Single nucleosome imaging reveals loose genome chromatin networks via active RNA polymerase II

Although chromatin organization and dynamics play a critical role in gene transcription, how they interplay remains unclear. To approach this issue, we investigated genome-wide chromatin behavior under various transcriptional conditions in living human cells using single-nucleosome imaging. While transcription by RNA polymerase II (RNAPII) is generally thought to need more open and dynamic chromatin, surprisingly, we found that active RNAPII globally constrains chromatin movements. RNAPII inhibition or its rapid depletion released the chromatin constraints and increased chromatin dynamics. Perturbation experiments of P-TEFb clusters, which are associated with active RNAPII, had similar results. Furthermore, chromatin mobility also increased in resting G0 cells and UV-irradiated cells, which are transcriptionally less active. Our results demonstrated that chromatin is globally stabilized by loose connections through active RNAPII, which is compatible with models of classical transcription factories or liquid droplet formation of transcription-related factors. Together with our computational modeling, we propose the existence of loose chromatin domain networks for various intra-/interchromosomal contacts via active RNAPII clusters/droplets.

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Ligand-induced native G-quadruplex stabilization impairs transcription initiation

Genome Research ◽

10.1101/gr.275431.121 ◽

2021 ◽

Author(s):

Conghui Li ◽

Honghong Wang ◽

Zhinang Yin ◽

Pingping Fang ◽

Ruijing Xiao ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Gene Transcription ◽

Transcription Initiation ◽

Regulatory Elements ◽

Gene Promoters ◽

Drug Induced ◽

Transcriptional Regulatory Elements ◽

Genome Wide ◽

Self Association

G-quadruplexes (G4s) are noncanonical DNA secondary structures formed through the self-association of guanines, and G4s are distributed widely across the genome. G4 participates in multiple biological processes including gene transcription, and G4-targeted ligands serve as potential therapeutic agents for DNA-targeted therapies. However, genome-wide studies of the exact roles of G4s in transcriptional regulation are still lacking. Here, we establish a sensitive G4-CUT&Tag method for genome-wide profiling of native G4s with high resolution and specificity. We find that native G4 signals are cell type–specific and are associated with transcriptional regulatory elements carrying active epigenetic modifications. Drug-induced promoter-proximal RNA polymerase II pausing promotes nearby G4 formation. In contrast, G4 stabilization by G4-targeted ligands globally reduces RNA polymerase II occupancy at gene promoters as well as nascent RNA synthesis. Moreover, ligand-induced G4 stabilization modulates chromatin states and impedes transcription initiation via inhibition of general transcription factors loading to promoters. Together, our study reveals a reciprocal genome-wide regulation between native G4 dynamics and gene transcription, which will deepen our understanding of G4 biology toward therapeutically targeting G4s in human diseases.

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Faculty Opinions recommendation of Genome-wide analyses reveal RNA polymerase II located upstream of genes poised for rapid response upon S. cerevisiae stationary phase exit.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1025401.300733 ◽

2005 ◽

Author(s):

Michael Meisterernst

Keyword(s):

Rna Polymerase ◽

Stationary Phase ◽

Rna Polymerase Ii ◽

Rapid Response ◽

Genome Wide

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The roles of nucleolar structure and function in the subcellular location of the HIV-1 Rev protein

Journal of Cell Science ◽

10.1242/jcs.108.8.2811 ◽

1995 ◽

Vol 108 (8) ◽

pp. 2811-2823 ◽

Cited By ~ 3

Author(s):

M. Dundr ◽

G.H. Leno ◽

M.L. Hammarskjold ◽

D. Rekosh ◽

C. Helga-Maria ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Critical Role ◽

Regulation Of Expression ◽

Rna Transcription ◽

Nucleolar Structure ◽

Class 1 ◽

Protein B23 ◽

Hiv 1 ◽

Rev Protein

The human immunodeficiency virus 1 (HIV-1) Rev transactivator protein plays a critical role in the regulation of expression of structural proteins by controlling the pathway of mRNA transport. The Rev protein is located predominantly in the nucleoli of HIV-1 infected or Rev-expressing cells. Previous studies demonstrated that the Rev protein forms a specific complex in vitro with protein B23 which is suggested to be a nucleolar receptor and/or carrier for the Rev protein. To study the role of the nucleolus and nucleolar proteins in Rev function, transfected COS-7 or transformed CMT3 cells expressing the Rev protein were examined for subcellular locations of Rev and other proteins using indirect immunofluorescence and immunoelectron microscopy. One day after transfection the Rev protein was found in most cells only in the nucleolar dense fibrillar and granular components where it colocalized with protein B23. These were designated class 1 cells. In a second class of cells Rev and B23 accumulated in the nucleoplasm as well as in nucleoli. Treatment of class 1 cells with actinomycin D (AMD) under conditions that blocked only RNA polymerase I transcription caused Rev to completely redistribute from nucleoli to the cytoplasm. Simultaneously, protein B23 was partially released from nucleoli, mostly into the nucleoplasm, with detectable amounts in the cytoplasm. In cells recovering from AMD treatment in the presence of cycloheximide Rev and B23 showed coincident relocation to nucleoli. Class 2 cells were resistant to AMD-induced Rev redistribution. Selective inhibition of RNA polymerase II transcription by alpha-amanitin or by DRB did not cause Rev to be released into the cytoplasm suggesting that active preribosomal RNA transcription is required for the nucleolar location of Rev. However, treatment with either of the latter two drugs at higher doses and for longer times caused partial disruption of nucleoli accompanied by translocation of the Rev protein to the cytoplasm. These results suggest that the nucleolar location of Rev depends on continuous preribosomal RNA transcription and a substantially intact nucleolar structure.

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Genome-wide regulations of the pre-initiation complex formation and elongating RNA polymerase II by an E3 ubiquitin ligase, San1.

Molecular and Cellular Biology ◽

10.1128/mcb.00368-21 ◽

2021 ◽

Author(s):

Priyanka Barman ◽

Rwik Sen ◽

Amala Kaja ◽

Jannatul Ferdoush ◽

Shalini Guha ◽

...

Keyword(s):

Quality Control ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Ubiquitin Ligase ◽

Nuclear Protein ◽

Protein Quality ◽

Initiation Complex ◽

Pol Ii ◽

Intrinsically Disordered ◽

Genome Wide

San1 ubiquitin ligase is involved in nuclear protein quality control via its interaction with intrinsically disordered proteins for ubiquitylation and proteasomal degradation. Since several transcription/chromatin regulatory factors contain intrinsically disordered domains and can be inhibitory to transcription when in excess, San1 might be involved in transcription regulation. To address this, we analyzed the role of San1 in genome-wide association of TBP [that nucleates pre-initiation complex (PIC) formation for transcription initiation] and RNA polymerase II (Pol II). Our results reveal the roles of San1 in regulating TBP recruitment to the promoters and Pol II association with the coding sequences, and hence PIC formation and coordination of elongating Pol II, respectively. Consistently, transcription is altered in the absence of San1. Such transcriptional alteration is associated with impaired ubiquitylation and proteasomal degradation of Spt16 and gene association of Paf1, but not the incorporation of centromeric histone, Cse4, into the active genes in Δsan1 . Collectively, our results demonstrate distinct functions of a nuclear protein quality control factor in regulating the genome-wide PIC formation and elongating Pol II (and hence transcription), thus unraveling new gene regulatory mechanisms.

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Mammalian RNA polymerase II core promoters: insights from genome-wide studies

Nature Reviews Genetics ◽

10.1038/nrg2026 ◽

2007 ◽

Vol 8 (6) ◽

pp. 424-436 ◽

Cited By ~ 314

Author(s):

Albin Sandelin ◽

Piero Carninci ◽

Boris Lenhard ◽

Jasmina Ponjavic ◽

Yoshihide Hayashizaki ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Core Promoters ◽

Genome Wide

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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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The telomeric Cdc13–Stn1–Ten1 complex regulates RNA polymerase II transcription

Nucleic Acids Research ◽

10.1093/nar/gkz279 ◽

2019 ◽

Vol 47 (12) ◽

pp. 6250-6268 ◽

Cited By ~ 2

Author(s):

Olga Calvo ◽

Nathalie Grandin ◽

Antonio Jordán-Pla ◽

Esperanza Miñambres ◽

Noelia González-Polo ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Genome Stability ◽

Elongation Factor ◽

Yeast Saccharomyces Cerevisiae ◽

Replication Fork Progression ◽

Genome Wide ◽

Length Regulation ◽

Transcription Regulators ◽

Rna Polymerase Ii Transcription

Abstract Specialized telomeric proteins have an essential role in maintaining genome stability through chromosome end protection and telomere length regulation. In the yeast Saccharomyces cerevisiae, the evolutionary conserved CST complex, composed of the Cdc13, Stn1 and Ten1 proteins, largely contributes to these functions. Here, we report genetic interactions between TEN1 and several genes coding for transcription regulators. Molecular assays confirmed this novel function of Ten1 and further established that it regulates the occupancies of RNA polymerase II and the Spt5 elongation factor within transcribed genes. Since Ten1, but also Cdc13 and Stn1, were found to physically associate with Spt5, we propose that Spt5 represents the target of CST in transcription regulation. Moreover, CST physically associates with Hmo1, previously shown to mediate the architecture of S-phase transcribed genes. The fact that, genome-wide, the promoters of genes down-regulated in the ten1-31 mutant are prefentially bound by Hmo1, leads us to propose a potential role for CST in synchronizing transcription with replication fork progression following head-on collisions.

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