scholarly journals ENL initiates multivalent phase separation of the super elongation complex (SEC) in controlling rapid transcriptional activation

2020 ◽  
Vol 6 (14) ◽  
pp. eaay4858 ◽  
Author(s):  
Chenghao Guo ◽  
Zhuanzhuan Che ◽  
Junjie Yue ◽  
Peng Xie ◽  
Shaohua Hao ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Liquid Droplet ◽  
Critical Role ◽  
Elongation Factor ◽  
Elongation Complex ◽  
Intrinsically Disordered ◽  
Super Elongation Complex

Release of paused RNA polymerase II (Pol II) requires incorporation of the positive transcription elongation factor b (P-TEFb) into the super elongation complex (SEC), thus resulting in rapid yet synchronous transcriptional activation. However, the mechanism underlying dynamic transition of P-TEFb from inactive to active state remains unclear. Here, we found that the SEC components are able to compartmentalize and concentrate P-TEFb via liquid-liquid phase separation from the soluble inactive HEXIM1 containing the P-TEFb complex. Specifically, ENL or its intrinsically disordered region is sufficient to initiate the liquid droplet formation of SEC. AFF4 functions together with ENL in fluidizing SEC droplets. SEC droplets are fast and dynamically formed upon serum exposure and required for rapid transcriptional induction. We also found that the fusion of ENL with MLL can boost SEC phase separation. In summary, our results suggest a critical role of multivalent phase separation of SEC in controlling transcriptional pause release.

scholarly journals Experimental Evidence of Intrinsic Disorder and Amyloid Formation by the Henipavirus W Proteins

2022 ◽  
Vol 23 (2) ◽  
pp. 923
Author(s):  
Giulia Pesce ◽  
Frank Gondelaud ◽  
Denis Ptchelkine ◽  
Juliet F. Nilsson ◽  
Christophe Bignon ◽  
...  
Keyword(s):  
Phase Separation ◽  
Critical Role ◽  
Limited Proteolysis ◽  
Size Exclusion ◽  
Amyloid Formation ◽  
Type I ◽  
Human Pathogens ◽  
Intrinsically Disordered ◽  
P Gene

Henipaviruses are severe human pathogens within the Paramyxoviridae family. Beyond the P protein, the Henipavirus P gene also encodes the V and W proteins which share with P their N-terminal, intrinsically disordered domain (NTD) and possess a unique C-terminal domain. Henipavirus W proteins antagonize interferon (IFN) signaling through NTD-mediated binding to STAT1 and STAT4, and prevent type I IFN expression and production of chemokines. Structural and molecular information on Henipavirus W proteins is lacking. By combining various bioinformatic approaches, we herein show that the Henipaviruses W proteins are predicted to be prevalently disordered and yet to contain short order-prone segments. Using limited proteolysis, differential scanning fluorimetry, analytical size exclusion chromatography, far-UV circular dichroism and small-angle X-ray scattering, we experimentally confirmed their overall disordered nature. In addition, using Congo red and Thioflavin T binding assays and negative-staining transmission electron microscopy, we show that the W proteins phase separate to form amyloid-like fibrils. The present study provides an additional example, among the few reported so far, of a viral protein forming amyloid-like fibrils, therefore significantly contributing to enlarge our currently limited knowledge of viral amyloids. In light of the critical role of the Henipavirus W proteins in evading the host innate immune response and of the functional role of phase separation in biology, these studies provide a conceptual asset to further investigate the functional impact of the phase separation abilities of the W proteins.

Allosteric transcription stimulation by RNA polymerase II super elongation complex

2021 ◽  
Author(s):  
Ying Chen ◽  
Seychelle M. Vos ◽  
Christian Dienemann ◽  
Momchil Ninov ◽  
Henning Urlaub ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Complex ◽  
Super Elongation Complex

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.

scholarly journals Mediator subunit MED1 is required for E2A-PBX1–mediated oncogenic transcription and leukemic cell growth

2021 ◽  
Vol 118 (6) ◽  
pp. e1922864118 ◽  
Author(s):  
Yu-Ling Lee ◽  
Keiichi Ito ◽  
Wen-Chieh Pi ◽  
I-Hsuan Lin ◽  
Chi-Shuen Chu ◽  
...  
Keyword(s):  
Cell Growth ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Critical Role ◽  
Gene Activation ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Specific Gene ◽  
Aberrant Expression ◽  
Transcriptional Machinery

The chimeric transcription factor E2A-PBX1, containing the N-terminal activation domains of E2A fused to the C-terminal DNA-binding domain of PBX1, results in 5% of pediatric acute lymphoblastic leukemias (ALL). We recently have reported a mechanism for RUNX1-dependent recruitment of E2A-PBX1 to chromatin in pre-B leukemic cells; but the subsequent E2A-PBX1 functions through various coactivators and the general transcriptional machinery remain unclear. The Mediator complex plays a critical role in cell-specific gene activation by serving as a key coactivator for gene-specific transcription factors that facilitates their function through the RNA polymerase II transcriptional machinery, but whether Mediator contributes to aberrant expression of E2A-PBX1 target genes remains largely unexplored. Here we show that Mediator interacts directly with E2A-PBX1 through an interaction of the MED1 subunit with an E2A activation domain. Results of MED1 depletion by CRISPR/Cas9 further indicate that MED1 is specifically required for E2A-PBX1–dependent gene activation and leukemic cell growth. Integrated transcriptome and cistrome analyses identify pre-B cell receptor and cell cycle regulatory genes as direct cotargets of MED1 and E2A-PBX1. Notably, complementary biochemical analyses also demonstrate that recruitment of E2A-PBX1 to a target DNA template involves a direct interaction with DNA-bound RUNX1 that can be further stabilized by EBF1. These findings suggest that E2A-PBX1 interactions with RUNX1 and MED1/Mediator are of functional importance for both gene-specific transcriptional activation and maintenance of E2A-PBX1–driven leukemia. The MED1 dependency for E2A-PBX1–mediated gene activation and leukemogenesis may provide a potential therapeutic opportunity by targeting MED1 in E2A-PBX1+ pre-B leukemia.

scholarly journals Transient DNA Binding Induces RNA Polymerase II Compartmentalization During Herpesviral Infection Distinct From Phase Separation

2018 ◽  
Author(s):  
David T McSwiggen ◽  
Anders S Hansen ◽  
Hervé Marie-Nelly ◽  
Sheila Teves ◽  
Alec B Heckert ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Protein Interactions ◽  
Viral Dna ◽  
Herpes Simplex Virus 1 ◽  
Pol Ii ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Simplex Virus

SummaryDuring lytic infection, Herpes Simplex Virus 1 generates replication compartments (RCs) in host nuclei that efficiently recruit protein factors, including host RNA Polymerase II (Pol II). Pol II and other cellular factors form hubs in uninfected cells that are proposed to phase separate via multivalent protein-protein interactions mediated by their intrinsically disordered regions. Using a battery of live cell microscopic techniques, we show that although RCs superficially exhibit many characteristics of phase separation, the recruitment of Pol II instead derives from nonspecific interactions with the viral DNA. We find that the viral genome remains nucleosome-free, profoundly affecting the way Pol II explores RCs by causing it to repetitively visit nearby binding sites, thereby creating local Pol II accumulations. This mechanism, distinct from phase separation, allows viral DNA to outcompete host DNA for cellular proteins. Our work provides new insights into the strategies used to create local molecular hubs in cells.

scholarly journals The AFF4 scaffold binds human P-TEFb adjacent to HIV Tat

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Ursula Schulze-Gahmen ◽  
Heather Upton ◽  
Andrew Birnberg ◽  
Katherine Bao ◽  
Seemay Chou ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Regulatory Proteins ◽  
Tat Protein ◽  
Elongation Complex ◽  
Cyclin T1 ◽  
Subunit Interface ◽  
Hiv Tat ◽  
Gene Transcripts ◽  
Hiv 1

Human positive transcription elongation factor b (P-TEFb) phosphorylates RNA polymerase II and regulatory proteins to trigger elongation of many gene transcripts. The HIV-1 Tat protein selectively recruits P-TEFb as part of a super elongation complex (SEC) organized on a flexible AFF1 or AFF4 scaffold. To understand this specificity and determine if scaffold binding alters P-TEFb conformation, we determined the structure of a tripartite complex containing the recognition regions of P-TEFb and AFF4. AFF4 meanders over the surface of the P-TEFb cyclin T1 (CycT1) subunit but makes no stable contacts with the CDK9 kinase subunit. Interface mutations reduced CycT1 binding and AFF4-dependent transcription. AFF4 is positioned to make unexpected direct contacts with HIV Tat, and Tat enhances P-TEFb affinity for AFF4. These studies define the mechanism of scaffold recognition by P-TEFb and reveal an unanticipated intersubunit pocket on the AFF4 SEC that potentially represents a target for therapeutic intervention against HIV/AIDS.

Crystal structure of the human transcription elongation factor DSIF hSpt4 subunit in complex with the hSpt5 dimerization interface

2009 ◽  
Vol 425 (2) ◽  
pp. 373-380 ◽  
Author(s):  
Sabine Wenzel ◽  
Berta M. Martins ◽  
Paul Rösch ◽  
Birgitta M. Wöhrl
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Structural Similarity ◽  
Transcription Elongation Factor

The eukaryotic transcription elongation factor DSIF [DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole) sensitivity-inducing factor] is composed of two subunits, hSpt4 and hSpt5, which are homologous to the yeast factors Spt4 and Spt5. DSIF is involved in regulating the processivity of RNA polymerase II and plays an essential role in transcriptional activation of eukaryotes. At several eukaryotic promoters, DSIF, together with NELF (negative elongation factor), leads to promoter-proximal pausing of RNA polymerase II. In the present paper we describe the crystal structure of hSpt4 in complex with the dimerization region of hSpt5 (amino acids 176–273) at a resolution of 1.55 Å (1 Å=0.1 nm). The heterodimer shows high structural similarity to its homologue from Saccharomyces cerevisiae. Furthermore, hSpt5-NGN is structurally similar to the NTD (N-terminal domain) of the bacterial transcription factor NusG. A homologue for hSpt4 has not yet been found in bacteria. However, the archaeal transcription factor RpoE” appears to be distantly related. Although a comparison of the NusG-NTD of Escherichia coli with hSpt5 revealed a similarity of the three-dimensional structures, interaction of E. coli NusG-NTD with hSpt4 could not be observed by NMR titration experiments. A conserved glutamate residue, which was shown to be crucial for dimerization in yeast, is also involved in the human heterodimer, but is substituted for a glutamine residue in Escherichia coli NusG. However, exchanging the glutamine for glutamate proved not to be sufficient to induce hSpt4 binding.

Critical Role of Vertical Phase Separation in Small-Molecule Organic Solar Cells

2018 ◽  
Vol 10 (15) ◽  
pp. 12913-12920 ◽  
Author(s):  
Jin Fang ◽  
Dan Deng ◽  
Zaiyu Wang ◽  
Muhammad Abdullah Adil ◽  
Tong Xiao ◽  
...  
Keyword(s):  
Solar Cells ◽  
Phase Separation ◽  
Small Molecule ◽  
Organic Solar Cells ◽  
Critical Role

scholarly journals DIPG-10. THE ONCOGENIC ROLE OF THE SUPER ELONGATION COMPLEX IN H3K27M-MUTANT DIFFUSE MIDLINE GLIOMAS

2018 ◽  
Vol 20 (suppl_2) ◽  
pp. i50-i50
Author(s):  
Nathan A Dahl ◽  
Sujatha Venkataraman ◽  
Ilango Balakrishnan ◽  
Krishna Madhavan ◽  
Susan Fosmire ◽  
...  
Keyword(s):  
Elongation Complex ◽  
Super Elongation Complex

scholarly journals DBC1, p300, HDAC3, and Siah1 coordinately regulate ELL stability and function for expression of its target genes

2020 ◽  
Vol 117 (12) ◽  
pp. 6509-6520 ◽  
Author(s):  
Subham Basu ◽  
Mahesh Barad ◽  
Dipika Yadav ◽  
Arijit Nandy ◽  
Bidisha Mukherjee ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Rna Polymerase Ii ◽  
Target Genes ◽  
Mononuclear Cells ◽  
Elongation Factor ◽  
Diabetes Patient ◽  
Elongation Complex ◽  
Peripheral Blood Mononuclear ◽  
And Function

Among all of the Super Elongation Complex (SEC) components, ELL1 (also known as ELL) is the only bona fide elongation factor that directly stimulates transcription elongation by RNA polymerase II. However, the mechanism(s) of functional regulation of ELL1 (referred to as ELL hereafter), through its stabilization, is completely unknown. Here, we report a function of human DBC1 in regulating ELL stability involving HDAC3, p300, and Siah1. Mechanistically, we show that p300-mediated site-specific acetylation increases, whereas HDAC3-mediated deacetylation decreases, ELL stability through polyubiquitylation by the E3 ubiquitin ligase Siah1. DBC1 competes with HDAC3 for the same binding sites on ELL and thus increases its acetylation and stability. Knockdown of DBC1 reduces ELL levels and expression of a significant number of genes, including those involved in glucose metabolism. Consistently, Type 2 diabetes patient-derived peripheral blood mononuclear cells show reduced expression of DBC1 and ELL and associated key target genes required for glucose homeostasis. Thus, we describe a pathway of regulating stability and functions of key elongation factor ELL for expression of diverse sets of genes, including ones that are linked to Type 2 diabetes pathogenesis.

Sign in / Sign up

Export Citation Format

Share Document