Transcriptional Regulation Study Of The Human PNRC Gene

PNRC (Proline-rich Nuclear Receptor Coactivator) was previously identified using bovine SF-1 (steroidogenic factor 1) as the bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. PNRC was found to interact with the ligand-binding domains of all the nuclear receptors tested, including ERα, ERβ, PR, GR, TR, RAR, and RXR, in a ligand-dependent manner. PNRC was also found to interact in a ligand-independent manner with orphan receptors such as SF1 and ERRα. Unlike most of the coactivators that interact with nuclear receptors through their LXXLL motif, this new coactivator interacts with nuclear receptors through a proline-rich Src homology domain-3 (SH3)-binding motif, S-D (E)-P-P-S-P-S. In addition to functioning as a nuclear receptor coactivator, PNRC also plays a role in the growth factor/Ras-signaling pathway through its interaction with Grb2, a central adaptor protein in Ras pathway. Significantly reduced PNRC mRNA expression in stomach, colorectal, and hepatocellular carcinomas and in breast cancer was observed in comparison with the corresponding non-cancerous tissues. In addition, time-dependent induction of mRNA expression of PNRC was observed during 5-azacytidine treatment. These data suggest a possibility of PNRC as a tumor-related gene and that the regulation of PNRC expression plays a role in human multistage carcinogenesis. However, very little is known about the regulation of the expression of PNRC gene itself. To better understand the molecular mechanisms that regulate the expression of PNRC gene, we carried out this study and the major results are summarized below:1. The transcriptional initiation site and the minimal promoter region of the human PNRC gene were identified in this study. Using 5'RACE approach, we identified a major transcriptional initiation site at a nucleotide G, 27 nt more 5'upstream from the 5'end of previous published longest PNRC cDNAs (NM 006813). The sequences in the 2100 bp 5′flanking region from this transcriptional start site of PNRC gene was then analyzed for promoter prediction (http://www.fruitfly.org/cgi-bin/seq_tools/promoter.pl) and for putative transcriptional factor binding site analyses (TRANSFAC Professional 8.1 software in the Biobase Biological Database) (www. biobase.de/pages/products/databases. Htm l # transfac). To identify the promoter sequences of the human PNRC gene, a series of luciferase reporter constructs containing various 5′flanking region deletions of PNRC gene were generated by PCR and they were transient transfected into HepG2 cell by Liposome. Functional assays showed that the minimal promoter region of the human PNRC gene was mapped in the region from nucleotides -123 to +27, and the predicted transcription factor binding sites of NFY and RFX1 were located in this region.2. Chromatin immunoprecipitation (ChIP) analysis and electrophoretic mobility shift assay (EMSA) revealed that transcription factor NFY and RFX1 interacted with the–123 ~ +27 promoter region of the human PNRC gene in vivo and in vitro.3. The results of the co-transfection experiments showed that the transcriptional activity of human PNRC promoter was found to be regulated negatively by transcription factors NFY and RFX1, through binding two NFY putative binding sites and the RFX1-binding sequence within PNRC promoter region. RT-PCR and Western blot further demonstrated that overexpression of NFY or RFX1 negatively regulated PNRC expression in HepG2 cells.4. After prediction of CpG island in promoter of PNRC gene using"MethPrimer"software, the status of methylation of CpG island in promoter of PNRC gene was analyzed using bisulfite sequencing PCR (BSP) and sequence assay. The results revealed that the CpG island in promoter of PNRC was methylated only in breast cancer MCF-7 cells but not in normal breast MCF-10A cells.5. MCF-7 cells transfected with the luciferase report plasmid that contains the promoter sequence of PNRC were treated with methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) and the promoter activity of PNRC was detected by luciferase assay. The expression of PNRC was analyzed by RT-PCR, Northern hybridization and Western blot after MCF-7 cells were treated with 5-Aza-CdR. The results showed that the promoter activity of PNRC in MCF-7 cells was levated upon the treatment of 5-Aza-CdR, and the levels of mRNA and protein of PNRC in MCF-7 cells were increased dramatically in response to the treatment of 5-Aza- CdR.In summary, in this study we have cloned and characterized the 5′flanking region of the human PNRC gene. Potential transcriptional start sites were determined by 5′RACE analysis. The minimal promoter region of the human PNRC gene was mapped in the region from nucleotides -123 to +27. The transcriptional activity of human PNRC promoter was found to be regulated negatively by transcription factors NFY and RFX1, through binding two NFY putative binding sites and the RFX1-binding sequence within PNRC promoter region. In addition, we also investigated the epigenetic regulation of PNRC gene. The results from this study suggest that hypermethylation of CpG island in promoter of PNRC is probably responsible for PNRC expression silencing in breast cancer cells, and the methyltransferase inhibitor such as 5-Aza-dc can effectively activate the expression of PNRC. The information generated from this study provides a solid base for further study of the transcriptional regulation of PNRC gene and the development of a novel strategy in breast cancer diagnosis and treatment by detecting and modulating the DNA methylation of PNRC gene.