【摘要】：巴豆�；揎検窃诎投辊；D(zhuǎn)移酶作用下將巴豆�；鶑陌投辊�-輔酶A轉(zhuǎn)移到賴氨酸殘基上的一種新型翻譯后修飾。其它實(shí)驗(yàn)室及我們實(shí)驗(yàn)室的研究表明,乙�；D(zhuǎn)移酶CBP/p300可以催化組蛋白巴豆酰化,組蛋白去乙�；窰DAC1、HDAC2、HDAC3、HDAC8以及SIRT1也具有組蛋白去巴豆�；富钚�。我們用小鼠的胚胎干細(xì)胞CGR8為材料,體外誘導(dǎo)分化形成擬胚體,檢測(cè)組蛋白乙酰化和巴豆�；桨l(fā)現(xiàn)胚胎干細(xì)胞分化后組蛋白乙酰化修飾和巴豆�；揎椝矫黠@下降。為了研究巴豆酰化修飾在胚胎干細(xì)胞中的作用,我們的策略是利用實(shí)驗(yàn)室前期篩選得到的具有去巴豆�；珕适Я巳ヒ阴；富钚缘腍D AC1突變體(HDAC1-VRPP),在胚胎干細(xì)胞中選擇性降低組蛋白巴豆�；揎棥Ｒ虼宋覀�?cè)谛∈笈咛ジ杉?xì)胞CGR8中構(gòu)建了可誘導(dǎo)表達(dá)野生型HDAC1和突變型HDAC1-VRPP的穩(wěn)系。我們通過(guò)蛋白印跡方法和RT-PCR方法分別檢測(cè)了Dox誘導(dǎo)野生型HDAC1和突變型HDAC1-VRPP表達(dá)不同時(shí)間對(duì)胚胎干性因子Oct4、Sox2和Nanog蛋白水平的影響和不同胚層的標(biāo)記基因在轉(zhuǎn)錄水平的變化,發(fā)現(xiàn)誘導(dǎo)野生型和突變型的HDAC1都能導(dǎo)致胚胎干性因子蛋白水平的下降和分化標(biāo)志基因表達(dá)不同程度的上調(diào),表明選擇性去巴豆酰化修飾誘導(dǎo)胚胎干細(xì)胞分化。通過(guò)顯微鏡觀察,我們也證實(shí)了 Dox誘導(dǎo)表達(dá)HDAC1-WT及HDAC1-VRP P九天后胚胎干細(xì)胞的克隆團(tuán)形成明顯變小。以上結(jié)果表明胚胎干細(xì)胞具有相比分化細(xì)胞而言更高水平的組蛋白巴豆�；揎�,并且發(fā)現(xiàn)高水平的組蛋白巴豆�；揎棇�(duì)維持胚胎干細(xì)胞的干性具有重要作用。
[Abstract]:Crotonyl modification is a new post-translational modification that transfers crotonyl from crotonyl-coenzyme A to lysine residues under the action of crotonyl transferase. Studies in other laboratories and in our laboratory have shown that acetyltransferase CBP/p300 can catalyze crotonylation of histone, histone deacetylase HDAC1,HDAC2,HDAC3,HDAC8 and SIRT1 also have histone debutamylase activity. Mouse embryonic stem cells (CGR8) were used as materials to induce differentiation into embryoid bodies in vitro. Histone acetylation and crotonylation levels were detected and the levels of histone acetylation and crotonylation modification were significantly decreased after differentiation of embryonic stem cells. In order to study the role of crotonyl modification in embryonic stem cells, our strategy is to use the HD AC1 mutants (HDAC1-VRPP), which have desuccinylation but have lost deacetylase activity, which were obtained from pre-laboratory screening. Selective reduction of histone crotonylation modification in embryonic stem cells. Therefore, we constructed a stable line in mouse embryonic stem cell CGR8 that could induce the expression of wild type HDAC1 and mutant HDAC1-VRPP. The expression of wild type HDAC1 and mutant HDAC1-VRPP induced by Dox were detected by Western blot and RT-PCR, respectively. The effects of Sox2 and Nanog protein levels and the changes of marker genes in different embryo layers at transcription level showed that both wild-type and mutant HDAC1 could induce the decrease of embryonic dry-factor protein level and the up-regulation of differentiation marker gene expression. The results showed that selective decrotonylation could induce the differentiation of embryonic stem cells. By microscope observation, we also confirmed that the colony formation of embryonic stem cells induced by Dox induced expression of HDAC1-WT and HDAC1-VRP P was significantly smaller after 9 days. These results suggest that embryonic stem cells have higher levels of histone crotonylation than differentiated cells and that high levels of histone crotonylation play an important role in maintaining the dryness of embryonic stem cells.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q75

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1 高申楠;組蛋白巴豆�；揎棇�(duì)小鼠胚胎干細(xì)胞干性調(diào)控的研究[D];華東師范大學(xué);2017年

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