Cloning and primarily function study of two novel putative N5-glutamine methyltransferase (Hemk) splice variants from mouse stem cells.
Methylation is one of epigenetic mechanisms regulating gene expression. The methylation pattern is determined during embryogenesis and passed over to differentiating cells and tissues. Beginning with the ESTs which were highly expressed in undifferentiated human ES cells and using homology research in mouse dbEST database, we cloned two novel putative (N (5))-glutamine methyltransferase (Hemk) splice variants termed mHemk1 and mHemk2 (Genbank accession number AY456393 and AY583759). Sequence analysis revealed that mHemk1 and mHemk2 cDNAs are 1,792 bp and 1,696 bp in length respectively. The deduced proteins have 214 amino acid residues (mHemk1) and 138 residues (mHemk2) in length and both share significant homology with (N (5))-glutamine methyltransferase (Hemk proteins) in database. Northern blot and RT-PCR analysis showed that mHemk mRNAs were abundantly expressed in undifferentiated ES cells, testis and brain, weakly expressed in differentiated ES cells and kidney, and not expressed in muscle, heart, placenta, pancreas, lung and stomach. Immunohistochemical analysis further revealed that the protein was most abundant in undifferentiated ES cells. The green fluorescent protein produced by pEGFP-C3/mHemk1 was detected mainly in the nucleus of COS7 cell lines after 24 h post-transfection. RNA interference (RNAi)-mediated knock-down method was established. Cell cycle analysis suggests that the cell proliferation decreases after RNAi with mHemk1. In vitro bioactivity assay showed that no evidence for a DNA adenine-methyltransferase activity was detected. The accumulating functional information from Hemk homology proteins in bacteria and yeast suggests that it may be an uncharacterized new mammalian N(5)-glutamine methyltransferase.
Nie
Dong-Song
DS
Institute of Human Reproduction and Stem Cell Engineering, Central South University, 88 Xiangya Road, Changsha, 410078 Hunan, People's Republic of China.
Liu
Yong-Bo
YB
Lu
Guang-Xiu
GX
D000818:Animals; D019556:COS Cells; D002453:Cell Cycle / Q000502:physiology; D048708:Cell Growth Processes / Q000502:physiology; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D019175:DNA Methylation; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D005973:Glutamine / Q000378:metabolism; D007150:Immunohistochemistry; D042541:Intracellular Space / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D020033:Protein Isoforms; D011496:Protein Methyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D020033:Protein Isoforms; D011994:Recombinant Proteins; D005973:Glutamine; D011496:Protein Methyltransferases