Correction: Temporal Dissection of Rate Limiting Transcriptional Events Using Pol II ChIP and RNA Analysis of Adrenergic Stress Gene Activation

AbstractThe coordinated regulation of gene expression at the transcriptional level is fundamental to organismal development and homeostasis. Inducible systems are invaluable when studying transcription because the regulatory process can be triggered instantaneously, allowing the tracking of ordered mechanistic events. Here, we use Precision Run-On sequencing (PRO-seq) to examine the genome-wide Heat Shock (HS) response in Drosophila and the function of two key transcription factors on the immediate transcription activation or repression of all genes regulated by HS. We identify the primary HS response genes and the rate-limiting steps in the transcription cycle that GAGA-Associated Factor (GAF) and HS Factor (HSF) regulate. We demonstrate that GAF acts upstream of promoter-proximally paused RNA Polymerase II (Pol II) formation, likely at the step of chromatin opening, and that GAF-facilitated Pol II pausing is critical for HS activation. In contrast, HSF is dispensable for establishing or maintaining Pol II pausing, but is critical for the release of paused Pol II into the gene body at a subset of highly-activated genes. Additionally, HSF has no detectable role in the rapid HS-repression of thousands of genes.

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The heat shock response: A case study of chromatin dynamics in gene regulation

Biochemistry and Cell Biology ◽

10.1139/bcb-2012-0075 ◽

2013 ◽

Vol 91 (1) ◽

pp. 42-48 ◽

Cited By ~ 11

Author(s):

Sheila S. Teves ◽

Steven Henikoff

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Heat Shock ◽

Heat Shock Response ◽

Control Of Gene Expression ◽

Pol Ii ◽

Chromatin Dynamics ◽

Regulatory Processes ◽

Shock Response ◽

Rate Limiting

Recent studies in transcriptional regulation using the Drosophila heat shock response system have elucidated many of the dynamic regulatory processes that govern transcriptional activation and repression. The classic view that the control of gene expression occurs at the point of RNA polymerase II (Pol II) recruitment is now giving way to a more complex outlook of gene regulation. Promoter chromatin dynamics coordinate with transcription factor binding to maintain the promoters of active genes accessible. For a large number of genes, the rate-limiting step in Pol II progression occurs during its initial elongation, where Pol II transcribes 30–50 bp and pauses for further signals. These paused genes have unique genic chromatin architecture and dynamics compared with genes where Pol II recruitment is rate limiting for expression. Further elongation of Pol II along the gene causes nucleosome turnover, a continuous process of eviction and replacement, which suggests a potential mechanism for Pol II transit along a nucleosomal template. In this review, we highlight recent insights into transcription regulation of the heat shock response and discuss how the dynamic regulatory processes involved at each transcriptional stage help to generate faithful yet highly responsive gene expression.

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CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide

Molecular and Cellular Biology ◽

10.1128/mcb.01993-06 ◽

2007 ◽

Vol 27 (5) ◽

pp. 1631-1648 ◽

Cited By ~ 104

Author(s):

Igor Chernukhin ◽

Shaharum Shamsuddin ◽

Sung Yun Kang ◽

Rosita Bergström ◽

Yoo-Wook Kwon ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Gene Activation ◽

Ctcf Binding ◽

Pol Ii ◽

Genome Wide ◽

Target Sites ◽

On Chip

ABSTRACT CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. “Serial” chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the β-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.

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Widespread Backtracking by RNA Pol II Is a Major Effector of Gene Activation, 5′ Pause Release, Termination, and Transcription Elongation Rate

Molecular Cell ◽

10.1016/j.molcel.2018.10.031 ◽

2019 ◽

Vol 73 (1) ◽

pp. 107-118.e4 ◽

Cited By ~ 21

Author(s):

Ryan M. Sheridan ◽

Nova Fong ◽

Angelo D’Alessandro ◽

David L. Bentley

Keyword(s):

Transcription Elongation ◽

Gene Activation ◽

Elongation Rate ◽

Rna Pol Ii ◽

Pol Ii

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Scalable control of developmental timetables by epigenetic switching networks

Journal of The Royal Society Interface ◽

10.1098/rsif.2021.0109 ◽

2021 ◽

Vol 18 (180) ◽

pp. 20210109

Author(s):

Phuc Nguyen ◽

Nicholas A. Pease ◽

Hao Yuan Kueh

Keyword(s):

Regulatory Networks ◽

Regulatory Gene ◽

Gene Activation ◽

Mathematical Framework ◽

Switching Networks ◽

Lineage Specification ◽

Differentiated Cells ◽

Population Sizes ◽

Gene Regulatory ◽

Rate Limiting

During development, progenitor cells follow timetables for differentiation that span many cell generations. These developmental timetables are robustly encoded by the embryo, yet scalably adjustable by evolution, facilitating variation in organism size and form. Epigenetic switches, involving rate-limiting activation steps at regulatory gene loci, control gene activation timing in diverse contexts, and could profoundly impact the dynamics of gene regulatory networks controlling developmental lineage specification. Here, we develop a mathematical framework to model regulatory networks with genes controlled by epigenetic switches. Using this framework, we show that such epigenetic switching networks uphold developmental timetables that robustly span many cell generations, and enable the generation of differentiated cells in precisely defined numbers and fractions. Changes to epigenetic switching networks can readily alter the timing of developmental events within a timetable, or alter the overall speed at which timetables unfold, enabling scalable control over differentiated population sizes. With their robust, yet flexibly adjustable nature, epigenetic switching networks could represent central targets on which evolution acts to manufacture diversity in organism size and form.

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RelA Ser276 Phosphorylation Is Required for Activation of a Subset of NF-κB-Dependent Genes by Recruiting Cyclin-Dependent Kinase 9/Cyclin T1 Complexes

Molecular and Cellular Biology ◽

10.1128/mcb.01152-07 ◽

2008 ◽

Vol 28 (11) ◽

pp. 3623-3638 ◽

Cited By ~ 119

Author(s):

David E. Nowak ◽

Bing Tian ◽

Mohammad Jamaluddin ◽

Istvan Boldogh ◽

Leoncio A. Vergara ◽

...

Keyword(s):

Gene Expression ◽

Target Gene ◽

Gene Activation ◽

Elongation Factor ◽

Genetic Network ◽

Transcriptional Elongation ◽

Cyclin Dependent Kinase ◽

Pol Ii ◽

Target Gene Expression ◽

Cyclin T1

ABSTRACT NF-κB plays a central role in cytokine-inducible inflammatory gene expression. Previously we empirically determined the identity of 92 members of the genetic network under direct NF-κB/RelA control that show marked heterogeneity in magnitude of transcriptional induction and kinetics of peak activation. To investigate this network further, we have applied a recently developed two-step chromatin immunoprecipitation assay that accurately reflects association and disassociation of RelA binding to its chromatin targets. Although inducible RelA binding occurs with similar kinetics on all NF-κB-dependent genes, serine 276 (Ser276)-phosphorylated RelA binding is seen primarily on a subset of genes that are rapidly induced by tumor necrosis factor (TNF), including Gro-β, interleukin-8 (IL-8), and IκBα. Previous work has shown that TNF-inducible RelA Ser276 phosphorylation is controlled by a reactive oxygen species (ROS)-protein kinase A signaling pathway. To further understand the role of phospho-Ser276 RelA in target gene expression, we inhibited its formation by ROS scavengers and antioxidants, treatments that disrupt phospho-Ser276 formation but not the translocation and DNA binding of nonphosphorylated RelA. Here we find that phospho-Ser276 RelA is required only for activation of IL-8 and Gro-β, with IκBα being unaffected. These data were confirmed in experiments using RelA−/− murine embryonic fibroblasts reconstituted with a RelA Ser276Ala mutation. In addition, we observe that phospho-Ser276 RelA binds the positive transcription elongation factor b (P-TEFb), a complex containing the cyclin-dependent kinase 9 (CDK-9) and cyclin T1 subunits. Inhibition of P-TEFb activity by short interfering RNA (siRNA)-mediated knockdown shows that the phospho-Ser276 RelA-P-TEFb complex is required for IL-8 and Gro-β gene activation but not for IκBα gene activation. These studies indicate that TNF induces target gene expression by heterogeneous mechanisms. One is mediated by phospho-Ser276 RelA formation and chromatin targeting of P-TEFb controlling polymerase II (Pol II) recruitment and carboxy-terminal domain phosphorylation on the IL-8 and Gro-β genes. The second involves a phospho-Ser276 RelA-independent activation of genes preloaded with Pol II, exemplified by the IκBα gene. Together, these data suggest that the binding kinetics, selection of genomic targets, and mechanisms of promoter induction by RelA are controlled by a phosphorylation code influencing its interactions with coactivators and transcriptional elongation factors.

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Progressive histone alterations and proinflammatory gene activation: consequences of heme protein/iron-mediated proximal tubule injury

AJP Renal Physiology ◽

10.1152/ajprenal.00683.2009 ◽

2010 ◽

Vol 298 (3) ◽

pp. F827-F837 ◽

Cited By ~ 20

Author(s):

Richard A. Zager ◽

Ali C. M. Johnson

Keyword(s):

Renal Failure ◽

Proximal Tubule ◽

Blood Urea Nitrogen ◽

Histone Modifications ◽

Gene Activation ◽

Mrna Levels ◽

Pol Ii ◽

Tnf Α ◽

Proinflammatory Gene ◽

Tgf Β1

Rhabdomyolysis (Fe)-induced acute renal failure (ARF) causes renal inflammation, and, with repetitive insults, progressive renal failure can result. To gain insights into these phenomena, we assessed the impact of a single episode of glycerol-induced rhabdomyolysis on proinflammatory/profibrotic [TNF-α, monocyte chemoattractant protein-1 (MCP-1), and transforming growth factor-β1 (TGF-β1)] gene expression and the time course of these changes. CD-1 mice were studied 1–7 days after glycerol injection. Normal mice served as controls. RNA polymerase II (Pol II) binding to the TNF-α, MCP-1, and TGF-β1 genes, “gene-activating” histone modifications [histone 3 lysine 4 (H3K4) trimethylation (H3K4m3) and histone 2 variant H2A.Z], and cognate mRNA levels were assessed. Results were contrasted to changes in anti-inflammatory heme oxygenase-1 (HO-1). Glycerol produced severe ARF (blood urea nitrogen ∼150–180 mg/dl) followed by marked improvement by day 7 (blood urea nitrogen ∼40 mg/dl). Early increases in TNF-α, MCP-1, and TGF-β1 mRNAs, Pol II gene binding, and H3K4m3/H2A.Z levels were observed. These progressed with time, despite resolution of azotemia. Comparable early HO-1 changes were observed. However, HO-1 mRNA normalized by day 7, and progressive Pol II binding/histone alterations did not occur. Fe-mediated injury to cultured proximal tubule (HK-2) cells recapitulated these in vivo results. Hence, this in vitro model was used for mechanistic assessments. On the basis of these studies, it was determined that 1) the H3K4m3/H2A.Z increases are early events (i.e., they precede mRNA increases), 2) subsequent mRNA elevations reflect transcription, not mRNA stabilization (actinomycin D assessments), and 3) increased transcription, per se, helps sustain elevated H2A.Z levels. We conclude that 1) Fe/glycerol-induced tubular injury causes sustained proinflammatory gene activation, 2) decreasing HO-1 expression, as reflected by mRNA levels, may facilitate this proinflammatory state, and 3) gene-activating histone modifications are early injury events and progressively increase at selected proinflammatory genes. Thus they may help sustain a proinflammatory state, despite resolving ARF.

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Framework estimation of stochastic gene activation using transcription average level

10.1101/2021.09.01.458497 ◽

2021 ◽

Author(s):

Liang Chen ◽

Genghong Lin ◽

Feng Jiao

Keyword(s):

Single Cell ◽

Gene Activation ◽

Mouse Fibroblast ◽

Dynamic Features ◽

Efficient Procedure ◽

Tumor Necrosis Factor Induction ◽

Transcription Data ◽

Rate Limiting ◽

Necrosis Factor ◽

Rule Out

Gene activation is usually a non-Markovian process that has been modeled as various frameworks that consist of multiple rate-limiting steps. Understanding the exact activation framework for a gene of interest is a central problem for single-cell studies. In this paper, we focus on the dynamical data of the average transcription level M(t), which is typically neglected when deciphering gene activation. Firstly, the smooth trend lines of M(t) data present rich, visually dynamic features. Secondly, tractable analysis of M(t) allows the establishment of bijections between M(t) dynamics and system parameter regions. Because of these two clear advantages, we can rule out frameworks that fail to capture M(t) features and we can further test potential competent frameworks by fitting M(t) data. We implemented this procedure to determine an exact activation framework for a large number of mouse fibroblast genes under tumor necrosis factor induction; the cross-talk between the signaling and basal pathways is crucial to trigger the first peak of M(t), while the following damped gentle M(t) oscillation is regulated by the multi-step basal pathway. Moreover, the fitted parameters for the mouse genes tested revealed two distinct regulation scenarios for transcription dynamics. Taken together, we were able to develop an efficient procedure for using traditional M(t) data to estimate the gene activation frameworks and system parameters. This procedure, together with sophisticated single-cell transcription data, may facilitate a more accurate understanding of stochastic gene activation.

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BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ

10.1101/2020.12.12.422337 ◽

2020 ◽

Author(s):

Jaehyeon Jeong ◽

Keunsoo Kang ◽

Doo Sin Jo ◽

Anh T Cong ◽

Donguk Kim ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Gene Activation ◽

Strand Break ◽

Protein Coding ◽

Pol Ii ◽

Dna Strand Break ◽

Biochemical Analyses ◽

Dna Strand ◽

Inducible Genes

RNA polymerase II (Pol II)-dependent transcription in stimulus-inducible genes requires topoisomerase IIβ (TOP2B)-mediated DNA strand break and the activation of DNA damage response signaling in humans. Here, we report a novel function of the breast cancer 1 (BRCA1)-BRCA1 associated ring domain 1 (BARD1) complex, in this process. We found that BRCA1 is phosphorylated at S1524 by the kinases ATM and ATR during gene activation and that this event is essential for productive transcription. Our in vitro biochemical analyses showed TOP2B and BARD1 interaction and colocalization in the EGR1 transcription start site (TSS) and that the BRCA1-BARD1 complex ubiquitinates TOP2B, which appears to stabilize TOP2B protein in the cell and binding to DNA. Intriguingly, BRCA1 phosphorylation at S1524 controls this interaction. In addition, genomic analyses indicated colocalization between TOP2B and BRCA1 in a large number of protein-coding genes. Together, these findings reveal the novel function of the BRCA1-BARD1 complex in gene expression and in the regulation of TOP2B during Pol II transcription.

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