Project description:Histone variant H2A.Z is a critical player in setting up the chromatin environment that mediates transcription and other activities on chromatin. However, how H2A.Z is incorporated to specific chromatin regions is not clear. To examine the potential role of sequence-specific transcription factors in targeting H2A.Z, we screened for genome-wide H2A.Z-interacting proteins in vivo using a novel technique called bait Protein-Protein Interaction-sequencing (bPPI-seq). Among the hundreds of H2A.Z-interacting proteins identified by bPPI-seq, we show that a zinc-finger transcription factor, Osr1 interacts with H2A.Z both in vitro and in vivo and co-localizes with H2A.Z on chromatin. Knockdown of Osr1 compromised H2A.Z deposition to hundreds of chromatin sites enriched with Osr1 binding motifs. Furthermore, Osr1 and H2A.Z co-regulate the expression of numerous target genes. These results indicate that Osr1 directly interacts with H2A.Z, mediates its incorporation to a large number of target sites and regulates gene expression. Our data indicate that bPPI-seq can be widely applied to identify unbiasedly interacting proteins under physiologic conditions.
Project description:DNA-protein interactions regulate critical biological processes. Identifying proteins that bind to specific, functional genomic loci is essential for understanding the underlying regulatory mechanisms on a molecular level. Here, we describe a novel co-binding-mediated protein profiling (CMPP) strategy to investigate the interactome of DNA G-quadruplexes (G4s) in cellular chromatin. CMPP involves cell-permeable, functionalized G4-ligand probes that bind endogenous G4s and subsequently crosslink to co-binding G4-interacting proteins in situ. We show the robustness of CMPP on proximity labelling of a G4 binding protein in vitro. Employing this approach in live cells, we identify hundreds of putative G4-interacting proteins from various functional classes. Next, we observe high G4 binding affinity and selectivity for several G4 interactors in vitro and confirm direct G4 interactions for one of the top candidates in chromatin. Our studies provide a chemical approach to map protein interactions of specific nucleic acid features in living cells.
Project description:We have combined biochemical purification of Mediator from chromatin with ChIP-sequencing to reveal Mediator occupupancy to DNA globally and to identify proteins interacting specifically with Mediator in chromatin. We find that Mediator occupy strong chromosomally interacting domain (CID) boundaries and nearly all tRNA genes. Purification of Mediator from chromatin shows that it interacts with proteins and protein complexes that have been shown to interact with CID boundaries such as RSC, Ssu72 and histone H4. We also show specific interaction between Mediator and the Arp2/Arp3, CPF, CF 1A and LSm, complexes in chromatin. These factors are involved in mRNA 3'-end processing, gene looping, actin assembly and mRNA decay.
Project description:DDX39A and DDX56 recombinant proteins were assayed using commercial protein microarrays in order to detect potential interaction partners.