ABSTRACT
Chromatin accessibility is a hallmark of active transcription and entails ATP-dependent nucleosome remodeling, which is carried out by complexes such as Brahma-associated factor (BAF). However, the mechanistic links between transcription, nucleosome remodeling and chromatin accessibility are unclear. Here, we used a chemical-genetic approach coupled with time-resolved chromatin profiling to dissect the interplay between RNA Polymerase II (RNAPII), BAF and DNA-sequence-specific transcription factors in mouse embryonic stem cells. We show that BAF dynamically unwraps and evicts nucleosomes at accessible chromatin regions, while RNAPII promoter-proximal pausing stabilizes BAF chromatin occupancy and enhances ATP-dependent nucleosome eviction by BAF. We find that although RNAPII and BAF dynamically probe both transcriptionally active and Polycomb-repressed genomic regions, pluripotency transcription factor chromatin binding confers locus specificity for productive chromatin remodeling and nucleosome eviction by BAF. Our study suggests a paradigm for how functional synergy between dynamically acting chromatin factors regulates locus-specific nucleosome organization and chromatin accessibility.
Subject(s)
Nucleosomes , Transcription Factors , Animals , Mice , Transcription Factors/genetics , Transcription Factors/metabolism , Nucleosomes/genetics , Chromatin/genetics , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Chromatin Assembly and Disassembly/genetics , Adenosine TriphosphateABSTRACT
In eukaryotic genomes, transcriptional machinery and nucleosomes compete for binding to DNA sequences; thus, a crucial aspect of gene regulatory element function is to modulate chromatin accessibility for transcription factor (TF) and RNA polymerase binding. Recent structural studies have revealed multiple modes of TF engagement with nucleosomes, but how initial "pioneering" results in steady-state DNA accessibility for further TF binding and RNA polymerase II (RNAPII) engagement has been unclear. Even less well understood is how distant sites of open chromatin interact with one another, such as when developmental enhancers activate promoters to release RNAPII for productive elongation. Here, we review evidence for the centrality of the conserved SWI/SNF family of nucleosome remodeling complexes, both in pioneering and in mediating enhancer-promoter contacts. Consideration of the nucleosome unwrapping and ATP hydrolysis activities of SWI/SNF complexes, together with their architectural features, may reconcile steady-state TF occupancy with rapid TF dynamics observed by live imaging.
Subject(s)
Nucleosomes , Transcription Factors , Nucleosomes/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Chromatin , DNA-Binding Proteins/genetics , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Epigenesis, Genetic , Chromatin Assembly and DisassemblyABSTRACT
The INO80 complex stood out in a large family of ATP-dependent chromatin remodelers because of its ATPase domain binding and translocating on DNA at the edge of nucleosomes, rather than at two helical turns from the center of DNA that is wrapped around nucleosomes. This unique property of INO80 was thought to account for its singular role in nucleosome placement at gene promoters in a DNA-sequence dependent manner that is crucial for transcription regulation. Now, we uncover INO80 functions differently than previously thought with its ATPase domain translocating on DNA close to the center of nucleosomes, like other remodelers. Our discovery also reveals the physical properties of the first ~36 bp of DNA on the entry side of nucleosomes is the main determinant for the DNA specificity of INO80 rather than the properties of the extranucleosomal DNA. The DNA sequence sensitive step of INO80 is after DNA is displaced from the histone octamer on the entry side of nucleosomes and 20 bp of DNA are moved out the exit side. We find the ATPase domain and Arp5 subunit of INO80 are likely involved in INO80's DNA specificity and the mechanism of INO80 remodeling is substantially different than originally proposed.
ABSTRACT
We uncover a tumor-suppressive process in urothelium called transcriptional-translational conflict caused by deregulation of the central chromatin remodeling component ARID1A. Loss of Arid1a triggers an increase in a nexus of pro-proliferation transcripts, but a simultaneous inhibition of the eukaryotic elongation factor 2 (eEF2), which results in tumor suppression. Resolution of this conflict through enhancing translation elongation speed enables the efficient and precise synthesis of a network of poised mRNAs resulting in uncontrolled proliferation, clonogenic growth, and bladder cancer progression. We observe a similar phenomenon in patients with ARID1A-low tumors, which also exhibit increased translation elongation activity through eEF2. These findings have important clinical implications because ARID1A-deficient, but not ARID1A-proficient, tumors are sensitive to pharmacologic inhibition of protein synthesis. These discoveries reveal an oncogenic stress created by transcriptional-translational conflict and provide a unified gene expression model that unveils the importance of the crosstalk between transcription and translation in promoting cancer.
Subject(s)
Chromatin , Urinary Bladder Neoplasms , Humans , Urinary Bladder Neoplasms/geneticsABSTRACT
Chromatin accessibility is a hallmark of active transcription and requires ATP-dependent nucleosome remodeling by Brahma-Associated Factor (BAF). However, the mechanistic link between transcription, nucleosome remodeling, and chromatin accessibility is unclear. Here, we used a chemical-genetic approach to dissect the interplay between RNA Polymerase II (RNAPII), BAF, and DNA-sequence-specific transcription factors (TFs) in mouse embryonic stem cells. By time-resolved chromatin profiling with acute transcription block at distinct stages, we show that RNAPII promoter-proximal pausing stabilizes BAF chromatin occupancy and enhances nucleosome eviction by BAF. We find that RNAPII and BAF probe both transcriptionally active and Polycomb-repressed genomic regions and provide evidence that TFs capture transient site exposure due to nucleosome unwrapping by BAF to confer locus specificity for persistent chromatin remodeling. Our study reveals the mechanistic basis of cell-type-specific chromatin accessibility. We propose a new paradigm for how functional synergy between dynamically acting chromatin factors regulates nucleosome organization.
ABSTRACT
Advanced prostate cancers comprise distinct phenotypes, but tumor classification remains clinically challenging. Here, we harnessed circulating tumor DNA (ctDNA) to study tumor phenotypes by ascertaining nucleosome positioning patterns associated with transcription regulation. We sequenced plasma ctDNA whole genomes from patient-derived xenografts representing a spectrum of androgen receptor active (ARPC) and neuroendocrine (NEPC) prostate cancers. Nucleosome patterns associated with transcriptional activity were reflected in ctDNA at regions of genes, promoters, histone modifications, transcription factor binding, and accessible chromatin. We identified the activity of key phenotype-defining transcriptional regulators from ctDNA, including AR, ASCL1, HOXB13, HNF4G, and GATA2. To distinguish NEPC and ARPC in patient plasma samples, we developed prediction models that achieved accuracies of 97% for dominant phenotypes and 87% for mixed clinical phenotypes. Although phenotype classification is typically assessed by IHC or transcriptome profiling from tumor biopsies, we demonstrate that ctDNA provides comparable results with diagnostic advantages for precision oncology. SIGNIFICANCE: This study provides insights into the dynamics of nucleosome positioning and gene regulation associated with cancer phenotypes that can be ascertained from ctDNA. New methods for classification in phenotype mixtures extend the utility of ctDNA beyond assessments of somatic DNA alterations with important implications for molecular classification and precision oncology. This article is highlighted in the In This Issue feature, p. 517.
Subject(s)
Circulating Tumor DNA , Prostatic Neoplasms , Male , Humans , Circulating Tumor DNA/genetics , Nucleosomes/genetics , Precision Medicine , Prostatic Neoplasms/pathology , Gene Expression Regulation, Neoplastic , PhenotypeABSTRACT
Mapping the epigenome is key to describe the relationship between chromatin landscapes and the control of DNA-based cellular processes such as transcription. Cleavage under targets and release using nuclease (CUT&RUN) is an in situ chromatin profiling strategy in which controlled cleavage by antibody-targeted Micrococcal Nuclease solubilizes specific protein-DNA complexes for paired-end DNA sequencing. When applied to budding yeast, CUT&RUN profiling yields precise genome-wide maps of histone modifications, histone variants, transcription factors, and ATP-dependent chromatin remodelers, while avoiding cross-linking and solubilization issues associated with the most commonly used chromatin profiling technique Chromatin Immunoprecipitation (ChIP). Furthermore, targeted chromatin complexes cleanly released by CUT&RUN can be used as input for a subsequent native immunoprecipitation step (CUT&RUN.ChIP) to simultaneously map two epitopes in single molecules genome-wide. The intrinsically low background and high resolution of CUT&RUN and CUT&RUN.ChIP allows for identification of transient genomic features such as dynamic nucleosome-remodeling intermediates. Starting from cells, one can perform CUT&RUN or CUT&RUN.ChIP and obtain purified DNA for sequencing library preparation in 2 days.
Subject(s)
Epigenome , Saccharomycetales , Chromatin , Chromatin Immunoprecipitation , DNA/genetics , Endonucleases/genetics , Micrococcal Nuclease , Nucleosomes/genetics , Saccharomycetales/geneticsABSTRACT
Over the last 3 decades ATP-dependent chromatin remodelers have been thought to recognize chromatin at the level of single nucleosomes rather than higher-order organization of more than one nucleosome. We show the yeast ISW1a remodeler has such higher-order structural specificity, as manifested by large allosteric changes that activate the nucleosome remodeling and spacing activities of ISW1a when bound to dinucleosomes. Although the ATPase domain of Isw1 docks at the SHL2 position when ISW1a is bound to either mono- or di-nucleosomes, there are major differences in the interactions of the catalytic subunit Isw1 with the acidic pocket of nucleosomes and the accessory subunit Ioc3 with nucleosomal DNA. By mutational analysis and uncoupling of ISW1a's dinucleosome specificity, we find that dinucleosome recognition is required by ISW1a for proper chromatin organization at promoters; as well as transcription regulation in combination with the histone acetyltransferase NuA4 and histone H2A.Z exchanger SWR1.
Subject(s)
Adenosine Triphosphatases/metabolism , Chromatin Assembly and Disassembly , DNA-Binding Proteins/metabolism , Nucleosomes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Transcription Factors/chemistry , Animals , Gene Expression Regulation , Histone Acetyltransferases/metabolism , Histones/metabolism , Saccharomyces cerevisiae/metabolism , Salmon , Transcription Factors/metabolism , XenopusABSTRACT
Gene regulation in eukaryotes requires the controlled access of sequence-specific transcription factors (TFs) to their sites in a chromatin landscape dominated by nucleosomes. Nucleosomes are refractory to TF binding, and often must be removed from regulatory regions. Recent genomic studies together with in vitro measurements suggest that the nucleosome barrier to TF binding is modulated by dynamic nucleosome unwrapping governed by ATP-dependent chromatin remodelers. Genome-wide occupancy and the regulation of subnucleosomal intermediates have gained recent attention with the application of high-resolution approaches for precision mapping of protein-DNA interactions. We summarize here recent findings on nucleosome substructures and TF binding dynamics, and highlight how unwrapped nucleosomal intermediates provide a novel signature of active chromatin.
Subject(s)
Epigenome/genetics , Nucleosomes/genetics , Nucleosomes/metabolism , Humans , Transcription Factors/metabolismABSTRACT
The classic view of nucleosome organization at active promoters is that two well-positioned nucleosomes flank a nucleosome-depleted region (NDR). However, this view has been recently disputed by contradictory reports as to whether wider (â³150 bp) NDRs instead contain unstable, micrococcal nuclease-sensitive ("fragile") nucleosomal particles. To determine the composition of fragile particles, we introduce CUT&RUN.ChIP, in which targeted nuclease cleavage and release is followed by chromatin immunoprecipitation. We find that fragile particles represent the occupancy of the RSC (remodeling the structure of chromatin) nucleosome remodeling complex and RSC-bound, partially unwrapped nucleosomal intermediates. We also find that general regulatory factors (GRFs) bind to partially unwrapped nucleosomes at these promoters. We propose that RSC binding and its action cause nucleosomes to unravel, facilitate subsequent binding of GRFs, and constitute a dynamic cycle of nucleosome deposition and clearance at the subset of wide Pol II promoter NDRs.
Subject(s)
Chromatin Assembly and Disassembly , Chromatin Immunoprecipitation/methods , DNA, Fungal/metabolism , DNA-Binding Proteins/metabolism , Micrococcal Nuclease/metabolism , Nucleosomes/enzymology , Promoter Regions, Genetic , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Transcription Factors/metabolism , Binding Sites , DNA, Fungal/genetics , DNA-Binding Proteins/genetics , Gene Expression Regulation, Fungal , Nucleic Acid Conformation , Nucleosomes/genetics , Protein Binding , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors/geneticsABSTRACT
Nuclear actin and actin-related proteins (Arps) are key components of chromatin remodeling and modifying complexes. Although Arps are essential for the functions of chromatin remodelers, their specific roles and mechanisms are unclear. Here we define the nucleosome binding interfaces and functions of the evolutionarily conserved Arps in the yeast INO80 chromatin remodeling complex. We show that the N-terminus of Arp8, C-terminus of Arp4 and the HSA domain of Ino80 bind extranucleosomal DNA 37-51 base pairs from the edge of nucleosomes and function as a DNA-length sensor that regulates nucleosome sliding by INO80. Disruption of Arp8 and Arp4 binding to DNA uncouples ATP hydrolysis from nucleosome mobilization by disengaging Arp5 from the acidic patch on histone H2A-H2B and the Ino80-ATPase domain from the Super-helical Location (SHL) -6 of nucleosomes. Our data suggest a functional interplay between INO80's Arp8-Arp4-actin and Arp5 modules in sensing the DNA length separating nucleosomes and regulating nucleosome positioning.
Subject(s)
Actins/metabolism , Chromatin Assembly and Disassembly , DNA, Fungal/metabolism , Microfilament Proteins/metabolism , Nuclear Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Actins/chemistry , Binding Sites , Microfilament Proteins/chemistry , Nuclear Proteins/chemistry , Nucleosomes/metabolism , Protein Binding , Protein Domains , Saccharomyces cerevisiae Proteins/chemistryABSTRACT
ATP-dependent chromatin remodellers modulate nucleosome dynamics by mobilizing or disassembling nucleosomes, as well as altering nucleosome composition. These chromatin remodellers generally function by translocating along nucleosomal DNA at the H3-H4 interface of nucleosomes. Here we show that, unlike other remodellers, INO80 translocates along DNA at the H2A-H2B interface of nucleosomes and persistently displaces DNA from the surface of H2A-H2B. DNA translocation and DNA torsional strain created near the entry site of nucleosomes by INO80 promotes both the mobilization of nucleosomes and the selective exchange of H2A.Z-H2B dimers out of nucleosomes and replacement by H2A-H2B dimers without any additional histone chaperones. We find that INO80 translocates and mobilizes H2A.Z-containing nucleosomes more efficiently than those containing H2A, partially accounting for the preference of INO80 to replace H2A.Z with H2A. Our data suggest that INO80 has a mechanism for dimer exchange that is distinct from other chromatin remodellers including its paralogue SWR1.
Subject(s)
Adenosine Triphosphatases/genetics , Chromatin Assembly and Disassembly/genetics , DNA, Fungal/genetics , Histones/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Adenosine Triphosphatases/metabolism , Chromatin/metabolism , Saccharomyces cerevisiae/metabolismABSTRACT
The SWI/SNF chromatin remodeling complex is highly conserved from yeast to human, and aberrant SWI/SNF complexes contribute to human disease. The Snf5/SMARCB1/INI1 subunit of SWI/SNF is a tumor suppressor frequently lost in pediatric rhabdoid cancers. We examined the effects of Snf5 loss on the composition, nucleosome binding, recruitment, and remodeling activities of yeast SWI/SNF. The Snf5 subunit is shown by crosslinking-mass spectrometry (CX-MS) and subunit deletion analysis to interact with the ATPase domain of Snf2 and to form a submodule consisting of Snf5, Swp82, and Taf14. Snf5 promotes binding of the Snf2 ATPase domain to nucleosomal DNA and enhances the catalytic and nucleosome remodeling activities of SWI/SNF. Snf5 is also required for SWI/SNF recruitment by acidic transcription factors. RNA-seq analysis suggests that both the recruitment and remodeling functions of Snf5 are required in vivo for SWI/SNF regulation of gene expression. Thus, loss of SNF5 alters the structure and function of SWI/SNF.
Subject(s)
Chromatin Assembly and Disassembly/physiology , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/metabolism , Transcription Factors/metabolism , Adenosine Triphosphatases/metabolism , Cell Nucleus/metabolism , Fungal Proteins/metabolism , Gene Expression/physiology , Nucleosomes/metabolism , Protein Subunits/metabolism , Yeasts/metabolismABSTRACT
Stalling of RNA Polymerase II (RNAPII) on chromatin during transcriptional stress results in polyubiquitination and degradation of the largest subunit of RNAPII, Rpb1, by the ubiquitin proteasome system (UPS). Here, we report that the ATP-dependent chromatin remodeling complex INO80 is required for turnover of chromatin-bound RNAPII in yeast. INO80 interacts physically and functionally with Cdc48/p97/VCP, a component of UPS required for degradation of RNAPII. Cells lacking INO80 are defective in Rpb1 degradation and accumulate tightly bound ubiquitinated Rpb1 on chromatin. INO80 forms a ternary complex with RNAPII and Cdc48 and targets Rpb1 primed for degradation. The function of INO80 in RNAPII turnover is required for cell growth and survival during genotoxic stress. Our results identify INO80 as a bona fide component of the proteolytic pathway for RNAPII degradation and suggest that INO80 nucleosome remodeling activity promotes the dissociation of ubiquitinated Rpb1 from chromatin to protect the integrity of the genome.