【作者】 王建芳；

【导师】 贾存灵；

【作者基本信息】 西北农林科技大学 ， 动物营养与饲料科学， 2021， 硕士

【摘要】 绒山羊作为重要的经济动物之一,以生产优质羊绒而著称,如何提高羊绒产量和绒毛品质一直以来备受研究者和生产者的关注。毛囊发育的周期变化直接影响羊绒产量和绒毛品质,这一过程受到多种基因和信号通路的共同调控,表观遗传修饰在协调体内基因表达情况,响应环境变化中发挥着至关重要的作用。在多种表观遗传修饰的研究中,有关组蛋白修饰对毛囊周期变化的影响多见于小鼠上,在绒山羊皮肤毛囊发育中的研究未见报道。H3K27me3作为重要的组蛋白修饰之一,对维持哺乳动物毛囊正常发育具有重要调控作用,因此,本研究主要探究H3K27me3对绒山羊毛囊周期再生调控的分子机制,以期为绒山羊绒毛提质增效提供理论依据。本研究选用3只成年陕北白绒山羊作为研究对象,分别采集其在生长期、退行期和休止期的皮肤组织样,利用染色质免疫共沉淀——测序技术(chromatin immunoprecipitation followed by sequencing,ChIP-seq)分析H3K27me3在不同时期对功能基因的修饰情况,通过筛选差异结合位点并进行功能注释和通路富集分析,进一步探究H3K27me3在毛囊再生周期中的调控作用;利用RNA-seq技术分析不同时期基因在转录水平上的差异;联合分析ChIP-seq和RNA-seq数据,筛选受H3K27me3直接作用的潜在靶基因,分析H3K27me3对毛囊发育相关基因的调控机制。主要试验结果如下:1.ChIP-seq分析结果表明,H3K27me3主要富集在基因的TSS区域,在陕北白绒山羊毛囊周期发育过程中存在动态修饰作用,生长期表现出高甲基化状态,退行期表现出低甲基化状态,休止期的甲基化状态介于生长期和退行期;对差异结合位点进行分析并对位于promoter区的peaks进行基因注释,结果发现生长期和退行期间共有7946个差异修饰基因(differentially modified genes,DMGs),生长期与休止期间有2419个DMGs,休止期与退行期间有397个DMGs。GO和KEGG富集分析表明,这些DMGs能够参与细胞、生物调控、发育和代谢等过程以及刺激响应,并参与MAPK、Hippo、Rap1和Focal adhesion等多条与毛囊周期发育相关的信号通路。2.RNA-seq分析结果表明,陕北白绒山羊毛囊不同发育时期共有1301个差异表达基因(differentially expressed genes,DEGs),其中,生长期和退行期间有1147个DEGs,生长期和休止期间有61个DEGs,休止期和退行期间有178个DEGs。GO和KEGG功能注释分析结果表明,这些DEGs同样也参与响应刺激、生物调控、发育和代谢等过程,以及PI3K-Akt、Wnt、AMPK和Rap1信号通路以及Focal adhesion等与毛囊周期发育相关通路,发现了潜在参与绒山羊毛囊周期发育的关键基因,包括SOX9、LHX2、LGR5、DBP、NR1D1、SOCS3和NR4A1等。3.ChIP-seq和RNA-seq联合分析共获得550个受H3K27me3直接调控的潜在靶基因,主要为生长期和退行期间差异表达的基因。相对于退行期,组蛋白高甲基化修饰主要发生在生长期,受到修饰的DEGs有273个在生长期低表达,274个在生长期高表达,低表达的DEGs中的高差异基因COL1A1、NR4A1、SOCS3、ETS1以及SERPINE1与细胞凋亡有关,这些基因更利于生长期毛囊的再生和退行期毛囊细胞稳态的维持。生长期高表达的基因KRT17、SOX9、LGR5、LHX2、DBP和NR1D1与细胞增殖和毛囊周期发育有关,通过预测H3K27me3结合位点的转录因子结合基序,发现H3K27me3可能通过影响Sp/KLF家族抑制因子在靶基因上的结合而影响基因的表达。综上所述,陕北白绒山羊毛囊的周期性发育与H3K27me3对毛囊发育相关基因的动态修饰作用有关,表现在生长期H3K27me3高修饰,退行期低修饰,休止期H3K27me3修饰作用逐渐上升,这一特点对维持绒山羊毛囊在整个发育周期的有序变化至关重要。本研究结果为深入探究绒山羊毛囊生长发育的调控机制提供了新思路,为改善绒山羊绒毛品质提供科学依据。更多还原

【Abstract】 Cashmere goats,as one of the important economic animals,are famous to produce cashmere.It has been the focus on improving the yield and quality of cashmere for researchers and producers.The cyclic development of hair follicles directly affects cashmere production and quality,which is regulated by multiple genes and signaling pathways.Epigenetic modifications play a crucial role in coordinating changes of gene expression in vivo and responding to environmental changes.Among various epigenetic modifications,the effects of histone modifications on the changes of hair follicle cycle have been reprted in mice,but it was few for cashmere goats.H3K27me3,as one of the important histone modifications,has an important regulatory role in maintaining normal hair follicle development in mammals.Therefore,this study mainly explored the roles of H3K27me3 in hair follicle cyclic regeneration of cashmere goats.The results will provide highlights for improving the quality and efficiency of cashmere goats.In this study,three adult Shaanbei white cashmere goats were selected as the research objects,and their skin tissue samples were collected in anagen,catagen and telogen,respectively.Chromatin immunoprecipitation followed by sequencing(ChIP-seq)technology was used to analyze the modification ability of H3K27me3 for functional genes in different development periods of hair follicle,screen differential binding sites and annotate genes function.RNA-seq technology was used to analyze the genes differences in different development periods of hair follicle.Combined analysis of ChIP-seq and RNA-seq,potential target genes directly affected by H3K27me3 were screened and the regulatory mechanism of H3K27me3 on genes related to hair follicle development was analyzed.The main experimental results were as follows:1.ChIP-seq analysis showed that H3K27me3 was mainly enriched in the TSS region of genes and had a dynamic modification mode during the development cycle of hair follicle of Shaanbei White Cashmere Goats.It appeared a hypermethylated state for H3K27me3 modification in anagen,a hypomethylated state in catagen and Intermediate level in telogen.By the analysis of differential binding sites and gene annotation of peaks located in promoter region,it revealed a total of 7946 differentially modified genes(DMGs)between anagen and catagen,2419 DMGs between anagen and telogen,and 397 DMGs between catagen and telogen.These DMGs could participate in cellular,bioregulatory,developmental and metabolic processes,and stimulus response,as well as were involved in multiple signaling pathways related to hair follicle cycle development such as MAPK,Hippo,Rap1 and Focal adhesion by GO and KEGG enrichment analysis.2.RNA-seq analysis showed 1301 differentially expressed genes(DEGs)in different developmental periods of the hair follicle of Shaanbei White Cashmere Goats.Among them,there were 1147 DEGs between anagen and catagen,61 DEGs between anagen and telogen,and 178 DEGs between catagen and telogen.GO and KEGG functional annotation analysis showed that these DEGs were also involved in response to stimulation,bioregulation,development and metabolism,as well as PI3K-Akt,Wnt,AMPK and Rap1 signaling pathways and Focal adhesion,which were related to hair follicle cycle development.The key genes potentially involved in hair follicle cycle development were identified,including SOX9,LHX2,LGR5,DBP,NR1D1,SOCS3 and NR4A1.3.Combined ChIP-seq and RNA-seq analysis,a total of 550 potential target genes directly regulated by H3K27me3 were found,which were mainly from the difference between anagen and catagen.Compared with catagen,histone hypermethylation modifications occurred mainly in anagen,where 273 DEGs were low-expressed and 274 were high-expressed.The highly differentially expressed genes,COL1A1,NR4A1,SOCS3,ETS1,and SERPINE1,were lowly-expressed and associated with apoptosis.These genes were more conducive to the regeneration of hair follicles during anagen and the maintenance of follicle cell homeostasis in catagen.The genes KRT17,SOX9,LGR5,LHX2,DBP and NR1D1,highly expressed in anagen,were associated with cell proliferation and hair follicle cycle development.Through predicting the transcription factor binding motifs at the H3K27me3 binding sites,it was found that H3K27me3 might affect gene expression by influencing the binding of Sp/KLF family repressors on target genes.In summary,the cyclic development of hair follicles in Shaanbei white cashmere goats had been associated with gene expression changes by the dynamic modification of H3K27me3 on hair follicle development,which showed high modification of H3K27me3 in anagen,low modification in catagen,and gradually increasing modification of H3K27me3 in telogen.This feature was essential to maintain the orderly changes of hair follicles throughout the development cycle in goats.This study provided a new idea to investigate the regulatory mechanism of hair follicle growth and development in cashmere goats,and provided a scientific basis for improving the quality of cashmere.更多还原