Project description:We performed a DNA pull-down assay to identify DNA-binding proteins interacting with the cas6 promoter in the M. tuberculosis Δorn strain
Project description:We performed a DNA pull-down assay to identify DNA-binding proteins interacting with the cas6 promoter in the M. tuberculosis Δorn strain
Project description:We performed DNA-pull down experiments using biotin-labeled and unlabeled promoter sequences of Rsf1, a susceptibility factor in rice against root-knot nematode infection, to capture interacting nuclear proteins from rice roots at different time points post-infection (0, 1, and 3 days). Captured proteins were analyzed by LC-MS/MS to identify potential transcriptional regulators or binding partners of the Rsf1 promoter.
Project description:We have designed a zebrafish genomic microarray to identify DNA-protein interactions in the proximal promoter regions of over 11,000 zebrafish genes. Using these microarrays, together with chromatin immunoprecipitation with an antibody directed against tri-methylated lysine 4 of Histone H3, we demonstrate the feasibility of this method in zebrafish. This approach will allow investigators to determine the genomic binding locations of DNA interacting proteins during development and expedite the assembly of the genetic networks that regulate embryogenesis. Keywords: ChIP-chip
Project description:Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the leading cause of death among all infectious diseases. There are 11 eukaryotic-like serine/threonine protein kinases (STPKs) in Mtb, which are thought to play pivotal roles in cell growth, signal transduction and pathogenesis. However, their underlying mechanisms of action remain largely uncharacterized. In this study, using a Mtb proteome microarray, we have globally identified the binding proteins in Mtb for all of the STPKs, and constructed the first STPK protein interaction (KPI) map that includes 492 binding proteins and 1,027 interactions. Bioinformatics analysis showed that the interacting proteins reflect diverse functions, including roles in two-component system, transcription, protein degradation, and cell wall integrity. Functional investigations confirmed that PknG regulates cell wall integrity through key components of peptidoglycan (PG) biosynthesis, e.g., MurC. The global STPK-KPIs network constructed here is expected to serve as a rich resource for understanding the key signaling pathways in Mtb, thus facilitating drug development and effective control of Mtb.
Project description:G-quadruplexes (G4s) are noncanonical DNA secondary structures formed through the self-association of guanines, and they are distributed widely across the genome. G4 participates in multiple biological processes including gene transcription, and G4-targeted ligands serve as potential therapeutic agents for DNA-targeted therapies. However, genome-wide studies of the exact roles of G4s in transcriptional regulation are still lacking. We found that drug-induced promoter-proximal RNA polymerase II pausing promotes nearby G4 formation, and oppositely, G4 stabilization by G4-targeted ligands globally reduces RNA polymerase II occupancy at gene promoters as well as nascent RNA synthesis. To study the underlying mechanisms by which native G4 affects transcriptional regulation, we annealed the biotin-labeled core promoter DNA to form G4s and performed pull-down assays with nuclear extraction proteins in the presence or absence of TMPyP4. Mass spectrometry analysis was performed to identify the interacting proteins with G4-forming core promoter DNA.
Project description:The metabolome profiles of S. cerevisiae mutants where the promoter of genes predicted to code for peroxisomally localized proteins are swapped with the TEF2 promoter were measured by flow-injection analysis.
Project description:Tuberculosis caused by Mycobacterium tuberculosis (Mtb) infection remains a huge global public health problem. One striking characteristic of Mtb is its ability to adapt to hypoxia, and thus ensuing transition to dormant state for persistent infection, but how the hypoxia responses of Mtb is regulated remains largely unknown. Here, we performed a quantitative acetylome analysis to compare the acetylation profile of Mtb under aeration and hypoxia, and showed that 377 acetylation sites in 269 proteins of Mtb were significantly change under hypoxia. Especially, deacetylation of Dormancy Survival Regulator (DosR) at K182 promoted the hypoxia response of Mtb and enhanced transcription of DosR-targeted genes. Mechanistically, recombinant DosRK182R protein demonstrated enhanced DNA-binding activity in comparison with DosRK182Q protein. Moreover, Rv0998 was identified as an acetyltransferase that mediates the acetylation of DosR at K182. Deletion of Rv0998 also promoted the adaption of Mtb to hypoxia and transcription of DosR-targeted genes. Mice infected with Mtb strain containing acetylation-defective DosRK182R or lacking Rv0998 had much lower bacterial counts, and less severe histopathological impairments compared with those infected with the wild-type strain. Our findings suggest that hypoxia induces the deacetylation of DosR, which in turn increases its DNA binding ability to promote the transcription of target genes, allowing Mtbto transit to dormancy under hypoxia.
Project description:We performed CovR ChIP-seq analysis in the emm1 strain MGAS2221 and its CovS kinase deficient derivative strain 2221-CovS-E281A. We identified that CovR bound in the promoter regions of nearly all virulence factor encoding genes in the CovR regulon. Additionally, direct CovR binding was observed for numerous genes encoding proteins involved in amino acid metabolism, but we found limited direct CovR binding to genes encoding other transcriptional regulators. The consensus sequence AATRANAAAARVABTAAA was present in the promoters of genes directly regulated by CovR, and mutations of highly conserved positions within this motif relieved CovR repression of the hasA and M2221_0187 promoters. Analysis of strain 2221-CovS-E281A revealed that binding of CovR at repressed, but not activated, promoters is highly dependent on CovR~P state. CovR repressed virulence factor encoding genes could be grouped dependent on how variation in CovR~P differentially impacted DNA binding and gene transcript levels.