Abstract
Abstract: :
Purpose: Previously, we have shown that pinin (Pnn) is involved in the regulation of epithelial cell adhesion. Pnn and its associated proteins of the SR–family, which are involved in mRNA splicing events, co–localize and interact in nuclear speckles. Interestingly, Pnn can also up regulate E–cadherin gene activity through its binding to the co–repressor CtBP, a multifunctional transcriptional regulator, which targets E–cadherin promoter. Transcriptional co–repressors bridge basal transcription machinery to enzymatic complexes capable of maintaining the silenced chromatin state within the gene regulatory regions. We are now addressing the mechanism by which Pnn may impact both transcription and mRNA processing. Methods: Pnn expression was knocked–down in HCET cells by expressing a short hairpin RNAi from pU6–vector. In vivo interaction studies from transiently transfected cells were carried out using anti–Flag or anti–myc affinity agarose. Pol II/Pnn complex was immuno–isolated from HeLa nuclear extracts using anti–Pol II. Chromatin immunoprecipitation (ChIP) were performed using the following antibodies: anti–HA, anti–acetylated histone H3, anti–polymerase 2, anti–dimethylated histone H3 at lysine9 (H3K9). For the PCR reactions primers spanning E–cadherin promoter region or GAPDH promoter region were utilized. Results: Knocking down Pnn expression with shRNA results in a down regulation of E–cadherin and loss of epithelial cell adhesion. In addition, the effect of Pnn–RNAi could be rescued by transfection of Pnn vectors carrying conserved substitutions within the RNAi target. These results are consistent with Pnn’s role in regulating epithelial cell–cell adhesion. ChIP analyses revealed that Pnn is associated with E–cadherin promoter but not GAPDH promoter. In addition, Pnn expressing cells exhibited enhanced acetylation of H4, reduction in dimethylation at H3K9, and increased presence of Pol II at the E–cadherin promoter. Conclusions: These data suggest that expression of Pnn is linked to increase in cell–cell adhesion and elevation in E–cadherin. Furthermore, the presence of Pnn at the E–cadherin promoter region may promote transcriptionally favorable local chromatin state along with the recruitment of transcriptional machinery associated with activation of the epithelial gene expression. Thus, Pnn may play a fundamental role in transcriptional regulation in addition to its previously reported role in RNA splicing, adding Pnn to the growing list of proteins that functionally bridge transcription and mRNA processing. (Supported by NIH grant EY07883)
Keywords: cell adhesions/cell junctions • cornea: epithelium • gene/expression