Project description:Identifying the mechanism action of SMARCB1 in totally different cell lines. Determine whether it is general role or context dependent role.
Project description:SMARCB1 (Snf5/Ini1/Baf47) is a potent tumor suppressor, the loss of which serves as the diagnostic feature in Malignant Rhabdoid Tumors (MRT) and Atypical Teratoid/Rhabdoid Tumors (AT/RT), two highly aggressive forms of pediatric neoplasms. Here, we restore Smarcb1 expression in cells derived from Smarcb1-deficient tumors which developed in Smarcb1-heterozygous p53-/- mice. Profiling Smarcb1 dependent gene expression we find genes which are dependent on Smarcb1 expression to be enriched for ECM and cell adhesion functions. We identify Igfbp7, which is related to the insulin-like growth factor binding proteins family, as a downstream target of Smarcb1 transcriptional activity, and show that re-introduction of Igfbp7 alone can hinder tumor development. Two cancer cell lines, 167 and 365, derived from Smarcb1-deficient tumors which developed in Smarcb1-heterozygous p53-/- mice were re-infected with a retro-viral vector for Smarcb1 re-expression or an empty retro-viral vector as control. Total-RNA was collected 3 days post infection so as to enrich for direct targets of Smarcb1 transcriptionaly regulated genes
Project description:In humans, there are eleven subtypes of linker histones that exhibit cell- and tissue-specific expression. Linker histone H1 proteins bind to both the core histones and linker DNA of chromatin fibers; and not only participate in control of gene activity but also serve to stabilize higher order chromatin structure. To determine the potential roles of linker histones in differentiation, we examined the global distribution of linker histone subtype H1.5 in human IMR90 fibroblasts and H1 embryonic stem cells (hESCs). Surprisingly, H1.5 binds to and represses a large fraction of gene family clusters in fully differentiated cell types representing all three embryonic germ layers. Little or no H1.5 enrichment at gene family clusters was detected in undifferentiated hESCs or partially differentiated somatic cells. We also found that SIRT1 histone deacetylase and H3K9me2, a repressive histone modification, are also enriched at gene family cluster in IMR90 cells, likely generating a stably repressive chromatin domain. To find out the mechanism of H1.5 targeting, H1.5 or SIRT1 was depleted in IMR90 cells by siRNA, and the binding patterns of SIRT1 and H1.5 were examined. In H1.5 knockdown cells, SIRT1 binding pattern was changed dramatically, and this changed pattern highly correlates to SIRT1 distribution in hESC. However, depletion of SIRT1 could not change the global binding pattern of H1.5. Depletion of H1.5 or SIRT1 leads to up-regulation of ~50% gene family clusters. However, the sets of gene family clusters that are affected by these two factors are different, suggesting H1.5 and SIRT1 may regulate gene transcription via different pathways.