Project description:Proteome analysis of affinity-purified materials prepared from nucleosome/chromatin fractions of HEK293T cells expressing various constructs.
Project description:Proteome analysis of affinity-purified materials prepared from chromatin fractions of HEK293T cells expressing various constructs. HEK293T cells transiently expressing various constructs were subjected to fanChIP (fractionation-assisted chromatin immunoprecipitation) method described in our previous paper (Okuda et al. 2014 Nucleic Acids Research 42;7 p4241-4256).
Project description:Sequence-specific transcription factors (TFs) regulate gene expression by binding to cognate motifs in promoters and enhancers. However, predicting genomic TF binding events and their quantitative contribution to expression remains a major challenge. In principle, the binding and enhancer activity of specific sites in vivo might depend on: (i) latent properties of the motif instance, (ii) cooperative interactions with other TFs that bind in the immediate vicinity, and (iii) the chromatin state of the sites in the genome. Here, we used massively parallel reporter assays (MPRA) involving 32,115 natural and synthetic enhancers, together with high-throughput in vivo assays, to systematically dissect the contributions of motif affinity, cooperative interactions, and chromatin accessibility to the binding and regulatory activity of genomic sequences that contain motifs for PPARγ, a TF that serves as a key regulator of adipogenesis. We show that PPARγ binding and enhancer activity are governed by distinct features. Genomic PPARγ binding to motif sites is largely governed by on larger-scale features, such as chromatin accessibility, whereas the degree to which a PPARγ motif site enhances transcriptional activity depends on the sequence immediately surround the motif. We detect and functionally validate a network of TFs comprised of multiple functional classes that collaborate with PPARγ to drive transcription. We extensively perturb this network, revealing functional cooperativity among classes of TFs that does not depend on precise positioning. Together, these results present a clear picture of how chromatin and TFs from distinct functional classes interact with PPARγ to determine binding and enhancer activity, and provide a paradigm for studying any TF.
Project description:Nonsense-mediated mRNA decay (NMD) is a conserved RNA degradation pathway that is involved in development, resistance to viral infections and evolution. Upf1, a core NMD factor, is associated with a large variety of cellular RNA in complex ribonucleoprotein particles. We characterized NMD complexes previously by affinity purification of tagged protein followed by mass spectrometry. Here, we extend our observations to proteins that are present in NMD complexes, but for whom the interaction with NMD factors is mediated by RNA. We purified tagged versions of Lsm1, Lsm7, Pat1, Dhh1 and Pab1 and assessed the presence and amount of NMD and RNA-related proteins in each purification. As expected from our previously published results, Upf1 was present in these purifications, suggesting novel hypotheses about the role of Pab1 and the Lsm1-7 complexes in RNA degradation.