Drosophila Brahma complex remodels nucleosome organizations in multiple aspects

Abstract ATP-dependent chromatin remodeling complexes regulate nucleosome organizations. In Drosophila, gene Brm encodes the core Brahma complex, the ATPase subunit of SWI/SNF class of chromatin remodelers. Its role in modulating the nucleosome landscape in vivo is unclear. In this study, we knocked down Brm in Drosophila third instar larvae to explore the changes in nucleosome profiles and global gene transcription. The results show that Brm knockdown leads to nucleosome occupancy changes throughout the entire genome with a bias in occupancy decrease. In contrast, the knockdown has limited impacts on nucleosome position shift. The knockdown also alters another important physical property of nucleosome positioning, fuzziness. Nucleosome position shift, gain or loss and fuzziness changes are all enriched in promoter regions. Nucleosome arrays around the 5′ ends of genes are reorganized in five patterns as a result of Brm knockdown. Intriguingly, the concomitant changes in the genes adjacent to the Brahma-dependent remodeling regions have important roles in development and morphogenesis. Further analyses reveal abundance of AT-rich motifs for transcription factors in the remodeling regions.

Download Full-text

The chromatin remodelers RSC and ISW1 display functional and chromatin-based promoter antagonism

eLife ◽

10.7554/elife.06073 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 44

Author(s):

Timothy J Parnell ◽

Alisha Schlichter ◽

Boris G Wilson ◽

Bradley R Cairns

Keyword(s):

Nucleosome Occupancy ◽

Histone H4 ◽

Genetic Screens ◽

Chromatin Remodelers ◽

Nucleosome Arrays

ISWI family chromatin remodelers typically organize nucleosome arrays, while SWI/SNF family remodelers (RSC) typically disorganize and eject nucleosomes, implying an antagonism that is largely unexplored in vivo. Here, we describe two independent genetic screens for rsc suppressors that yielded mutations in the promoter-focused ISW1a complex or mutations in the ‘basic patch’ of histone H4 (an epitope that regulates ISWI activity), strongly supporting RSC-ISW1a antagonism in vivo. RSC and ISW1a largely co-localize, and genomic nucleosome studies using rsc isw1 mutant combinations revealed opposing functions: promoters classified with a nucleosome-deficient region (NDR) gain nucleosome occupancy in rsc mutants, but this gain is attenuated in rsc isw1 double mutants. Furthermore, promoters lacking NDRs have the highest occupancy of both remodelers, consistent with regulation by nucleosome occupancy, and decreased transcription in rsc mutants. Taken together, we provide the first genetic and genomic evidence for RSC-ISW1a antagonism and reveal different mechanisms at two different promoter architectures.

Download Full-text

Arabidopsis FORGETTER1 mediates stress-induced chromatin memory through nucleosome remodeling

eLife ◽

10.7554/elife.17061 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 49

Author(s):

Krzysztof Brzezinka ◽

Simone Altmann ◽

Hjördis Czesnick ◽

Philippe Nicolas ◽

Michal Gorka ◽

...

Keyword(s):

Gene Expression ◽

Heat Stress ◽

Nucleosome Occupancy ◽

Promoter Regions ◽

Nucleosome Remodeling ◽

Chromatin Remodelers ◽

Stress Memory ◽

Active Memory ◽

Nucleosome Dynamics ◽

Sessile Organisms

Plants as sessile organisms can adapt to environmental stress to mitigate its adverse effects. As part of such adaptation they maintain an active memory of heat stress for several days that promotes a more efficient response to recurring stress. We show that this heat stress memory requires the activity of the FORGETTER1 (FGT1) locus, with fgt1 mutants displaying reduced maintenance of heat-induced gene expression. FGT1 encodes the Arabidopsis thaliana orthologue of Strawberry notch (Sno), and the protein globally associates with the promoter regions of actively expressed genes in a heat-dependent fashion. FGT1 interacts with chromatin remodelers of the SWI/SNF and ISWI families, which also display reduced heat stress memory. Genomic targets of the BRM remodeler overlap significantly with FGT1 targets. Accordingly, nucleosome dynamics at loci with altered maintenance of heat-induced expression are affected in fgt1. Together, our results suggest that by modulating nucleosome occupancy, FGT1 mediates stress-induced chromatin memory.

Download Full-text

Active nucleosome positioning beyond intrinsic biophysics is revealed by in vitro reconstitution

Biochemical Society Transactions ◽

10.1042/bst20110730 ◽

2012 ◽

Vol 40 (2) ◽

pp. 377-382 ◽

Cited By ~ 14

Author(s):

Philipp Korber

Keyword(s):

Dna Sequence ◽

Nucleosome Occupancy ◽

Nucleosome Positioning ◽

Major Theme ◽

Promoter Regions ◽

Genome Wide ◽

Nucleosome Formation ◽

Dna Binders

Genome-wide nucleosome maps revealed well-positioned nucleosomes as a major theme in eukaryotic genome organization. Promoter regions often show a conserved pattern with an NDR (nucleosome-depleted region) from which regular nucleosomal arrays emanate. Three mechanistic contributions to such NDR-array-organization and nucleosome positioning in general are discussed: DNA sequence, DNA binders and DNA-templated processes. Especially, intrinsic biophysics of DNA sequence preferences for nucleosome formation was prominently suggested to explain the majority of nucleosome positions (‘genomic code for nucleosome positioning’). Nonetheless, non-histone factors that bind DNA with high or low specificity, such as transcription factors or remodelling enzymes respectively and processes such as replication, transcription and the so-called ‘statistical positioning’ may be involved too. Recently, these models were tested for yeast by genome-wide reconstitution. DNA sequence preferences as probed by SGD (salt gradient dialysis) reconstitution generated many NDRs, but only few individual nucleosomes, at their proper positions, and no arrays. Addition of a yeast extract and ATP led to dramatically more in vivo-like nucleosome positioning, including regular arrays for the first time. This improvement depended essentially on the extract and ATP but not on transcription or replication. Nucleosome occupancy and close spacing were maintained around promoters, even at lower histone density, arguing for active packing of nucleosomes against the 5′ ends of genes rather than statistical positioning. A first extract fractionation identified a direct, specific, necessary, but not sufficient role for the RSC (remodels the structure of chromatin) remodelling enzyme. Collectively, nucleosome positioning in yeast is actively determined by factors beyond intrinsic biophysics, and in steady-state rather than at equilibrium.

Download Full-text

The Active Rather than Intrinsic Mechanism of Nucleosome Depletion by Poly(dA:dT) Tracts

10.20944/preprints202104.0579.v1 ◽

2021 ◽

Author(s):

Toby Barnes ◽

Philipp Korber

Keyword(s):

Atp Hydrolysis ◽

Nucleosome Occupancy ◽

Nucleosome Assembly ◽

Chromatin Remodelers ◽

Intrinsic Mechanism ◽

Energy Penalty ◽

Exclusion Mechanism ◽

Species Specific

Poly(dA:dT) tracts cause nucleosome depletion in many species, e.g., at promoters and replication origins. Their intrinsic biophysical sequence properties make them stiff and unfavorable for nucleosome assembly, as probed by in vitro nucleosome reconstitution. The mere correlation between nucleosome depletion over poly(dA:dT) tracts in vitro and in vivo inspired an intrinsic nucleosome exclusion mechanism in vivo. However, we compile here published and new evidence that this correlation does not reflect mechanistic causation. 1) Nucleosome depletion over poly(dA:dT) in vivo is not universal, e.g., very weak in S. pombe. 2) The energy penalty for incorporating poly(dA:dT) tracts into nucleosomes is modest (<10%) relative to ATP hydrolysis energy abundantly invested by chromatin remodelers. 3) Nucleosome depletion over poly(dA:dT) is much stronger in vivo than in vitro if monitored without MNase and 4) actively maintained in vivo. 5) S. cerevisiae promoters evolved a biased poly(dA) versus poly(dT) distribution. 6) Nucleosome depletion over poly(dA) is directional in vivo. 7) The ATP dependent chromatin remodeler RSC preferentially and directionally displaces nucleosomes towards 5’ of poly(dA). Especially bias and directionality would not be expected for an intrinsic mechanism. Together, this argues for a mechanism where active and species-specific read out of intrinsic sequence properties, e.g., by remodelers, shapes nucleosome occupancy.

Download Full-text

DNA-Encoded Chromatin Structural Intron Boundary Signals Identify Conserved Genes with Common Function

International Journal of Genomics ◽

10.1155/2015/167578 ◽

2015 ◽

Vol 2015 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Justin A. Fincher ◽

Gary S. Tyson ◽

Jonathan H. Dennis

Keyword(s):

Nucleosome Occupancy ◽

Consensus Sequence ◽

Physical Property ◽

Mrna Splicing ◽

Conserved Genes ◽

V Genes ◽

Chromatin Structural ◽

Intron Boundary ◽

Exon Boundary

The regulation of metazoan gene expression occurs in part by pre-mRNA splicing into mature RNAs. Signals affecting the efficiency and specificity with which introns are removed have not been completely elucidated. Splicing likely occurs cotranscriptionally, with chromatin structure playing a key regulatory role. We calculated DNA encoded nucleosome occupancy likelihood (NOL) scores at the boundaries between introns and exons across five metazoan species. We found that (i) NOL scores reveal a sequence-based feature at the introns on both sides of the intron-exon boundary; (ii) this feature is not part of any recognizable consensus sequence; (iii) this feature is conserved throughout metazoa; (iv) this feature is enriched in genes sharing similar functions: ATPase activity, ATP binding, helicase activity, and motor activity; (v) genes with these functions exhibit different genomic characteristics; (vi)in vivonucleosome positioning data confirm ontological enrichment at this feature; and (vii) genes with this feature exhibit unique dinucleotide distributions at the intron-exon boundary. The NOL scores point toward a physical property of DNA that may play a role in the mechanism of pre-mRNA splicing. These results provide a foundation for identification of a new set of regulatory DNA elements involved in splicing regulation.

Download Full-text

Ubiquitylome Analysis Reveals a Central Role for the Ubiquitin-Proteasome System in Plant Innate Immunity

Plant Physiology ◽

10.1093/plphys/kiab011 ◽

2021 ◽

Author(s):

Xiyu Ma ◽

Chao Zhang ◽

Do Young Kim ◽

Yanyan Huang ◽

Elizabeth Chatt ◽

...

Keyword(s):

Plant Immunity ◽

High Sensitivity ◽

Ubiquitin Proteasome System ◽

Immune Recognition ◽

Atpase Subunit ◽

Ample Evidence ◽

Network Analyses ◽

Affinity Enrichment ◽

Regulatory Loop

Abstract Protein ubiquitylation profoundly expands proteome functionality and diversifies cellular signaling processes, with recent studies providing ample evidence for its importance to plant immunity. To gain a proteome-wide appreciation of ubiquitylome dynamics during immune recognition, we employed a two-step affinity enrichment protocol based on a 6His-tagged ubiquitin (Ub) variant coupled with high sensitivity mass spectrometry to identify Arabidopsis proteins rapidly ubiquitylated upon plant perception of the microbe-associated molecular pattern (MAMP) peptide flg22. The catalog from 2-week-old seedlings treated for 30 minutes with flg22 contained 690 conjugates, 64 Ub footprints, and all seven types of Ub linkages, and included previously uncharacterized conjugates of immune components. In vivo ubiquitylation assays confirmed modification of several candidates upon immune elicitation, and revealed distinct modification patterns and dynamics for key immune components, including poly- and monoubiquitylation, as well as induced or reduced levels of ubiquitylation. Gene ontology and network analyses of the collection also uncovered rapid modification of the Ub-proteasome system itself, suggesting a critical auto-regulatory loop necessary for an effective MAMP-triggered immune response and subsequent disease resistance. Included targets were UBIQUITIN-CONJUGATING ENZYME 13 (UBC13) and proteasome component REGULATORY PARTICLE NON-ATPASE SUBUNIT 8b (RPN8b), whose subsequent biochemical and genetic analyses implied negative roles in immune elicitation. Collectively, our proteomic analyses further strengthened the connection between ubiquitylation and flg22-based immune signaling, identified components and pathways regulating plant immunity, and increased the database of ubiquitylated substrates in plants.

Download Full-text

Activation of MAT2A-RIP1 signaling axis reprograms monocytes in gastric cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001364 ◽

2021 ◽

Vol 9 (2) ◽

pp. e001364

Author(s):

Yan Zhang ◽

Hui Yang ◽

Jun Zhao ◽

Ping Wan ◽

Ye Hu ◽

...

Keyword(s):

Gastric Cancer ◽

Cell Metabolism ◽

Vital Role ◽

Methionine Metabolism ◽

Promoter Regions ◽

Normal Tissues ◽

Methionine Cycle

BackgroundThe activation of tumor-associated macrophages (TAMs) facilitates the progression of gastric cancer (GC). Cell metabolism reprogramming has been shown to play a vital role in the polarization of TAMs. However, the role of methionine metabolism in function of TAMs remains to be explored.MethodsMonocytes/macrophages were isolated from peripheral blood, tumor tissues or normal tissues from healthy donors or patients with GC. The role of methionine metabolism in the activation of TAMs was evaluated with both in vivo analyses and in vitro experiments. Pharmacological inhibition of the methionine cycle and modulation of key metabolic genes was employed, where molecular and biological analyses were performed.ResultsTAMs have increased methionine cycle activity that are mainly attributed to elevated methionine adenosyltransferase II alpha (MAT2A) levels. MAT2A modulates the activation and maintenance of the phenotype of TAMs and mediates the upregulation of RIP1 by increasing the histone H3K4 methylation (H3K4me3) at its promoter regions.ConclusionsOur data cast light on a novel mechanism by which methionine metabolism regulates the anti-inflammatory functions of monocytes in GC. MAT2A might be a potential therapeutic target for cancer cells as well as TAMs in GC.

Download Full-text

Rice protein-binding microarrays: a tool to detect cis-acting elements near promoter regions in rice

Planta ◽

10.1007/s00425-021-03572-w ◽

2021 ◽

Vol 253 (2) ◽

Author(s):

Joung Sug Kim ◽

SongHwa Chae ◽

Kyong Mi Jun ◽

Gang-Seob Lee ◽

Jong-Seong Jeon ◽

...

Keyword(s):

Dna Binding ◽

Protein Binding ◽

Gene Networks ◽

Upstream Region ◽

Transcriptional Level ◽

Cis Elements ◽

Promoter Regions ◽

Entire Genome ◽

Binding Motifs ◽

Dna Binding Motifs

Abstract Main conclusion The present study showed that a rice (Oryza sativa)-specific protein-binding microarray (RPBM) can be applied to analyze DNA-binding motifs with a TF where binding is evaluated in extended natural promoter regions. The analysis may facilitate identifying TFs and their downstream genes and constructing gene networks through cis-elements. Abstract Transcription factors (TFs) regulate gene expression at the transcriptional level by binding a specific DNA sequence. Thus, predicting the DNA-binding motifs of TFs is one of the most important areas in the functional analysis of TFs in the postgenomic era. Although many methods have been developed to address this challenge, many TFs still have unknown DNA-binding motifs. In this study, we designed RPBM with 40-bp probes and 20-bp of overlap, yielding 49 probes spanning the 1-kb upstream region before the translation start site of each gene in the entire genome. To confirm the efficiency of RPBM technology, we selected two previously studied TFs, OsWOX13 and OsSMF1, and an uncharacterized TF, OsWRKY34. We identified the ATTGATTG and CCACGTCA DNA-binding sequences of OsWOX13 and OsSMF1, respectively. In total, 635 and 932 putative feature genes were identified for OsWOX13 and OsSMF1, respectively. We discovered the CGTTGACTTT DNA-binding sequence and 195 putative feature genes of OsWRKY34. RPBM could be applicable in the analysis of DNA-binding motifs for TFs where binding is evaluated in the promoter and 5′ upstream CDS regions. The analysis may facilitate identifying TFs and their downstream genes and constructing gene networks through cis-elements.

Download Full-text

Prrx1 promotes stemness and angiogenesis via activating TGF-β/smad pathway and upregulating proangiogenic factors in glioma

Cell Death and Disease ◽

10.1038/s41419-021-03882-7 ◽

2021 ◽

Vol 12 (6) ◽

Author(s):

Zetao Chen ◽

Yihong Chen ◽

Yan Li ◽

Weidong Lian ◽

Kehong Zheng ◽

...

Keyword(s):

Stem Cells ◽

Therapeutic Strategy ◽

Critical Role ◽

Glioma Stem Cells ◽

Promoter Regions ◽

Aberrant Expression ◽

Smad Pathway ◽

Tgf Β1

AbstractGlioma is one of the most lethal cancers with highly vascularized networks and growing evidences have identified glioma stem cells (GSCs) to account for excessive angiogenesis in glioma. Aberrant expression of paired-related homeobox1 (Prrx1) has been functionally associated with cancer stem cells including GSCs. In this study, Prrx1 was found to be markedly upregulated in glioma specimens and elevated Prrx1 expression was inversely correlated with prognosis of glioma patients. Prrx1 potentiated stemness acquisition in non-stem tumor cells (NSTCs) and stemness maintenance in GSCs, accompanied with increased expression of stemness markers such as SOX2. Prrx1 also promoted glioma angiogenesis by upregulating proangiogenic factors such as VEGF. Consistently, silencing Prrx1 markedly inhibited glioma proliferation, stemness, and angiogenesis in vivo. Using a combination of subcellular proteomics and in vitro analyses, we revealed that Prrx1 directly bound to the promoter regions of TGF-β1 gene, upregulated TGF-β1 expression, and ultimately activated the TGF-β/smad pathway. Silencing TGF-β1 mitigated the malignant behaviors induced by Prrx1. Activation of this pathway cooperates with Prrx1 to upregulate the expression of stemness-related genes and proangiogenic factors. In summary, our findings revealed that Prrx1/TGF-β/smad signal axis exerted a critical role in glioma stemness and angiogeneis. Disrupting the function of this signal axis might represent a new therapeutic strategy in glioma patients.

Download Full-text

Circadian clock protein Rev-erbα regulates neuroinflammation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1812405116 ◽

2019 ◽

Vol 116 (11) ◽

pp. 5102-5107 ◽

Cited By ~ 36

Author(s):

Percy Griffin ◽

Julie M. Dimitry ◽

Patrick W. Sheehan ◽

Brian V. Lananna ◽

Chun Guo ◽

...

Keyword(s):

Circadian Clock ◽

Microglial Activation ◽

Time Of Day ◽

Resting State Functional Connectivity ◽

Promoter Regions ◽

Clock Component ◽

Glial Cultures ◽

Inflammatory Phenotype

Circadian dysfunction is a common attribute of many neurodegenerative diseases, most of which are associated with neuroinflammation. Circadian rhythm dysfunction has been associated with inflammation in the periphery, but the role of the core clock in neuroinflammation remains poorly understood. Here we demonstrate that Rev-erbα, a nuclear receptor and circadian clock component, is a mediator of microglial activation and neuroinflammation. We observed time-of-day oscillation in microglial immunoreactivity in the hippocampus, which was disrupted in Rev-erbα−/− mice. Rev-erbα deletion caused spontaneous microglial activation in the hippocampus and increased expression of proinflammatory transcripts, as well as secondary astrogliosis. Transcriptomic analysis of hippocampus from Rev-erbα−/− mice revealed a predominant inflammatory phenotype and suggested dysregulated NF-κB signaling. Primary Rev-erbα−/− microglia exhibited proinflammatory phenotypes and increased basal NF-κB activation. Chromatin immunoprecipitation revealed that Rev-erbα physically interacts with the promoter regions of several NF-κB–related genes in primary microglia. Loss of Rev-erbα in primary astrocytes had no effect on basal activation but did potentiate the inflammatory response to lipopolysaccharide (LPS). In vivo, Rev-erbα−/− mice exhibited enhanced hippocampal neuroinflammatory responses to peripheral LPS injection, while pharmacologic activation of Rev-erbs with the small molecule agonist SR9009 suppressed LPS-induced hippocampal neuroinflammation. Rev-erbα deletion influenced neuronal health, as conditioned media from Rev-erbα–deficient primary glial cultures exacerbated oxidative damage in cultured neurons. Rev-erbα−/− mice also exhibited significantly altered cortical resting-state functional connectivity, similar to that observed in neurodegenerative models. Our results reveal Rev-erbα as a pharmacologically accessible link between the circadian clock and neuroinflammation.

Download Full-text