Mechanistic Study Of Regulation Of Glc7 Activity By SAS Complex

Telomere is located at the end of heterochromatin and is composed of some simple short DNA repeats and specially recognized proteins.As a typical heterochromatin structure,telomere plays an important role in maintaining chromosome stability and controlling cell division cycle.In addition,telomere also plays an important role in cell response to DNA damage and delaying cell aging.It has been found that telomere length and structure are regulated by cell metabolism under certain conditions.More and more studies have shown that histone post-translational modifications are also crucial for the regulation of telomere silencing.As a very important post-translational modification,histone H3 threonine 11(H3T11)phosphorylation is involved in the regulation of many important cellular life processes.Phosphorylation of H3T11(H3p T11)catalyzed by pyruvate kinase Pyk1 in SESAME complex can maintain telomere silencing by regulating the expression of telomere silencing protein Sir2.In Saccharomyces cerevisiae,the serine/threonine phosphatase Glc7 has been identified to be able to dephosphorylate H3T11.However,the research on the role of Glc7 in the regulation of telomere silencing is more focused on its role as a catalytic subunit in the formation of complex with telomere proteins,it is unknown whether Glc7 interacts with other modifying enzymes and how it functions as a phosphatase.Telomere silencing is regulated by a variety of post-translational modifications,and many reports have suggested that these post-translational modifications act independently.However,more and more studies have shown that there is combinatorial regulation between histone post-translational modifications,that is,there can also be cross-talk between modifications occurring at two or more different amino acids.Therefore,it is rare to investigate whether H3 p T11 interacts with other post-translational modifications and functions.In this study,Saccharomyces cerevisiae was used to screen for other modifying enzymes that physically interact with phosphatase Glc7,and to investigate the function of the crosstalk between the interacting modifying enzymes and the histone posttranslational modifications they affect in the regulation of telomere heterochromatin.The phosphatase Glc7 physically interacts with the acetyltransferase SAS complex,and Sas2 is able to inhibit the interaction between Glc7 and histones,resulting in the reduction of H3 p T11 levels.In addition,Ch IP-seq analysis found that the distribution of H3 p T11 and H4 lysine 16(H4K16)acetylation showed an opposite trend.Sas2 can regulate the binding of Glc7 and H3 p T11 on telomere and autophagy genes.In conclusion,our study provides important clues for the dynamic regulation of telomere silencing and autophagy by exploring the crosstalk between modifying enzyme interactions and histone posttranslational modifications based on the regulation of modifying enzyme activities.The specific research results are as follows:1.The phosphatase Glc7 interacts physically with the acetyltransferase Sas2.Through extensive immunoprecipitation screening,we found the modification enzyme Sas2 that interacted with Glc7.The interaction between Glc7 and Sas2 was confirmed by protein immunoprecipitation of Sas2 and Glc7 immunoprecipitation of Sas2,as well as yeast cell dot plate assay.2.SAS complex-catalyzed acetylation of H4K16 inhibits the expression of H3T11 phosphorylation.We analyzed the data related to the birth letter and found that the distribution of H4K16 ac and H3 p T11 showed a negative correlation.The expression of H4K16 ac was then examined in the mutant glc7-ts,as well as H3T11 A and H3T11 D,and H4K16 ac levels were found to be unchanged.When H3 p T11 was detected in sas2Δ,sas4Δ and sas5Δ mutants,it was found that the level of H3 p T11 was significantly increased,but neither the transcription nor protein levels of Glc7 were significantly changed.A significant increase in H3 p T11 levels was similarly observed in the acetylation inactivation mutants H4K16 A and H4K16 R.Taken together,we found that H4K16 ac catalyzed by the SAS complex was able to inhibit H3 p T11but did not affect the levels of the interacting phosphatase Glc7.In summary,we found that SAS complex-catalyzed H4K16 ac was able to inhibit H3 p T11 without affecting the level of its interacting phosphatase Glc7.3.Sas2-catalyzed H4K16 ac promotes the binding of Glc7 to histone proteins.We purified E.coli Glc7-His protein and performed histone co-immunoprecipitation(HIP)assay in vitro.Western blots was used to detect the interaction between Glc7 and histone H3,H4,H2 A and H2 B.Glc7 binding to histones was found to be significantly reduced in the sas2Δand H4K16 R mutants,and in this way the level of H3 p T11 was suppressed.4.Sas2 promotes the binding of Glc7 to autophagy genes and telomere genes.We have demonstrated that Sas2 and Glc7 interact with each other,and the distribution in the telomere region is opposite.Then,using Ch IP-q PCR technology,Ch IP:Glc7 in mutant sas2Δ,we found that the binding of Glc7 to both autophagy genes and telomere genes was significantly reduced.This indicates that Sas2 can specifically recruit Glc7 to bind to telomere genes,thus destroying telomere silencing.5.Sas2 inhibits the binding of H3 p T11 to autophagy genes and telomere genes.Glc7 is a phosphatase that dephosphorylates H3T11 in yeast.To prove whether the recruitment of Glc7 by Sas2 to regulate telomere silencing and autophagy is realized through its dephosphorylation function,we detected the binding of H3 p T11 on autophagy genes and telomere genes in mutant sas2Δ by Ch IP-q PCR.In contrast to Glc7 binding,the binding of H3 p T11 to autophagy genes and telomere genes decreased.According to the above studies,our results suggest an interaction between the phosphatase Glc7 and the acetyltransferase SAS complex;Sas2 decreased the level of H3 p T11 by inhibiting the binding of Glc7 to histone.Taking telomere silencing and autophagy as the starting point,the regulatory mechanism of Sas2 as an acetyltransferase on phosphatase Glc7 was studied.Sas2 regulates gene silencing by promoting the binding of Glc7 to autophagy genes and telomere genes and then affecting H3 p T11.It also indicates the crosstalk between two histone posttranslational modifications H4K16 ac and H3 p T11 during the regulation of telomere silencing.Overall,our study identified the role of crosstalk between the two histone modification enzymes and the post-translational modifications they affect in the regμLation of telomere heterochromatin and autophagy.It is extremely important to study the mechanism of regulating H3T11 phosphorylation for regulating telomere heterochromatin and delaying cell senescence.