2017.10.12，單革教授實(shí)驗(yàn)室在Developmental Cell發(fā)表文章，報(bào)導(dǎo)了其實(shí)驗(yàn)室最新的研究成果。他們發(fā)現(xiàn)秀麗線蟲(chóng)中兩個(gè)高度保守的轉(zhuǎn)錄因子UNC-30和UNC-55，共調(diào)控包括cAMP通路、microRNA和lncRNA等在內(nèi)的數(shù)以千計(jì)的靶基因的表達(dá)，從而調(diào)控D型運(yùn)動(dòng)神經(jīng)元的發(fā)育和分化。
為了獲知這兩個(gè)轉(zhuǎn)錄因子在D型運(yùn)動(dòng)神經(jīng)元中的所有靶基因，該實(shí)驗(yàn)室首先利用CRISPR-Cas9技術(shù)將GFP分別插入到unc-30和unc-55基因的3’末端，得到表達(dá)UNC-30::GFP和UNC-55::GFP融合蛋白的秀麗線蟲(chóng)，再對(duì)利用所獲秀麗線蟲(chóng)進(jìn)行ChIP（染色質(zhì)免疫共沉淀）實(shí)驗(yàn)。通過(guò)對(duì)原有ChIP實(shí)驗(yàn)方法進(jìn)行改進(jìn)和優(yōu)化，克服了兩個(gè)轉(zhuǎn)錄因子秀麗線蟲(chóng)中表達(dá)量小，表達(dá)時(shí)間窗口短，表達(dá)的D型神經(jīng)元細(xì)胞占秀麗線蟲(chóng)細(xì)胞比例少（約占2%）等限制性瓶頸，成功的完成了ChIP實(shí)驗(yàn)、拿到了可供高通量測(cè)序（ChIP-seq）的DNA樣品。這也是第一次實(shí)現(xiàn)了在多細(xì)胞動(dòng)物中原位表達(dá)水平轉(zhuǎn)錄因子的ChIP-seq實(shí)驗(yàn)。
研究揭示這兩個(gè)轉(zhuǎn)錄因子各自均調(diào)控近2000多個(gè)基因的表達(dá)，而且非常有意思的是，它們共同調(diào)控1300多個(gè)編碼和非編碼基因的表達(dá)。這些靶基因在多個(gè)對(duì)神經(jīng)元發(fā)育和可塑性具有重要調(diào)控作用的信號(hào)通路中行使功能。其中一個(gè)通路是決定環(huán)腺苷酸（cAMP）濃度的cAMP代謝通路。為了直接觀測(cè)活體秀麗線蟲(chóng)細(xì)胞中的cAMP濃度變化，作者開(kāi)創(chuàng)性地將用于哺乳動(dòng)物細(xì)胞中檢測(cè)cAMP水平的探針改造為在活體秀麗線蟲(chóng)中也可以使用工具。進(jìn)一步的研究揭示這兩個(gè)在動(dòng)物界高度保守的轉(zhuǎn)錄因子通過(guò)調(diào)節(jié)神經(jīng)元中的基因表達(dá)，調(diào)控了細(xì)胞中cAMP的濃度，進(jìn)而在時(shí)間上影響D型運(yùn)動(dòng)神經(jīng)元發(fā)育和可塑性的進(jìn)程。另外，從全基因組水平上獲得了UNC-30和UNC-55調(diào)控的靶基因，為進(jìn)一步探索動(dòng)物細(xì)胞中轉(zhuǎn)錄因子調(diào)控網(wǎng)絡(luò)提供了重要的基礎(chǔ)。
文章的第一作者是博士生余斌。研究得到了科技部、中科院、國(guó)家基金委、以及中科大非編碼RNA功能及功能機(jī)理創(chuàng)新團(tuán)隊(duì)的經(jīng)費(fèi)支持。
Bin Yu, Xiaolin Wang, Shuai Wei, Tao Fu, Emmanuel Enoch Dzakah, Ahmed Waqas, Walter W. Walthall, Ge Shan. Convergent transcriptional programs regulate cAMP levels in C. elegans GABAergic motor neurons. Dev. Cell.?2017, Online.
論文鏈接：http://www.cell.com/developmental-cell/fulltext/S1534-5807(17)30765-7
SUMMARY
Both transcriptional regulation and signaling pathways play crucial roles in neuronal differentiation and plasticity. Caenorhabditis elegans?possesses 19 GABAergic motor neurons (MNs) called D MNs, which are divided into two subgroups as DD and VD. DD but not VD MNs reverse their cellular polarity in a developmental process called respecification. UNC-30 and UNC-55 are two critical transcription factors in D MNs. By chromatin immunoprecipitation with CRISPR/cas9 knock-in of GFP fusion, we uncovered the global targets of UNC-30 and UNC-55. UNC-30 and UNC-55 are largely converged to regulate over 1,300 noncoding and coding genes, and genes in multiple biological processes including the cAMP metabolism are co-regulated. Increase of cAMP levels may serve as a timing signal for respecification, whereas UNC-55 regulates genes such as pde-4 to keep the cAMP levels low in VD. Other genes modulating DD respecification such as lin-14, irx-1, and oig-1 are also found to affect cAMP levels.
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