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Biomedical subjects

Yuki Yamaguchi

Publications and source records attributed to Yuki Yamaguchi.

At least 37 records · Page 2Linked to original sources
PubMed2004

Attenuation of estrogen receptor alpha-mediated transcription through estrogen-stimulated recruitment of a negative elongation factor.

Estrogen receptor alpha (ERalpha) signaling is paramount for normal mammary gland development and function and the repression of breast cancer. ERalpha function in gene regulation is mediated by a number of coactivators and corepressors, most of which are known to modify chromatin structure and/or influence the assembly of the regulatory complexes at the level of transcription initiation. Here we describe a novel mechanism of attenuating the ERalpha activity. We show that cofactor of BRCA1 (COBRA1), an integral subunit of the human negative elongation factor (NELF), directly binds to ERalpha and represses ERalpha-mediated transcription. Reduction of the endogenous NELF proteins in breast cancer cells using small interfering RNA results in elevated ERalpha-mediated transcription and enhanced cell proliferation. Chromatin immunoprecipitation reveals that recruitment of COBRA1 and the other NELF subunits to endogenous ERalpha-responsive promoters is greatly stimulated upon estrogen treatment. Interestingly, COBRA1 does not affect the estrogen-dependent assembly of transcription regulatory complexes at the ERalpha-regulated promoters. Rather, it causes RNA polymerase II (RNAPII) to pause at the promoter-proximal region, which is consistent with its in vitro biochemical activity. Therefore, our in vivo work defines the first corepressor of nuclear receptors that modulates ERalpha-dependent gene expression by stalling RNAPII. We suggest that this new level of regulation may be important to control the duration and magnitude of a rapid and reversible hormonal response.

Blotting, Northern↗
PubMed2004

Interferon regulatory factor 1 (IRF-1) and IRF-2 distinctively up-regulate gene expression and production of interleukin-7 in human intestinal epithelial cells.

Intestinal epithelial cell-derived interleukin (IL)-7 functions as a pleiotropic and nonredundant cytokine in the human intestinal mucosa; however, the molecular basis of its production has remained totally unknown. We here showed that human intestinal epithelial cells both constitutively and when induced by gamma interferon (IFN-gamma) produced IL-7, while several other factors we tested had no effect. Transcriptional regulation via an IFN regulatory factor element (IRF-E) on the 5' flanking region, which lacks canonical core promoter sequences, was pivotal for both modes of IL-7 expression. IRF-1 and IRF-2, the latter of which is generally known as a transcriptional repressor, were shown to interact with IRF-E and transactivate IL-7 gene expression in an IFN-gamma-inducible and constitutive manner, respectively. Indeed, tetracycline-inducible expression experiments revealed that both of these IRF proteins up-regulated IL-7 protein production, and their exclusive roles were further confirmed by small interfering RNA-mediated gene silencing systems. Moreover, these IRFs displayed distinct properties concerning the profile of IL-7 transcripts upon activation and expression patterns within human colonic epithelial tissues. These results suggest that the functional interplay between IRF-1 and IRF-2 serves as an elaborate and cooperative mechanism for timely as well as continuous regulation of IL-7 production that is essential for local immune regulation within human intestinal mucosa.

5' Untranslated Regions↗
PubMed2004

Elongation inhibition by DRB sensitivity-inducing factor is regulated by the A20 promoter via a novel negative element and NF-kappaB.

A20 is an immediate-early NF-kappaB target gene. Prior to NF-kappaB stimulation, the A20 promoter is bound by the polymerase II machinery to allow rapid transcription activation. Here we show that the basal A20 transcription is repressed at the level of elongation in a promoter-specific fashion. Immunodepletion in vitro and RNA interference in cultured cells suggest that the basal elongation inhibition is conferred by DRB sensitivity-inducing factor (DSIF). We have identified a negative upstream promoter element called ELIE that controls DSIF activity. Remarkably, following NF-kappaB stimulation, inhibition of the A20 promoter by DSIF persists, but it is now regulated by NF-kappaB rather than ELIE. Similar regulation by DSIF is shown for another NF-kappaB-responsive gene, the IkappaBalpha gene. These findings reveal an intimate and dynamic relationship between DSIF inhibition of elongation and promoter-bound transcription factors. The potential significance of the differential regulation of DSIF activity by cis-acting elements is discussed.

Base Sequence↗
PubMed2004

Human Spt6 stimulates transcription elongation by RNA polymerase II in vitro.

Recent studies have suggested that Spt6 participates in the regulation of transcription by RNA polymerase II (RNAPII). However, its underlying mechanism remains largely unknown. One possibility, which is supported by genetic and biochemical studies of Saccharomyces cerevisiae, is that Spt6 affects chromatin structure. Alternatively, Spt6 directly controls transcription by binding to the transcription machinery. In this study, we establish that human Spt6 (hSpt6) is a classic transcription elongation factor that enhances the rate of RNAPII elongation. hSpt6 is capable of stimulating transcription elongation both individually and in concert with DRB sensitivity-inducing factor (DSIF), comprising human Spt5 and human Spt4. We also provide evidence showing that hSpt6 interacts with RNAPII and DSIF in human cells. Thus, in vivo, hSpt6 may regulate multiple steps of mRNA synthesis through its interaction with histones, elongating RNAPII, and possibly other components of the transcription machinery.

Animals↗
PubMed2003

A novel hydrogen peroxide-induced phosphorylation and ubiquitination pathway leading to RNA polymerase II proteolysis.

DNA damage-induced ubiquitination of the largest subunit of RNA polymerase II, Rpb1, has been implicated in transcription-coupled repair for years. The studies so far, however, have been limited to the use of bulky helix-distorting DNA damages caused by UV light and cisplatin, which are corrected by the nucleotide excision repair pathway. Non-bulky, non-helix-distorting damages are caused at high frequency by reactive oxygen species in cells and corrected by the base excision repair pathway. Contrary to a classic view, we recently found that the second type of DNA lesions also causes RNA polymerase II stalling in vitro. In this paper, we show that hydrogen peroxide (H(2)O(2)) causes significant ubiquitination and proteasomal degradation of Rpb1 by mechanisms that are distinct from those employed after UV irradiation. UV irradiation and H(2)O(2) treatment cause characteristic changes in protein kinases phosphorylating the carboxyl-terminal domain at Ser-2 and -5. The H(2)O(2)-induced ubiquitination is likely dependent on unusual Ser-5 phosphorylation by ERK1/2. Moreover, the H(2)O(2)-induced ubiquitination occurs on transcriptionally engaged polymerases without the help of Cockayne syndrome A and B proteins and von Hippel-Lindau tumor suppressor proteins, which are all required for the UV-induced ubiquitination. These results suggest that stalled polymerases are recognized and ubiquitinated differentially depending on the types of DNA lesions. Our findings may have general implications in the basic mechanism of transcription-coupled nucleotide excision repair and base excision repair.

Cell Death↗
PubMed2003

NELF and DSIF cause promoter proximal pausing on the hsp70 promoter in Drosophila.

NELF and DSIF collaborate to inhibit elongation by RNA polymerase IIa in extracts from human cells. A multifaceted approach was taken to investigate the potential role of these factors in promoter proximal pausing on the hsp70 gene in Drosophila. Immunodepletion of DSIF from a Drosophila nuclear extract reduced the level of polymerase that paused in the promoter proximal region of hsp70. Depletion of one NELF subunit in salivary glands using RNA interference also reduced the level of paused polymerase. In vivo protein-DNA cross-linking showed that NELF and DSIF associate with the promoter region before heat shock. Immunofluorescence analysis of polytene chromosomes corroborated the cross-linking result and showed that NELF, DSIF, and RNA polymerase IIa colocalize at the hsp70 genes, small heat shock genes, and many other chromosomal locations. Finally, following heat shock induction, DSIF and polymerase but not NELF were strongly recruited to chromosomal puffs harboring the hsp70 genes. We propose that NELF and DSIF cause polymerase to pause in the promoter proximal region of hsp70. The transcriptional activator, HSF, might cause NELF to dissociate from the elongation complex. DSIF continues to associate with the elongation complex and could serve a positive role in elongation.

Animals↗
PubMed2003

Mutations of the PAX6 gene detected in patients with a variety of optic-nerve malformations.

The PAX6 gene is involved in ocular morphogenesis and is expressed in the developing central nervous system and numerous ocular tissues during development. PAX6 mutations have been detected in various ocular anomalies, including aniridia, Peters anomaly, corneal dystrophy, congenital cataracts, and foveal hypoplasia. However, it has not been identified in patients with optic-nerve malformations. Here, we identified novel mutations in eight pedigrees with optic-nerve malformations, including coloboma, morning glory disc anomaly, optic-nerve hypoplasia/aplasia, and persistent hyperplastic primary vitreous. A functional assay demonstrated that each mutation decreased the transcriptional activation potential of PAX6 through the paired DNA-binding domain. PAX6 and PAX2 are each thought to downregulate the expression of the other. Four of the detected mutations affected PAX6-mediated transcriptional repression of the PAX2 promoter in a reporter assay. Because PAX2 gene mutations were detected in papillorenal syndrome, alternation of PAX2 function by PAX6 mutations may affect phenotypic manifestations of optic-nerve malformations.

Adult↗
PubMed2003

NTP-driven translocation by human RNA polymerase II.

We report a "running start, two-bond" protocol to analyze elongation by human RNA polymerase II (RNAP II). In this procedure, the running start allowed us to measure rapid rates of elongation and provided detailed insight into the RNAP II mechanism. Formation of two bonds was tracked to ensure that at least one translocation event was analyzed. By using this method, RNAP II is stalled briefly at a defined template position before restoring the next NTP. Significantly, slow reaction steps are identified both before and after phosphodiester bond synthesis, and both of these steps can be highly dependent on the next templated NTP. The initial and final NTP-driven events, however, are not identical, because the slow step after chemistry, which includes translocation and pyrophosphate release, is regulated differently by elongation factors hepatitis delta antigen and transcription factor IIF. Because recovery from a stall and the processive transition from one bond to the next can be highly NTP-dependent, we conclude that translocation can be driven by the incoming substrate NTP, a model fully consistent with the RNAP II elongation complex structure.

Adenosine Triphosphate↗
PubMed2003

Evaluation of the impact of bioaugmentation and biostimulation by in situ hybridization and microelectrode.

Three rotating disk biofilm reactors were operated to evaluate whether bioaugmentation and biostimulation can be used to improve the start-up of microbial nitrification. The first reactor was bioaugmented during start-up period with an enrichment culture of nitrifying bacteria, the second reactor received a synthetic medium containing NH(4)(+) and NO(2)(-) to facilitate concomitant proliferation of ammonia- and nitrite-oxidizing bacteria, and the third reactor was used as a control. To evaluate the effectiveness of bioaugmentation and biostimulation approaches, time-dependent developments of nitrifying bacterial community and in situ nitrifying activity in biofilms were monitored by fluorescence in situ hybridization (FISH) technique and microelectrode measurements of NH(4)(+), NO(2)(-), NO(3)(-), and O(2). In situ hybridization results revealed that addition of the enrichment culture of nitrifying bacteria significantly facilitated development of dense nitrifying bacterial populations in the biofilm shortly after, which led to a rapid start-up and enhancement of in situ nitrification activity. The inoculated bacteria could proliferate and/or survive in the biofilm. In addition, the addition of nitrifying bacteria increased the abundance of nitrifying bacteria in the surface of the biofilm, resulting in the higher nitrification rate. On the other hand, the addition of 2.1mM NO(2)(-) did not stimulate the growth of nitrite-oxidizing bacteria and did inhibit the proliferation of ammonia-oxidizing bacteria instead. Thus, the start-up of NO(2)(-) oxidation was unchanged, and the start-up of NH(4)(+) oxidation was delayed. In all the three biofilm reactors, data sets of time series analyses on population dynamics of nitrifying bacteria determined by FISH, in situ nitrifying activities determined by microelectrode measurements, and the reactor performances revealed an approximate agreement between the appearance of nitrifying bacteria and the initiation of nitrification activity, suggesting that the combination of these techniques was a very powerful monitoring tool to evaluate the effectiveness of bioaugmentation and biostimulation strategies.

Biofilms↗
PubMed2003

Mechanism of H-8 inhibition of cyclin-dependent kinase 9: study using inhibitor-immobilized matrices.

BACKGROUND: Positive transcription elongation factor b (P-TEFb), which phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII), is comprised of the catalytic subunit cyclin-dependent kinase 9 (CDK9) and the regulatory subunit cyclin T. The kinase activity and transcriptional activation potential of P-TEFb is sensitive to various compounds, including H-8, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB), and flavopiridol. RESULTS: We investigated the molecular mechanism of the H-8 inhibition of CDK9 using matrices to which H-9, an amino derivative of H-8, was immobilized. CDK9 bound specifically to H-9, and this interaction was competitively inhibited by ATP and DRB, but not by flavopiridol. Mutational analyses demonstrated that the central region of CDK9, which encompasses the T-loop region, was important for its binding to H-9. CONCLUSIONS: H-9-immobilized latex beads are useful for trapping CDK9 and a subset of kinases from crude cell extracts. The flavopiridol-binding region of CDK9 is most likely different from its H-9-binding region. These biochemical data support previously reported observations which were based on crystallographic data.

Adenosine Triphosphate↗
PubMed2003

Structure-function analysis of human Spt4: evidence that hSpt4 and hSpt5 exert their roles in transcriptional elongation as parts of the DSIF complex.

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.

Amino Acid Sequence↗
PubMed2003

A rapid purification method for human RNA polymerase II by two-step affinity chromatography.

The molecular dissection of transcription mechanisms is greatly facilitated by constructing and manipulating defined transcription systems in vitro. This approach requires highly purified transcription factors. A major enzyme participating in the transcription reaction is RNA polymerase II (RNAPII), which is composed of at least 12 subunits (RPB1-12). Due to its complex structure, it is difficult to prepare highly pure RNAPII by the conventional purification procedure. We transfected HeLa cells with a plasmid expressing RPB3 with a double FLAG-histidine tag on its amino-terminus. A high yielding clone was isolated and its extracts were subjected to immunoaffinity purification and then Co(2+) affinity chromatography. This resulted in a preparation of RNAPII complexes that consisted of all the core subunits, including the double-tagged RPB3 protein. Transcription reactions with oligo (dC)-tailed templates and transcription assays involving general transcription factors revealed that the double-tagged RNAPII complexes are active and functional in basal and activated transcription. Our method is superior to the conventionally used purification procedure in that the final preparation is markedly more pure (92% versus 40%), and the procedures are much less time-consuming. Thus, this two-step affinity purification method is an uncomplicated and effective method by which active and functional RNAPII can be prepared.

Cell Line↗
PubMed2003

Human transcription elongation factor NELF: identification of novel subunits and reconstitution of the functionally active complex.

The multisubunit transcription elongation factor NELF (for negative elongation factor) acts together with DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) sensitivity-inducing factor (DSIF)/human Spt4-Spt5 to cause transcriptional pausing of RNA polymerase II (RNAPII). NELF activity is associated with five polypeptides, A to E. NELF-A has sequence similarity to hepatitis delta antigen (HDAg), the viral protein that binds to and activates RNAPII, whereas NELF-E is an RNA-binding protein whose RNA-binding activity is critical for NELF function. To understand the interactions of DSIF, NELF, and RNAPII at a molecular level, we identified the B, C, and D proteins of human NELF. NELF-B is identical to COBRA1, recently reported to associate with the product of breast cancer susceptibility gene BRCA1. NELF-C and NELF-D are highly related or identical to the protein called TH1, of unknown function. NELF-B and NELF-C or NELF-D are integral subunits that bring NELF-A and NELF-E together, and coexpression of these four proteins in insect cells resulted in the reconstitution of functionally active NELF. Detailed analyses using mutated recombinant complexes indicated that the small region of NELF-A with similarity to HDAg is critical for RNAPII binding and for transcriptional pausing. This study defines several important protein-protein interactions and opens the way for understanding the mechanism of DSIF- and NELF-induced transcriptional pausing.

Amino Acid Sequence↗
PubMed2002

Effects of endogenous DNA base lesions on transcription elongation by mammalian RNA polymerase II. Implications for transcription-coupled DNA repair and transcriptional mutagenesis.

The blockage of transcription elongation by RNA polymerase II (pol II) is thought to be a trigger for transcription-coupled repair in the pathway of nucleotide excision repair. Purified pol II and oligo(dC)-tailed templates containing a single non-bulky DNA lesion on the transcribed strand such as an apurinic/apyrimidinic (AP) site, uracil, or 8-oxoguanine (8-oxoG) were used for transcription elongation assays. In this system pol II could bypass both the AP site and uracil without pausing and insert cytosine opposite the AP site and either guanine or adenine opposite to uracil. Thus, the AP site on the DNA templates could lead to correct transcription only if depurination at guanine occurred, whereas uracil generated either the correct transcriptional product or an incorrect one with a G:C to A:T transition. In the case of 8-oxoG, pol II stalled at the lesion, but sometimes bypassed it and inserted a cytosine residue or the incorrect adenine residue leading to a G:C to T:A transversion. These findings indicate that 8-oxoG lesions caused a blockage of transcription elongation and/or the misincorporation of a ribonucleotide by pol II, implying the initiation of transcription-coupled repair of 8-oxoG and/or transcriptional mutagenesis.

Adenine↗
PubMed2002

Spatial redox regulation of a critical cysteine residue of NF-kappa B in vivo.

Reduction-oxidation (redox) regulation has been implicated in the activation of the transcription factor NF-kappaB. However, the significance and mechanism of the redox regulation remain elusive, mainly due to the technical limitations caused by rapid proton transfer in redox reactions and by the presence of many redox molecules within cells. Here we establish versatile methods for measuring redox states of proteins and their individual cysteine residues in vitro and in vivo, involving thiol-modifying reagents and LC-MS analysis. Using these methods, we demonstrate that the redox state of NF-kappaB is spatially regulated by its subcellular localization. While the p65 subunit and most cysteine residues of the p50 subunit are reduced similarly in the cytoplasm and in the nucleus, Cys-62 of p50 is highly oxidized in the cytoplasm and strongly reduced in the nucleus. The reduced form of Cys-62 is essential for the DNA binding activity of NF-kappaB. Several lines of evidence suggest that the redox factor Ref-1 is involved in Cys-62 reduction in the nucleus. We propose that the Ref-1-dependent reduction of p50 in the nucleus is a necessary step for NF-kappaB activation. This study also provides the first example of a drug that inhibits the redox reaction between two specific proteins.

Cysteine↗