Remodeling Of H3K27me3-marked Silencer In Gastric Intestinal Metaplasia And Prelimilary Characterization Of A Transcriptional Regulator ATOH1

Gastric intestinal metaplasia(IM)is a precancerous lesion in which normal gastric cell lineages are replaced by cells resembling intestinal epithelial morphology.The molecular mechanisms underlying gastric intestinal metaplasia have not yet been fully elucidated.Previous studies have shown that intestinal metaplastic epithelium possesses a distinct transcriptome profile from normal gastric epithelium.Due to the fewer alterations in the genome sequence of IM than gastric cancer(Copy number changes and mutation burdens are extremely low),it is suggested that epigenetic disorders may be be one of the significant causes of gastric intestinalization.The representative non-coding regulatory elements(NCREs),silencer,plays an important role in mediating the inhibition of targeted genes within independent manners out of time and space,which is marked by the trimethylation of lysine27 on histone H3(H3K27me3)catalyzed by Poly-comb repressive complex 2(PRC2).H3K27me3-associated silencer regions can recruit inhibitory transcription factors,which promote chromatin remodeling or block activator binding to these regions to turn off targeted gene expression programs that regulate biological processes such as cell differentiation/cell fate determination,organ development,and tumorigenesis.Currently,the role of silencer-mediated epigenetic regulation during gastric intestinalization is unknown.Significant cellular morphological and functional transitions occur in metaplastic lesions,suggesting that silencer-associated epigenetic alterations may play an important role in this process.In addition,gastric IM tissues highly express CDX1,CDX2,ATOH1 and other factors that determine the cell fate of intestinal metaplastic cell lineages,and these transcription factors may play an critical role in the trans-differentiation of gastric mucosal cells into intestinal metaplastic cells.Although it has been found that CDX2 can transcriptionally up-regulate ATOH1 expression,the role of ATOH1 in intestinal metaplasia of gastric mucosa and the epigenetic regulatory mechanism of CDX1 and over-expression of other transcription factors in gastric IM remain unclear.Therefore,in this study,CUT&Tag-sequencing was applied for capturing H3K27me3-marked sequences to systematically characterize the genome-wide distribution of silencers in the pathological process of gastric IM.RNA-seq data was integrated to explore the regulatory mechanism of H3K27me3-marked silencers on target genes associated with IM.It provides a novel insight for understanding the mechanism of gastric intestinalization and exploitation of preventive strategies of gastric cancer.A list of experimental methods and materials,results and main conclusions are as followed.1.Gastric IM is characterized by genome-wide remodeling of silencers.We captured silencer genomic regions by H3K27me3 CUT&Tag-sequencing on a genome-wide scale and evaluated silencer signal intensity.Annotation of functional regions on the genome by Ch IPseeker revealed that H3K27me3-marked silencers were mainly distributed in the distal intergenic regions and non-coding introns,which were conserved in normal and IM tissues;however,silencer signal intensity was significantly down-regulated in IM tissues(P < 0.05).H3K27me3-marked silencer features within IM tissues also differed evidently from those of normal tissues by principle component analysis(PCA).2.H3K27me3-marked silencer remodeling is associated with the activation of IM driver gene CDX1,as well as multiple metabolic processes.BETA tool was employed to integrate transcriptomic data to predict differential gene expression potentially regulated by differential H3K27me3-marked silencer regions,and silencer remodeling-associated target genes with up-and down-regulation expression levels were identified,such as CDX1,a driver gene of gastric IM.KEGG enrichment analysis revealed that these target genes were closely related to multiple metabolic processes in IM,including aberrant activation of lipid metabolism and pyruvate metabolism and inhibition of gastric acid secretion.3.Loss of H3K27me3-markeed silencer might recruit transcription factors ATOH1 and ONECUT2 to regulate aberrant gene expression profiles in IM tissues.Since loss of H3K27me3-marked silencer signaling leading to the transcriptional activation of metaplastic genes,Homer was applied for predicting transcription factor(TF)binding motifs enriched in the region with loss of H3K27me3-marked silencer signaling,including TAL1,SOX9,TWIST2,ONECUT2,ISL1,TFAP2 A,BHLHA5,and ATOH1.By analyzing the expression levels of these transcription factors in IM tissues,we identified two highly expressed candidate transcription factors,ATOH1 and ONECUT2.Comparing binding motifs of ATOH1 and ONECUT2 with the H3K27me3-maarked silencer-loss regions,we constructed a transcriptional regulatory network,containing several intestinal differentiation-related genes,including CDX1,HOXB13,and SAT2 B,etc.In addition,ATOH1 regulates greater numbers of intestinal differentiated genes than ONECUT2,and thus ATOH1 may play a more essential role in gastric IM.Results from IGV showed that loss of H3K27me3-marked silencer signaling on upstream of the CDX1 transcription start site(TSS)may recruit ATOH1 and thus promote the up-regulation of CDX1 expression level.4.The transcription factor ATOH1 may activate the CDX1 expression to regulate IM.We enforced the over-expression of ATOH1 in GES-1 cell lines and using q PCR to detected m RNA levels of target genes.The expression levels of ATOH1 and CDX1 were found to be positively correlated(P < 0.0005).In addition,the expression level of MUC2,a typical biomarker of gastric IM,was also significantly up-regulated(P < 0.0001).Experimental results of q PCR assays and immunohistochemistry(IHC)staining both indicated that IM tissue highly expressed ATOH1(P < 0.0001)and CDX1(P < 0.0001).As a consequence,ATOH1 might facilitate the transcription of CDX1 to regulate the pathological process of gastric intestinalization,but further experimental verification is needed.In conclusion,a common epigenetic feature in gastric IM tissues was revealed for the first time via utilized multi-omic sequencing and integrated analysis.Silencer remodeling was associated with differential expression of multiple gastric metaplastic genes,potentially affecting multiple metabolic processes.Combined TF motif enrichment analysis and differential gene expression,the silencer-associated TFs,ATOH1 and ONECUT2,were identified as candidate transcription factors and may establish the transcription regulatory net.Meanwhile,in vitro experiments verified that ATOH1 promotes the expression of CDX1.The above results indicate that H3K27me3-marked silencer-mediated gene transcriptional regulation is a key molecular mechanism in gastric intestinalization,and the correlation of transcription factor ATOH1 with the activation of CDX1,the novel and potential downstream regulatory mechanism of ATOH1 promoting the initiation of gastric IM.