Roles Of Human Heart Developmental Candidate Genes Znf382 And Cxxc5

Human heart development is a complex process and regulated by a series of transcription factors.Although many of the heart transcription regulatory factors have been identified,there are still a lot of them to be identified,and the molecular mechanism of regulations to be lucubrated.ZNF382 is a human heart developmental candidate gene cloned by our lab(NM₀32825),and the gene is widespread expressed in different periods of human embryos and only expressed in the heart in adult tissues. its expression pattern of ZNF382 indicates that it could play a potential role in the regulation of heart and muscles development.The author continues to study its role in the development of heart and muscle.RT-PCR assay showed that after the differentiation of C2C12 cells was induced,the mRNA of the endogenetic MHC and ZNF382 were time-dependent upregulated.Report gene transcription activity analysis indicated that ZNF382 alone can not activate the transcription activity of MCK(MCK4800-luc).But when transfecting tegother with MyoD and E12,ZNF382 could activate the MCK transcription activity,suggesting that ZNF382,MyoD and E12 are coordinated to regulate the muscle cell differentiation.In order to identify which protein interacts with ZNF382,the yeast two-hybrid system was emploied to screen and identify its interacting proteins.A MYL2 protein interacted with ZNF382 from adult heart library was isolated.MYL2 encodes the regulatory light chain associated with cardiac myosin beta(or slow)heavy chain,suggesting that ZNF382 with MYL2 together might regulate heart development related diseases.We first use of subcellular location experiment to detect the locations of ZNF382 and MYL2 in HEK293 cells.The results showed that ZNF382 alone located in the nucleus,MYL2 in both the nucleus and cytoplasm.When the two proteins transfected together into the HEK293 cells,most of MYL2 proteins were observed in the nucleus,which indicated that the two proteins interact in vivo.To further examine whether ZNF382 and MYL2 are interacted, co-immunoprecipitation experiments were carried out.MYC-labeled MYL2 could be precipitated by FLAG-labeled ZNF382,and the FLAG-labeled ZNF382 could also be precipitated by MYC-labeled MYL2. The results show that there exist indeed interactions in vivo between the two.The studies have shown that Ca⁺ triggers the phosphorylation of regulatory light chain that in turn triggers its contraction.Mutations in MYL2 are associated with mid-left ventricular chamber type hypertrophic cardiomyopathy.It is interesting to focus in further to study whether ZNF382 together with MYL2 play a role in the left ventricular cavity ventricular hypertrophy cardiomyopathy.In addition,we also cloned a new human gene CXXC5 with zf-CXXC type of the zinc finger.It encodes 322 amino acids.It is highly conservative in evolution of species.RT-PCR analysis showed that CXXC5 is widespread expressed in adult and embryonic tissues.CXXC5 is located in the nucleus and cytoplasm.Overexpression of CXXC5 in cells could lead to nuclear condensation,suggesting that the gene may be involved in the process of apoptosis.In the HEK293 cells line,we first detected cells with the overexpression of CXXC5 by FACS to examine whether it is involved in the apoptosis.The results showed that a large number of cells were staged in the sub-G1.Then we detected cells with the overexpression of CXXC5 by TUNEL staining.The results showed that lots of cells with the overexpression of CXXC5 were TUNEL positive.These results suggest that CXXC5 is indeed involved to the apoptosis.In order to study the underlying mechnism of CXXC5 inducing apoptosis,we used reporter system and Western blot to analysis whether CXXC5 is involved in apoptosis signaling pathway.According to the key factor caspases roles of apoptosis,two separable pathways,receptor and the mitochondrial pathways,are related to the caspase activation.TNF-a signaling is one of the receptor pathways and can induce activation of prosurvival pathways(NF-κB and JNK)and death signaling.Our results showed that overexpression of CXXC5 inhibited p53 transcription activity, but activated TNF-a transcriptive activity.Among the TNF-a subpathways,the overexpression of CXXC5 inhibited the transcriptive activities of c-jun,AP-1 and NF-κ3,but activated the activities of caspase-8 and caspase-3.These results suggest that CXXC5 may induce apoptosis through the activation of TNF-a caspases pathway.