scholarly journals Dynamic and Selective Low-Complexity Domain Interactions Revealed by Live-Cell Single-Molecule Imaging

2017 ◽  
Author(s):  
Shasha Chong ◽  
Claire Dugast-Darzacq ◽  
Zhe Liu ◽  
Peng Dong ◽  
Gina M. Dailey ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Low Complexity ◽  
Live Cell ◽  
Single Molecule Imaging ◽  
High Concentration ◽  
Pol Ii ◽  
Intrinsically Disordered ◽  
Dynamic Display

AbstractMany eukaryotic transcription factors (TFs) contain intrinsically disordered low-complexity domains (LCDs) but how they perform transactivation functions remains unclear. Recent studies report that TF-LCDs can undergo hydrogel formation or liquid-liquid phase separation in vitro. Here, live-cell single-molecule imaging reveals that TF-LCDs form local high concentration interaction hubs at synthetic and endogenous genomic loci. TF-LCD hubs stabilize DNA binding, recruit RNA polymerase II (Pol II) and activate transcription. LCD-LCD interactions within hubs are highly dynamic, display selectivity with binding partners, and are differentially sensitive to disruption by hexanediols. These findings suggest that under physiological conditions, rapid reversible and multivalent LCD-LCD interactions occur between TFs and the Pol II machinery, which underpins a central mechanism for transactivation and plays a key role in gene expression and disease.

scholarly journals Imaging dynamic and selective low-complexity domain interactions that control gene transcription

Science ◽  
2018 ◽  
Vol 361 (6400) ◽  
pp. eaar2555 ◽  
Author(s):  
Shasha Chong ◽  
Claire Dugast-Darzacq ◽  
Zhe Liu ◽  
Peng Dong ◽  
Gina M. Dailey ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Transcriptional Control ◽  
Low Complexity ◽  
Single Molecule Imaging ◽  
High Concentration ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Intrinsically Disordered ◽  
Dynamic Display

Many eukaryotic transcription factors (TFs) contain intrinsically disordered low-complexity sequence domains (LCDs), but how these LCDs drive transactivation remains unclear. We used live-cell single-molecule imaging to reveal that TF LCDs form local high-concentration interaction hubs at synthetic and endogenous genomic loci. TF LCD hubs stabilize DNA binding, recruit RNA polymerase II (RNA Pol II), and activate transcription. LCD-LCD interactions within hubs are highly dynamic, display selectivity with binding partners, and are differentially sensitive to disruption by hexanediols. Under physiological conditions, rapid and reversible LCD-LCD interactions occur between TFs and the RNA Pol II machinery without detectable phase separation. Our findings reveal fundamental mechanisms underpinning transcriptional control and suggest a framework for developing single-molecule imaging screens for drugs targeting gene regulatory interactions implicated in disease.

scholarly journals RNA polymerase II clustering through CTD phase separation

2018 ◽  
Author(s):  
M. Boehning ◽  
C. Dugast-Darzacq ◽  
M. Rankovic ◽  
A. S. Hansen ◽  
T. Yu ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Low Complexity ◽  
Initiation Factor ◽  
Published Data ◽  
Pol Ii ◽  
Intrinsically Disordered ◽  
Ctd Phosphorylation

The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is an intrinsically disordered low-complexity region that is critical for pre-mRNA transcription and processing. The CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast. Here we report that human and yeast CTDs undergo cooperative liquid phase separation at increasing protein concentration, with the shorter yeast CTD forming less stable droplets. In human cells, truncation of the CTD to the length of the yeast CTD decreases Pol II clustering and chromatin association whereas CTD extension has the opposite effect. CTD droplets can incorporate intact Pol II and are dissolved by CTD phosphorylation with the transcription initiation factor IIH kinase CDK7. Together with published data, our results suggest that Pol II forms clusters/hubs at active genes through interactions between CTDs and with activators, and that CTD phosphorylation liberates Pol II enzymes from hubs for promoter escape and transcription elongation.

Release of human TFIIB from actively transcribing complexes is triggered upon synthesis of 7 nt and 9 nt RNAs

2019 ◽  
Author(s):  
Elina Ly ◽  
Abigail E. Powell ◽  
James A. Goodrich ◽  
Jennifer F. Kugel
Keyword(s):  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Trigger Points ◽  
Pol Ii ◽  
Transcription System ◽  
Recognition Element ◽  
General Transcription Factors ◽  
Early Transcription ◽  
Promoter Dna

AbstractRNA polymerase II (Pol II) and its general transcription factors assemble on the promoters of mRNA genes to form large macromolecular complexes that initiate transcription in a regulated manner. During early transcription these complexes undergo dynamic rearrangement and disassembly as Pol II moves away from the start site of transcription and transitions into elongation. One step in disassembly is the release of the general transcription factor TFIIB, although the mechanism of release and its relationship to the activity of transcribing Pol II is not understood. We developed a single molecule fluorescence transcription system to investigate TFIIB release in vitro. Leveraging our ability to distinguish active from inactive complexes, we found that nearly all transcriptionally active complexes release TFIIB during early transcription. Release is not dependent on the contacts TFIIB makes with its recognition element in promoter DNA. We identified two different points in early transcription at which release is triggered, reflecting heterogeneity across the population of actively transcribing complexes. TFIIB releases after both trigger points with similar kinetics, suggesting the rate of release is independent of the molecular transformations that prompt release. Together our data support the model that TFIIB release is important to maintain the transcriptional activity of Pol II as initiating complexes transition into elongation complexes.

scholarly journals Cockayne syndrome B protein acts as an ATP-dependent processivity factor that helps RNA polymerase II overcome nucleosome barriers

2020 ◽  
Vol 117 (41) ◽  
pp. 25486-25493 ◽  
Author(s):  
Jun Xu ◽  
Wei Wang ◽  
Liang Xu ◽  
Jia-Yu Chen ◽  
Jenny Chong ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Neurological Diseases ◽  
Transcription Elongation ◽  
Cockayne Syndrome ◽  
Stable Complex ◽  
Loss Of Function ◽  
Pol Ii ◽  
Chromatin Remodelers ◽  
Processivity Factor

While loss-of-function mutations in Cockayne syndrome group B protein (CSB) cause neurological diseases, this unique member of the SWI2/SNF2 family of chromatin remodelers has been broadly implicated in transcription elongation and transcription-coupled DNA damage repair, yet its mechanism remains largely elusive. Here, we use a reconstituted in vitro transcription system with purified polymerase II (Pol II) and Rad26, a yeast ortholog of CSB, to study the role of CSB in transcription elongation through nucleosome barriers. We show that CSB forms a stable complex with Pol II and acts as an ATP-dependent processivity factor that helps Pol II across a nucleosome barrier. This noncanonical mechanism is distinct from the canonical modes of chromatin remodelers that directly engage and remodel nucleosomes or transcription elongation factors that facilitate Pol II nucleosome bypass without hydrolyzing ATP. We propose a model where CSB facilitates gene expression by helping Pol II bypass chromatin obstacles while maintaining their structures.

scholarly journals Phase-Separated Transcriptional Condensates Accelerate Target-Search Process Revealed by Live-Cell Single-Molecule Imaging

Cell Reports ◽  
2020 ◽  
Vol 33 (2) ◽  
pp. 108248 ◽  
Author(s):  
Samantha Kent ◽  
Kyle Brown ◽  
Chou-hsun Yang ◽  
Njood Alsaihati ◽  
Christina Tian ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cell ◽  
Search Process ◽  
Single Molecule Imaging ◽  
Target Search

scholarly journals Direct Interaction between the Subunit RAP30 of Transcription Factor IIF (TFIIF) and RNA Polymerase Subunit 5, Which Contributes to the Association between TFIIF and RNA Polymerase II

2001 ◽  
Vol 276 (15) ◽  
pp. 12266-12273 ◽  
Author(s):  
Wenxiang Wei ◽  
Dorjbal Dorjsuren ◽  
Yong Lin ◽  
Weiping Qin ◽  
Takahiro Nomura ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Middle Part ◽  
Wild Type ◽  
Binding Region ◽  
Pol Ii ◽  
Transcription Factor Iif ◽  
B Virus

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30in vitrousing purified recombinant proteins andin vivoin COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47–120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101–170) and the N-terminus (aa 1–100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding inin vitroandin vivoassays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.

scholarly journals Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Linda S. Forero-Quintero ◽  
William Raymond ◽  
Tetsuya Handa ◽  
Matthew N. Saxton ◽  
Tatsuya Morisaki ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Computational Models ◽  
Spatial Organization ◽  
Fluorescent Antibody ◽  
Single Gene ◽  
Single Copy ◽  
Living Cells ◽  
Live Cell

AbstractThe carboxyl-terminal domain of RNA polymerase II (RNAP2) is phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to different phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as Serine 5 phosphorylated RNAP2 clusters that remain separated in both space and time from nascent mRNA synthesis. Computational models determine that 5 to 40 RNAP2 cluster around the promoter during a typical transcriptional burst, with most phosphorylated at Serine 5 within 6 seconds of arrival and roughly half escaping the promoter in ~1.5 minutes. Taken together, our data provide live-cell support for the notion of efficient transcription clusters that transiently form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.

scholarly journals Regulation of P-TEFb Elongation Complex Activity by CDK9 Acetylation

2007 ◽  
Vol 27 (13) ◽  
pp. 4641-4651 ◽  
Author(s):  
Junjiang Fu ◽  
Ho-Geun Yoon ◽  
Jun Qin ◽  
Jiemin Wong
Keyword(s):  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Transcriptional Elongation ◽  
Elongation Complex ◽  
Complex Activity ◽  
Interacting Protein ◽  
Pol Ii ◽  
Carboxy Terminal

ABSTRACT P-TEFb, comprised of CDK9 and a cyclin T subunit, is a global transcriptional elongation factor important for most RNA polymerase II (pol II) transcription. P-TEFb facilitates transcription elongation in part by phosphorylating Ser2 of the heptapeptide repeat of the carboxy-terminal domain (CTD) of the largest subunit of pol II. Previous studies have shown that P-TEFb is subjected to negative regulation by forming an inactive complex with 7SK small RNA and HEXIM1. In an effort to investigate the molecular mechanism by which corepressor N-CoR mediates transcription repression, we identified HEXIM1 as an N-CoR-interacting protein. This finding led us to test whether the P-TEFb complex is regulated by acetylation. We demonstrate that CDK9 is an acetylated protein in cells and can be acetylated by p300 in vitro. Through both in vitro and in vivo assays, we identified lysine 44 of CDK9 as a major acetylation site. We present evidence that CDK9 is regulated by N-CoR and its associated HDAC3 and that acetylation of CDK9 affects its ability to phosphorylate the CTD of pol II. These results suggest that acetylation of CDK9 is an important posttranslational modification that is involved in regulating P-TEFb transcriptional elongation function.

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

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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