组蛋白甲基转移酶SETDB1调控雄性生殖干细胞存活的分子机制研究

发布时间：2018-05-28 04:42

本文选题：精原干细胞 + 性原细胞　；参考：《西北农林科技大学》2017年博士论文

【摘要】：具有干细胞特性的性原细胞及精原细胞统称为雄性生殖干细胞,这类干细胞可以自我更新维持干细胞库,也可以分化产生精子。精原干细胞(spermatogonial stem cells,SSCs)是唯一可以将亲本遗传信息传递给后代的成体干细胞。SSCs可以分化为精原细胞并起始精子发生,这个过程需要严密的基因表达调控。基因的表达调控受到组蛋白修饰的影响。组蛋白H3和H4的赖氨酸残基上可以发生三种甲基化修饰,包括一、二和三甲基化。通常H3K4和H3K36的甲基化与基因的转录激活相关,而H3K9,H3K27和H4K20的甲基化与转录抑制相关。这些组蛋白的甲基化修饰受到甲基转移酶或去甲基化酶的催化。组蛋白甲基转移酶SETDB1(SET domain,bifurcated 1),也称为ESET,可通过H3K9me2/3调控异染色质的形成,抑制基因表达。SETDB1对于胚胎干细胞的维持、神经细胞的存活具有重要作用。本研究利用蛋白质免疫共沉淀、染色质免疫共沉淀、组织免疫荧光等技术研究了SETDB1调控小鼠精原干细胞存活的分子机制,以及SETDB1对猪性原细胞存活的调控作用。主要结果如下:(1)Setdb1敲低可引起C18-4细胞线粒体膜电位的下降,细胞膜磷脂酰丝氨酸的外翻及凋亡晚期DNA的断裂,且Setdb1-KD诱导了促凋亡基因Bax、Apaf1、p53、Caspase9表达的上调,抑凋亡基因XIAP表达下调。说明Setdb1干扰可以通过调节凋亡相关基因的表达调控细胞凋亡。(2)Setdb1-KD可激活PTEN,进而抑制AKT及FOXO1的磷酸化。Setdb1及Pten的双敲低可挽救细胞凋亡的发生,且反转了由于Setdb1-KD诱导的凋亡及通路相关蛋白的表达变化。说明了PTEN/AKT/FOXO1通路参与了Setdb1-KD诱导的小鼠精原干细胞凋亡。(3)SETDB1可与AKT相互作用,且SETDB1可以增强AKT对下游靶蛋白FOXO1的调控,抑制FOXO1的入核,从而抑制其自身启动子的转录活性。Setdb1-KD可诱导FOXO1进核,并激活促凋亡基因Bim和Puma的表达。说明SETDB1可以协同AKT调控FOXO1活性并影响下游靶基因的表达。(4)H3K9me3-ChIP结果显示,Setdb1-KD导致了基因Pten、Foxo3和Bim启动子区域H3K9me3水平的降低,而Foxo1、Puma、Bax和Bcl2启动子区域的H3K9me3无显著变化。另外,SETDB1-ChIP显示,SETDB1可以直接结合到Pten、Bim、Puma和Bax基因启动子区域。结果说明,SETDB1只负责部分基因启动子上H3K9me3修饰,且SETDB1可以通过直接作用于Pten及Bim启动子区域催化H3K9me3修饰,从而调控小鼠精原干细胞的存活。(5)在猪睾丸发育过程中,SETDB1的表达量随着猪的发育逐渐升高,而H3K9me3的表达水平在出生7天猪睾丸中表达量最高。在7天和两月龄猪的睾丸组织中,SETDB1在性原细胞和精原干细胞中呈胞质分布,而H3K9me3呈核周分布。在成年猪睾丸组织中,SETDB1定位于精原干细胞和分化的精原细胞的细胞核,而H3K9me3在精原干细胞中呈核周分布,在分化的精原细胞中呈斑点状分布。(6)为了研究SETDB1在猪性原细胞中的功能,通过差异贴壁富集性原细胞,富集后的性原细胞纯度可达到76.5%±3.9%。通过Western及RT-PCR结果发现,SETDB1主要表达于性原细胞。说明SETDB1在调控猪性原细胞的存活上可能发挥着重要功能。(7)SETDB1干扰引起猪性原细胞凋亡,且凋亡的发生不依赖于H3K9me3;另外,SETDB1可与催化H3K27甲基化的甲基转移酶EZH2结合,且SETDB1敲低引起H3K27me3水平的降低,说明SETDB1干扰调控猪性原细胞的凋亡是通过H3K27me3水平调控的。综上所述,通过PTEN/AKT/FOXO1通路及H3K9me3水平,Setdb1-KD诱导小鼠精原干细胞的凋亡;SETDB1协同AKT参与对下游靶蛋白FOXO1的活性调控。同时,SETDB1-KD也诱导猪性原细胞凋亡,但是H3K9me3水平并未改变,而与SETDB1结合的组蛋白甲基转移酶EZH2催化的H3K27me3水平的降低有关。本研究揭示了表观遗传机制和凋亡信号通路之间的关系,并填补了表观遗传调控雄性生殖干细胞存活研究领域的空缺,为今后雄性不育的研究提供了新的思路。
[Abstract]:The stem cells and spermatogonial cells, which have the characteristics of stem cells, are called male reproductive stem cells. These types of stem cells can self renew and maintain a stem cell bank and can produce sperm. Spermatogonial stem cells (SSCs) is the only adult stem cell that can transmit the genetic information to the offspring,.SSCs can be differentiated into Spermatogonial cells and initiating spermatogenesis, this process requires strict regulation of gene expression. The regulation of gene expression is affected by histone modification. Three kinds of methylation modification can occur on the lysine residues of histone H3 and H4, including one, two and trimethylation. The methylation of H3K4 and H3K36 is usually associated with gene transcription activation, and H3K 9, the methylation of H3K27 and H4K20 is related to transcriptional inhibition. Methylation modification of these histones is catalyzed by methyltransferase or demethylation. Histone methyltransferase SETDB1 (SET domain, bifurcated 1), also known as ESET, can regulate the formation of heterochromatin through H3K9me2/3 and inhibit the gene expression of.SETDB1 for embryonic stem cells. In this study, the molecular mechanism of SETDB1 regulating the survival of mouse spermatogonial stem cells and the regulation of SETDB1 on the survival of porcine spermatogonial cells were studied by protein immunoprecipitation, chromatin immunofluorescence, and tissue immunofluorescence. The main results are as follows: (1) C18- in Setdb1 can cause C18- The decrease of mitochondrial membrane potential in 4 cells, ectropion of phosphatidylserine in cell membrane and rupture of late apoptosis DNA, and Setdb1-KD induced up regulation of apoptosis gene Bax, Apaf1, p53, Caspase9 expression and down regulation of apoptosis gene XIAP expression. It shows that Setdb1 interference can regulate apoptosis by regulating the expression of apoptosis related genes. (2) Setdb1-KD Activation of PTEN, and then inhibition of AKT and FOXO1 phosphorylation.Setdb1 and Pten double knock low can save the occurrence of apoptosis, and reverse the Setdb1-KD induced apoptosis and the expression of pathway related proteins. It shows that PTEN/AKT/FOXO1 pathway participates in Setdb1-KD induced mouse spermatogonial stem cell apoptosis. (3) SETDB1 can interact with AKT And SETDB1 can enhance the regulation of AKT to the downstream target protein FOXO1, inhibit the nucleation of FOXO1, inhibit the transcriptional activity.Setdb1-KD of its own promoter, induce FOXO1 entry and activate the expression of Bim and Puma of the apoptotic gene. It shows that SETDB1 can regulate FOXO1 activity with AKT and influence the expression of the downstream target gene. (4) H3K9me3-ChIP results Setdb1-KD resulted in a decrease in the H3K9me3 level in the promoter region of the gene Pten, Foxo3 and Bim, while H3K9me3 in Foxo1, Puma, Bax and Bcl2 promoter region did not change significantly. Me3 modification, and SETDB1 can regulate the survival of mouse spermatogonial stem cells by catalyzing H3K9me3 modification directly in Pten and Bim promoter regions. (5) during the development of pig testicles, the expression of SETDB1 increased gradually with the development of pigs, while the expression level of H3K9me3 was highest in the 7 days of pig testicles at birth. In 7 days and 2 month old, the expression level of H3K9me3 was the highest. In the testis of pigs, SETDB1 is distributed in the cytoplasm of the proogenic and spermatogonial stem cells, and the H3K9me3 is perinuclear. In adult pig testis, SETDB1 is located in the nucleus of the spermatogonial stem cells and the differentiated spermatogonial cells, while H3K9me3 is distributed in the spermatogonial stem cells and is distributed in the differentiated spermatogonial cells. (6) In order to study the function of SETDB1 in porcine proto cells, the purity of the enriched primary cells can reach 76.5% + 3.9%. through Western and RT-PCR through differentially adhered preconcentration cells. SETDB1 is mainly expressed in the primary cells. It shows that SETDB1 may play an important role in regulating the survival of porcine primitive cells. (7) SETDB1 interference. It causes the apoptosis of porcine procells, and the occurrence of apoptosis is not dependent on H3K9me3; in addition, SETDB1 can be combined with the methyltransferase EZH2 that catalyzes H3K27 methylation, and the SETDB1 knockdown causes the decrease of H3K27me3 level. It indicates that the apoptosis of the porcine primary cells by SETDB1 interference is regulated by the level of H3K27me3. In summary, the PTEN/AKT/FOXO1 pathway is described. And H3K9me3 level, Setdb1-KD induced the apoptosis of mouse spermatogonial stem cells; SETDB1 coordinated AKT to regulate the activity of FOXO1 in the downstream target protein. At the same time, SETDB1-KD also induced the apoptosis of porcine proogenic cells, but the H3K9me3 level did not change, but the decrease of H3K27me3 level catalyzed by the histone methyltransferase, which was combined with SETDB1, was related to the decrease of the level of H3K27me3. It reveals the relationship between epigenetic and apoptotic signaling pathways, and fills the vacancy in the study of epigenetic regulation of male reproductive stem cell survival, and provides a new way of thinking for the future research of male infertility.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q23

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