Of. Sirt7 Gene Start The Regulatory Mechanism Of The Sub And Its Protein Stability Studies

The chromatin silencing factor Sir2(silent information regulator-2) catalyses NAD+-dependent histone deacetylation to regulate genomic stability and cellular senescence in budding yeast.Sirtuins in mammals constitute a family of seven genes (SIRT1-SIRT7) recently been proposed to be involved in the control of critical metabolic pathways as well as apoptosis, stress responses, DNA repair, cell cycle, genomic stability and gene expression.SIRT7is the only Sirtuin located in nucleolus, and is the only Sirtuin protein for which a clear enzymatic activity has remained elusive. Previous studies suggest sirt7is an activator of RNA polymerse1transcription. It over expression enhances rDNA transcription. An elevated Sirt7expression has been detected in several human cancers such as breast cancer. The study of SIRT7regulation is valuable for us to understand the exact roles of SIRT7in tumorgensis and metabolic pathways.Here we try to definite the SIRT7gene promoter and investigate the role of ubiquitin-mediated proteasomal degradation in regulation of SIRT7levels. We also examine the sirt7for its cellular localization; protein deacetylase activity.These works will develop our understanding of the role of SIRT7.1SIRT7mRNA ubiquitously expressed.The mRNA level of SIRT7gene was detected by Northern blotting or RT-PCR. Data shows SIRT7mRNA was expressed in all mouse tissues examined, and was most abundant in liver. Human SIRT7mRNA was also expressed in several cell lines such as HEK293T, MCF7, Huh7, HepG2, and HeLa except H1299cells.2The transcription site of SIRT7was determined by5'-RACE assay.Using SIRT7primer and5'RACE outer primer, we amplified a650bp fragment by nest PCR from total mouse live RNA and HL60RNA The data showed that there is only one transcription initiation sites on sirt7gene, and the site is47bp apart from sirt7translation start site ATG. Characterization of the5'-flanking genomic region, which precedes the sirt7transcription initiation site, revealed a TATA-and GC-box less promoter that lacks CpG islands.3Functional identification of the SIRT7gene promoter.A2.1Kb5'-flangking region of human Sirt7was cloned and characterized. Promoter deletion assays suggested the constructed (-312/+30) contained a conserved C/EBPα binding site can efficiently drove luciferase activity.4Anticancer agent doxorubicin can induce the SIRT7degradation and the degradation is mediated by26s proteasome. SIRT7can polyubiquitination of SIRT7in vivo.The SIRT7protein levels but not mRNA levels are decreased by doxorubicin treatment. And the SIRT7is a short-lived protein and the effect on the SIRT7protein degradation is MG132dose and time dependent. SIRT7might be regulated by ubiquitin-mediated proteasomal degradation.5Cellular localization of Sirt7protein.Exogenous expressed Flag-tagged full length human SIRT7locate in nucleus but not in nucleoli in HEK293T and MCF7by immnofluorescence analysis. And it mainly concentrates near nuclear envelope. During M phase, when nucleoli disintegrate, SIRT7did not bind to the condensed mitotic chromatin. In contrast, GFP-SIRT7is mainly located in nucleoli. During M phase, EGFP-SIRT7remained bound to the condensed mitotic chromatin. Antibodies were raised against the amino acids363-379of mSIRT7. And we demonstrate the antibody can specifically recognize the mSIRT7protein. Using anti-SIRT7C-terminal antibodies for immunofluorescence co focal Microscopy, we found SIRT7proteins mainly locate in nucleus of HEK293T, Huh7, MCF7cells. 6The deaceylation activity of SIRT7We Purify GST-fusion SIRT7protein and total histone and set up the vitro deacetylation assay. Data shows SIRT7does not deacetylase H4K16in vitro. SIRT7don't show deacetylase activity on histone H3K9Ac, H3K14Ac, H4K5AC,H4K8AC, H4K12Ac, H4K16Ac in MCF7cells. SIRT7does not deacetylate p53in MCF7cells.