Figure 1
From: Novel p63 target genes involved in paracrine signaling and keratinocyte differentiation
Identification of novel direct p63 target genes. (a) Rapidly growing cultures of NHEKs were analyzed for p63 isoforms by comparison with protein markers generated by transfection of the indicated expression vectors in H1299 cells. Total protein was isolated and expression of p63 isoforms was analyzed by western blot. (b) NHEKs were chemically crosslinked and quantitative chromatin immunoprecipitation used to determine the relative binding of p63 to a negative control region (con) and to three previously published p63 binding sites located in the p21, MDM2, and Jagged-1. (c) p63-bound genes in NHEKs were overlayed with p63 target genes identified in ME180 cells.17 Bootstrap distribution statistical analysis was performed to compare the significance of gene overlap between the two datasets. (d) NHEKs were transduced with a retrovirus-expressing shRNAs specific to GFP (con) or the DNA-binding domain of p63 (sip63). Cells were puromycin selected for 48 h and harvested for western analysis of p63 and actin. (e) RNA was isolated and submitted, in duplicate, for Affymetrix microarray analysis to identify genes with expression patterns dependent on the presence of p63. Data shown are representative of at least three independent experiments. p63-bound genes identified in the ChIP dataset (left circle) were overlayed with genes exhibiting p63-dependent expression in microarray analyses (right circle). Eighty-two genes, as annotated by the GeneSpring 7 software, were present in both datasets and were chosen for further biological analyses
