Structure-Function Analysis Of Myocardial Ischemic Preconditioning-Upregulated Gene Mipu1

Zinc fingers are of importance by binding to specific DNA sequences with characteristics of forming a secondary structure in which cysteines and/or histidines coordinate zinc ions. Zinc finger proteins function by binding to double-stranded DNA (dsDNA), single-stranded DNA or RNA in transcriptional control and splicing of RNA. According to the amino acid residues that coordinate zinc ion, zinc finger proteins are classified into a lot of sub-families, among which the subfamily of C2H2 (Kruippel) zinc fmger proteins is the largest, with zinc finger CX2CX3FX5LX2HX3H and conserved linker sequence TGEKP(Y/F)â…©(whereâ…©denotes any amino acid). C2H2 zinc finger proteins can be further divided into 4 groups by their domains at the N-terminus, including FAâ…©(finger-associated boxes), FAR (finger-associated repeats), POZ (pox virus and zinc fingers, also known as Zin) and KRAB (Kruppel-associated box).Zinc finger proteins containing KRAB domain (also known as KRAB-containing zinc finger proteins) account for about one-thirds (209) of total zinc finger proteins (799) arranged in human genome, forming the largest family of transcriptional factors. 220 of KRAB-containing zinc finger proteins express differentially in different tissures and developmental stages, functioning importantly in the control of embryo development, cellular differentiation, cellular transformation and cell cycle.Mipul is a new gene cloned in our lab from transiently ischemic and reperfused rat heart (Genbank accession number AY221750) that was up-regulated after transient myocardial ischemia/reperfusion. It has an open reading frame of 1827 bp, encoding a 608 amino acid polypeptide with an N-terminal KRAB domain and 14 C-terminal C2H2 ZinC fingers. Preliminary study has shown that over-expression of Mipul in C2C12 myogenic cells were more resistant to oxidative injury when compared with the control, as indicated by lower LDH release and apoptosis occurrence in Mipul-overexpressed cells than in the control cells, and that overexpression of Mipul could alleviate cellular growth arrest in the stress induced by a seral withdrawal. The present study is to clarify the function and structure-function relationship of Mipul.In order to investigate the DNA binding activity of Mipul, we first constructed more than ten Mipul bacterial and eukaryotic expression plasmids. Using the bacterial expression plasmid pGEX-Mipul we prepared Mipul fusion protein GST-Mipul. GST-Mipul was immobilized on glutathione-bound Sepharose beads to capture specific oligonucleotide sequences from a random oligonucleotide library, and a consensus sequence (5'-TGTCTTATCGAA-3') was obtained.To determine if the consensus was specific Mipul DNA binding site, a probe containing the consensus sequence was synthesized and ³²p end- labelled. EMSA showed that GST-Mipul can bind to the probe in a protein concentration dependent and zinc dependent manner while GST can not. Mutation analysis showed that nonlabelled wild type (wt) probe was good competitor, but the nonlabelled mutant probe with putative core base substitution at the consensus sequence could not compete with the wt labeled probe. These results suggest that the consensus sequence 5'-TGTCTTATCGAA-3' is specific Mipul DNA binding site (MDBS), in which CTTA is the core sequence.To further study the Mipul-DNA interaction, a truncated Mipul fusion protein GST-ZF containing 14 zinc fingers (ZF) but without the KRAB domain and the linker was expressed and purified. A probe containing MDBS was also synthesized and biotin-labelled. Target detection assay showed that GST-Mipul and GST-ZF can interact with the probe in a zinc dependent manner while GST protein can not, which is consistent with the results above in EMSA, suggesting that the consensus sequence is Mipul DNA binding site, and the zinc finger region of Mipul is necessary and sufficient for its DNA binding activity.To further study the structure-function relationship of Mipul, two more truncated Mipul fusion proteins were prepared: GST-ZF1 which consisted of zinc fingers 1-8, and GST-ZF2 which was composed of 9-14 zinc fingers. Target detection assay also showed that GST-ZF2 can bind to above biotin-labelled probe while GST-ZF1 can not, suggesting that the last 6 zinc fingers of Mipul is required and sufficient for its DNA binding.To clarify the potential function of Mipul as a transcription factor, an oligonucleotide fragment containing 3 MDBSs was chemically synthesized and inserted into pGL3-promoter vector to produce pGL3-promoter-MDBS, and the mutant reporter plasmid pGL3-promoter-MDBS^mut was also constructed with mutant core sequence (TCTTA→AGCGC). Co-transfection of Raw 264.7 cells with pGL3-promoter-MDBS and pcDNA3.1-Mipul showed that Mipul overexpression reduced the reporter activity of pGL3-promoter-MDBS reproducibly, but Co-transfection of Raw 264.7 cells with pGL3-promoter-MDBS^mut and pcDNA3.1-Mipul showed that Mipul overexpression failed to reduce the reporter activity of pGL3-promoter-MDBS^mut, suggesting that Mipul suppressed promoters containing MDBS.Full-length ORF (Mipul) or the KRAB region plus the linker sequence (KRAB) or 14 zinc fingers (ZF) of Mipulwas fused with EGFP, respectively. C₂C₁₂ cells were transfected with the pEGFP recombinant plasmids overnight, and observed under a fluorescence microscopy. Although the cells transfected with pEGFP empty vector or pEGFP-ZF had a generalized fluorescence distribution in the cytoplasm and nucleus, the cells transfected with either pEGFP-Mipul or pEGFP-KRAB showed distinct nuclear fluorescence localization. Thus, the ZF domain was not required for nuclear localization, whereas the presence of the KRAB region and/or the remaining 57 amino acids of the linker region appeared to be sufficient for directing nuclear localization of Mipul.We further analyzed the possible target genes of Mipul and found that many apoptosis-related genes contain MDBS or its core sequence at their promoters by using bioinformatic techniques. It was found that Bax, an important proapoptotic gene, contains several MDBSs, and biotin-labelled Bax promoter could bind to GST-ZF and GST-ZF2.It was concluded that:â‘ Mipul is a DNA binding protein that binds to the specific sequence 5'-TGTCTTATCGAA-3', with CTTA as the core sequence;â‘¡Mipul is a nuclear protein that is localized to the nucleus through its KRAB domain or the linker adjacent to its zinc finger region; â‘¢Mipul is a transcriptional repressor that may repress apoptosis by repressing the expression of proapoptotic genes which may up-regulate in myocardial ischemia-reperfusion injury.