PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC

AbstractThe C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay.

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PHF3 regulates neuronal gene expression through the new Pol II CTD reader domain SPOC

10.1101/2020.02.11.943159 ◽

2020 ◽

Author(s):

Lisa-Marie Appel ◽

Vedran Franke ◽

Melania Bruno ◽

Irina Grishkovskaya ◽

Aiste Kasiliauskaite ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Mrna Stability ◽

Embryonic Stem ◽

Pol Ii ◽

Knock Out ◽

Phd Finger ◽

Neuronal Gene ◽

Binding Factor ◽

Neuronal Genes ◽

Global Increase

SUMMARYThe C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a new CTD-binding factor that negatively regulates transcription and mRNA stability. The PHF3 SPOC domain preferentially binds to CTD repeats phosphorylated on Serine-2 and PHF3 tracks with Pol II across the length of genes. PHF3 competes with TFIIS for Pol II binding through its TFIIS-like domain (TLD), thus inhibiting TFIIS-mediated rescue of backtracked Pol II. PHF3 knock-out or PHF3 SPOC deletion in human cells result in gene upregulation and a global increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 is a prominent effector of neuronal gene regulation at the interface of transcription elongation and mRNA decay.

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FUS driven circCNOT6L biogenesis in mouse and human spermatozoa supports zygote development

Cellular and Molecular Life Sciences ◽

10.1007/s00018-021-04054-8 ◽

2021 ◽

Author(s):

Teresa Chioccarelli ◽

Geppino Falco ◽

Donato Cappetta ◽

Antonella De Angelis ◽

Luca Roberto ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Rna Binding ◽

Cannabinoid Receptor ◽

Rna Binding Proteins ◽

Embryonic Stem ◽

Circular Rna ◽

Physical Interaction ◽

Knock Out ◽

Maternal Contribution ◽

Zygote Development

AbstractCircular RNA (circRNA) biogenesis requires a backsplicing reaction, promoted by inverted repeats in cis-flanking sequences and trans factors, such as RNA-binding proteins (RBPs). Among these, FUS plays a key role. During spermatogenesis and sperm maturation along the epididymis such a molecular mechanism has been poorly explored. With this in mind, we chose circCNOT6L as a study case and wild-type (WT) as well as cannabinoid receptor type-1 knock-out (Cb1−/−) male mice as animal models to analyze backsplicing mechanisms. Our results suggest that spermatozoa (SPZ) have an endogenous skill to circularize mRNAs, choosing FUS as modulator of backsplicing and under CB1 stimulation. A physical interaction between FUS and CNOT6L as well as a cooperation among FUS, RNA Polymerase II (RNApol2) and Quaking (QKI) take place in SPZ. Finally, to gain insight into FUS involvement in circCNOT6L biogenesis, FUS expression was reduced through RNA interference approach. Paternal transmission of FUS and CNOT6L to oocytes during fertilization was then assessed by using murine unfertilized oocytes (NF), one-cell zygotes (F) and murine oocytes undergoing parthenogenetic activation (PA) to exclude a maternal contribution. The role of circCNOT6L as an active regulator of zygote transition toward the 2-cell-like state was suggested using the Embryonic Stem Cell (ESC) system. Intriguingly, human SPZ exactly mirror murine SPZ.

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A Minor Subset of Super Elongation Complexes Plays a Predominant Role in Reversing HIV-1 Latency

Molecular and Cellular Biology ◽

10.1128/mcb.00994-15 ◽

2016 ◽

Vol 36 (7) ◽

pp. 1194-1205 ◽

Cited By ~ 17

Author(s):

Zichong Li ◽

Huasong Lu ◽

Qiang Zhou

Keyword(s):

Rna Polymerase Ii ◽

Cellular Level ◽

Transcriptional Elongation ◽

Predominant Role ◽

Drug Induced ◽

Pol Ii ◽

Knock Out ◽

Future Design ◽

Latent Hiv ◽

Hiv 1

Promoter-proximal pausing by RNA polymerase II (Pol II) is a key rate-limiting step in HIV-1 transcription and latency reversal. The viral Tat protein recruits human super elongation complexes (SECs) to paused Pol II to overcome this restriction. Despite the recent progress in understanding the functions of different subsets of SECs in controlling cellular and Tat-activated HIV transcription, little is known about the SEC subtypes that help reverse viral latency in CD4+T cells. Here, we used the CRISPR-Cas9 genome-editing tool to knock out the gene encoding the SEC subunit ELL2, AFF1, or AFF4 in Jurkat/2D10 cells, a well-characterized HIV-1 latency model. Depletion of these proteins drastically reduced spontaneous and drug-induced latency reversal by suppressing HIV-1 transcriptional elongation. Surprisingly, a low-abundance subset of SECs containing ELL2 and AFF1 was found to play a predominant role in cooperating with Tat to reverse latency. By increasing the cellular level/activity of these Tat-friendly SECs, we could potently activate latent HIV-1 without using any drugs. These results implicate the ELL2/AFF1-SECs as an important target in the future design of a combinatorial therapeutic approach to purge latent HIV-1.

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An H3K9/S10 methyl-phospho switch modulates Polycomb and Pol II binding at repressed genes during differentiation

Molecular Biology of the Cell ◽

10.1091/mbc.e13-10-0628 ◽

2014 ◽

Vol 25 (6) ◽

pp. 904-915 ◽

Cited By ~ 24

Author(s):

Pierangela Sabbattini ◽

Marcela Sjoberg ◽

Svetlana Nikic ◽

Alberto Frangini ◽

Per-Henrik Holmqvist ◽

...

Keyword(s):

Stem Cells ◽

Rna Polymerase Ii ◽

Embryonic Stem ◽

Epigenetic Silencing ◽

Aurora B ◽

Developmentally Regulated ◽

Pol Ii ◽

Differentiated Cells ◽

Silent Genes ◽

The Relationship

Methylated histones H3K9 and H3K27 are canonical epigenetic silencing modifications in metazoan organisms, but the relationship between the two modifications has not been well characterized. H3K9me3 coexists with H3K27me3 in pluripotent and differentiated cells. However, we find that the functioning of H3K9me3 is altered by H3S10 phosphorylation in differentiated postmitotic osteoblasts and cycling B cells. Deposition of H3K9me3/S10ph at silent genes is partially mediated by the mitogen- and stress-activated kinases (MSK1/2) and the Aurora B kinase. Acquisition of H3K9me3/S10ph during differentiation correlates with loss of paused S5 phosphorylated RNA polymerase II, which is present on Polycomb-regulated genes in embryonic stem cells. Reduction of the levels of H3K9me3/S10ph by kinase inhibition results in increased binding of RNAPIIS5ph and the H3K27 methyltransferase Ezh1 at silent promoters. Our results provide evidence of a novel developmentally regulated methyl-phospho switch that modulates Polycomb regulation in differentiated cells and stabilizes repressed states.

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Mediator and RNA polymerase II clusters associate in transcription-dependent condensates

Science ◽

10.1126/science.aar4199 ◽

2018 ◽

Vol 361 (6400) ◽

pp. 412-415 ◽

Cited By ~ 351

Author(s):

Won-Ki Cho ◽

Jan-Hendrik Spille ◽

Micca Hecht ◽

Choongman Lee ◽

Charles Li ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Spatial Organization ◽

Embryonic Stem ◽

Light Sheet ◽

Gene Control ◽

Pol Ii ◽

Stable Clusters ◽

Large Clusters

Models of gene control have emerged from genetic and biochemical studies, with limited consideration of the spatial organization and dynamics of key components in living cells. We used live-cell superresolution and light-sheet imaging to study the organization and dynamics of the Mediator coactivator and RNA polymerase II (Pol II) directly. Mediator and Pol II each form small transient and large stable clusters in living embryonic stem cells. Mediator and Pol II are colocalized in the stable clusters, which associate with chromatin, have properties of phase-separated condensates, and are sensitive to transcriptional inhibitors. We suggest that large clusters of Mediator, recruited by transcription factors at large or clustered enhancer elements, interact with large Pol II clusters in transcriptional condensates in vivo.

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RNA polymerase II assembly and mRNA decay regulation are mediated and interconnected via CTD Ser5P phosphatase Rtr1 in Saccharomyces cerevisiae

10.1101/2020.12.23.424112 ◽

2020 ◽

Author(s):

A.I. Garrido-Godino ◽

A. Cuevas-Bermúdez ◽

F. Gutiérrez-Santiago ◽

M.C. Mota-Trujillo ◽

F. Navarro

Keyword(s):

Rna Polymerase Ii ◽

Mrna Stability ◽

Transcription Initiation ◽

Mrna Decay ◽

Rna Pol Ii ◽

Pol Ii ◽

Cytoplasmic Rna ◽

Ctd Phosphatase ◽

Rule Out

ABSTRACTRtr1 is an RNA pol II CTD-phosphatase that influences gene expression by acting during the transition from transcription initiation to elongation, and during transcription termination. Rtr1 has been proposed as an RNA pol II import factor in RNA pol II biogenesis, and participating in mRNA decay by autoregulating the turnover of its own mRNA. In addition, the interaction of Rtr1 with RNA pol II depends on the phosphorylation state of CTD, which also influences Rpb4/7 dissociation during transcription. In this work, we demonstrate that Rtr1 acts in RNA pol II assembly, likely in a final cytoplasmic RNA pol II biogenesis step, and mediates the Rpb4 association with the rest of the enzyme, However, we do not rule out discard a role in the Rpb4 association with RNA pol II in the nucleus. This role of Rtr1 interplays RNA pol II biogenesis and mRNA decay regulation. In fact, RTR1 deletion alters RNA pol II assembly and leads to the chromatin association of RNA pol II lacking Rpb4, in addition to whole RNA pol II, decreasing mRNA-Rpb4 imprinting and, consequently, increasing mRNA stability. Notably, the RPB5 overexpression that overcomes RNA pol II assembly and the defect in Rpb4 binding to chromatin-associated RNA pol II partially suppresses the mRNA stability defect of rtr1Δ cells. Our data also indicate that Rtr1 mediates mRNA decay regulation more broadly than previously proposed in cooperation with Rpb4 and Dhh1. Interestingly, these data include new layers in the crosstalk between mRNA synthesis and decay.

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TAF8 regions important for TFIID lobe B assembly, or for TAF2 interactions, are required for embryonic stem cell survival

10.1101/2021.07.06.451281 ◽

2021 ◽

Author(s):

Elisabeth Scheer ◽

Jie Luo ◽

Frank Ruffenach ◽

Jean-Marie Garnier ◽

Isabelle Kolb-Cheynel ◽

...

Keyword(s):

Stem Cell ◽

Embryonic Stem Cell ◽

Cell Survival ◽

Rna Polymerase Ii ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cell ◽

Gene Promoters ◽

Pol Ii ◽

Histone Fold ◽

Stem Cell Survival

The human general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). In eukaryotic cells, TFIID is thought to nucleate RNA polymerase II (Pol II) preinitiation complex formation on all protein coding gene promoters and thus, be crucial for Pol II transcription. TFIID is composed of three lobes, named A, B and C. Structural studies showed that TAF8 forms a histone fold pair with TAF10 in lobe B and participates in connecting lobe B to lobe C. In the present study, we have investigated the requirement of the different regions of TAF8 for in vitro TFIID assembly, and the importance of certain TAF8 regions for mouse embryonic stem cell (ESC) viability. We have identified a TAF8 region, different from the histone fold domain of TAF8, important for assembling with the 5TAF core complex in lobe B, and four regions of TAF8 each individually required for interacting with TAF2 in lobe C. Moreover, we show that the 5TAF core-interacting TAF8 domain, and the proline rich domain of TAF8 that interacts with TAF2, are both required for mouse embryonic stem cell survival. Thus, our study demonstrates that distinct TAF8 regions involved in connecting lobe B to lobe C are crucial for TFIID function and consequent ESC survival.

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Histone H2Bub1 deubiquitylation is essential for mouse development, but does not regulate global RNA polymerase II transcription

10.1101/2021.02.23.432458 ◽

2021 ◽

Author(s):

Fang Wang ◽

Farrah El-Saafin ◽

Tao Ye ◽

Matthieu Stierle ◽

Luc Negroni ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Embryonic Stem ◽

Cellular Systems ◽

Strong Increase ◽

Modular Organization ◽

Saga Complex ◽

Mouse Development ◽

Pol Ii ◽

Primary Mouse

AbstractCo-activator complexes dynamically deposit post-translational modifications (PTMs) on histones, or remove them, to regulate chromatin accessibility and/or to create/erase docking surfaces for proteins that recognize histone PTMs. SAGA (Spt-Ada-Gcn5 Acetyltransferase) is an evolutionary conserved multisubunit co-activator complex with modular organization. The deubiquitylation module (DUB) of mammalian SAGA complex is composed of the ubiquitin-specific protease 22 (USP22) and three adaptor proteins, ATXN7, ATXN7L3 and ENY2, which are all needed for the full activity of the USP22 enzyme to remove monoubiquitin (ub1) from histone H2B. Two additional USP22-related ubiquitin hydrolases (called USP27X or USP51) have been described to form alternative DUBs with ATXN7L3 and ENY2, which can also deubiquitylate H2Bub1. Here we report that USP22 and ATXN7L3 are essential for normal embryonic development of mice, however their requirements are not identical during this process, as Atxn7l3−/− embryos show developmental delay already at embryonic day (E) 7.5, while Usp22−/− embryos are normal at this stage, but die at E14.5. Global histone H2Bub1 levels were only slightly affected in Usp22 null embryos, in contrast H2Bub1 levels were strongly increased in Atxn7l3 null embryos and derived cell lines. Our transcriptomic analyses carried out from wild type and Atxn7l3−/− mouse embryonic stem cells (mESCs), or primary mouse embryonic fibroblasts (MEFs) suggest that the ATXN7L3-related DUB activity regulates only a subset of genes in both cell types. However, the gene sets and the extent of their deregulation were different in mESCs and MEFs. Interestingly, the strong increase of H2Bub1 levels observed in the Atxn7l3−/− mESCs, or Atxn7l3−/− MEFs, does not correlate with the modest changes in RNA Polymerase II (Pol II) occupancy and lack of changes in Pol II elongation observed in the two Atxn7l3−/− cellular systems. These observations together indicate that deubiquitylation of histone H2Bub1 does not directly regulate global Pol II transcription elongation.

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SUPT3H-less SAGA coactivator can assemble and function without significantly perturbing RNA polymerase II transcription in mammalian cells

10.1101/2021.07.09.451791 ◽

2021 ◽

Author(s):

Veronique Fischer ◽

Elisabeth Scheer ◽

Elisabeth Lata ◽

Bastien Morlet ◽

Damien Plassard ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Mammalian Cells ◽

Embryonic Stem ◽

Gene Promoters ◽

Saga Complex ◽

Self Renewal ◽

Pol Ii ◽

Histone Fold ◽

Specific Subset

Coactivator complexes regulate chromatin accessibility and transcription. SAGA (Spt-Ada-Gcn5 Acetyltransferase) is an evolutionary conserved coactivator complex. The core module scaffolds the entire SAGA complex and adopts a histone octamer-like structure, which consists of six histone fold domain (HFD)-containing proteins forming three histone fold (HF) pairs, to which the double HFD-containing SUPT3H adds an HF pair. Spt3, the yeast ortholog of SUPT3H, interacts genetically and biochemically with the TATA binding protein (TBP) and contributes to global RNA polymerase II (Pol II) transcription. Here we demonstrate that i) SAGA purified from human U2OS or mouse embryonic stem cells (mESC) can assemble without SUPT3H; ii) SUPT3H is not essential for mESC survival, iii) SUPT3H is required for mESC growth and self-renewal, and iv) the loss of SUPT3H from mammalian cells affects the transcription of only a specific subset of genes. Accordingly, in the absence of SUPT3H no major change in TBP accumulation at gene promoters was observed. Thus, SUPT3H is not required for the assembly of SAGA, TBP recruitment, or overall Pol II transcription, but plays a role in mESC growth and self-renewal. Our data further suggest that yeast and mammalian SAGA complexes contribute to transcription regulation by distinct mechanisms.

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Rpb4 and Puf3 imprint and post-transcriptionally control the stability of a common set of mRNAs in yeast

10.1101/2020.07.25.220095 ◽

2020 ◽

Author(s):

A.I. Garrido-Godino ◽

I. Gupta ◽

F. Gutiérrez-Santiago ◽

A.B. Martínez-Padilla ◽

A. Alekseenko ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Mrna Stability ◽

Molecular Mechanisms ◽

Rna Binding ◽

Large Population ◽

Dependent Manner ◽

Mrna Synthesis ◽

Rna Pol Ii ◽

Pol Ii ◽

The Stability

ABSTRACTGene expression involving RNA polymerase II is regulated by the concerted interplay between mRNA synthesis and degradation, crosstalk in which mRNA decay machinery and transcription machinery respectively impact transcription and mRNA stability. Rpb4, and likely dimer Rpb4/7, seem the central components of the RNA pol II governing these processes. In this work we unravel the molecular mechanisms participated by Rpb4 that mediate the posttranscriptional events regulating mRNA imprinting and stability. By RIP-Seq, we analyzed genome-wide the association of Rpb4 with mRNAs and demonstrated that it targeted a large population of more than 1400 transcripts. A group of these mRNAs was also the target of the RNA binding protein, Puf3. We demonstrated that Rpb4 and Puf3 physically, genetically, and functionally interact and also affect mRNA stability, and likely the imprinting, of a common group of mRNAs. Furthermore, the Rpb4 and Puf3 association with mRNAs depends on one another. We also demonstrated, for the first time, that Puf3 associates with chromatin in an Rpb4-dependent manner. Our data also suggest that Rpb4 could be a key element of the RNA pol II that coordinates mRNA synthesis, imprinting and stability in cooperation with RBPs.

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