Mediator subunit MED31 is required for radial patterning of Arabidopsis roots

Stem cell specification in multicellular organisms relies on the precise spatiotemporal control of RNA polymerase II (Pol II)-dependent gene transcription, in which the evolutionarily conserved Mediator coactivator complex plays an essential role. In Arabidopsis thaliana, SHORTROOT (SHR) and SCARECROW (SCR) orchestrate a transcriptional program that determines the fate and asymmetrical divisions of stem cells generating the root ground tissue. The mechanism by which SHR/SCR relays context-specific regulatory signals to the Pol II general transcription machinery is unknown. Here, we report the role of Mediator in controlling the spatiotemporal transcriptional output of SHR/SCR during asymmetrical division of stem cells and ground tissue patterning. The Mediator subunit MED31 interacted with SCR but not SHR. Reduction of MED31 disrupted the spatiotemporal activation of CYCLIND6;1 (CYCD6;1), leading to defective asymmetrical division of stem cells generating ground tissue. MED31 was recruited to the promoter of CYCD6;1 in an SCR-dependent manner. MED31 was involved in the formation of a dynamic MED31/SCR/SHR ternary complex through the interface protein SCR. We demonstrate that the relative protein abundance of MED31 and SHR in different cell types regulates the dynamic formation of the ternary complex, which provides a tunable switch to strictly control the spatiotemporal transcriptional output. This study provides valuable clues to understand the mechanism by which master transcriptional regulators control organ patterning.

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Structural basis of transcriptional stalling and bypass of abasic DNA lesion by RNA polymerase II

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1722050115 ◽

2018 ◽

Vol 115 (11) ◽

pp. E2538-E2545 ◽

Cited By ~ 19

Author(s):

Wei Wang ◽

Celine Walmacq ◽

Jenny Chong ◽

Mikhail Kashlev ◽

Dong Wang

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Dna Lesions ◽

Structural Motif ◽

Structural Basis ◽

Dependent Manner ◽

Abasic Site ◽

Structural Explanation ◽

Pol Ii ◽

Abasic Sites

Abasic sites are among the most abundant DNA lesions and interfere with DNA replication and transcription, but the mechanism of their action on transcription remains unknown. Here we applied a combined structural and biochemical approach for a comprehensive investigation of how RNA polymerase II (Pol II) processes an abasic site, leading to slow bypass of lesion. Encounter of Pol II with an abasic site involves two consecutive slow steps: insertion of adenine opposite a noninstructive abasic site (the A-rule), followed by extension of the 3′-rAMP with the next cognate nucleotide. Further studies provided structural insights into the A-rule: ATP is slowly incorporated into RNA in the absence of template guidance. Our structure revealed that ATP is bound to the Pol II active site, whereas the abasic site is located at an intermediate state above the Bridge Helix, a conserved structural motif that is cirtical for Pol II activity. The next extension step occurs in a template-dependent manner where a cognate substrate is incorporated, despite at a much slower rate compared with nondamaged template. During the extension step, neither the cognate substrate nor the template base is located at the canonical position, providing a structural explanation as to why this step is as slow as the insertion step. Taken together, our studies provide a comprehensive understanding of Pol II stalling and bypass of the abasic site in the DNA template.

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Pausing sites of RNA polymerase II on actively transcribed genes are enriched in DNA double-stranded breaks

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011665 ◽

2020 ◽

Vol 295 (12) ◽

pp. 3990-4000 ◽

Cited By ~ 3

Author(s):

Sandeep Singh ◽

Karol Szlachta ◽

Arkadi Manukyan ◽

Heather M. Raimer ◽

Manikarna Dinda ◽

...

Keyword(s):

Transcriptional Regulation ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Topoisomerase I ◽

Cell Types ◽

Dna Breaks ◽

Transcription Start ◽

Pol Ii ◽

Transcription Start Sites

DNA double-stranded breaks (DSBs) are strongly associated with active transcription, and promoter-proximal pausing of RNA polymerase II (Pol II) is a critical step in transcriptional regulation. Mapping the distribution of DSBs along actively expressed genes and identifying the location of DSBs relative to pausing sites can provide mechanistic insights into transcriptional regulation. Using genome-wide DNA break mapping/sequencing techniques at single-nucleotide resolution in human cells, we found that DSBs are preferentially located around transcription start sites of highly transcribed and paused genes and that Pol II promoter-proximal pausing sites are enriched in DSBs. We observed that DSB frequency at pausing sites increases as the strength of pausing increases, regardless of whether the pausing sites are near or far from annotated transcription start sites. Inhibition of topoisomerase I and II by camptothecin and etoposide treatment, respectively, increased DSBs at the pausing sites as the concentrations of drugs increased, demonstrating the involvement of topoisomerases in DSB generation at the pausing sites. DNA breaks generated by topoisomerases are short-lived because of the religation activity of these enzymes, which these drugs inhibit; therefore, the observation of increased DSBs with increasing drug doses at pausing sites indicated active recruitment of topoisomerases to these sites. Furthermore, the enrichment and locations of DSBs at pausing sites were shared among different cell types, suggesting that Pol II promoter-proximal pausing is a common regulatory mechanism. Our findings support a model in which topoisomerases participate in Pol II promoter-proximal pausing and indicated that DSBs at pausing sites contribute to transcriptional activation.

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The Myc Transactivation Domain Promotes Global Phosphorylation of the RNA Polymerase II Carboxy-Terminal Domain Independently of Direct DNA Binding

Molecular and Cellular Biology ◽

10.1128/mcb.01828-06 ◽

2007 ◽

Vol 27 (6) ◽

pp. 2059-2073 ◽

Cited By ~ 85

Author(s):

Victoria H. Cowling ◽

Michael D. Cole

Keyword(s):

Dna Binding ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Target Genes ◽

Dependent Manner ◽

Transactivation Domain ◽

Pol Ii ◽

Terminal Domain ◽

Ctd Phosphorylation

ABSTRACT Myc is a transcription factor which is dependent on its DNA binding domain for transcriptional regulation of target genes. Here, we report the surprising finding that Myc mutants devoid of direct DNA binding activity and Myc target gene regulation can rescue a substantial fraction of the growth defect in myc −/− fibroblasts. Expression of the Myc transactivation domain alone induces a transcription-independent elevation of the RNA polymerase II (Pol II) C-terminal domain (CTD) kinases cyclin-dependent kinase 7 (CDK7) and CDK9 and a global increase in CTD phosphorylation. The Myc transactivation domain binds to the transcription initiation sites of these promoters and stimulates TFIIH binding in an MBII-dependent manner. Expression of the Myc transactivation domain increases CDK mRNA cap methylation, polysome loading, and the rate of translation. We find that some traditional Myc transcriptional target genes are also regulated by this Myc-driven translation mechanism. We propose that Myc transactivation domain-driven RNA Pol II CTD phosphorylation has broad effects on both transcription and mRNA metabolism.

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Antagonistic cotranscriptional regulation through ARGONAUTE1 and the THO/TREX complex orchestrates FLC transcriptional output

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2113757118 ◽

2021 ◽

Vol 118 (47) ◽

pp. e2113757118

Author(s):

Congyao Xu ◽

Xiaofeng Fang ◽

Tiancong Lu ◽

Caroline Dean

Keyword(s):

Rna Polymerase Ii ◽

Transcription Initiation ◽

Transcriptional Control ◽

Antisense Transcript ◽

Elongation Rate ◽

Pol Ii ◽

Alternative Processing ◽

Floral Repressor ◽

Related Proteins ◽

Transcriptional Output

Quantitative transcriptional control is essential for physiological and developmental processes in many organisms. Transcriptional output is influenced by cotranscriptional processes interconnected to chromatin regulation, but how the functions of different cotranscriptional regulators are integrated is poorly understood. The Arabidopsis floral repressor locus FLOWERING LOCUS C (FLC) is cotranscriptionally repressed by alternative processing of the antisense transcript COOLAIR. Proximal 3′-end processing of COOLAIR resolves a cotranscriptionally formed R-loop, and this process physically links to a histone-modifying complex FLD/SDG26/LD. This induces a chromatin environment locally that determines low transcription initiation and a slow elongation rate to both sense and antisense strands. Here, we show that ARGONAUTE1 (AGO1) genetically functions in this cotranscriptional repression mechanism. AGO1 associates with COOLAIR and influences COOLAIR splicing dynamics to promote proximal COOLAIR, R-loop resolution, and chromatin silencing. Proteomic analyses revealed physical associations between AGO1, subunits of RNA Polymerase II (Pol II), the splicing-related proteins—the spliceosome NineTeen Complex (NTC) and related proteins (NTR)—and the THO/TREX complex. We connect these activities by demonstrating that the THO/TREX complex activates FLC expression acting antagonistically to AGO1 in COOLAIR processing. Together these data reveal that antagonistic cotranscriptional regulation through AGO1 or THO/TREX influences COOLAIR processing to deliver a local chromatin environment that determines FLC transcriptional output. The involvement of these conserved cotranscriptional regulators suggests similar mechanisms may underpin quantitative transcriptional regulation generally.

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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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RNA polymerase II pausing regulates a quiescence-dependent transcriptional program, priming cells for cell cycle reentry

10.1101/250910 ◽

2018 ◽

Cited By ~ 1

Author(s):

Hardik P. Gala ◽

Debarya Saha ◽

Nisha Venugopal ◽

Ajoy Aloysius ◽

Jyotsna Dhawan

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Gene Networks ◽

Adult Stem Cells ◽

Transcriptional Networks ◽

Elongation Complex ◽

Pol Ii ◽

Pol Ii Pausing

AbstractAdult stem cells persist in mammalian tissues by entering a state of reversible arrest or quiescence associated with low transcription. Using cultured myoblasts and primary muscle stem cells, we show that RNA synthesis is strongly repressed in G0, returning within minutes of activation. We investigate the underlying mechanism and reveal a role for promoter-proximal RNAPol II pausing: by mapping global Pol II occupancy using ChIP-seq, in conjunction with RNA-seq to identify repressed transcriptional networks unique to G0. Strikingly, Pol II pausing is enhanced in G0 on genes encoding regulators of RNA biogenesis (Ncl, Rps24, Ctdp1), and release of pausing is critical for cell cycle re-entry. Finally, we uncover a novel, unexpected repressive role of the super-elongation complex component Aff4 in G0-specific stalling. We propose a model wherein Pol II pausing restrains transcription to maintain G0, preconfigures gene networks required for the G0-G1 transition, and sets the timing of their transcriptional activation.

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Human Mediator Enhances Activator-Facilitated Recruitment of RNA Polymerase II and Promoter Recognition by TATA-Binding Protein (TBP) Independently of TBP-Associated Factors

Molecular and Cellular Biology ◽

10.1128/mcb.23.17.6229-6242.2003 ◽

2003 ◽

Vol 23 (17) ◽

pp. 6229-6242 ◽

Cited By ~ 41

Author(s):

Shwu-Yuan Wu ◽

Tianyuan Zhou ◽

Cheng-Ming Chiang

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Associated Factors ◽

Binding Protein ◽

Tata Binding Protein ◽

Dependent Manner ◽

Basal Transcription ◽

Pol Ii ◽

Promoter Recognition ◽

Stimulation Of

ABSTRACT Mediator is a general cofactor implicated in the functions of many transcriptional activators. Although Mediator with different protein compositions has been isolated, it remains unclear how Mediator facilitates activator-dependent transcription, independent of its general stimulation of basal transcription. To define the mechanisms of Mediator function, we isolated two forms of human Mediator complexes (Mediator-P.5 and Mediator-P.85) and demonstrated that Mediator-P.5 clearly functions by enhancing activator-mediated recruitment of RNA polymerase II (pol II), whereas Mediator-P.85 works mainly by stimulating overall basal transcription. The coactivator function of Mediator-P.5 was not impaired when TATA-binding protein (TBP) was used in place of TFIID, but it was abolished when another general cofactor, PC4, was omitted from the reaction or when Mediator-P.5 was added after pol II entry into the preinitiation complex. Moreover, Mediator- P.5 is able to enhance TBP binding to the TATA box in an activator-dependent manner. Our data provides biochemical evidence that Mediator functions by facilitating activator-mediated recruitment of pol II and also promoter recognition by TBP, both of which can occur in the absence of TBP-associated factors in TFIID.

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RNA polymerase II pausing as a context-dependent reader of the genome

Biochemistry and Cell Biology ◽

10.1139/bcb-2015-0045 ◽

2016 ◽

Vol 94 (1) ◽

pp. 82-92 ◽

Cited By ~ 16

Author(s):

Adam Scheidegger ◽

Sergei Nechaev

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Cell Types ◽

Gene Promoters ◽

Cell Type ◽

Pol Ii ◽

Cell Type Specific ◽

Pol Ii Pausing ◽

Context Dependent ◽

Specific Regulation

The RNA polymerase II (Pol II) transcribes all mRNA genes in eukaryotes and is among the most highly regulated enzymes in the cell. The classic model of mRNA gene regulation involves recruitment of the RNA polymerase to gene promoters in response to environmental signals. Higher eukaryotes have an additional ability to generate multiple cell types. This extra level of regulation enables each cell to interpret the same genome by committing to one of the many possible transcription programs and executing it in a precise and robust manner. Whereas multiple mechanisms are implicated in cell type-specific transcriptional regulation, how one genome can give rise to distinct transcriptional programs and what mechanisms activate and maintain the appropriate program in each cell remains unclear. This review focuses on the process of promoter-proximal Pol II pausing during early transcription elongation as a key step in context-dependent interpretation of the metazoan genome. We highlight aspects of promoter-proximal Pol II pausing, including its interplay with epigenetic mechanisms, that may enable cell type-specific regulation, and emphasize some of the pertinent questions that remain unanswered and open for investigation.

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SETD5 modulates homeostasis of hematopoietic stem cells by mediating RNA Polymerase II pausing in cooperation with HCF-1

Leukemia ◽

10.1038/s41375-021-01481-1 ◽

2021 ◽

Author(s):

Mengke Li ◽

Chen Qiu ◽

Yujie Bian ◽

Deyang Shi ◽

Bichen Wang ◽

...

Keyword(s):

Stem Cells ◽

Rna Polymerase ◽

Hematopoietic Stem Cells ◽

Rna Polymerase Ii ◽

Critical Role ◽

Whole Body ◽

Hematopoietic Stem ◽

Pol Ii

AbstractSETD5 mutations were identified as the genetic causes of neurodevelopmental disorders. While the whole-body knockout of Setd5 in mice leads to embryonic lethality, the role of SETD5 in adult stem cell remains unexplored. Here, a critical role of Setd5 in hematopoietic stem cells (HSCs) is identified. Specific deletion of Setd5 in hematopoietic system significantly increased the number of immunophenotypic HSCs by promoting HSC proliferation. Setd5-deficient HSCs exhibited impaired long-term self-renewal capacity and multiple-lineage differentiation potentials under transplantation pressure. Transcriptome analysis of Setd5-deficient HSCs revealed a disruption of quiescence state of long-term HSCs, a cause of the exhaustion of functional HSCs. Mechanistically, SETD5 was shown to regulate HSC quiescence by mediating the release of promoter-proximal paused RNA polymerase II (Pol II) on E2F targets in cooperation with HCF-1 and PAF1 complex. Taken together, these findings reveal an essential role of SETD5 in regulating Pol II pausing-mediated maintenance of adult stem cells.

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FACT-mediated maintenance of chromatin integrity during transcription is essential for viability of mammalian stem cells

10.1101/2021.06.21.449309 ◽

2021 ◽

Author(s):

Imon Goswami ◽

Poorva Sandlesh ◽

Aimee Stablewski ◽

Ilya Toshkov ◽

Mikhail Magnitov ◽

...

Keyword(s):

Stem Cells ◽

Mammalian Cells ◽

Transcription Elongation ◽

Cell Types ◽

Chromatin Accessibility ◽

Conditional Knockout ◽

Dependent Manner ◽

Chromatin Integrity ◽

Prominent Response

Replication and transcription machineries access DNA through unwrapping it from histone core. Preservation of nucleosome structure during replication is a focus of intensive research, while maintenance of nucleosomes during transcription is less studied. Histone chaperone FACT is involved in transcription elongation, although whether it opens nucleosomes for polymerase or protects core histones from loss during passage of polymerase is still unclear. We used conditional knockout model of Ssrp1, subunit of FACT complex, to deplete FACT in mice and monitored consequences of FACT loss to establish the functional relevance of FACT in mammals. FACT loss was lethal for mice at all ages due to failure of hematopoietic and intestinal tissues. In these tissues, only the progenitors completely vanished upon FACT loss, while number of some other cell types were changed up and down. Using isolated stem cells of several tissues we showed that FACT loss was toxic only for stem cells, but not for cells which were differentiated in vitro. Chromatin accessibility in stem cells was increased genome wide upon FACT depletion in transcription dependent manner. The most prominent response to the loss of chromatin integrity upon FACT depletion in cells was activation of interferon signaling, followed by accumulation of immune cells in sensitive organs. Thus, in undifferentiated mammalian cells FACT keeps chromatin stable during transcription elongation and this function of FACT is essential for viability of stem cells.

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