A Valuable Extension♦ : A Progesterone Receptor Co-activator (JDP2) Mediates Activity through Interaction with Residues in the Carboxyl-terminal Extension of the DNA Binding Domain
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PROVIDER: S-EPMC2782059 | biostudies-literature | 2009 Sep
REPOSITORIES: biostudies-literature
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Project description:Sigma factors, the specificity subunits of RNA polymerase, are involved in interactions with promoter DNA, the core subunits of RNA polymerase, and transcription factors. The bacteriophage T4-encoded activator, MotA, is one such factor, which engages the C terminus of the Escherichia coli housekeeping sigma factor, σ(70). MotA functions in concert with a phage-encoded co-activator, AsiA, as a molecular switch. This process, termed sigma appropriation, inhibits host transcription while activating transcription from a class of phage promoters. Previous work has demonstrated that MotA contacts the C terminus of σ(70), H5, a region that is normally bound within RNA polymerase by its interaction with the β-flap tip. To identify the specific σ(70) residues responsible for interacting with MotA and the β-flap tip, we generated single substitutions throughout the C terminus of σ(70). We find that MotA targets H5 residues that are normally engaged by the β-flap. In two-hybrid assays, the interaction of σ(70) with either the β-flap tip or MotA is impaired by alanine substitutions at residues Leu-607, Arg-608, Phe-610, Leu-611, and Asp-613. Transcription assays identify Phe-610 and Leu-611 as the key residues for MotA/AsiA-dependent transcription. Phe-610 is a crucial residue in the H5/β-flap tip interaction using promoter clearance assays with RNA polymerase alone. Our results show how the actions of small transcriptional factors on a defined local region of RNA polymerase can fundamentally change the specificity of polymerase.
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Project description:BackgroundCrosstalk between the aryl hydrocarbon receptor (AhR) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling is called the "AhR-Nrf2 gene battery", which works synergistically in detoxification to support cell survival. Nrf2-dependent phase II gene promoters are controlled by coordinated recruitment of the AhR to adjacent dioxin responsive element (DRE) and Nrf2 recruitment to the antioxidative response element (ARE). The molecular interaction between AhR and Nrf2 members, and the regulation of each target, including phase I and II gene complexes, and their mediators are poorly understood.MethodsKnockdown and forced expression of AhR-Nrf2 battery members were used to examine the molecular interactions between the AhR-Nrf2 axis and AhR promoter activation. Sequential immunoprecipitation, chromatin immunoprecipitation, and histology were used to identify each protein complex recruited to their respective cis-elements in the AhR promoter. Actin fiber distribution, cell spreading, and invasion were examined to identify functional differences in the AhR-Jdp2 axis between wild-type and Jdp2 knockout cells. The possible tumorigenic role of Jdp2 in the AhR-Nrf2 axis was examined in mutant Kras-Trp53-driven pancreatic tumors.ResultsCrosstalk between AhR and Nrf2 was evident at the transcriptional level. The AhR promoter was activated by phase I ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) through the AhR-Jdp2-Nrf2 axis in a time- and spatial transcription-dependent manner. Jdp2 was a bifunctional activator of DRE- and ARE-mediated transcription in response to TCDD. After TCDD exposure, Jdp2 activated the AhR promoter at the DRE and then moved to the ARE where it activated the promoter to increase reactive oxygen species (ROS)-mediated functions such as cell spreading and invasion in normal cells, and cancer regression in mutant Kras-Trp53-driven pancreatic tumor cells.ConclusionsJdp2 plays a critical role in AhR promoter activation through the AhR-Jdp2-Nrf2 axis in a spatiotemporal manner. The AhR functions to maintain ROS balance and cell spreading, invasion, and cancer regression in a mouse model of mutant Kras-Trp53 pancreatic cancer. These findings provide new insights into the roles of Jdp2 in the homeostatic regulation of oxidative stress and in the antioxidation response in detoxification, inflammation, and cancer progression.
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Project description:Results of in vitro assays identified residues in the C-terminal domain (CTD) of human immunodeficiency virus type 1 (HIV-1) integrase (IN) important for IN-IN and IN-DNA interactions, but the potential roles of these residues in virus replication were mostly unknown. Sixteen CTD residues were targeted here, generating 24 mutant viruses. Replication-defective mutants were typed as class I (blocked at integration) or class II (additional reverse transcription and/or assembly defects). Most defective viruses (15 of 17) displayed reverse transcription defects. In contrast, replication-defective HIV-1(E246K) synthesized near-normal cDNA levels but processing of Pr55(gag) was largely inhibited in virus-producing cells. Because single-round HIV-1(E246K.Luc(R-)) transduced cells at approximately 8% of the wild-type level, we concluded that the late-stage processing defect contributed significantly to the overall replication defect of HIV-1(E246K). Results of complementation assays revealed that the CTD could function in trans to the catalytic core domain (CCD) in in vitro assays, and we since determined that certain class I and class II mutants defined a novel genetic complementation group that functioned in cells independently of IN domain boundaries. Seven of eight novel Vpr-IN mutant proteins efficiently trans-complemented class I active-site mutant virus, demonstrating catalytically active CTD mutant proteins during infection. Because most of these mutants inefficiently complemented a class II CCD mutant virus, the majority of CTD mutants were likely more defective for interactions with cellular and/or viral components that affected reverse transcription and/or preintegration trafficking than the catalytic activity of the IN enzyme.
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Project description:Pseudomonas aeruginosa is an important bacterial opportunistic pathogen, presenting a significant threat towards individuals with underlying diseases such as cystic fibrosis. The transcription factor AmrZ regulates expression of multiple P. aeruginosa virulence factors. AmrZ belongs to the ribbon-helix-helix protein superfamily, in which many members function as dimers, yet others form higher order oligomers. In this study, four independent approaches were undertaken and demonstrated that the primary AmrZ form in solution is tetrameric. Deletion of the AmrZ C-terminal domain leads to loss of tetramerization and reduced DNA binding to both activated and repressed target promoters. Additionally, the C-terminal domain is essential for efficient AmrZ-mediated activation and repression of its targets.
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Project description:Jak/STAT is an important signaling pathway mediating multiple events in development. We describe participation of metazoan co-activator SAYP/PHF10 in this pathway downstream of STAT. The latter, via its activation domain, interacts with the conserved core of SAYP. STAT is associated with the SAYP-containing co-activator complex BTFly and recruits BTFly onto genes. SAYP is necessary for stimulating STAT-driven transcription of numerous genes. Mutation of SAYP leads to maldevelopments similar to those observed in STAT mutants. Thus, SAYP is a novel co-activator mediating the action of STAT.
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Project description:Utilizing a homologous gene replacement in order to retain the native promoter and 5' and 3' untranslated messenger regions (and thereby ensure physiological validity), we have shown that deletion of the N-terminal 389 amino acids of the transcriptional activator AREA does not result in nitrogen metabolite derepression in Aspergillus nidulans. Our results provide no evidence for a modulating interaction involving the N terminus of AREA and contrast with those of H. K. Lamb, A. L. Dodds, D. R. Swatman, E. Cairns, and A. R. Hawkins (J. Bacteriol. 179:6649-6656, 1997), who used nontargeted ectopic copies of a construct containing a heterologous promoter and untranslated regions. Results obtained with this deletion mutant, nevertheless, provide further evidence for the dispensability of large portions of AREA.
| S-EPMC107639 | biostudies-literature
Project description:Cyclin-dependent kinase 12 (CDK12) phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) but its roles in transcription beyond the expression of DNA damage response genes remain unclear. Here, we have used TT-seq and mNET-seq to monitor the direct effects of rapid CDK12 inhibition on transcription activity and CTD phosphorylation in human cells. CDK12 inhibition causes a genome-wide defect in transcription elongation and a global reduction of CTD Ser2 and Ser5 phosphorylation. The elongation defect is explained by the loss of the elongation factors LEO1 and CDC73, part of PAF1 complex, and SPT6 from the newly-elongating pol II. Our results indicate that CDK12 is a general activator of pol II transcription elongation and indicate that it targets both Ser2 and Ser5 residues of the pol II CTD.
2020-06-03 | GSE115290 | GEO
Project description:Cyclin-dependent kinase 12 (CDK12) phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) but its roles in transcription beyond the expression of DNA damage response genes remain unclear. Here, we have used TT-seq and mNET-seq to monitor the direct effects of rapid CDK12 inhibition on transcription activity and CTD phosphorylation in human cells. CDK12 inhibition causes a genome-wide defect in transcription elongation and a global reduction of CTD Ser2 and Ser5 phosphorylation. The elongation defect is explained by the loss of the elongation factors LEO1 and CDC73, part of PAF1 complex, and SPT6 from the newly-elongating pol II. Our results indicate that CDK12 is a general activator of pol II transcription elongation and indicate that it targets both Ser2 and Ser5 residues of the pol II CTD.
2020-06-18 | GSE133156 | GEO
Project description:Listeria monocytogenes is a facultative intracellular pathogen that secretes the cytolysin listeriolysin O (LLO), which enables the bacteria to cross the phagosomal membrane. L. monocytogenes regulates LLO activity in the phagosome and minimizes its activity in the host cytosol. Mutants that fail to compartmentalize LLO activity are cytotoxic and have attenuated virulence. Here, we showed that residues N478 and V479 of LLO are required for LLO hemolytic activity and bacterial virulence. A single N478A mutation (LLON478A) significantly increased the hemolytic activity of LLO at a neutral pH, while no difference was observed at the optimum acidic pH, compared with wild-type LLO. Conversely, the mutant LLOV479A exhibited lower hemolytic activity at the acidic pH, but not at the neutral pH. The double mutant LLON478AV479A showed a greater decrease in hemolytic activity at both the acidic and neutral pHs. Interestingly, strains producing LLON478A or LLOV479A lysed erythrocytes similarly to the wild-type strain. Surprisingly, bacteria-secreting LLON478AV479A had barely detectable hemolytic activity, but exhibited host cell cytotoxicity, escaped from the phagosome, grew intracellularly, and spread cell-to-cell with the same efficiency as the wild-type strain, but were highly attenuated in virulence in mice. These data demonstrate that these two residues are required for LLO hemolytic activity and pathogenicity in mice, but not for escape from the phagosome and cell-to-cell spreading. The finding that the nearly non-hemolytic LLON478AV479A mutant grew intracellularly indicates that mutagenesis of a virulence determinant is a novel approach for the development of live vaccine strains.
| S-EPMC5671954 | biostudies-literature
Project description:IntroductionPuberty is a period of increased susceptibility to factors that cause increased breast cancer risk in adulthood. Mammary end buds (EBs) that develop during puberty are believed to be the targets of breast cancer initiation. Whereas the role of estrogen (E) has been extensively studied in pubertal mammary gland development, the role of progesterone (P) during puberty is less defined.MethodsPubertal and prepubertal ovariectomized mice were treated with vehicle control (C), E, P, or E+P. Mammary glands from these mice were analyzed for changes in morphology, proliferation, and expression of the downstream targets amphiregulin (AREG) and receptor activator of NF-?B ligand (RANKL).ResultsP, acting specifically through the progesterone receptor, induced increases in mammary gland proliferation and EB formation that were associated with increased AREG expression in ducts and EBs. E, acting specifically through the estrogen receptor, produced similar responses also mediated by AREG. Blocking AREG action by treatment with an EGFR inhibitor completely abrogated the effect of P on EB formation and proliferation and significantly reduced proliferation within ducts. P also increased expression of RANKL, primarily in ducts. Treatment with RANK-Fc, an inhibitor of RANKL, reduced P-dependent proliferation in ducts and to a lesser extent in EB, but did not cause EB regression.ConclusionsThese results demonstrate a novel P-specific effect through AREG to cause EB formation and proliferation in the developing mammary gland both before and during puberty. Thus, hormones and/or factors in addition to E that upregulate AREG can promote mammary gland development and have the potential to affect breast cancer risk associated with pubertal mammary gland development.
| S-EPMC3738150 | biostudies-literature