Transcriptional Factor HP1BP3 Negatively Regulates HSP70 Transcription In Crassostrea Hongkongensis

Hsp70s are important members in heat shock protein family and the approximate size is 70 kDa.They are widely present in all cellular species as highly conserved molecular chaperones,playing critical roles in assisting the proper folding and assembly of the nascent polypeptide chains,and restoring the denatured protein or promoting their degradation after a variety of intense stresses or injury.Under stressed conditions,the expression level of HSP70 is upregulated significantly and rapidly,as is known to be achieved by interacting with various regulatory factors controlling the transcriptional level.Although the activation of the Hsp70 gene is regulated by the Heat Shock Factors,the mechanisms of negative transcriptional regulation of inducting Hsp70 expression remain largely unknown.Therefore,we screened the potential transcriptional factors in Crassostrea hongkongensis HSP70?ChHSP70?,and then identified as well as verified the regulation of the transcriptional factors on ChHsp70.The main contents of this study are as follows:1.ChHP1BP3 was identified as one of a transcription factor in Crassostrea hongkongensis HSP70?ChHSP70?by DNA-affinity purification and mass spectrometry analysis,which was a chromatin-related protein with dynamic combination and interact with nucleosomal DNA directly.The full-length cDNA sequence of ChHp1bp3 was determined by the technique of RACE?Rapid-Amplification of cDNA Ends?,which was 1920 bp in length and contained a 1434 bp coding region as well as 177 bp and 309 bp 5?and 3?untranslated regions;A globular domain,which has been previously reported to interact with nucleosomal DNA was predicted by the method of three-dimensional modeling.2.The ability of ChHP1BP3 to interact with ChHsp70 promoter region was detected by the EMSA?Electrophoretic Mobility Shift Assay?technique using a FITC-labeled ChHsp70 promoter core region and His6-ChHP1BP3 expressed and purified from Escherichia coli.The results showed that ChHsp70 was combine with His6-ChHP1BP3 specifically.The expression of ChHsp70 mRNA was significantly enhanced after the degradation of ChHp1bp3 mRNA by RNA interference in hemocytes of C.hongkongensis.However,the overexpression of ChHp1bp3 via the detection of dual-luciferase activity in the heterologous HEK293 T cells was correlated with fluctuation in ChHsp70 transcription.3.Quantitative RT-PCR was employed to reveal the correlation of the transcriptions between ChHsp70 and ChHp1bp3 in their mRNA expressions in response to external stresses given by heat,CdCl₂ and NP.The results indicated that the highest peaks was induced by heat,CdCl₂ and NP at 3 h,2 d and 4 d.ChHsp70 transcriptions were postponed by all the tested stresses,and a different induction pattern was observed for CdCl₂ and NP treatment since two different induction peaks were shown.This indicated the distinctive expression patterns of ChHp1bp3 upon physical and chemical stresses suggested that the mechanisms that mediate ChHp1bp3 induction might be stress-specific.While there is a plausible correlation between ChHsp70 and ChHp1bp3 in the stress-induced genetic regulatory pathway.In summary,This study discovered a novel role of HP1BP3 as a negative regulator in controlling the Hsp70 transcription in C.hongkongensis,and contributed to a better understanding of the complex regulatory mechanisms governing Hsp70 transcription.