The Characteristics Analysis Of Transcriptional Regulation Of Receptor Interacting Protein Kinase 3

Objective: To investigate interaction between the core promoter region of receptor interacting protein kinase 3(RIPK3) and transcription factor. To study the relationship of Cp G island methylation and gene expression levels.Methods:(1)Afterbioinformatics analysis of the RIPK3 promoter, using luciferase(Luc) assays to determine the effect of Exon I, Intron I and 5’UTR on RIPK3 promoter activity. Luc reporters driven bydifferent lengthswere constructed with a series of5’deletion promoter region of the RIPK3 promoter and the promoter-less control(p GL4-Basic) were transfected into 293 T cells. The Luc reporters driven by the functional RIPK3 promoter were transfected into 293 T cells, as compared to various promoter truncates by missing or mutating GC-Boxes.(2) The functional RIPK3 promoter were co-transfected with the increasing doses of Sp1, Sp3, a dominant negative mutant of Sp1[Sp1-DN, encoding DNA binding domain amino acids(aa) 592–778] and a dominant negative mutant of Sp3[Sp3-DN, encoding DNA binding domain amino acids(aa) 399–654]plasmids for Luc assay in 293 T cells.Sp1, Sp3, Sp1-DN and Sp3-DN plasmids were transfected into 293 T cells to examine the expression of their proteins by Western blotting.Treatment with an inhibitor(mithramycin) of Sp1 function that disrupts GC-box to figure out wether it can block the RIPK3 promoter activity.(3) For the EMSA analysis, nuclear extracts(Nu) from 293 T cells were incubated with Biotin-labeled oligo nucleotides(Biotin-probe) spanning the GC-rich region of the RIPK3 promoter. Competition reactions were performed with 200 X of unlabeled cold competitor(cold), Sp1 antibody or Sp3 antibody.(4) Co-transfection of Sp1-si RNAs or Sp3-si RNAs with the RIPK3 promoter construct resulted in a decrease or increase in promoter activity when compared with co-transfection of the si RNA control. Sp1-si RNAs or Sp3-si RNAs were transfected into293 T cells to examine the expression of Sp1 or Sp3 by western blotting. To investigate wether combined knockdown of Sp1 and Sp3 using an equal ratio(1:1) of si Sp1 and si Sp3 resulted in a knockdown of endogenous RIPK3 protein in HT-29 cells.(5)Using Bisulfite sequencing PCR(BSP) to figure out methylation status of individual cloned DNA fragmentsof two colorectal cell lines with or without5-aza-2’-deoxycytidine(5-aza-Cd R).To observethe RIPK3 changes which were treated with5-aza-Cd R aloneby western blotting in HCT-116 cells.Results:(1) 5’UTRplays a major role in RIPK3 promoter activation. GC-box “2” is essential for activation of the RIPK3 promoter.(2) Sp1 and Sp3 increase RIPK3 promoter activity in cultured 293 T cells.An inhibitor of Sp1-like transcription factors(mithramycin)blocks Sp1/Sp3-dependent RIPK3 promoter activity in 293 T cells.(3) EMSA test indicates that Sp1/Sp3 binds to the GC-box of the RIPK3 promoter.(4) RIPK3 promoter activity is decreased by Sp1 si RNAsand Sp3 si RNAs in 293 T cells. Knockdown of transcription factors Sp1/Sp3 decreases endogenous RIPK3 protein in HT-29 cells.(5) The cell line lacking methylated bands by BSP were nearly all unmethylated at all 13 Cp G dinucleotides in all cloned alleles. The cell line not lacking methylated bands by BSP were nearly all methylated at all 13 Cp G dinucleotides in all cloned alleles. The RIPK3 protein increasing were also observed by western blotting in HCT-116 cells that were treated with 5-aza-Cd R alone.Conclusion: The present study identified a proximal core sequence in the RIPK3 promoter that is composed of one enriched Sp1 binding motif and established Sp1 and Sp3 as the major RIPK3 transactivators, providing a novel understanding of the mechanisms of RIPK3 gene transcriptional regulation.Given that increases of RIPK3 expression have been implicated in a wide range of pathophysiologic states including stroke, sepsis, tumors,atherosclerosis and eye diseases, blockade of Sp1-like transcription factors or changing methylation status of Cp G dinucleotides represents a novel direction in therapeutic strategies.