scholarly journals Stochastic transcription in the p53-mediated response to DNA damage is modulated by burst frequency

2019 ◽  
Author(s):  
Dhana Friedrich ◽  
Laura Friedel ◽  
Andreas Herrmann ◽  
Stephan Preibisch ◽  
Alexander Loewer
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Burst Frequency ◽  
Time Resolved ◽  
Mammalian Cell Lines ◽  
Lysine Residues ◽  
Acetylation State ◽  
Specific Regulation ◽  
Discontinuous Transcription ◽  
Target Promoters

ABSTRACTDiscontinuous transcription has been described for different mammalian cell lines and numerous promoters. However, our knowledge of how the activity of individual promoters is adjusted by dynamic signaling inputs from transcription factor is limited. To address this question, we characterized the activity of selected target genes that are regulated by pulsatile accumulation of the tumor suppressor p53 in response to ionizing radiation. We performed time resolved measurements of gene expression at the single cell level by smFISH and used the resulting data to inform a mathematical model of promoter activity. We found that p53 target promoters are regulated by frequency modulation of stochastic bursting and can be grouped along three archetypes of gene expression. The occurrence of these archetypes cannot solely be explained by nuclear p53 abundance or promoter binding of total p53. Instead, we provide evidence that the time-varying acetylation state of p53’s C-terminal lysine residues is critical for gene-specific regulation of stochastic bursting.

scholarly journals PRC1 drives Polycomb-mediated gene repression by controlling transcription initiation and burst frequency

2020 ◽  
Author(s):  
Paula Dobrinić ◽  
Aleksander T. Szczurek ◽  
Robert J. Klose
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Transcriptional Control ◽  
Target Genes ◽  
Gene Repression ◽  
Burst Frequency ◽  
Chromatin Domains ◽  
Time Resolved ◽  
Drive Gene

AbstractThe Polycomb repressive system plays a fundamental role in controlling gene expression during mammalian development. To achieve this, Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) bind target genes and use histone modification-dependent feedback mechanisms to form Polycomb chromatin domains and repress transcription. The interrelatedness of PRC1 and PRC2 activity at these sites has made it difficult to discover the specific components of Polycomb chromatin domains that drive gene repression and to understand mechanistically how this is achieved. Here, by exploiting rapid degron-based approaches and time-resolved genomics we kinetically dissect Polycomb-mediated repression and discover that PRC1 functions independently of PRC2 to counteract RNA polymerase II binding and transcription initiation. Using single-cell gene expression analysis, we reveal that PRC1 acts uniformly within the cell population, and that repression is achieved by controlling transcriptional burst frequency. These important new discoveries provide a mechanistic and conceptual framework for Polycomb-dependent transcriptional control.

New insights into promoter–enhancer communication mechanisms revealed by dynamic single-molecule imaging

2021 ◽  
Author(s):  
Jieru Li ◽  
Alexandros Pertsinidis
Keyword(s):  
Gene Expression ◽  
Single Molecule ◽  
Target Genes ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Single Molecule Imaging ◽  
Cell Type ◽  
Regulatory Information ◽  
Cell Type Specific ◽  
Target Promoters

Establishing cell-type-specific gene expression programs relies on the action of distal enhancers, cis-regulatory elements that can activate target genes over large genomic distances — up to Mega-bases away. How distal enhancers physically relay regulatory information to target promoters has remained a mystery. Here, we review the latest developments and insights into promoter–enhancer communication mechanisms revealed by live-cell, real-time single-molecule imaging approaches.

scholarly journals The MarR-type regulator MalR is involved in stress-responsive cell envelope remodeling inCorynebacterium glutamicum

2019 ◽  
Author(s):  
Max Hünnefeld ◽  
Marcus Persicke ◽  
Jörn Kalinowski ◽  
Julia Frunzke
Keyword(s):  
Gene Expression ◽  
Antibiotic Treatment ◽  
Target Genes ◽  
Cell Envelope ◽  
Affinity Purification ◽  
Stress Protein ◽  
Bacterial Cells ◽  
Phage Gene ◽  
A Genome ◽  
Target Promoters

1AbstractIt is the enormous adaptive capacity of microorganisms, which is key to their competitive success in nature, but also challenges antibiotic treatment of human diseases. To deal with a diverse set of stresses, bacteria are able to reprogram gene expression using a wide variety of transcription factors. Here, we focused on the MarR-type regulator MalR conserved in theCorynebacterineae, including the prominent pathogensCorynebacterium diphtheriaeandMycobacterium tuberculosis. In several corynebacterial species, themalRgene forms an operon with a gene encoding a universal stress protein (uspA). Chromatin-affinity purification and sequencing (ChAP-Seq) analysis revealed that MalR binds more than 60 target promoters in theC. glutamicumgenome as well as in the large cryptic prophage CGP3. Overproduction of MalR caused severe growth defects and an elongated cell morphology. ChAP-Seq data combined with a global transcriptome analysis of themalRoverexpression strain emphasized a central role of MalR in cell envelope remodeling in response to environmental stresses. Prominent MalR targets are for example involved in peptidoglycan biosynthesis and synthesis of branched-chain fatty acids. Phenotypic microarrays suggest an altered sensitivity of a ΔmalRmutant towards several β-lactam antibiotics. We furthermore revealed MalR as a repressor of several prophage genes suggesting that MalR may be involved in the control of stress-responsive induction of the large CGP3 element. In conclusion, our results emphasize MalR as a regulator involved in stress-responsive remodeling of the cell envelope ofC. glutamicumand suggest a link between cell envelope stress and the control of phage gene expression.ImportanceBacteria live in changing environments that force the cells to be highly adaptive. The cell envelope represents both, a barrier against harsh external conditions and an interaction interface. The dynamic remodeling of the cell envelope as a response towards, e.g. antibiotic treatment represents a major challenge in the treatment of diseases. Members of the MarR family of regulators are known to contribute to an adaptation of bacterial cells towards antibiotic stress. However, our knowledge on this adaptive response was so far restricted to a small number of well-described target genes. In this study, we performed a genome-wide profiling of DNA-binding of the MarR-type regulator MalR ofC. glutamicum, which is conserved in several coryne- and mycobacterial species. By binding to more than 60 different target promoters, MalR is shaping a global reprogramming of gene expression conferring a remodeling of the cell envelope in response to stress.

scholarly journals Time-Resolved Gene Expression Analysis Monitors the Regulation of Inflammatory Mediators and Attenuation of Adaptive Immune Response by Vitamin D

2022 ◽  
Vol 23 (2) ◽  
pp. 911
Author(s):  
Andrea Hanel ◽  
Carsten Carlberg
Keyword(s):  
Gene Expression ◽  
Vitamin D ◽  
Immune Response ◽  
Immune System ◽  
Calcium Binding ◽  
Target Genes ◽  
Mononuclear Cells ◽  
Adaptive Immune Response ◽  
Time Resolved ◽  
Adaptive Immune

Peripheral blood mononuclear cells (PBMCs) belong to the innate and adaptive immune system and are highly sensitive and responsive to changes in their systemic environment. In this study, we focused on the time course of transcriptional changes in freshly isolated human PBMCs 4, 8, 24 and 48 h after onset of stimulation with the active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). Taking all four time points together, 662 target genes were identified and segregated either by time of differential gene expression into 179 primary and 483 secondary targets or by driver of expression change into 293 direct and 369 indirect targets. The latter classification revealed that more than 50% of target genes were primarily driven by the cells' response to ex vivo exposure than by the nuclear hormone and largely explained its down-regulatory effect. Functional analysis indicated vitamin D’s role in the suppression of the inflammatory and adaptive immune response by down-regulating ten major histocompatibility complex class II genes, five alarmins of the S100 calcium binding protein A family and by affecting six chemokines of the C-X-C motif ligand family. Taken together, studying time-resolved responses allows to better contextualize the effects of vitamin D on the immune system.

scholarly journals Differential activation of P-TEFb complexes in the development of cardiomyocyte hypertrophy following activation of distinct GPCRs

2020 ◽  
Author(s):  
Ryan D. Martin ◽  
Yalin Sun ◽  
Sarah MacKinnon ◽  
Luca Cuccia ◽  
Viviane Pagé ◽  
...  
Keyword(s):  
Gene Expression ◽  
Large Scale ◽  
Target Genes ◽  
Elongation Factor ◽  
Neonatal Rat ◽  
Cardiomyocyte Hypertrophy ◽  
Rat Cardiomyocytes ◽  
Hypertrophic Response ◽  
Specific Regulation ◽  
Brd4 Inhibition

AbstractPathological cardiac hypertrophy is driven by neurohormonal activation of specific G protein-coupled receptors (GPCRs) in cardiomyocytes and is accompanied by large-scale changes in cardiomyocyte gene expression. These transcriptional changes require activity of positive transcription elongation factor b (P-TEFb), which is recruited to target genes by the bromodomain protein Brd4 or the Super Elongation Complex (SEC). Here we describe GPCR-specific regulation of these P-TEFb complexes and a novel mechanism for activating Brd4 in primary neonatal rat cardiomyocytes. The SEC was required for the hypertrophic response downstream of either the α1-adrenergic receptor (α1-AR) or the endothelin receptor (ETR). In contrast, Brd4 inhibition selectively impaired the α1-AR response. This was corroborated by the finding that activation of α1-AR, but not ETR, increased Brd4 occupancy at promoters and super enhancers of hypertrophic genes. Transcriptome analysis demonstrated that activation of both receptors initiated similar gene expression programs, but that Brd4 inhibition attenuated hypertrophic genes more robustly following α1-AR activation. Finally, we show that protein kinase A (PKA) is required for α1-AR stimulation of Brd4 chromatin occupancy. The differential role of the Brd4/P-TEFb complex in response to distinct GPCR pathways has potential clinical implications as therapies targeting this complex are currently being explored for heart failure.

scholarly journals Mechanisms of enhancer action: the known and the unknown

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Anil Panigrahi ◽  
Bert W. O’Malley
Keyword(s):  
Gene Expression ◽  
Differential Gene Expression ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Cell Types ◽  
Regulatory Sequences ◽  
Quantitative Expression ◽  
Full Complement ◽  
Differential Gene ◽  
Target Promoters

AbstractDifferential gene expression mechanisms ensure cellular differentiation and plasticity to shape ontogenetic and phylogenetic diversity of cell types. A key regulator of differential gene expression programs are the enhancers, the gene-distal cis-regulatory sequences that govern spatiotemporal and quantitative expression dynamics of target genes. Enhancers are widely believed to physically contact the target promoters to effect transcriptional activation. However, our understanding of the full complement of regulatory proteins and the definitive mechanics of enhancer action is incomplete. Here, we review recent findings to present some emerging concepts on enhancer action and also outline a set of outstanding questions.

scholarly journals Epigenetic evidence of an Ac/Dc axis by VPA and SAHA

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Sebastian Lunke ◽  
Scott Maxwell ◽  
Ishant Khurana ◽  
Harikrishnan K.N. ◽  
Jun Okabe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Histone Acetylation ◽  
Histone Modifications ◽  
Target Genes ◽  
Hdac Inhibitors ◽  
Histone Deacetylation ◽  
Lysine Methylation ◽  
Blocking Voltage ◽  
Lysine Residues ◽  
Voltage Gated Ion Channels

Abstract Background Valproic acid (VPA) is one of the most commonly used anti-epileptic drugs with pharmacological actions on GABA and blocking voltage-gated ion channels. VPA also inhibits histone deacetylase (HDAC) activity. Suberoylanilide hydroxamic acid is also a member of a larger class of compounds that inhibit HDACs. At the time of this article, there are 123 active international clinical trials for VPA (also known as valproate, convulex, divalproex, and depakote) and SAHA (vorinostat, zolinza). While it is well known that VPA and SAHA influence the accumulation of acetylated lysine residues on histones, their true epigenetic complexity remains poorly understood. Results Primary human cells were exposed to VPA and SAHA to understand the extent of histone acetylation (H3K9/14ac) using chromatin immunoprecipitation followed by sequencing (ChIP-seq). Because histone acetylation is often associated with modification of lysine methylation, we also examined H3K4me3 and H3K9me3. To assess the influence of the HDAC inhibitors on gene expression, we used RNA sequencing (RNA-seq). ChIP-seq reveals a distribution of histone modifications that is robust and more broadly regulated than previously anticipated by VPA and SAHA. Histone acetylation is a characteristic of the pharmacological inhibitors that influenced gene expression. Surprisingly, we observed histone deacetylation by VPA stimulation is a predominant signature following SAHA exposure and thus defines an acetylation/deacetylation (Ac/Dc) axis. ChIP-seq reveals regionalisation of histone acetylation by VPA and broader deacetylation by SAHA. Independent experiments confirm H3K9/14 deacetylation of NFκB target genes by SAHA. Conclusions The results provide an important framework for understanding the Ac/Dc axis by highlighting a broader complexity of histone modifications by the most established and efficacious anti-epileptic medication in this class, VPA and comparison with the broad spectrum HDAC inhibitor, SAHA.

Exogenous application of RNAs as a silencing tool for discovering gene function.

2021 ◽  
pp. 14-24
Author(s):  
Barbara Molesini ◽  
Tiziana Pandolfini
Keyword(s):  
Gene Expression ◽  
Gene Function ◽  
Rna Silencing ◽  
Target Genes ◽  
Specific Gene ◽  
Transcriptional Level ◽  
Fruit Setting ◽  
Organ Tissue ◽  
Specific Regulation ◽  
Time Specific

Abstract This chapter focuses on the importance of the RNA silencing technique in unraveling the function of genes by inhibiting gene expression at the post-transcriptional level, and is particularly appropriate for studying developmental processes such as fruit setting and growth that require a tight organ/tissue and time-specific regulation of the expression of target genes. Some methods used for establishing the function of a specific gene altering gene expression at either the genomic or post-transcriptional level are also presented.

scholarly journals The ability of POU family transcription factors to activate or repress gene expression is dependent on the spacing and context of their specific response elements

1996 ◽  
Vol 314 (2) ◽  
pp. 439-443 ◽  
Author(s):  
Sally J. DAWSON ◽  
Yu-Zhen LIU ◽  
Bernd RODEL ◽  
Tarik MÖRÖY ◽  
David S. LATCHMAN
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Herpes Simplex ◽  
Binding Sites ◽  
Target Genes ◽  
Gene Promoter ◽  
Specific Response ◽  
Response Elements ◽  
Simplex Virus ◽  
Target Promoters

The different forms of the Oct-2 and Brn-3 POU family transcription factors can have distinct effects on their target promoters involving both the activation and repression of gene expression. To investigate the requirements for these effects we have altered both the context and spacing of the two TAATGARAT binding sites for these factors within the herpes simplex virus immediate-early 3 gene promoter. We show that the activation of this promoter by Brn-3a and its repression by Brn-3b is dependent on the correct spacing of these binding sites. In contrast, repression of the promoter by Oct-2.4 and Oct-2.5 is dependent on both the spacing and context of these sites with the requirements for repression by Oct-2.4 or Oct-2.5 being different. These effects are discussed in the context of the mechanisms by which POU factors activate or repress their target genes.

scholarly journals Hepatocyte-specific PPARA expression exclusively promotes agonist-induced cell proliferation without influence from nonparenchymal cells

2017 ◽  
Vol 312 (3) ◽  
pp. G283-G299 ◽  
Author(s):  
Chad N. Brocker ◽  
Jiang Yue ◽  
Donghwan Kim ◽  
Aijuan Qu ◽  
Jessica A. Bonzo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Lipid Metabolism ◽  
Target Genes ◽  
Elevated Serum ◽  
Hepatocyte Proliferation ◽  
Peroxisome Proliferator ◽  
Wild Type ◽  
Nonparenchymal Cells ◽  
Specific Regulation

Peroxisome proliferator-activated receptor-α (PPARA) is a nuclear transcription factor and key mediator of systemic lipid metabolism. Prolonged activation in rodents causes hepatocyte proliferation and hepatocellular carcinoma. Little is known about the contribution of nonparenchymal cells (NPCs) to PPARA-mediated cell proliferation. NPC contribution to PPARA agonist-induced hepatomegaly was assessed in hepatocyte ( Ppara△Hep)- and macrophage ( Ppara△Mac)-specific Ppara null mice. Mice were treated with the agonist Wy-14643 for 14 days, and response of conditional null mice was compared with conventional knockout mice ( Ppara−/−). Wy-14643 treatment caused weight loss and severe hepatomegaly in wild-type and Ppara△Mac mice, and histological analysis revealed characteristic hepatocyte swelling; Ppara△Hep and Ppara−/− mice were protected from these effects. Ppara△Mac serum chemistries, as well as aspartate aminotransferase and alanine aminotransferase levels, matched wild-type mice. Agonist-treated Ppara△Hep mice had elevated serum cholesterol, phospholipids, and triglycerides when compared with Ppara−/− mice, indicating a possible role for extrahepatic PPARA in regulating circulating lipid levels. BrdU labeling confirmed increased cell proliferation only in wild-type and Ppara△Mac mice. Macrophage PPARA disruption did not impact agonist-induced upregulation of lipid metabolism, cell proliferation, or DNA damage and repair-related gene expression, whereas gene expression was repressed in Ppara△Hep mice. Interestingly, downregulation of inflammatory cytokines IL-15 and IL-18 was dependent on macrophage PPARA. Cell type-specific regulation of target genes was confirmed in primary hepatocytes and Kupffer cells. These studies conclusively show that cell proliferation is mediated exclusively by PPARA activation in hepatocytes and that Kupffer cell PPARA has an important role in mediating the anti-inflammatory effects of PPARA agonists.

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