Project description:This SuperSeries is composed of the following subset Series:; GSE10562: Induction of ERDNp63a via Tamoxifen in primary keratinocytes; GSE10563: Primary keratinocytes treated with Tamoxifen; GSE10564: Silencing of p63 (trp63) in primary keratinocytes via siRNA oligo transfection. Experiment Overall Design: Refer to individual Series
Project description:Human primary keratinocytes were depleted of GRHL3 by siRNA and induced to differentiated for 2 days by addition of Calcium Primary normal human keratinocytes were transfected with GRHL3 or scrambled control siRNA using RNAi max (Life Technologies). 24 hours post transfection medium was raised to 1.8mM to induce differentiation. Cells were collected 48 hours later.
Project description:Human primary keratinocytes were depleted of MLL2 by siRNA and induced to differentiated for 2 days by addition of Calcium Primary normal human keratinocytes were transfected with MLL2 or scrambled control siRNA using RNAi max (Life Technologies). 24 hours post transfection medium was raised to 1.8mM calcium to induce differentiation. Cells were collected 48 hours later.
Project description:Cellular binary fate decisions require the progeny to silence genes associated with the alternative fate. The major subsets of alpha:beta T cells have been extensively studied as a model system for fate decisions. While the transcription factor RUNX3 is required for the initiation of Cd4 silencing in CD8 T cell progenitors, it is not required to maintain the silencing of Cd4 and other helper T lineage genes. The other runt domain containing protein, RUNX1, silences Cd4 in an earlier T cell progenitor, but this silencing is reversed whereas the gene silencing after RUNX3 expression is not reverse. Therefore, we hypothesized that RUNX3 and not RUNX1 recruits other factors that maintains the silencing of helper T lineage genes in CD8 T cells. To this end, we performed a proteomics screen of RUNX1 and RUNX3 to determine candidate silencing factors.
Project description:We identified p63 target genes and binding sites responsible for ectodermal defects by genome-wide profiling of p63 binding using ChIP-seq and expression analysis in human primary keratinocytes from patients with p63 mutations. As proof of principle, we identified a novel de novo microdeletion causing limb defects (SHFM1) that includes a p63 binding site functioning as a cis-regulatory element to control expression of the distally located DLX5/DLX6 genes essential for limb development. Our data demonstrate that target genes and regulatory elements detected in this study can serve as powerful tools to identify causative mutations of unresolved ectodermal disorders. ChIP-seq profiles of p63 in primary human keratinocytes established from two different normal individuals.
Project description:<p>Transcription factor p63 is a key regulator of epidermal keratinocyte proliferation and differentiation. Mutations in the p63 DNA-binding domain are associated with Ectrodactyly Ectodermal Dysplasia Cleft Lip/Palate (EEC) syndrome. Underlying molecular mechanism of these mutations however remain unclear. Here we characterized the transcriptome and epigenome of p63 mutant keratinocytes derived from EEC patients. The transcriptome of p63 mutant keratinocytes deviated from the normal epidermal cell identity. Epigenomic analyses showed an altered enhancer landscape in p63 mutant keratinocytes contributed by loss of p63-bound active enhancers and by unexpected gain of enhancers. The gained enhancers were frequently bound by deregulated transcription factors such as RUNX1. Reversing RUNX1 overexpression partially rescued deregulated gene expression and the altered enhancer landscape. Our findings identify an unreported disease mechanism whereby mutant p63 rewires the enhancer landscape and affects epidermal cell identity, consolidating the pivotal role of p63 in controlling the enhancer landscape of epidermal keratinocytes.</p>
