increased production of TE transcripts, readily converted in p4-siRNAs by the RdDM machinery.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0455-z) contains supplementary material, which is available to authorized users.

Automation Highlights from the Literature

Automation Highlights from the Literature

Functions of the proteasome on chromatin.

The proteasome is a large self-compartmentalized protease complex that recognizes, unfolds, and destroys ubiquitylated substrates. Proteasome activities are required for a host of cellular functions, and it has become clear in recent years that one set of critical actions of the proteasome occur on chromatin. In this review, we discuss some of the ways in which proteasomes directly regulate the structure and function of chromatin and chromatin regulatory proteins, and how this influences gene transcription. We discuss lingering controversies in the field, the relative importance of proteolytic versus non-proteolytic proteasome activities in this process, and highlight areas that require further investigation. Our intention is to show that proteasomes are involved in major steps controlling the expression of the genetic information, that proteasomes use both proteolytic mechanisms and ATP-dependent protein remodeling to accomplish this task, and that much is yet to be learned about the full spectrum of ways that proteasomes influence the genome.

Long noncoding RNAs: an emerging link between gene regulation and nuclear organization.

Mammalian genomes encode thousands of long noncoding RNAs (lncRNAs) that play important roles in diverse biological processes. As a class, lncRNAs are generally enriched in the nucleus and, specifically, within the chromatin-associated fraction. Consistent with their localization, many lncRNAs have been implicated in the regulation of gene expression and in shaping 3D nuclear organization. In this review, we discuss the evidence that many nuclear-retained lncRNAs can interact with various chromatin regulatory proteins and recruit them to specific sites on DNA to regulate gene expression. Furthermore, we discuss the role of specific lncRNAs in shaping nuclear organization and their emerging mechanisms. Based on these examples, we propose a model that explains how lncRNAs may shape aspects of nuclear organization to regulate gene expression.

Multiplexed analysis of protein-ligand interactions by fluorescence anisotropy in a microfluidic platform.

Homogeneous assay platforms for measuring protein-ligand interactions are highly valued due to their potential for high-throughput screening. However, the implementation of these multiplexed assays in conventional microplate formats is considerably expensive due to the large amounts of reagents required and the need for automation. We implemented a homogeneous fluorescence anisotropy-based binding assay in an automated microfluidic chip to simultaneously interrogate >2300 pairwise interactions. We demonstrated the utility of this platform in determining the binding affinities between chromatin-regulatory proteins and different post-translationally modified histone peptides. The microfluidic chip assay produces comparable results to conventional microtiter plate assays, yet requires 2 orders of magnitude less sample and an order of magnitude fewer pipetting steps. This approach enables one to use small samples for medium-scale screening and could ease the bottleneck of large-scale protein purification.