Exploration Of Mechanism Of Indirect Gene Regulation By Histone Acetyltransferase Gcn5

Chromatin regulatory factors are divided into chromatin remodeling factors and chromatin modification factors,which play crucial roles in gene transcription.Analysis of the RNA-seq and Ch IP-seq data showed that many chromatin regulatory factors regulate gene expression in an unknown indirect manner.Revealing the molecular basis of this indirect gene regulation will undoubtedly help understand their precise roles in gene regulation and associated biological processes.Here,we aimed to study the mechanism by which histone modifying enzymes regulate gene expression by modulating the transcription of histone methyltransferases,SET1.Through unbiased screening of the histone H3/H4 mutant library,we identified 14 histone substitution mutations with reduced levels of Set1 and H3K4 trimethylation(H3K4me3)and2 mutations with increased levels Set1 and H3K4me3,which locate primarily at 3 structure clusters.Among these substitutions,the H3K14 A mutant has substantially reduced SET1 transcription and H3K4me3.Further studies showed that H3K14 is acetylated by histone acetyltransferase Gcn5 at SET1 promoter,which then promotes SET1 transcription to maintain normal H3K4me3 levels.In contrast,the histone deacetylase Rpd3 deacetylases H3K14 to repress SET1 transcription and hence reduce H3K4me3 levels,establishing a dynamic crosstalk between H3K14 ac and H3K4me3.By promoting the transcription of SET1 and maintaining H3K4me3 levels,Gcn5 regulates the transcription of a subset genes in an indirect manner.Collectively,we propose a model wherein Gcn5 promotes the expression of chromatin modifiers to regulate histone crosstalk and gene transcription.The specific research contents are as follows.1.Unbiased screen of histone H3/H4 mutant library for histone mutants that have the same trend changes of H3K4me3 and Set1.We examined the H3K4me3 and Set1 levels in histone H3/H4 mutant library by Western blots.Through unbiased screening,we identified 13 mutants with reduced Set1 and H3K4me3 levels and 2 mutants with increased Set1 and H3K4me3.Next,we examined SET1 m RNA levels in these 15 mutants.As Spp1 is specifically required for H3K4me3 as a subunit of Set1 C,we also determined SPP1 transcription.Our results showed that 6 mutants regulate H3K4me3 by affecting SET1 transcription level,which were further confirmed by transforming these mutants with Set1 overexpression vector.2.Gcn5 and Rpd3 dynamically regulate H3K14 ac to regulate Set1 and H3K4me3After analyzing the screening results,we focused on how H3K14 mutants regulate SET1 transcription and Set1-catalyzed H3K4me3.screening the known acetyltransferase(HAT)mutants and deacetylase(HDAC)mutants for altered H3K4me3 and Set1,we found that Gcn5 and Rpd3 dynamically modulate H3K14 ac to regulate SET1 transcription and H3K4me3.By analyzing RNA-seq and Ch IP-seq data,we propose that Gcn5 and Rpd3 regulate SET1 transcription by directly binding to SET1 and modulating H3K14 ac at SET1 gene promoter.3.Gcn5 indirectly regulate gene transcription by modulating SET1 transcriptionBy analyzing the dataset for Gcn5 RNA-seq and Ch IP-seq,we found that among 1226 genes regulated by Gcn5,only 265 genes were occupied by Gcn5,that 961 genes were indirectly regulated by Gcn5.Among these genes,114 genes were co-regulated by Gcn5 and Set1 and 78 genes were occupied by Set1.We randomly chose 4 genes: YIL015 W,YKL138C,YKR080 W and YMR189 W and confirmed their indirect regulation by Gcn5 by RT-q PCR and Ch IP-q PCR.We further confirmed the results in Set1 overexpression strains.Finally,we proposed the model by which Gcn5 regulates gene expression by modulating SET1 transcription.