Project description:To determine the global gene occupancy by Wiskott - Aldrich syndrome Protein (WASP) we perform ChIP-seq assay in two lymphoblastoid cell lines. We identify WASP-enriched genes, including several WASP-interaction genes previously reported; in addition, our results suggest the implication of WASP in diverse cellular process
Project description:To investigate a role of nuclear WASp in T cell development we performed WASp chromatin immunoprecipitation and deep sequencing (ChIP-Seq) in thymocytes and spleen CD4+ T cells. To pre-process raw ChIP-Seq data, the total number of reads were normalized and aligned against the mouse genome. WASp was enriched at transcription start sites of a large number of protein-coding genes. Many of the WASp-enriched genes were associated with RNA Polymerase II-enriched genes and active epigenetic marks of transcription; H3K4m3, H3K9a, H3K27a, and with the epigenetic mark for active enhancers H3K4m1. To study the distribution of overactive WASpI296T in the thymocyte genome and to identify regions enriched in WASpI296T binding, we performed second round of ChIP-Seq analysis using the WASp F-8 antibody. To detect differences in gene enrichment between thymocytes expressing wildtype WASp or WASpI296T, we applied stringent conditions and subtracted common genes between the two samples. Using this approach, we identify 70 WASpI296T-enriched genes. Functional clustering of these genes revealed that WASpI296T was associated with RNA Polymerase II genes in 11 functional groups of genes.thymocytes and spleen CD4+ T cells. WASp was enriched at transcription start sites of a large number of protein-coding genes.
Project description:Wiskott-Aldrich syndrome (WAS), which is caused by mutations in the gene encoding WASP, manifests in a wide range of hematologic and immune dysfunctions and predisposition to cancer development 1. WASP is most known as a cytoplasmic effector of actin cytoskeleton rearrangement. However, defective actin polymerization cannot explain many aspects of WAS pathogenesis. Incomplete knowledge of WASP function precludes in-depth understanding of the underlying mechanisms of WAS, and therefore hampers the development of effective therapies. Here we generated induced pluripotent stem cells (iPSCs) from WAS patients (WAS-iPSC) and corresponding isogenic iPSCs wherein the mutations were corrected by targeted genome editing. Hematopoietic cells differentiated from WAS-iPSCs not only recapitulated known disease phenotypes, but also revealed novel nuclear functions of WASP. WASP deficiency causes differential expression of genes involved in many aspects of immune cell function, most prominently cell proliferation. It also leads to a large number of alternative splicing events that are highly enriched in cell cycle regulators. Proteomic analyses revealed that WASP physically interacted with nuclear body components, nuclear structural proteins, chromatin modifying complexes, and many RNA-binding proteins including multiple splicing factors. We show that WASP physically interacts with SRSF2 and is important for nuclear speckle organization. WASP regulates cell cycle progression by physically interacting with EZH2 and thereby influencing epigenetic silencing of its target gene CDKN2A (aka p16). Together, these observations unveil novel functions of WASP in regulation of RNA splicing and cell cycle. The binding of WASP to SRSF2 and EZH2 and the misregulation of these two frequently mutated genes in hematologic malignancies2 in WAS cells provide a possible mechanism for the frequent occurance of malignancy in WAS patients. Furthermore, WAS-associated splicing and proliferation abnormalities could serve as diagnosis tools for patients at risk for cancer and may be exploited as therapeutic targets.
Project description:First brain methylome and transcriptomic data of two simple eusocial insects: Polistes canadensis paper wasp and Dinoponera quadriceps dinosaur ant
Project description:The pleiotropic RTK Kit can provide cytoskeletal signals that define cell shape, positioning and migration, but the underlying mechanisms are less well understood. Here we provide evidence that Kit signals through WASP (Wiskott-Aldrich Syndrome Protein), the central hematopoietic actin nucleation- promoting factor and regulator of the cytoskeleton. KL-mediated gene expression in WT and WASP-deficient BMMCs was compared and revealed that approximately 30% of all Kit-induced changes were WASP-dependent. The results indicate that Kit signaling through WASP is necessary for normal Kit-mediated filopodia formation, cell survival and gene expression and provide new insight in the mechanism how WASP exerts a strong selective pressure in hematopoiesis.