Effect Of Nhp6A And Nhp6B On Cells Growth And ELF1 Expression

The high-mobility nucleosome-binding protein(Nhp6)is a type of small molecule protein commonly found in eukaryotes.In yeast,Nhp6 is divided into two similar homologous proteins,Nhp6 A and Nhp6 B,which bind non-specifically at the bend of the DNA,playing an important role in promoting RNA polymerase III transcription,promoting RNA polymerase II to form a transcription initiation complex,and promoting the formation of chromatin modification complexes.The transcription elongation factor ELF1(elongation factor 1)is an DNA-binding protein which is evolutionarily conserved and structurally similar that is involved in transcription elongation and the formation of chromosome structures.Both Nhp6 and ELF1 play a role in transcriptional process and chromosome modification.Our research group found that there was a correlation between ELF1 and Nhp6 in yeast by GST pull-down technique in the previous study,but no other studies have reported the interaction relationshipbetween these two types of proteins.Therefore,it is not clear whether there is a certain degree of regulation between the two types of proteins.To know whether Nhp6 A and Nhp6 B have an effect on the normal growth of yeast Y258,Our research prepared the mutants nhp6a(-)and nhp6b(-)by long-flanking homology region PCR(LFH-PCR)mediated gene disruption methods,Meanwhile,It was found that when the sequence lengths of the upstream and downstream homologous regions of the NHP6 A and NHP6 B disruption cassettes were in the range of 200 bp to 300 bp,knock-out efficiency was highest.Based on yeast-induced expression technology,our research constructed pEGH-NHP6 A and pEGH-NHP6 B expression vectors respectively,and prepared overexpressed yeast Y258(pEGH),nhp6a(+)and nhp6b(+).In this study,the rescue yeasts nhp6a(r)and nhp6b(r)were prepared based on the mutants nhp6a(-)and nhp6b(-).Finally,the effects of Nhp6 A and Nhp6 B on the normal growth of yeast were examined by dripping plate experiments and detecting Cell growth curve to observe the growth status of the above different yeasts under normal conditions.The results showed that the mutant yeasts nhp6a(-)and nhp6b(-)both survived,but nhp6a(-)grew more slowly than the wild-type yeast,whereas the rescue yeast nhp6a(r)resumed normal growth.There was no significant difference in growth status between nhp6b(-)and nhp6b(r)compared to wild-type yeast.In summary,Nhp6 is not necessary for yeast survival,but Nhp6 A is more important for normal yeast growth than Nhp6 B.The results also showed that the expression of yeast nhp6a(+)and nhp6b(+)grew more slowly than the wild type yeastY258(pEGH),indicating that excessive expression of nhp6 a and nhp6 b could inhibit the normal growth of yeast.Therefore,the expression of Nhp6 A and Nhp6 B can only maintain the normal growth of yeast in a certain range,and no expression or overexpression bothe can inhibit the normal growth of yeast.To understand the role of Nhp6 in the regulation of ELF1 in yeast,this study through the Real-time PCR to detect the change of the expression of the ELF1 in the wildtype yeast Y258,the mutant yeasts nhp6a(-)and nhp6b(-),the rescue yeast nhp6a(r)and nhp6b(r)and the overexpressing yeasts nhp6a(+)and nhp6b(+).The results showed that compared with the wildtype yeast,the expression level of ELF1 showed an increasing trend with the increase ofNhp6 A expression,indicating that in yeast,Nhp6 A played a positive regulatory role on ELF1,while Nhp6 B expression was no significant effect on ELF1,indicating that Nhp6 B had no regulatory effect on ELF1.In conclusion,this study preliminarily explored the effect of Nhp6 A and Nhp6 B on normal growth of yeast,and the regulation of Nhp6 A and Nhp6 B on the transcription extension factor ELF1 in yeast.Both Nhp6 and ELF1 play a role in transcriptional process and chromosome modification.Therefore,this study can provide a basis for further study of the interaction between Nhp6 and ELF1 in yeast transcription and chromosome modification.