The Experts below are selected from a list of 1326 Experts worldwide ranked by ideXlab platform
Hiroshi Handa - One of the best experts on this subject based on the ideXlab platform.
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DSIF and NELF interact with Integrator to specify the correct post-transcriptional fate of snRNA genes
Nature communications, 2014Co-Authors: Junichi Yamamoto, Hiroshi Handa, Takashi Narita, Yuri Hagiwara, Kunitoshi Chiba, Tomoyasu Isobe, Yuki YamaguchiAbstract:The elongation factors DSIF and NELF have established roles in polymerase pausing, elongation and 3'-end processing of replication-dependent histone mRNAs. Here the authors demonstrate that DSIF and NELF form a complex with Integrator and allow proper 3'-processing of snRNA transcripts by preventing the recruitment of CstF.
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DSIF Restricts NF-κB Signaling by Coordinating Elongation with mRNA Processing of Negative Feedback Genes
Cell reports, 2012Co-Authors: Gil Diamant, Yuki Yamaguchi, Hiroshi Handa, Liat Amir-zilberstein, Rivka DiksteinAbstract:NF-κB is central for immune response and cell survival, and its deregulation is linked to chronic inflammation and cancer through poorly defined mechanisms. IκBα and A20 are NF-κB target genes and negative feedback regulators. Upon their activation by NF-κB, DSIF is recruited, P-TEFb is released, and their elongating polymerase II (Pol II) C-terminal domain (CTD) remains hypophosphorylated. We show that upon DSIF knockdown, mRNA levels of a subset of NF-κB targets are not diminished; yet much less IκBα and A20 protein are synthesized, and NF-κB activation is abnormally prolonged. Further analysis of IκBα and A20 mRNA revealed that a significant portion is uncapped, unspliced, and retained in the nucleus. Interestingly, the Spt5 C-terminal repeat (CTR) domain involved in elongation stimulation through P-TEFb is dispensable for IκBα and A20 regulation. These findings assign a function for DSIF in cotranscriptional mRNA processing when elongating Pol II is hypophosphorylated and define DSIF as part of the negative feedback regulation of NF-κB.
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DSIF, the Paf1 complex, and Tat-SF1 have nonredundant, cooperative roles in RNA polymerase II elongation
Genes & development, 2009Co-Authors: Yexi Chen, Yuki Yamaguchi, Yuta Tsugeno, Junichi Yamamoto, Tomoko Yamada, Mitsuhiro Nakamura, Koji Hisatake, Hiroshi HandaAbstract:Transcription elongation factor DSIF/Spt4-Spt5 is capable of promoting and inhibiting RNA polymerase II elongation and is involved in the expression of various genes. While it has been known for many years that DSIF inhibits elongation in collaboration with the negative elongation factor NELF, how DSIF promotes elongation is largely unknown. Here, an activity-based biochemical approach was taken to understand the mechanism of elongation activation by DSIF. We show that the Paf1 complex (Paf1C) and Tat-SF1, two factors implicated previously in elongation control, collaborate with DSIF to facilitate efficient elongation. In human cells, these factors are recruited to the FOS gene in a temporally coordinated manner and contribute to its high-level expression. We also show that elongation activation by these factors depends on P-TEFb-mediated phosphorylation of the Spt5 C-terminal region. A clear conclusion emerging from this study is that a set of elongation factors plays nonredundant, cooperative roles in elongation. This study also shows unambiguously that Paf1C, which is generally thought to have chromatin-related functions, is involve directlyd in elongation control.
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Role of human transcription elongation factor DSIF in the suppression of senescence and apoptosis.
Genes to cells : devoted to molecular & cellular mechanisms, 2009Co-Authors: Toshiharu Komori, Yuki Yamaguchi, Naoto Inukai, Tomoko Yamada, Hiroshi HandaAbstract:DSIF is an evolutionarily conserved, ubiquitously expressed, heterodimeric transcription elongation factor composed of two subunits, Spt4 and Spt5. Previous biochemical studies have shown that DSIF positively and negatively regulates RNA polymerase II elongation in collaboration with other protein factors. While several data suggest that DSIF is a 'general' elongation factor, there is also evidence that DSIF exerts a tissue- and gene-specific function. Here we sought to address the question of whether physiological functions of DSIF are general or specific, by using a sophisticated knockdown approach and gene expression microarray analysis. We found that Spt5 is essential for cell growth of various human cell lines and that Spt5 knockdown causes senescence and apoptosis. However, Spt5 knockdown affects a surprisingly small number of genes. In Spt5 knockdown cells, the p53 signaling pathway is activated and mediates part of the knockdown-induced transcriptional change, but apoptotic cell death occurs in the absence of p53. Structure-function analysis of Spt5 shows that the C-terminal approximately 300 amino acid residues are not required to support cell proliferation. These results suggest that one of the functions of Spt5 is to suppress senescence and apoptosis, and that this function is exerted through its association with Spt4 and Pol II.
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DSIF contributes to transcriptional activation by DNA-binding activators by preventing pausing during transcription elongation
Nucleic acids research, 2007Co-Authors: Wenyan Zhu, Yuki Yamaguchi, Tadashi Wada, Sachiko Okabe, Takuya Taneda, Hiroshi HandaAbstract:The transcription elongation factor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF) regulates RNA polymerase II (RNAPII) processivity by promoting, in concert with negative elongation factor (NELF), promoter-proximal pausing of RNAPII. DSIF is also reportedly involved in transcriptional activation. However, the role of DSIF in transcriptional activation by DNA-binding activators is unclear. Here we show that DSIF acts cooperatively with a DNA-binding activator, Gal4-VP16, to promote transcriptional activation. In the absence of DSIF, Gal4-VP16-activated transcription resulted in frequent pausing of RNAPII during elongation in vitro. The presence of DSIF reduced pausing, thereby supporting Gal4-VP16-mediated activation. We found that DSIF exerts its positive effects within a short time-frame from initiation to elongation, and that NELF does not affect the positive regulatory function of DSIF. Knockdown of the gene encoding the large subunit of DSIF, human Spt5 (hSpt5), in HeLa cells reduced Gal4-VP16-mediated activation of a reporter gene, but had no effect on expression in the absence of activator. Together, these results provide evidence that higher-level transcription has a stronger requirement for DSIF, and that DSIF contributes to efficient transcriptional activation by preventing RNAPII pausing during transcription elongation.
Yuki Yamaguchi - One of the best experts on this subject based on the ideXlab platform.
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DSIF and NELF interact with Integrator to specify the correct post-transcriptional fate of snRNA genes
Nature communications, 2014Co-Authors: Junichi Yamamoto, Hiroshi Handa, Takashi Narita, Yuri Hagiwara, Kunitoshi Chiba, Tomoyasu Isobe, Yuki YamaguchiAbstract:The elongation factors DSIF and NELF have established roles in polymerase pausing, elongation and 3'-end processing of replication-dependent histone mRNAs. Here the authors demonstrate that DSIF and NELF form a complex with Integrator and allow proper 3'-processing of snRNA transcripts by preventing the recruitment of CstF.
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DSIF Restricts NF-κB Signaling by Coordinating Elongation with mRNA Processing of Negative Feedback Genes
Cell reports, 2012Co-Authors: Gil Diamant, Yuki Yamaguchi, Hiroshi Handa, Liat Amir-zilberstein, Rivka DiksteinAbstract:NF-κB is central for immune response and cell survival, and its deregulation is linked to chronic inflammation and cancer through poorly defined mechanisms. IκBα and A20 are NF-κB target genes and negative feedback regulators. Upon their activation by NF-κB, DSIF is recruited, P-TEFb is released, and their elongating polymerase II (Pol II) C-terminal domain (CTD) remains hypophosphorylated. We show that upon DSIF knockdown, mRNA levels of a subset of NF-κB targets are not diminished; yet much less IκBα and A20 protein are synthesized, and NF-κB activation is abnormally prolonged. Further analysis of IκBα and A20 mRNA revealed that a significant portion is uncapped, unspliced, and retained in the nucleus. Interestingly, the Spt5 C-terminal repeat (CTR) domain involved in elongation stimulation through P-TEFb is dispensable for IκBα and A20 regulation. These findings assign a function for DSIF in cotranscriptional mRNA processing when elongating Pol II is hypophosphorylated and define DSIF as part of the negative feedback regulation of NF-κB.
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DSIF, the Paf1 complex, and Tat-SF1 have nonredundant, cooperative roles in RNA polymerase II elongation
Genes & development, 2009Co-Authors: Yexi Chen, Yuki Yamaguchi, Yuta Tsugeno, Junichi Yamamoto, Tomoko Yamada, Mitsuhiro Nakamura, Koji Hisatake, Hiroshi HandaAbstract:Transcription elongation factor DSIF/Spt4-Spt5 is capable of promoting and inhibiting RNA polymerase II elongation and is involved in the expression of various genes. While it has been known for many years that DSIF inhibits elongation in collaboration with the negative elongation factor NELF, how DSIF promotes elongation is largely unknown. Here, an activity-based biochemical approach was taken to understand the mechanism of elongation activation by DSIF. We show that the Paf1 complex (Paf1C) and Tat-SF1, two factors implicated previously in elongation control, collaborate with DSIF to facilitate efficient elongation. In human cells, these factors are recruited to the FOS gene in a temporally coordinated manner and contribute to its high-level expression. We also show that elongation activation by these factors depends on P-TEFb-mediated phosphorylation of the Spt5 C-terminal region. A clear conclusion emerging from this study is that a set of elongation factors plays nonredundant, cooperative roles in elongation. This study also shows unambiguously that Paf1C, which is generally thought to have chromatin-related functions, is involve directlyd in elongation control.
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Role of human transcription elongation factor DSIF in the suppression of senescence and apoptosis.
Genes to cells : devoted to molecular & cellular mechanisms, 2009Co-Authors: Toshiharu Komori, Yuki Yamaguchi, Naoto Inukai, Tomoko Yamada, Hiroshi HandaAbstract:DSIF is an evolutionarily conserved, ubiquitously expressed, heterodimeric transcription elongation factor composed of two subunits, Spt4 and Spt5. Previous biochemical studies have shown that DSIF positively and negatively regulates RNA polymerase II elongation in collaboration with other protein factors. While several data suggest that DSIF is a 'general' elongation factor, there is also evidence that DSIF exerts a tissue- and gene-specific function. Here we sought to address the question of whether physiological functions of DSIF are general or specific, by using a sophisticated knockdown approach and gene expression microarray analysis. We found that Spt5 is essential for cell growth of various human cell lines and that Spt5 knockdown causes senescence and apoptosis. However, Spt5 knockdown affects a surprisingly small number of genes. In Spt5 knockdown cells, the p53 signaling pathway is activated and mediates part of the knockdown-induced transcriptional change, but apoptotic cell death occurs in the absence of p53. Structure-function analysis of Spt5 shows that the C-terminal approximately 300 amino acid residues are not required to support cell proliferation. These results suggest that one of the functions of Spt5 is to suppress senescence and apoptosis, and that this function is exerted through its association with Spt4 and Pol II.
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DSIF contributes to transcriptional activation by DNA-binding activators by preventing pausing during transcription elongation
Nucleic acids research, 2007Co-Authors: Wenyan Zhu, Yuki Yamaguchi, Tadashi Wada, Sachiko Okabe, Takuya Taneda, Hiroshi HandaAbstract:The transcription elongation factor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF) regulates RNA polymerase II (RNAPII) processivity by promoting, in concert with negative elongation factor (NELF), promoter-proximal pausing of RNAPII. DSIF is also reportedly involved in transcriptional activation. However, the role of DSIF in transcriptional activation by DNA-binding activators is unclear. Here we show that DSIF acts cooperatively with a DNA-binding activator, Gal4-VP16, to promote transcriptional activation. In the absence of DSIF, Gal4-VP16-activated transcription resulted in frequent pausing of RNAPII during elongation in vitro. The presence of DSIF reduced pausing, thereby supporting Gal4-VP16-mediated activation. We found that DSIF exerts its positive effects within a short time-frame from initiation to elongation, and that NELF does not affect the positive regulatory function of DSIF. Knockdown of the gene encoding the large subunit of DSIF, human Spt5 (hSpt5), in HeLa cells reduced Gal4-VP16-mediated activation of a reporter gene, but had no effect on expression in the absence of activator. Together, these results provide evidence that higher-level transcription has a stronger requirement for DSIF, and that DSIF contributes to efficient transcriptional activation by preventing RNAPII pausing during transcription elongation.
Patrick Cramer - One of the best experts on this subject based on the ideXlab platform.
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Structure of activated transcription complex Pol II-DSIF-PAF-SPT6.
Nature, 2018Co-Authors: Seychelle M. Vos, Lucas Farnung, Marc Boehning, C. Wigge, Andreas Linden, Henning Urlaub, Patrick CramerAbstract:Gene regulation involves activation of RNA polymerase II (Pol II) that is paused and bound by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we show that formation of an activated Pol II elongation complex in vitro requires the kinase function of the positive transcription elongation factor b (P-TEFb) and the elongation factors PAF1 complex (PAF) and SPT6. The cryo-EM structure of an activated elongation complex of Sus scrofa Pol II and Homo sapiens DSIF, PAF and SPT6 was determined at 3.1 A resolution and compared to the structure of the paused elongation complex formed by Pol II, DSIF and NELF. PAF displaces NELF from the Pol II funnel for pause release. P-TEFb phosphorylates the Pol II linker to the C-terminal domain. SPT6 binds to the phosphorylated C-terminal-domain linker and opens the RNA clamp formed by DSIF. These results provide the molecular basis for Pol II pause release and elongation activation.
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Structure of paused transcription complex Pol II–DSIF–NELF.
Nature, 2018Co-Authors: Seychelle M. Vos, Lucas Farnung, Henning Urlaub, Patrick CramerAbstract:Metazoan gene regulation often involves the pausing of RNA polymerase II (Pol II) in the promoter-proximal region. Paused Pol II is stabilized by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we report the cryo-electron microscopy structure of a paused transcription elongation complex containing Sus scrofa Pol II and Homo sapiens DSIF and NELF at 3.2 A resolution. The structure reveals a tilted DNA-RNA hybrid that impairs binding of the nucleoside triphosphate substrate. NELF binds the polymerase funnel, bridges two mobile polymerase modules, and contacts the trigger loop, thereby restraining Pol II mobility that is required for pause release. NELF prevents binding of the anti-pausing transcription elongation factor IIS (TFIIS). Additionally, NELF possesses two flexible 'tentacles' that can contact DSIF and exiting RNA. These results define the paused state of Pol II and provide the molecular basis for understanding the function of NELF during promoter-proximal gene regulation.
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structure of paused transcription complex pol ii DSIF nelf
Nature, 2018Co-Authors: Seychelle M. Vos, Lucas Farnung, Henning Urlaub, Patrick CramerAbstract:Metazoan gene regulation often involves the pausing of RNA polymerase II (Pol II) in the promoter-proximal region. Paused Pol II is stabilized by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we report the cryo-electron microscopy structure of a paused transcription elongation complex containing Sus scrofa Pol II and Homo sapiens DSIF and NELF at 3.2 A resolution. The structure reveals a tilted DNA-RNA hybrid that impairs binding of the nucleoside triphosphate substrate. NELF binds the polymerase funnel, bridges two mobile polymerase modules, and contacts the trigger loop, thereby restraining Pol II mobility that is required for pause release. NELF prevents binding of the anti-pausing transcription elongation factor IIS (TFIIS). Additionally, NELF possesses two flexible 'tentacles' that can contact DSIF and exiting RNA. These results define the paused state of Pol II and provide the molecular basis for understanding the function of NELF during promoter-proximal gene regulation.
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Structure of a transcribing RNA polymerase II–DSIF complex reveals a multidentate DNA–RNA clamp
Nature Structural & Molecular Biology, 2017Co-Authors: Carrie Bernecky, Jürgen M Plitzko, Patrick CramerAbstract:Cryo-EM and X-ray crystallography to determine the mammalian RNA Pol II–DSIF complex structure maps DSIF's polymerase, DNA-template and transcript contacts that facilitate transcription elongation. During transcription, RNA polymerase II (Pol II) associates with the conserved elongation factor DSIF. DSIF renders the elongation complex stable and functions during Pol II pausing and RNA processing. We combined cryo-EM and X-ray crystallography to determine the structure of the mammalian Pol II–DSIF elongation complex at a nominal resolution of 3.4 Å. Human DSIF has a modular structure with two domains forming a DNA clamp, two domains forming an RNA clamp, and one domain buttressing the RNA clamp. The clamps maintain the transcription bubble, position upstream DNA, and retain the RNA transcript in the exit tunnel. The mobile C-terminal region of DSIF is located near exiting RNA, where it can recruit factors for RNA processing. The structure provides insight into the roles of DSIF during mRNA synthesis.
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Structure of a transcribing RNA polymerase II-DSIF complex reveals a multidentate DNA-RNA clamp.
Nature structural & molecular biology, 2017Co-Authors: Carrie Bernecky, Jürgen M Plitzko, Patrick CramerAbstract:During transcription, RNA polymerase II (Pol II) associates with the conserved elongation factor DSIF. DSIF renders the elongation complex stable and functions during Pol II pausing and RNA processing. We combined cryo-EM and X-ray crystallography to determine the structure of the mammalian Pol II-DSIF elongation complex at a nominal resolution of 3.4 A. Human DSIF has a modular structure with two domains forming a DNA clamp, two domains forming an RNA clamp, and one domain buttressing the RNA clamp. The clamps maintain the transcription bubble, position upstream DNA, and retain the RNA transcript in the exit tunnel. The mobile C-terminal region of DSIF is located near exiting RNA, where it can recruit factors for RNA processing. The structure provides insight into the roles of DSIF during mRNA synthesis.
Birgitta M. Wöhrl - One of the best experts on this subject based on the ideXlab platform.
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structure and nucleic acid binding properties of kow domains 4 and 6 7 of human transcription elongation factor DSIF
Scientific Reports, 2018Co-Authors: Philipp K Zuber, Kristian Schweimer, Paul Rösch, Lukas Hahn, Anne Reinl, Stefan H Knauer, Maxwell E Gottesman, Birgitta M. WöhrlAbstract:The human transcription elongation factor DSIF is highly conserved throughout all kingdoms of life and plays multiple roles during transcription. DSIF is a heterodimer, consisting of Spt4 and Spt5 that interacts with RNA polymerase II (RNAP II). DSIF binds to the elongation complex and induces promoter-proximal pausing of RNAP II. Human Spt5 consists of a NusG N-terminal (NGN) domain motif, which is followed by several KOW domains. We determined the solution structures of the human Spt5 KOW4 and the C-terminal domain by nuclear magnetic resonance spectroscopy. In addition to the typical KOW fold, the solution structure of KOW4 revealed an N-terminal four-stranded β-sheet, previously designated as the KOW3-KOW4 linker. In solution, the C-terminus of Spt5 consists of two β-barrel folds typical for KOW domains, designated KOW6 and KOW7. We also analysed the nucleic acid and RNAP II binding properties of the KOW domains. KOW4 variants interacted with nucleic acids, preferentially single stranded RNA, whereas no nucleic acid binding could be detected for KOW6-7. Weak binding of KOW4 to the RNAP II stalk, which is comprised of Rpb4/7, was also detected, consistent with transient interactions between Spt5 and these RNAP II subunits.
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Crystal structure of the human transcription elongation factor DSIF hSpt4 subunit in complex with the hSpt5 dimerization interface
The Biochemical journal, 2009Co-Authors: Sabine Wenzel, Paul Rösch, Berta M. Martins, Birgitta M. WöhrlAbstract:The eukaryotic transcription elongation factor DSIF [DRB (5,6-dichloro-1-beta-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 A (1 A=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.
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Crystal structure of the human transcription elongation factor DSIF hSpt4 subunit in complex with the hSpt5 dimerization interface
Biochemical Journal, 2009Co-Authors: Sabine Wenzel, Paul Rösch, Berta M. Martins, Birgitta M. WöhrlAbstract:The eukaryotic transcription elongation factor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) sensitivity inducing factor (DSIF) 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 the negative elongation factor NELF, leads to promoter-proximal pausing of RNA polymerase II. Here we describe the crystal structure of hSpt4 in complex with the dimerization region of hSpt5 (amino acids 176 to 273) at a resolution of 1.55 Å. The heterodimer shows high structural similarity to its homologue from Saccharomyces cerevisiae. Furthermore, hSpt5 is structurally similar to the N-terminal domain (NTD) 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 by a glutamine in Escherichia coli NusG. However, exchanging the glutamine to glutamate proved not to be sufficient to induce hSpt4 binding.
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the small hspt4 subunit of the human transcription elongation factor DSIF is a zn finger protein with α β type topology
Biochemical and Biophysical Research Communications, 2008Co-Authors: Sabine Wenzel, Kristian Schweimer, Paul Rösch, Birgitta M. WöhrlAbstract:Abstract The eukaryotic transcription elongation factor 5,6-dichloro-1-beta- d -ribofuranosylbenzimidazole (DRB) sensitivity inducing factor (DSIF), is involved in regulating the processivity of RNA polymerase II. DSIF plays also a role in transcriptional activation, and in concert with the negative elongation factor NELF causes promoter proximal pausing of RNA polymerase II. Furthermore, DSIF has also been implicated in regulating the transcription of the human immunodeficiency virus proviral DNA. Human DSIF is composed of the two subunits, hSpt4 (p14) and hSpt5 (p160), corresponding to the yeast homologs Spt4 and Spt5. Here we show the purification and characterization of the small subunit, hSpt4. We were able to purify the protein in a soluble form separately from the larger hSpt5 subunit. CD and NMR spectroscopy show that the purified protein hSpt4 exhibits an α/β topology with a well defined tertiary structure. Furthermore metal analysis by ICP-OES indicates that the protein contains a functional 4-Cys Zn-finger.
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The small hSpt4 subunit of the human transcription elongation factor DSIF is a Zn-finger protein with α/β type topology
Biochemical and biophysical research communications, 2008Co-Authors: Sabine Wenzel, Kristian Schweimer, Paul Rösch, Birgitta M. WöhrlAbstract:Abstract The eukaryotic transcription elongation factor 5,6-dichloro-1-beta- d -ribofuranosylbenzimidazole (DRB) sensitivity inducing factor (DSIF), is involved in regulating the processivity of RNA polymerase II. DSIF plays also a role in transcriptional activation, and in concert with the negative elongation factor NELF causes promoter proximal pausing of RNA polymerase II. Furthermore, DSIF has also been implicated in regulating the transcription of the human immunodeficiency virus proviral DNA. Human DSIF is composed of the two subunits, hSpt4 (p14) and hSpt5 (p160), corresponding to the yeast homologs Spt4 and Spt5. Here we show the purification and characterization of the small subunit, hSpt4. We were able to purify the protein in a soluble form separately from the larger hSpt5 subunit. CD and NMR spectroscopy show that the purified protein hSpt4 exhibits an α/β topology with a well defined tertiary structure. Furthermore metal analysis by ICP-OES indicates that the protein contains a functional 4-Cys Zn-finger.
Tadashi Wada - One of the best experts on this subject based on the ideXlab platform.
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DSIF contributes to transcriptional activation by DNA-binding activators by preventing pausing during transcription elongation
Nucleic acids research, 2007Co-Authors: Wenyan Zhu, Yuki Yamaguchi, Tadashi Wada, Sachiko Okabe, Takuya Taneda, Hiroshi HandaAbstract:The transcription elongation factor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF) regulates RNA polymerase II (RNAPII) processivity by promoting, in concert with negative elongation factor (NELF), promoter-proximal pausing of RNAPII. DSIF is also reportedly involved in transcriptional activation. However, the role of DSIF in transcriptional activation by DNA-binding activators is unclear. Here we show that DSIF acts cooperatively with a DNA-binding activator, Gal4-VP16, to promote transcriptional activation. In the absence of DSIF, Gal4-VP16-activated transcription resulted in frequent pausing of RNAPII during elongation in vitro. The presence of DSIF reduced pausing, thereby supporting Gal4-VP16-mediated activation. We found that DSIF exerts its positive effects within a short time-frame from initiation to elongation, and that NELF does not affect the positive regulatory function of DSIF. Knockdown of the gene encoding the large subunit of DSIF, human Spt5 (hSpt5), in HeLa cells reduced Gal4-VP16-mediated activation of a reporter gene, but had no effect on expression in the absence of activator. Together, these results provide evidence that higher-level transcription has a stronger requirement for DSIF, and that DSIF contributes to efficient transcriptional activation by preventing RNAPII pausing during transcription elongation.
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transcriptional pausing caused by nelf plays a dual role in regulating immediate early expression of the junb gene
Molecular and Cellular Biology, 2006Co-Authors: Masatoshi Aida, Yuki Yamaguchi, Tadashi Wada, Yexi Chen, Koichi Nakajima, Hiroshi HandaAbstract:Human 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) negatively regulate transcription elongation by RNA polymerase II (RNAPII) in vitro. However, the physiological roles of this negative regulation are not well understood. Here, by using a number of approaches to identify protein-DNA interactions in vivo, we show that DSIF- and NELF-mediated transcriptional pausing has a dual function in regulating immediate-early expression of the human junB gene. Before induction by interleukin-6, RNAPII, DSIF, and NELF accumulate in the promoter-proximal region of junB, mainly at around position +50 from the transcription initiation site. After induction, the association of these proteins with the promoter-proximal region continues whereas RNAPII and DSIF are also found in the downstream regions. Depletion of a subunit of NELF by RNA interference enhances the junB mRNA level both before and after induction, indicating that DSIF- and NELF-mediated pausing contributes to the negative regulation of junB expression, not only by inducing RNAPII pausing before induction but also by attenuating transcription after induction. These regulatory mechanisms appear to be conserved in other immediate-early genes as well.
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Structure-function analysis of human Spt4: evidence that hSpt4 and hSpt5 exert their roles in transcriptional elongation as parts of the DSIF complex.
Genes to cells : devoted to molecular & cellular mechanisms, 2003Co-Authors: Dong-ki Kim, Yuki Yamaguchi, Tadashi Wada, Akiko Furuya, Naoto Inukai, Tomoko Yamada, Hiroe Sato, Hiroshi HandaAbstract:Background: The human Spt4/Spt5 complex, termed DRB-sensitivity inducing factor (DSIF) is a dual regulator of transcription that stimulates, or, when cooperating with negative elongation factor (NELF), represses RNA polymerase II (RNAPII) elongation. Spt4 and Spt5 are also thought to be involved in mRNA capping, homologous DNA recombination, and transcription-coupled DNA repair. As a first step to understanding how these proteins regulate diverse cellular processes, we investigated the structure and function of hSpt4 in vitro. Results: Immunodepletion of hSpt5 from HeLa nuclear extracts resulted in the efficient co-depletion of hSpt4. Using DSIF-depleted nuclear extracts and a series of Spt4 mutants, we examined the amino acid sequence of hSpt4 which was important for hSpt5 binding and for transcriptional repression and activation by DSIF. Unexpectedly, the zinc finger of hSpt4, which is critical for the yeast counterpart to function in vivo, was dispensable for hSpt5 binding and for transcriptional regulation in vitro. Conclusion: These and other results suggest: (i) that the central region of hSpt4 is necessary and sufficient for its function in vitro and (ii) that there is no free hSpt4 or hSpt5 in cells. We propose that hSpt4 and hSpt5 exert their roles in transcriptional regulation, and possibly in other nuclear processes, as parts of the DSIF complex.
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Evidence that Negative Elongation Factor Represses Transcription Elongation through Binding to a DRB Sensitivity-Inducing Factor/RNA Polymerase II Complex and RNA
Molecular and cellular biology, 2002Co-Authors: Yuki Yamaguchi, Tadashi Wada, Naoto Inukai, Takashi Narita, Hiroshi HandaAbstract:Negative elongation factor (NELF) is a human transcription factor complex that cooperates with DRB sensitivity-inducing factor (DSIF)/hSpt4-hSpt5 to repress elongation by RNA polymerase II (RNAPII). NELF activity is associated with five polypeptides, including NELF-A, a candidate gene product for Wolf-Hirschhorn syndrome, and NELF-E, a putative RNA-binding protein with arginine-aspartic acid (RD) dipeptide repeats. Here we report several important findings regarding the DSIF/NELF-dependent elongation control. First, we have established an effective method for purifying the active NELF complex using an epitope-tagging technique. Second, the five polypeptides each are important and together are sufficient for its function in vitro. Third, NELF does not bind to either DSIF or RNAPII alone but does bind to the preformed DSIF/RNAPII complex. Fourth, NELF-E has a functional RNA-binding domain, whose mutations impair transcription repression without affecting known protein-protein interactions. Taken together, we propose that NELF causes RNAPII pausing through binding to the DSIF/RNAPII complex and to nascent transcripts. These results also have implications for how DSIF and NELF are regulated in a gene-specific manner in vivo.
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A highly purified RNA polymerase II elongation control system.
The Journal of biological chemistry, 2001Co-Authors: Dan B. Renner, Yuki Yamaguchi, Tadashi Wada, Hiroshi Handa, David H. PriceAbstract:Abstract Studying the sensitivity of transcription to the nucleotide analog 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole has led to the discovery of a number of proteins involved in the regulation of transcription elongation by RNA polymerase II. We have developed a highly purified elongation control system composed of three purified proteins added back to isolated RNA polymerase II elongation complexes. Two of the proteins, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) and negative elongation factor (NELF), act as negative transcription elongation factors by increasing the time the polymerase spent at pause sites. P-TEFb reverses the negative effect of DSIF and NELF through a mechanism dependent on its kinase activity. TFIIF is a general initiation factor that positively affects elongation by decreasing pausing. We show that TFIIF functionally competes with DSIF and NELF, and this competition is dependent on the relative concentrations of TFIIF and NELF.
