| As the carrier of genetic material,chromatin carries a variety of epigenetic information,among which,ATP-dependent chromatin remodeling plays an important role in epigenetics.The switch/sucrose nonfermenting(SWI/SNF)subfamily complex belongs to the ATP-dependent chromatin remodeling complex,which can hydrolyze ATP via the core catalytic subunit BRG1 or BRM,affect the interaction between histones and DNA,remodel chromatin and regulate transcription,thus changing the conformation of DNA.Generally speaking,most DNA molecules in chromatin are right-handed double helix,while there is also a small part of left-handed double helix called Z-DNA.Latest literature shows that Z-DNA is widespread in vivo and it’s important for the transcription initiation.Besides,it is found to be related with refractory diseases as well.However,the biological function of Z-DNA in cells has not been fully understood mainly due to the difficulty to directly observe DNA conformation at the cellular level.Infrared(IR)spectroscopy can detect the vibrational movement of chemical bonds in biomolecules,thereby generate characteristic spectra representing different components in cells,and provide a solution including identifying DNA conformational changes of biomolecules.Compared with other biological methods,Fourier Transform Infrared(FTIR)spectroscopy can detect biological samples without damage and has outstanding advantages in terms of speed,signal-tonoise ratio,resolution and reproducibility.When the composition and conformation of macromolecules such as nucleic acids in cells change,the corresponding peak shape,intensity,and frequency of infrared spectroscopy will also change accordingly.Therefore,the composition and conformational changes of macromolecules could be deduced based on the analysis of the infrared spectroscopy.This study regulated the catalytic subunit of SWI/SNF via knockdown/overexpression of BRG1/BRM in Hep G2 and He La cells,confirmed the response of Z-DNA transformation to the regulation of BRG1/BRM by detecting the change of DNA conformations via FTIR spectroscopy and verified the characterized results of the FTIR spectroscopy through chromatin immunoprecipitation(Ch IP)technology.This research established the direct method to detect and investigate the change of Z-DNA conformation after ATP-dependent tryptophan remodeling.The paper included three parts and obtained main results and conclusions as follows:(1)Z-DNA synthesized in vitro was characterized via FTIRa)In order to synthesize Z-DNA in vitro and characterize Z-DNA by infrared spectra,a large number of nucleic acids are needed to be precipitated.Therefore,the methods to precipitate nucleic acids were optimized and Z-DNA was prepared under the optimal condition.b)Z-DNA in vitro was characterized by FTIR and compared with natural B-DNA spectroscopy.It was found that Z-DNA contained the unique 930 cm-1 peak,which was significantly different from that of B-DNA,therefore,this special peak was used to distinguish the different two conformations,laying a foundation for the Z-DNA analysis.(2)Cellular Z-DNA of chromatin remodeling was characterized by FTIRThe catalytic subunit of the SWI/SNF family was regulated,i.e.knock down/overexpress BRG1/BRM in Hep G2 and He La cells.Under the condition of inhibiting/enhancing chromatin remodeling,the cell infrared spectra was collected to analyze the change of DNA conformation in cells.It was found that Z-DNA conformation was positively correlated with the level of BRG1/BRM.In addition,ZDNA transformation responded to SWI/SNF regulation and confirmed the effectiveness of the FTIR spectroscopy in identifying Z-DNA transformation in cells.(3)Verification of FTIR characterization resultsa)Z-DNA binding proteins ADAR1 and ZBP1 were analyzed with Western Blotting after BRG1/BRM gene silencing/overexpression,the results showed that knockdown BRG1/BRM decreased ADAR1 and ZBP1 expression,and overexpression of BRG1/BRM increased ADAR1 and ZBP1 expression,which indirectly indicated the decreasing/increasing of Z-DNA conformation,therefore,the characterization results of FTIR spectra were verified.b)Ch IP analysis showed that there was a specific binding between ADAR1 and Z-DNA in cells,and ADAR1 decreased/increased after BRG1/BRM knockdown or overexpression.In addition,Z-DNA nucleic acid obtained in Ch IP also reduced/increased accordingly,further confirmed the reliability of FTIR characterizing Z-DNA conformation. |
