Find A New Histone Lysine Demethylase And Methylated Lysine Recognition Protein

Lysine methylation, one participator of "histone code" hypothesis, plays an important role in chromatin-associated activities. Histone lysine methylation is catalyzed by histone lysine methyltransferases with SAM as a methyl donor. It functions when binding with methyl-lysine recognition proteins and can be erased by histone lysine demethylases. So far, demethylases targeting some lysine methylation sites have not been found. And discovering novel histone methyl-lysine recognition proteins will provide more comprehensive understanding of the functions of lysine methylation.In the first part of our study, we searched for novel histone demethylases. We found that ALKBH7, a member of AlkB protein family, could bind with bulk histones in GST pull-down assays. ALKBH7 can slightly reduce the signal of anti-H4K20me2 in demethylation assays with bulk histones as a substrate. Though we have tried throughly, we failed to enhance the activity presented in western blot. In mass spectrometry, we did not detect the H4K20me2 demethylase activity with H4K20me2 peptide as a substrate. Then our immunofluorescence and subcellular fractionation results proved that ALKBH7 is a mitochondrial protein. With custom anti-ALKBH7, endogenous immunoprecipitation was performed and putative ALKBH7-interacting proteins (e.g. NDUFS3 etc.) were identified with mass spectrometry. Considering the structural similarity of ALKBH7 and PHD2, we designed over 70 peptides of ALKBH7-interacting proteins, and screening with mass spectrometry. But no positive results turned out.In the second part of our study, we considered the possibility that FMR1 functions as a histone methyl-lysine recognition protein. We found that the N-terminus of FMR1 could bind with bulk histones in GST pull-down assays, which is dependent on post-translational modifications of histones and amino acid residues forming putative "hydrophobic box" of FMR1. We have tried peptide array, peptide pull-down etc. to search for the FMR1-binding methylation sites on histones, but no consistent results were obtained. By immunofluorescence and subcellular fractionation assays, we found FMR1 was associated with nucleus tightly, but ChIP-cloning assay showed that there was no DNA co-immunoprecipited with FMR1. We also found that when endogenous FMR1 was knocked down in HeLa cells, the level of protein P55 elevated, which provided a possible explaination for FMR1 knockdown-induced yH2AX suppression.The third part of our study investigated the efficacy of purified anti-CD3×anti-HER2 bispecific antibody in directing cytotoxicity of CIK cells towards tumor cells. We found that the efficacy is associated with the spontaneous cytotoxicity of CIK cells. When the spontaneous cytotoxicity is relatively weak, the purified bispecific antibody can enhance the cytototoxity stronger than the crude chemical conjugates. When the spontaneous cytotoxicity is strong enough, no obvious difference between purified and crude bispecific antibody was observed.