The Investigation On Inhibitor Of Histone Acetyltransferases

INHAT(inhibitor of histone acetyltransferases)widely exists in eukaryote cells possessing the characteristic of lower molecular weight, leucine-rich, acidic domain in C-terminal and inducibility and so on. INHAT has two subunits, one is TAF-1αor TAF-1β, the other one is pp32. INHAT mainly has three kown functions at present:(1)binding to histone; (2)inhibit histone acetylation; (3)inhibit related gene transcription.In this paper,we express and purify INHAT from E.coli and synthesize the radioactive 35S labelling INHAT in in vitro transcription and translation system,then we do the histone peptide pulldown assay based on the provided material above respetively, the two results simultaneously indicate INHAT can specially bind to H3K4Me2,but it does not bind to monomethylation and trimethylation of lysine-4 in H3.As we all known,the methylation of lysine-4 in H3 is a mark associated with active genes,but INHAT is associated with gene silencing which can inhibit gene transcription by the way of binding to histones,so we predict methylation of lysine-4 in H3 blocks the binding site of INHAT and histone H3, then probably promotes the downstream related gene transcription.In this paper,we construct five deletion mutants of pp32,then we do the GST pulldown assay,the result suggests the two subunits can interaction with each other and the interaction is dependent on the C-terminal acidic domain(150-249)of pp32.According to the immunofluorescence, it demonstrates the deletion mutant of pp32 which delete the C-terminal acidic domain(150-249)can not completely localize in the nucleus, it also can localize in the cytoplasm.