Nucleosome Landscape Research Articles

Remodeling of Individual Nucleosomes in Nucleosome Arrays.

Adenosin triphosphate (ATP)-dependent nucleosome remodeling factors sculpt the nucleosomal landscape of eukaryotic chromatin. They deposit, evict, or reposition nucleosomes along DNA in a process termed nucleosome sliding. Remodeling has traditionally been analyzed using mononucleosomes as a model substrate. In vivo, however, nucleosomes form extended arrays with regular spacing. Here we describe how regularly spaced nucleosome arrays can be reconstituted in vitro and how these arrays can be used to dissect remodeling in the test tube. We outline two assays. Both assays exploit the changes in the accessibility of DNA to restriction enzymes during the remodeling reaction. The first assay uses the restriction enzyme to cleave the restriction site as soon as it becomes accessible during remodeling. As such, this assay mostly reports the kinetic parameter of the "forward" reaction of nucleosome remodeling. In contrast, the second assay measures how fast a particular nucleosome in the array reaches its steady-state position.

NucMACC: An MNase-seq pipeline to identify structurally altered nucleosomes in the genome.

Micrococcal nuclease sequencing is the state-of-the-art method for determining chromatin structure and nucleosome positioning. Data analysis is complex due to the AT-dependent sequence bias of the endonuclease and the requirement for high sequencing depth. Here, we present the nucleosome-based MNase accessibility (nucMACC) pipeline unveiling the regulatory chromatin landscape by measuring nucleosome accessibility and stability. The nucMACC pipeline represents a systematic and genome-wide approach for detecting unstable ("fragile") nucleosomes. We have characterized the regulatory nucleosome landscape in Drosophila melanogaster, Saccharomyces cerevisiae, and mammals. Two functionally distinct sets of promoters were characterized, one associated with an unstable nucleosome and the other being nucleosome depleted. We show that unstable nucleosomes present intermediate states of nucleosome remodeling, preparing inducible genes for transcriptional activation in response to stimuli or stress. The presence of unstable nucleosomes correlates with RNA polymerase II proximal pausing. The nucMACC pipeline offers unparalleled precision and depth in nucleosome research and is a valuable tool for future nucleosome studies.

Genome wide nucleosome landscape shapes 3D chromatin organization

Genome wide nucleosome landscape shapes 3D chromatin organization

INO80-Dependent Remodeling of Transcriptional Regulatory Network Underlies the Progression of Heart Failure.

Progressive remodeling of cardiac gene expression underlies decline in cardiac function, eventually leading to heart failure. However, the major determinants of transcriptional network switching from normal to failed hearts remain to be determined. In this study, we integrated human samples, genetic mouse models, and genomic approaches, including bulk RNA sequencing, single-cell RNA sequencing, chromatin immunoprecipitation followed by high-throughput sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing, to identify the role of chromatin remodeling complex INO80 in heart homeostasis and dysfunction. The INO80 chromatin remodeling complex was abundantly expressed in mature cardiomyocytes, and its expression further increased in mouse and human heart failure. Cardiomyocyte-specific overexpression of Ino80, its core catalytic subunit, induced heart failure within 4 days. Combining RNA sequencing, chromatin immunoprecipitation followed by high-throughput sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing, we revealed INO80 overexpression-dependent reshaping of the nucleosomal landscape that remodeled a core set of transcription factors, most notably the MEF2 (Myocyte Enhancer Factor 2) family, whose target genes were closely associated with cardiac function. Conditional cardiomyocyte-specific deletion of Ino80 in an established mouse model of heart failure demonstrated remarkable preservation of cardiac function. In summary, our findings shed light on the INO80-dependent remodeling of the chromatin landscape and transcriptional networks as a major mechanism underlying cardiac dysfunction in heart failure, and suggest INO80 as a potential preventative or interventional target.

A chromatinized origin reduces the mobility of ORC and MCM through interactions and spatial constraint

Chromatin replication involves the assembly and activity of the replisome within the nucleosomal landscape. At the core of the replisome is the Mcm2-7 complex (MCM), which is loaded onto DNA after binding to the Origin Recognition Complex (ORC). In yeast, ORC is a dynamic protein that diffuses rapidly along DNA, unless halted by origin recognition sequences. However, less is known about the dynamics of ORC proteins in the presence of nucleosomes and attendant consequences for MCM loading. To address this, we harnessed an in vitro single-molecule approach to interrogate a chromatinized origin of replication. We find that ORC binds the origin of replication with similar efficiency independently of whether the origin is chromatinized, despite ORC mobility being reduced by the presence of nucleosomes. Recruitment of MCM also proceeds efficiently on a chromatinized origin, but subsequent movement of MCM away from the origin is severely constrained. These findings suggest that chromatinized origins in yeast are essential for the local retention of MCM, which may facilitate subsequent assembly of the replisome.

ISW1a modulates cohesin distribution in centromeric and pericentromeric regions.

Cohesin is a highly conserved, multiprotein complex whose canonical function is to hold sister chromatids together to ensure accurate chromosome segregation. Cohesin association with chromatin relies on the Scc2-Scc4 cohesin loading complex that enables cohesin ring opening and topological entrapment of sister DNAs. To better understand how sister chromatid cohesion is regulated, we performed a proteomic screen in budding yeast that identified the Isw1 chromatin remodeler as a cohesin binding partner. In addition, we found that Isw1 also interacts with Scc2-Scc4. Lack of Isw1 protein, the Ioc3 subunit of ISW1a or Isw1 chromatin remodeling activity resulted in increased accumulation of cohesin at centromeres and pericentromeres, suggesting that ISW1a may promote efficient translocation of cohesin from the centromeric site of loading to neighboring regions. Consistent with the role of ISW1a in the chromatin organization of centromeric regions, Isw1 was found to be recruited to centromeres. In its absence we observed changes in the nucleosomal landscape at centromeres and pericentromeres. Finally, we discovered that upon loss of RSC functionality, ISW1a activity leads to reduced cohesin binding and cohesion defect. Taken together, our results support the notion of a key role of chromatin remodelers in the regulation of cohesin distribution on chromosomes.

PGE2 alters chromatin through H2A.Z-variant enhancer nucleosome modification to promote hematopoietic stem cell fate

Prostaglandin E2 (PGE2) and 16,16-dimethyl-PGE2 (dmPGE2) are important regulators of hematopoietic stem and progenitor cell (HSPC) fate and offer potential to enhance stem cell therapies [C. Cutler et al. Blood 122, 3074-3081(2013); W. Goessling et al. Cell Stem Cell 8, 445-458 (2011); W. Goessling et al. Cell 136, 1136-1147 (2009)]. Here, we report that PGE2-induced changes in chromatin at enhancer regions through histone-variant H2A.Z permit acute inflammatory gene induction to promote HSPC fate. We found that dmPGE2-inducible enhancers retain MNase-accessible, H2A.Z-variant nucleosomes permissive of CREB transcription factor (TF) binding. CREB binding to enhancer nucleosomes following dmPGE2 stimulation is concomitant with deposition of histone acetyltransferases p300 and Tip60 on chromatin. Subsequent H2A.Z acetylation improves chromatin accessibility at stimuli-responsive enhancers. Our findings support a model where histone-variant nucleosomes retained within inducible enhancers facilitate TF binding. Histone-variant acetylation by TF-associated nucleosome remodelers creates the accessible nucleosome landscape required for immediate enhancer activation and gene induction. Our work provides a mechanism through which inflammatory mediators, such as dmPGE2, lead to acute transcriptional changes and modify HSPC behavior to improve stem cell transplantation.

The nucleosome unwrapping free energy landscape defines distinct regions of transcription factor accessibility and kinetics.

Transcription factors (TF) require access to target sites within nucleosomes to initiate transcription. The target site position within the nucleosome significantly influences TF occupancy, but how is not quantitatively understood. Using ensemble and single-molecule fluorescence measurements, we investigated the targeting and occupancy of the transcription factor, Gal4, at different positions within the nucleosome. We observe a dramatic decrease in TF occupancy to sites extending past 30 base pairs (bp) into the nucleosome which cannot be explained by changes in the TF dissociation rate or binding site orientation. Instead, the nucleosome unwrapping free energy landscape is the primary determinant of Gal4 occupancy by reducing the Gal4 binding rate. The unwrapping free energy landscape defines two distinct regions of accessibility and kinetics with a boundary at 30 bp into the nucleosome where the inner region is over 100-fold less accessible. The Gal4 binding rate in the inner region no longer depends on its concentration because it is limited by the nucleosome unwrapping rate, while the frequency of nucleosome rewrapping decreases because Gal4 exchanges multiple times before the nucleosome rewraps. Our findings highlight the importance of the nucleosome unwrapping free energy landscape on TF occupancy and dynamics that ultimately influences transcription initiation.

Structural mechanism of extranucleosomal DNA readout by the INO80 complex

The nucleosomal landscape of chromatin depends on the concerted action of chromatin remodelers. The INO80 remodeler specifically places nucleosomes at the boundary of gene regulatory elements, which is proposed to be the result of an ATP-dependent nucleosome sliding activity that is regulated by extranucleosomal DNA features. Here, we use cryo-electron microscopy and functional assays to reveal how INO80 binds and is regulated by extranucleosomal DNA. Structures of the regulatory A-module bound to DNA clarify the mechanism of linker DNA binding. The A-module is connected to the motor unit via an HSA/post-HSA lever element to chemomechanically couple the motor and linker DNA sensing. Two notable sites of curved DNA recognition by coordinated action of the four actin/actin-related proteins and the motor suggest how sliding by INO80 can be regulated by extranucleosomal DNA features. Last, the structures clarify the recruitment of YY1/Ies4 subunits and reveal deep architectural similarities between the regulatory modules of INO80 and SWI/SNF complexes.

Genome-Wide Identification of Open Chromatin in Plants Using MH-Seq.

Functional cis-regulatory elements (CREs) act as precise transcriptional switches for fine-tuning gene transcription. Identification of CREs is critical for understanding regulatory mechanisms of gene expression associated with various biological processes in eukaryotes. It is well known that CREs reside in open chromatin that exhibits hypersensitivity to enzyme cleavage and physical shearing. Currently, high-throughput methodologies, such as DNase-seq, ATAC-seq, and FAIRE-seq, have been widely applied in mapping open chromatin in various eukaryotic genomes. More recently, differential MNase (micrococcal nuclease) treatment has been successfully employed to map open chromatin in addition to profiling nucleosome landscape in both mammalian and plant species. We have developed a MNase hypersensitivity sequencing (MH-seq) technique in plants. The MH-seq procedure includes plant nuclei fixation and purification, differential treatments of purified nuclei with MNase, specific recovery of MNase-trimmed small DNA fragments within 20~100bp in length, and MH-seq library construction followed by Illumina sequencing and data analysis. MH-seq has been successfully applied for global identification of open chromatin in both Arabidopsis thaliana and maize. It has been proven to be an attractive alternative for profiling open chromatin. Thus, MH-seq is expected to be valuable in probing chromatin accessibility on a genome-wide scale for other plants with sequenced genomes. Moreover, MHS data allow to implement footprinting assays to unveil binding sites of transcription factors.

Multiplexing mechanical and translational cues on genes

The genetic code gives precise instructions on how to translate codons into amino acids. Due to the degeneracy of the genetic code—18 out of 20 amino acids are encoded for by more than one codon—more information can be stored in a basepair sequence. Indeed, various types of additional information have been discussed in the literature, e.g., the positioning of nucleosomes along eukaryotic genomes and the modulation of the translating efficiency in ribosomes to influence cotranslational protein folding. The purpose of this study is to show that it is indeed possible to carry more than one additional layer of information on top of a gene. In particular, we show how much translation efficiency and nucleosome positioning can be adjusted simultaneously without changing the encoded protein. We achieve this by mapping genes on weighted graphs that contain all synonymous genes, and then finding shortest paths through these graphs. This enables us, for example, to readjust the disrupted translational efficiency profile after a gene has been introduced from one organism (e.g., human) into another (e.g., yeast) without greatly changing the nucleosome landscape intrinsically encoded by the DNA molecule.

The TRIPLE PHD FINGERS proteins are required for SWI/SNF complex-mediated +1 nucleosome positioning and transcription start site determination in Arabidopsis

Eukaryotes have evolved multiple ATP-dependent chromatin remodelers to shape the nucleosome landscape. We recently uncovered an evolutionarily conserved SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeler complex in plants reminiscent of the mammalian BAF subclass, which specifically incorporates the MINUSCULE (MINU) catalytic subunits and the TRIPLE PHD FINGERS (TPF) signature subunits. Here we report experimental evidence that establishes the functional relevance of TPF proteins for the complex activity. Our results show that depletion of TPF triggers similar pleiotropic phenotypes and molecular defects to those found in minu mutants. Moreover, we report the genomic location of MINU2 and TPF proteins as representative members of this SWI/SNF complex and their impact on nucleosome positioning and transcription. These analyses unravel the binding of the complex to thousands of genes where it modulates the position of the +1 nucleosome. These targets tend to produce 5′-shifted transcripts in the tpf and minu mutants pointing to the participation of the complex in alternative transcription start site usage. Interestingly, there is a remarkable correlation between +1 nucleosome shift and 5′ transcript length change suggesting their functional connection. In summary, this study unravels the function of a plant SWI/SNF complex involved in +1 nucleosome positioning and transcription start site determination.

Dynamic nucleosome landscape elicits a noncanonical GATA2 pioneer model

Knowledge gaps remain on how nucleosome organization and dynamic reorganization are governed by specific pioneer factors in a genome-wide manner. In this study, we generate over three billons of multi-omics sequencing data to exploit dynamic nucleosome landscape governed by pioneer factors (PFs), FOXA1 and GATA2. We quantitatively define nine functional nucleosome states each with specific characteristic nucleosome footprints in LNCaP prostate cancer cells. Interestingly, we observe dynamic switches among nucleosome states upon androgen stimulation, accompanied by distinct differential (gained or lost) binding of FOXA1, GATA2, H1 as well as many other coregulators. Intriguingly, we reveal a noncanonical pioneer model of GATA2 that it initially functions as a PF binding at the edge of a nucleosome in an inaccessible crowding array. Upon androgen stimulation, GATA2 re-configures an inaccessible to accessible nucleosome state and subsequently acts as a master transcription factor either directly or recruits signaling specific transcription factors to enhance WNT signaling in an androgen receptor (AR)-independent manner. Our data elicit a pioneer and master dual role of GATA2 in mediating nucleosome dynamics and enhancing downstream signaling pathways. Our work offers structural and mechanistic insight into the dynamics of pioneer factors governing nucleosome reorganization.

Nuclear HMGB1 protects from nonalcoholic fatty liver disease through negative regulation of liver X receptor.

Dysregulations of lipid metabolism in the liver may trigger steatosis progression, leading to potentially severe clinical consequences such as nonalcoholic fatty liver diseases (NAFLDs). Molecular mechanisms underlying liver lipogenesis are very complex and fine-tuned by chromatin dynamics and multiple key transcription factors. Here, we demonstrate that the nuclear factor HMGB1 acts as a strong repressor of liver lipogenesis. Mice with liver-specific Hmgb1 deficiency display exacerbated liver steatosis, while Hmgb1-overexpressing mice exhibited a protection from fatty liver progression when subjected to nutritional stress. Global transcriptome and functional analysis revealed that the deletion of Hmgb1 gene enhances LXRα and PPARγ activity. HMGB1 repression is not mediated through nucleosome landscape reorganization but rather via a preferential DNA occupation in a region carrying genes regulated by LXRα and PPARγ. Together, these findings suggest that hepatocellular HMGB1 protects from liver steatosis development. HMGB1 may constitute a new attractive option to therapeutically target the LXRα-PPARγ axis during NAFLD.

The biogenesis and function of nucleosome arrays

Numerous chromatin remodeling enzymes position nucleosomes in eukaryotic cells. Aside from these factors, transcription, DNA sequence, and statistical positioning of nucleosomes also shape the nucleosome landscape. The precise contributions of these processes remain unclear due to their functional redundancy in vivo. By incisive genome engineering, we radically decreased their redundancy in Saccharomyces cerevisiae. The transcriptional machinery strongly disrupts evenly spaced nucleosomes. Proper nucleosome density and DNA sequence are critical for their biogenesis. The INO80 remodeling complex helps space nucleosomes in vivo and positions the first nucleosome over genes in an H2A.Z-independent fashion. INO80 requires its Arp8 subunit but unexpectedly not the Nhp10 module for spacing. Cells with irregularly spaced nucleosomes suffer from genotoxic stress including DNA damage, recombination and transpositions. We derive a model of the biogenesis of the nucleosome landscape and suggest that it evolved not only to regulate but also to protect the genome.

Nucleosome landscape reflects phenotypic differences in Trypanosoma cruzi life forms.

Trypanosoma cruzi alternates between replicative and nonreplicative life forms, accompanied by a shift in global transcription levels and by changes in the nuclear architecture, the chromatin proteome and histone posttranslational modifications. To gain further insights into the epigenetic regulation that accompanies life form changes, we performed genome-wide high-resolution nucleosome mapping using two T. cruzi life forms (epimastigotes and cellular trypomastigotes). By combining a powerful pipeline that allowed us to faithfully compare nucleosome positioning and occupancy, more than 125 thousand nucleosomes were mapped, and approximately 20% of them differed between replicative and nonreplicative forms. The nonreplicative forms have less dynamic nucleosomes, possibly reflecting their lower global transcription levels and DNA replication arrest. However, dynamic nucleosomes are enriched at nonreplicative regulatory transcription initiation regions and at multigenic family members, which are associated with infective-stage and virulence factors. Strikingly, dynamic nucleosome regions are associated with GO terms related to nuclear division, translation, gene regulation and metabolism and, notably, associated with transcripts with different expression levels among life forms. Finally, the nucleosome landscape reflects the steady-state transcription expression: more abundant genes have a more deeply nucleosome-depleted region at putative 5' splice sites, likely associated with trans-splicing efficiency. Taken together, our results indicate that chromatin architecture, defined primarily by nucleosome positioning and occupancy, reflects the phenotypic differences found among T. cruzi life forms despite the lack of a canonical transcriptional control context.

P53 affects epigenetic signature on SOCS1 promoter in response to TLR4 inhibition

Suppressor of cytokine signaling (SOCS1) functions as a negative regulator of toll-like receptor (TLR) induced inflammatory signaling. As silencing of SOCS1 is concomitant with elevated TLR4 levels in glioblastoma, we investigated the effect of TLR4 inhibition on SOCS1 expression. Pharmacological inhibition of TLR4 signaling by TAK242 or its siRNA-mediated knockdown in p53 mutant or wild-type glioma cells resulted in either increased or decreased SOCS1 expression and promoter activity, respectively. Genetic manipulation of p53 indicated that SOCS1 expression upon TLR4 inhibition is dependent on p53 mutational status. Increased SOCS1 level was concomitant with diminished nucleosomal occupancy around p53-binding site on SOCS1 promoter. This altered nucleosomal landscape was accompanied by (i) diminished nuclear H3K9me3 and (ii) increased JMJD2A and Brg1 levels. JMJD2A inhibition or ectopic expression of ATPase-deficient BRG1 prevented TAK242 mediated increase in SOCS1 expression. Recruitment of Brg1-p53-JMJD2A complex on p53 binding sites of SOCS1 promoter upon TLR4 inhibition was concomitant with increased SOCS1 expression in p53-mutant cells. The Cancer Genome Atlas (TCGA) dataset indicated an inverse correlation between TLR4 and SOCS1 levels in p53 mutant but not in p53WT GBM. Taken together, p53 mutational status regulates transcriptional plasticity of SOCS1 promoter through differential recruitment of chromatin remodelers and epigenetic regulators in response to TLR4 inhibition.

A Histone Cycle: a Systematic Overview on Histone (Octamer Protein) Replication

Genetic information plays an important role in each individual for their differentiation. DNA replication is a highly conserved process that accurately copies the genetic information from one generation to the next. The processes of chromatin disassembly and reassembly during DNA replication also have to be precisely regulated to ensure that the genetic material is compactly packaged to fit into the nucleus while also maintaining the epigenetic information that is carried by the histone proteins bound to the DNA, through cell divisions. So, for the synthesis of genome or gene a group of eight proteins collectively known as octamer proteins or histones plays a crucial role. Colloid of chromosomes is converts into the double stranded DNA structure with intermediate structure of histone proteins. As eukaryotic replication disrupts each nucleosome as the fork passes, followed by incorporation of newly synthesized histones into nucleosomes in the daughter genomes. For the cellular machinery to access the DNA, the chromatin must be unwound and the DNA cleared of histone proteins. In this review, we focus on the process of replication-coupled nucleosome assembly to understand how characteristic steady-state nucleosome landscapes are attained. Recent studies have begun to elucidate mechanisms involved in histone transfer during replication and maturation of the nucleosome landscape after disruption by replication. A fuller understanding of replication-coupled nucleosome assembly will be needed to explain how epigenetic information is replicated at every cell division. We also give details about regulation of histone in human and the DNA Damage Response.

Phenotypes from cell-free DNA.

Cell-free DNA (cfDNA) has the potential to enable non-invasive detection of disease states and progression. Beyond its sequence, cfDNA also represents the nucleosomal landscape of cell(s)-of-origin and captures the dynamics of the epigenome. In this review, we highlight the emergence of cfDNA epigenomic methods that assess disease beyond the scope of mutant tumour genotyping. Detection of tumour mutations is the gold standard for sequencing methods in clinical oncology. However, limitations inherent to mutation targeting in cfDNA, and the possibilities of uncovering molecular mechanisms underlying disease, have made epigenomics of cfDNA an exciting alternative. We discuss the epigenomic information revealed by cfDNA, and how epigenomic methods exploit cfDNA to detect and characterize cancer. Future applications of cfDNA epigenomic methods to act complementarily and orthogonally to current clinical practices has the potential to transform cancer management and improve cancer patient outcomes.

GATA Factor-Mediated Gene Regulation in Human Erythropoiesis.

SummaryErythroid commitment and differentiation are regulated by the coordinated action of a host of transcription factors, including GATA2 and GATA1. Here, we explored GATA-mediated transcriptional regulation through the integrative analysis of gene expression, chromatin modifications, and GATA factors' binding in human multipotent hematopoietic stem/progenitor cells, early erythroid progenitors, and late precursors. A progressive loss of H3K27 acetylation and a diminished usage of active enhancers and super-enhancers were observed during erythroid commitment and differentiation. GATA factors mediate transcriptional changes through a stage-specific interplay with regulatory elements: GATA1 binds different sets of regulatory elements in erythroid progenitors and precursors and controls the transcription of distinct genes during commitment and differentiation. Importantly, our results highlight a pivotal role of promoters in determining the transcriptional program activated upon erythroid differentiation. Finally, we demonstrated that GATA1 binding to a stage-specific super-enhancer sustains the expression of the KIT receptor in human erythroid progenitors.