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:The mouse lncRNA Braveheart (Bvht) as a non-coding transcript has been found to act in trans to regulate cardiovascular lineage commitment. However, the mechanism of Bvht action is still not clear. lncRNAs have been shown to regulate gene expression though cooperating with protein partners. Recently, we experimentally determine the secondary structure of Bvht containing a novel structural motif AGIL. AGIL motif deletion (BvhtdAGIL) in mouse embryonic stem cells prevents the transition from mesoderm cells to cardiac progenitors. To identify proteins that interact with the Bvht AGIL motif, we used a human protein microarray platform (Human ProtoArray, Life Technology). Full-length Bvht and BvhtdAGIL transcripts were generated by in vitro transcription and labeled with Cy5. 50pmol Cy5-labeled RNAs were individually incubated with the protein microarray.
Project description:DDX39A and DDX56 recombinant proteins were assayed using commercial protein microarrays in order to detect potential interaction partners.
Project description:Vacuoles and lysosomes are single-membrane-bound organelles involved in diverse functions such as intracellular digestion and storage or secretion of metabolites. To understand their origin, evolution and fundamental features, the identification of proteins comprising these compartments in missing links would be invaluable. So, we isolated the vacuoles from Cyanidioschyzon merolae, which is considered to be one of the most primitive photosynthetic eukaryotes, and identified 46 proteins by matrix-assisted laser desorption/ionization time of flight-mass spectrometry. Keywords: peptide mass fingerprinting, MALDI-TOF