Charaterization And Function Analysis Of A Novel HMT, SET07

Objective:Recent studies of histone methylation have yielded fundermental newinsights pertaining to the role of this modification in gene activation as well as in gene silencing.While a number of methylation sites are known to occur on histones,only limited information exists regarding the relevant enzymes that mediate these methylation events.We thus sought to identify native histone methyltransferase(HMT) activation from human tissues.Here we discribe the cloning ,characterization and function of SET07,a novel HMT.Methods:Using bioinformation tools,we got a new gene containing SET domain, andpredicted its biochemical characterization.We cloned its coding sequences from fetus spleen tissue. Its ORF,mapping and expression information,translation sequence,functional domain, protein characerization and potential role were tested with Northern Blot,Western Blot, RT-PCR and HMT analysis.In addition ,we constructed GST-tagged vector PGEX-4T-2-P2RP2 SET07 578bp for expression in E.coli and acquired antiserum against SET07 by immuning KunMing mouse.Results: A novel putative HMT of 3160bp was obtained through bioinformationmethods and was mapped in chromosom 2Pll+2,with ubiquitous distribution from tumour tissues to fetus.SET07 preotein is about 39KDa,with PI 9.57 and a transmembrane helices in 6-26.It lacks signal peptide and hydrophobic segment. As SET07 is highly similar to H4-K20,SET8 ,it is potentially a histone methyltransferase. We cloned 1117bp ,its coding sequences from fetus spleen tissue and namedSET07 .Its ORF of 1114bp started from +12 to +1055,encoding 347 amino acids,with SET domain in C terminus. Northern Blot proved that mRNA of SET07 was about 3387bp.SET07 was found in nuclei and cytoplasm by Western Blot. Compared with only one form of 49KDa in nuclei,SET07 has four forms from 39 to 49KDa in cytoplasm.HMT proved that SET07 can methylate H4,H2A/H2B,H3 of histone.SET07 is widely expressed in tumor cell line,early fetus tissues and normal endometrium.But 5' segments and 3' segments of SET07 was found absent in HK2 (a normal renal cell line) and gall bladder tissue respectively.The developmental expression pattern of SET07 showed that its maximal expreesion was observed in fetus 739 mouse.Then it is more and more restricted as development progress ,only found in skin tissue of newborn 739 mouse and testis tissue of mature 739 mouse.Conclusion: 1.We successfully cloned a novel HMT and named SET07,withGI15029566 in GenBank.2.We described its biochemical characterization and revealed that a 49KDa complex of SET07 functioned as histone methyltransferase.3.SET07 plays an important role in transcriptional regulation,,cell development and carcinogenesis.