Project description:Human ΔNp63-specific siRNA was obtained from Invitrogen (Carlsbad, CA; sense: 5′-ACAAUGCCCAGACUCAAUU-3′; antisense: 5′-AAUUGAGUCUGGGCAUUGU-3′). Scrambled sequence siRNA for the negative control was purchased from Invitrogen. Transfections were performed using Lipofectamine RNAiMAX (Invitrogen) in Opti-MEM (GIBCO) at 40 nmol/L according to the manufacturer's instructions. The culture medium was replaced at 6 h after siRNA transfection. mRNA was extracted from NHEKs transfected with ΔNp63-specific or scramble sequence siRNA at 72 h after transfection. Microarray slides were scanned using a 3D-GENE human 25k (TORAY, Tokyo, Japan) and microarray images were automatically analyzed with AROSTM, version 4.0 (Operon Biotechnologies, Tokyo, Japan).
Project description:TP63 (p63) is strongly expressed in lower-grade carcinomas of head-and-neck, skin, breast, urothelium, etc. to maintain the well-differentiated phenotype. TP63 has two transcription start sites at exon 1 and exon 3’ to produce TAp63 and DeltaNp63 isoforms, respectively. The major protein, DeltaNp63alpha, epigenetically activates genes essential for epidermal/craniofacial differentiation, including DeltaNp63 itself. To examine whether weakly expressed TAp63 has a specific role, we disrupted exon 1 by CRISPR-Cas9 homology directed repair in a head and neck squamous cell carcinoma (SCC) line. Surprisingly, TAp63 knockout cells, either by monoallelic GFP cassette insertion paired with a frameshift deletion allele or by biallelic GFP cassette insertion, caused DeltaNp63 silencing. Loss of keratinocyte-specific gene expression, switching of intermediate filaments from KRT(s) to VIM, and suppression of cell-cell and cell-matrix adhesion components indicated core events of epithelial mesenchymal transition. Most of the positively and negatively impacted genes including DeltaNp63 displayed local DNA methylation changes. Furthermore, DeltaNp63 expression was partially rescued by transfection of TAp63alpha followed by incubation with DNA methyltransferase inhibitor Zebularine. TAp63, as a minor part of TP63 gene, may possibly be involved in the auto-activation mechanism of DeltaNp63 by which keratinocyte-specific epigenome is maintained in SCC.
Project description:TP63 (p63) is strongly expressed in lower-grade carcinomas of head-and-neck, skin, breast, urothelium, etc. to maintain the well-differentiated phenotype. TP63 has two transcription start sites at exon 1 and exon 3’ to produce TAp63 and DeltaNp63 isoforms, respectively. The major protein, DeltaNp63alpha, functions as a core factor to organize super enhancers of genes essential for epidermal/craniofacial differentiation and for self-activation of DeltaNp63. To examine whether very weakly expressed TAp63 has a specific role, we disrupted exon 1 by CRISPR-Cas9 homology directed repair (HDR) in a head and neck squamous cell carcinoma (SCC) line. Surprisingly, TAp63 knockout cells, with either ‘monoallelic HDR and a frameshift deletion on the other allele’ or ‘biallelic HDR’, caused DeltaNp63 silencing. Loss of keratinocyte-specific gene expression, replacement of KRT5 with VIM, and transcriptional suppression of cell-cell and cell-matrix adhesion components indicated core events of epithelial mesenchymal transition. Most of the positively and negatively impacted genes including DeltaNp63 displayed local CpG methylation changes. Furthermore, DeltaNp63 expression was partially rescued by transfection of TAp63alpha, followed by incubation with DNA methyltransferase inhibitor Zebularine. This study suggests that TAp63 is indispensable for DeltaNp63 expression by which keratinocyte-specific epigenome is maintained in SCC.
Project description:We report the analysis of the transcriptomic program regulated by DeltaNp63. In short, RNA was extracted from SCC90 cells transfected with siRNA against ∆Np63 or non-specific scrambled siRNA, and total RNA was harvested from the cells using a TRIzol-mediated cell lysis approach. To identify the deregulated genes, RNA-Sequencing was performed on extracted total RNAs and the sequences obtained were selectively aligned on a chimeric genome composed of the human genome (hg38) using STAR version 2.5.3a.
Project description:A loss of StarPap would be predicted to result in a decrease in cellular levels of mRNAs which it polyadenylates. Moreover, if PIPKIalpha has a function relationship with StarPap, knockdown of PIPKIalpha should cause a decrease in a pool of target mRNAs which require both StarPap and PIPKIalpha for their maturation. To test this, we independently knocked down StarPap and PIPKIalpha, and performed microarray analysis of total polyadenylated mRNAs from each group. Experiment Overall Design: HEK293 cells were transfected with siRNA specific for StarPap or PIPKIalpha or control siRNA. N=3
Project description:Changes in the cellular transcriptome, proteome and phosphorylated proteome after knockdown of ERK5 protein in HCT116 cells using PROTACs and siRNA, respectively.