The Experts below are selected from a list of 2883 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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CTCF regulates NELF, DSIF and P-TEFb recruitment during transcription.
Transcription, 2015Co-Authors: Clelia Laitem, Yuki Yamaguchi, Hiroshi Handa, Justyna Zaborowska, Michael Tellier, Qing-fu Cao, Sylvain Egloff, Shona MurphyAbstract:CTCF is a versatile transcription Factor with well-established roles in chromatin organization and insulator function. Recent findings also implicate CTCF in the control of Elongation by RNA polymerase (RNAP) II. Here we show that CTCF knockdown abrogates RNAP II pausing at the early Elongation checkpoint of c-myc by affecting recruitment of DRB-sensitivity-inducing Factor (DSIF). CTCF knockdown also causes a termination defect on the U2 snRNA genes (U2), by affecting recruitment of Negative Elongation Factor (NELF). In addition, CTCF is required for recruitment of positive Elongation Factor b (P-TEFb), which phosphorylates NELF, DSIF, and Ser2 of the RNAP II CTD to activate Elongation of transcription of c-myc and recognition of the snRNA gene-specific 3' box RNA processing signal. These findings implicate CTCF in a complex network of protein:protein/protein:DNA interactions and assign a key role to CTCF in controlling RNAP II transcription through the Elongation checkpoint of the protein-coding c-myc and the termination site of the non-coding U2, by regulating the recruitment and/or activity of key players in these processes.
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Transcription Elongation Factors DSIF and NELF: promoter-proximal pausing and beyond.
Biochimica et biophysica acta, 2012Co-Authors: Yuki Yamaguchi, Hirotaka Shibata, Hiroshi HandaAbstract:DRB sensitivity-inducing Factor (DSIF) and Negative Elongation Factor (NELF) were originally identified as Factors responsible for transcriptional inhibition by 5,6-dichloro-1-beta-d-ribofuranosyl-benzimidazole (DRB) and were later found to control transcription Elongation, together with P-TEFb, at the promoter-proximal region. Although there is ample evidence that these Factors play roles throughout the genome, other data also suggest gene- or tissue-specific roles for these Factors. In this review, we discuss how these apparently conflicting data can be reconciled. In light of recent findings, we also discuss the detailed mechanism by which these Factors control the Elongation process at the molecular level. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.
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Promoter-proximal pausing and its release: Molecular mechanisms and physiological functions
Experimental cell research, 2010Co-Authors: Kunitoshi Chiba, Yuki Yamaguchi, Junichi Yamamoto, Hiroshi HandaAbstract:For a long time, not much attention had been paid to post-initiation steps in transcription, because it was widely believed that transcriptional control was brought about almost entirely through the regulation of transcription initiation. However, it has become clear that the process of Elongation is also tightly controlled by a collection of regulatory Factors called transcription Elongation Factors and contributes, for example, to rapid induction of immediate-early genes and to the control over the viral life cycle. Transcription Elongation has attracted attention also because this process is coupled with various RNA processing events. In this review, we discuss biochemical and physiological aspects of Elongation control, particularly focusing on the role of the Negative Elongation Factor NELF.
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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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Cellular dynamics of the Negative transcription Elongation Factor NELF.
Experimental cell research, 2009Co-Authors: Tetsu M.c. Yung, Takashi Narita, Yuki Yamaguchi, Toshiharu Komori, Hiroshi HandaAbstract:Negative Elongation Factor (NELF) is a transcription Factor discovered based on its biochemical activity to suppress transcription Elongation, and has since been implicated in various diseases ranging from neurological disorders to cancer. Besides its role in promoter-proximal pausing of RNA polymerase II during early stages of transcription, recently we found that it also plays important roles in the 3'-end processing of histone mRNA. Furthermore, NELF has been found to form a distinct subnuclear structure, which we named NELF bodies. These recent developments point to a wide range of potential functions for NELF, and, as most studies on NELF thus far had been carried out in vitro, here, we prepared a complete set of fusion protein constructs of NELF subunits and carried out a general cell biological study of the intracellular dynamics of NELF. Our data show that NELF subunits exhibit highly specific subcellular localizations, such as in NELF bodies or in midbodies, and some shuttle actively between the nucleus and cytoplasm. We further show that loss of NELF from cells can lead to enlarged and/or multiple nuclei. This work serves as a foundation and starting point for further cell biological investigations of NELF in the future.
Yuki Yamaguchi - One of the best experts on this subject based on the ideXlab platform.
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Repression of RNA polymerase II Elongation in vivo is critically dependent on the C-terminus of Spt5. PLoS One 2009
2016Co-Authors: Hui Chen, Yuki Yamaguchi, Xavier Contreras, Hiroshi H, Matija B. Peterlin, Su GuoAbstract:The stalling of RNA polymerase II (RNAPII) at the promoters of many genes, including developmental regulators, stress-responsive genes, and HIVLTR, suggests transcription Elongation as a critical regulatory step in addition to initiation. Spt5, the large subunit of the DRB sensitivity-inducing Factor (DSIF), represses or activates RNAPII Elongation in vitro. How RNAPII Elongation is repressed in vivo is not well understood. Here we report that CTR1 and CTR2CT, the two repeat-containing regions constituting the C-terminus of Spt5, play a redundant role in repressing RNAPII Elongation in vivo. First, mis-expression of Spt5 lacking CTR1 or CTR2CT is inconsequential, but mis-expression of Spt5 lacking the entire C-terminus (termed NSpt5) dominantly impairs embryogenesis in zebrafish. Second, NSpt5 de-represses the transcription of hsp70-4 in zebrafish embryos and HIVLTR in cultured human cells, which are repressed at the RNAPII Elongation step under non-inducible conditions. Third, NSpt5 directly associates with hsp70-4 chromatin in vivo and increases the occupancy of RNAPII, positive transcription Elongation Factor b (P-TEFb), histone H3 Lys 4 trimethylation (H3K4Me3), and surprisingly, the Negative Elongation Factor A (NELF-A) at the locus, indicating a direct action of NSpt5 on the Elongation repressed locus. Together, these results reveal a dominant activity of NSpt5 to de-repress RNAPII Elongation, and suggest that the C
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CTCF regulates NELF, DSIF and P-TEFb recruitment during transcription.
Transcription, 2015Co-Authors: Clelia Laitem, Yuki Yamaguchi, Hiroshi Handa, Justyna Zaborowska, Michael Tellier, Qing-fu Cao, Sylvain Egloff, Shona MurphyAbstract:CTCF is a versatile transcription Factor with well-established roles in chromatin organization and insulator function. Recent findings also implicate CTCF in the control of Elongation by RNA polymerase (RNAP) II. Here we show that CTCF knockdown abrogates RNAP II pausing at the early Elongation checkpoint of c-myc by affecting recruitment of DRB-sensitivity-inducing Factor (DSIF). CTCF knockdown also causes a termination defect on the U2 snRNA genes (U2), by affecting recruitment of Negative Elongation Factor (NELF). In addition, CTCF is required for recruitment of positive Elongation Factor b (P-TEFb), which phosphorylates NELF, DSIF, and Ser2 of the RNAP II CTD to activate Elongation of transcription of c-myc and recognition of the snRNA gene-specific 3' box RNA processing signal. These findings implicate CTCF in a complex network of protein:protein/protein:DNA interactions and assign a key role to CTCF in controlling RNAP II transcription through the Elongation checkpoint of the protein-coding c-myc and the termination site of the non-coding U2, by regulating the recruitment and/or activity of key players in these processes.
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Transcription Elongation Factors DSIF and NELF: promoter-proximal pausing and beyond.
Biochimica et biophysica acta, 2012Co-Authors: Yuki Yamaguchi, Hirotaka Shibata, Hiroshi HandaAbstract:DRB sensitivity-inducing Factor (DSIF) and Negative Elongation Factor (NELF) were originally identified as Factors responsible for transcriptional inhibition by 5,6-dichloro-1-beta-d-ribofuranosyl-benzimidazole (DRB) and were later found to control transcription Elongation, together with P-TEFb, at the promoter-proximal region. Although there is ample evidence that these Factors play roles throughout the genome, other data also suggest gene- or tissue-specific roles for these Factors. In this review, we discuss how these apparently conflicting data can be reconciled. In light of recent findings, we also discuss the detailed mechanism by which these Factors control the Elongation process at the molecular level. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.
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Promoter-proximal pausing and its release: Molecular mechanisms and physiological functions
Experimental cell research, 2010Co-Authors: Kunitoshi Chiba, Yuki Yamaguchi, Junichi Yamamoto, Hiroshi HandaAbstract:For a long time, not much attention had been paid to post-initiation steps in transcription, because it was widely believed that transcriptional control was brought about almost entirely through the regulation of transcription initiation. However, it has become clear that the process of Elongation is also tightly controlled by a collection of regulatory Factors called transcription Elongation Factors and contributes, for example, to rapid induction of immediate-early genes and to the control over the viral life cycle. Transcription Elongation has attracted attention also because this process is coupled with various RNA processing events. In this review, we discuss biochemical and physiological aspects of Elongation control, particularly focusing on the role of the Negative Elongation Factor NELF.
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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.
Birgitta M. Wöhrl - One of the best experts on this subject based on the ideXlab platform.
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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.
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structural studies on the rna recognition motif of nelf e a cellular Negative transcription Elongation Factor involved in the regulation of hiv transcription
Biochemical Journal, 2006Co-Authors: Jampani Nageswara Rao, Sabine Wenzel, Kristian Schweimer, Paul Rösch, Liane Neumann, Birgitta M. WöhrlAbstract:The Elongation of transcription of HIV RNA at the TAR (transactivation-response element) is highly regulated by positive and Negative Factors. The cellular Negative transcription Elongation Factor NELF (Negative Elongation Factor) was suggested to be involved in transcriptional regulation of HIV-1 (HIV type 1) by binding to the stem of the viral TAR RNA which is synthesized by cellular RNA polymerase II at the viral long terminal repeat. NELF is a heterotetrameric protein consisting of NELF A, B, C or the splice variant D, and E. In the present study, we determined the solution structure of the RRM (RNA-recognition motif) of the RNA-binding subunit NELF E and studied its interaction with the viral TAR RNA. Our results show that the separately expressed recombinant NELF E RRM has alpha-helical and beta-strand elements adopting a betaalphabetabetaalphabeta fold and is able to bind to TAR RNA. Fluorescence equilibrium titrations with fluorescently labelled double- and single-stranded oligoribonucleotides representing the TAR RNA stem imply that NELF E RRM binds to the single-stranded TAR RNAs with K(d) values in the low-micromolar range.
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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Functional interactions of RNA-capping enzyme with Factors that positively and Negatively regulate promoter escape by RNA polymerase II.
Proceedings of the National Academy of Sciences, 2004Co-Authors: Subhrangsu S. Mandal, Tadashi Wada, Hiroshi Handa, Aaron J Shatkin, Chun Chu, Danny ReinbergAbstract:Capping of the 5' ends of nascent RNA polymerase II transcripts is the first pre-mRNA processing event in all eukaryotic cells. Capping enzyme (CE) is recruited to transcription complexes soon after initiation by the phosphorylation of Ser-5 of the carboxyl-terminal domain of the largest subunit of RNA polymerase II. Here, we analyze the role of CE in promoter clearance and its functional interactions with different Factors that are involved in promoter clearance. FCP1-mediated dephosphorylation of the carboxyl-terminal domain results in a drastic decrease in cotranscriptional capping efficiency but is reversed by the presence of DRB sensitivity-inducing Factor (DSIF). These results suggest involvement of DSIF in CE recruitment. Importantly, CE relieves transcriptional repression by the Negative Elongation Factor, indicating a critical role of CE in the Elongation checkpoint control mechanism during promoter clearance. This functional interaction between CE and the Negative Elongation Factor documents a dynamic role of CE in promoter clearance beyond its catalytic activities.
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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, Naoto Inukai, Yuki Yamaguchi, Tadashi Wada, Akiko Furuya, 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, Naoto Inukai, Takashi Narita, Tadashi Wada, 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.
Sabine Wenzel - One of the best experts on this subject based on the ideXlab platform.
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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.
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structural studies on the rna recognition motif of nelf e a cellular Negative transcription Elongation Factor involved in the regulation of hiv transcription
Biochemical Journal, 2006Co-Authors: Jampani Nageswara Rao, Sabine Wenzel, Kristian Schweimer, Paul Rösch, Liane Neumann, Birgitta M. WöhrlAbstract:The Elongation of transcription of HIV RNA at the TAR (transactivation-response element) is highly regulated by positive and Negative Factors. The cellular Negative transcription Elongation Factor NELF (Negative Elongation Factor) was suggested to be involved in transcriptional regulation of HIV-1 (HIV type 1) by binding to the stem of the viral TAR RNA which is synthesized by cellular RNA polymerase II at the viral long terminal repeat. NELF is a heterotetrameric protein consisting of NELF A, B, C or the splice variant D, and E. In the present study, we determined the solution structure of the RRM (RNA-recognition motif) of the RNA-binding subunit NELF E and studied its interaction with the viral TAR RNA. Our results show that the separately expressed recombinant NELF E RRM has alpha-helical and beta-strand elements adopting a betaalphabetabetaalphabeta fold and is able to bind to TAR RNA. Fluorescence equilibrium titrations with fluorescently labelled double- and single-stranded oligoribonucleotides representing the TAR RNA stem imply that NELF E RRM binds to the single-stranded TAR RNAs with K(d) values in the low-micromolar range.
