| Chromatin remodeling complex in eukaryotic cells is a class of ATP-dependent chromatin structure regulating enzymes which can regulate the chromatin structure by changing the assembly,disassembly and rearrangement of nucleosomes,thereby changing transcription factors(TFs)accessibility to it's gene promoters.Intracellular chromatin remodeling complexes,according to the functional domains they contain,are roughly divided into four families: SWI / SNF,ISWI,CHD,and INO80.It has been found that certain chromatin remodeling complexes such as human INO80,SRCAP and yeast SWR1 have the H2 A.Z exchange activity of into or out of nucleosomes.H2 A.Z is a highly conserved variant of histone H2 A.Its distribution in the genome is closely related to gene expression regulation,DNA double-strand damage repair(DSB),and maintenance of chromatin stability.Our research group found in the previous study that the nuclear extracts protein components of HeLa cell separated and purified by various chromatography columns can exchange the histone variant H2 A.Z into nucleosomes.The results of protein mass spectrometry analysis showed that the main component of our protein was DNA-PKcs,suggesting that DNA-PKcs or protein complex containing DNA-PKcs may be a New chromatin structure regulating enzyme with H2 A.Z substitution activity.DNA-PKcs weights 465 KD,two regulatory subunits Ku70/Ku80 heterodimer forming DNA-PK complex,which has Serine/Threonine kinase activity.In cells,DNA-PK is involved in regulating many important biological processes.The absence or malfunction of this enzyme can lead to abnormal transcriptional regulation of certain genes,genome instability,abnormal polarization of the spindle,defects in cell cycle function,and even cause expression changes or mutations in certain oncogenic factors or tumor suppressors.This suggests the diversity and extensiveness of DNA-PK functions in cells.Does the DNA-PKcs' s H2 A.Z exchange activity relate to its known biological function? If so,what biological processes are involved? What is the specific regulating mechanism? The problem has yet to be resolved.In this paper,1)By using in vitro recombinant nucleosomes(mono-nucleosome),Flag-tagged H2 A.Z-H2 B dimers,and SRCAP complexes which known to have H2 A.Z displacing enzyme activity,we established an H2 A.Z replacement enzyme activity detection system in vitro.And though this system,we confirmed that both the protein components chromatographic separated from the HeLa cell nuclear extract and the anti-Flag immunopurified protein components purified from Flag-DNA-PKcs overexpressing cells have H2 A.Z in vitro replacement enzyme activity.This strongly supported the reliability of our previous data;2)In order to further study the correlation between the biological function of DNA-PKcs and its H2 A.Z replacement activity,we designed and constructed Plvx-zsGreen-shRNA and Plvx-puro-shRNA plasmids to knock down DNA-PKcs;3)Knockdown of DNA-PKcs in cells or treat cells with DNA-PKcs inhibitor NU7026 resulted in slower cell proliferation and increased number of apoptosis.The cycle of DNA-PKcs knockdown stable cell line appear to be delayed from G1 ? G2 phase.In addition,meganuclear and multinuclear phenomena appeared in those cells,indicating abnormal cell division;4)Chromatin extraction experiments showed that in cells with DNA-PKcs knockdown,H2 A.Z recruitment in chromatin was significantly reduced,suggesting DNA-PKcs can regulate the distribution of endogenous H2 A.Z on chromatin.At the same time,this regulation may directly affect the biological function of DNA-PKcs.The above research results provide a theoretical basis and experimental foundation for further study of the DNA-PKcs' effection on the distribution of histone variant H2 A.Z in the genome and may elaborate upon new biological mechanism of DNA-PKcs. |
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