劉建云, 張杰, 馬百成, 吳萍, 熊建軍
間充質(zhì)干細(xì)胞成骨分化早期染色質(zhì)開(kāi)放區(qū)域的動(dòng)態(tài)變化*
劉建云, 張杰, 馬百成, 吳萍, 熊建軍△
(九江學(xué)院基礎(chǔ)醫(yī)學(xué)院,江西 九江 332000)
在全基因組水平觀察骨髓間充質(zhì)干細(xì)胞(MSCs)成骨分化早期染色質(zhì)開(kāi)放區(qū)域的動(dòng)態(tài)變化。原代培養(yǎng)人骨髓MSCs至第6代,成骨誘導(dǎo)劑分別刺激0、3、5和7 d。收集各組細(xì)胞,運(yùn)用ATAC-seq技術(shù)(基于高通量測(cè)序的轉(zhuǎn)座酶可接近染色質(zhì)分析)檢測(cè)染色質(zhì)開(kāi)放區(qū)域,并對(duì)各組間染色質(zhì)開(kāi)放區(qū)域數(shù)量、開(kāi)放區(qū)域的DNA基序以及鄰近基因功能進(jìn)行生物信息學(xué)分析。在MSCs成骨定向分化早期的不同時(shí)點(diǎn),細(xì)胞染色質(zhì)開(kāi)放區(qū)域數(shù)量發(fā)生顯著動(dòng)態(tài)變化;各組功能性區(qū)域peak分布主要富集在轉(zhuǎn)錄起始點(diǎn)附近; motif分析顯示各組細(xì)胞間參與轉(zhuǎn)錄調(diào)控的活化DNA發(fā)生細(xì)微改變;新增染色質(zhì)開(kāi)放區(qū)域的生物學(xué)過(guò)程主要集中在各種GTP酶活性的調(diào)節(jié)、細(xì)胞外基質(zhì)組成、Wnt信號(hào)通路等;而新增的基因顯著性富集信號(hào)通路主要集中在Rap1信號(hào)通路、黏著斑、黏著連接等。在骨髓MSCs成骨定向分化早期,各時(shí)段的染色質(zhì)開(kāi)放區(qū)域發(fā)生了顯著的特征性改變。
間充質(zhì)干細(xì)胞;成骨分化;ATAC-seq;生物信息學(xué)
骨質(zhì)疏松癥是全球性公共健康問(wèn)題,以骨量減少、微觀結(jié)構(gòu)破壞、骨脆性增加和易發(fā)生骨折為顯著的臨床特征。經(jīng)典觀點(diǎn)認(rèn)為,骨質(zhì)疏松的發(fā)生與破骨細(xì)胞產(chǎn)生增多、活性增強(qiáng),致使骨量丟失與骨質(zhì)量下降有關(guān)[1]。新近研究證實(shí),來(lái)源于骨髓的間充質(zhì)干細(xì)胞(mesenchymal stem cells, MSCs)定向分化為成骨細(xì)胞(osteoblast, OB)和脂肪細(xì)胞(adipocyte, AD)的比例失衡(成骨細(xì)胞減少、脂肪細(xì)胞增多),是骨質(zhì)疏松發(fā)生發(fā)展的另一關(guān)鍵因素[2-4]。成骨誘導(dǎo)劑(地塞米松聯(lián)合β-甘油磷酸鈉、維生素C)是體外誘導(dǎo)MSCs向成骨細(xì)胞分化最經(jīng)典、最常用的方法。經(jīng)過(guò)21~28 d誘導(dǎo), MSCs由骨祖細(xì)胞經(jīng)前成骨細(xì)胞分化為成骨細(xì)胞,表達(dá)特異性成熟標(biāo)志物,如堿性磷酸酶(alkaline phosphatase, ALP)、I型膠原蛋白及骨鈣素(osteocalcin)等[5]。利用這一誘導(dǎo)模型,一些調(diào)控成骨分化的重要信號(hào)分子和通路得以發(fā)現(xiàn)和闡明。在MSCs的分化過(guò)程中伴隨著眾多基因的轉(zhuǎn)錄激活或抑制,表觀遺傳在轉(zhuǎn)錄水平動(dòng)態(tài)地調(diào)控相關(guān)基因的表達(dá),對(duì)細(xì)胞功能發(fā)揮了至關(guān)重要的作用[6]?;诟咄繙y(cè)序的轉(zhuǎn)座酶可接近染色質(zhì)分析(assay for transposase-accessible chromatin with high-throughput sequencing, ATAC-seq)是近年來(lái)興起的用于研究染色質(zhì)開(kāi)放性的表觀遺傳學(xué)工具,通過(guò)獲得染色質(zhì)上開(kāi)放區(qū)域的位置和活躍的調(diào)控序列,有助于在全基因組范圍內(nèi)鑒定激活的轉(zhuǎn)錄因子及其作用規(guī)律[7-8]。本項(xiàng)目以MSCs定向分化為成骨細(xì)胞的早期過(guò)程為研究對(duì)象,運(yùn)用ATAC-seq技術(shù)分析其中的染色質(zhì)開(kāi)放性變化,為研究成骨定向分化的調(diào)控機(jī)制及鑒定有效靶基因提供了新的切入點(diǎn)。
細(xì)胞來(lái)源于本實(shí)驗(yàn)室保存的人源性原代培養(yǎng)骨髓MSCs。培養(yǎng)環(huán)境: 37℃, 5% CO2。每隔48 h傳代1次。選擇第6代MSCs進(jìn)行成骨誘導(dǎo),誘導(dǎo)劑成分為:DMEM培養(yǎng)液中含10%胎牛血清、100 mmol/L地塞米松、10 mmol/L甘油磷酸鈉和50 μg/L維生素C。成骨誘導(dǎo)劑分別刺激0 d (MSC-0d)、3 d (OB-3d)、5 d (OB-5d)和7 d (OB-7d)后,收集細(xì)胞,在4℃環(huán)境下以500×離心5分鐘去除上清液,隨后以50 μL冰冷PBS洗滌1次,去除上清液,再以50 μL 冰冷lysis buffer懸浮細(xì)胞,立即離心10 min;去除上清液,進(jìn)行后續(xù)轉(zhuǎn)座反應(yīng)。
細(xì)胞置于冰上,使用轉(zhuǎn)座反應(yīng)體系(25 μL 2× reaction buffer from Nextera kit、2.5 μL Nextera Tn5 transposase from Nextera kit和22.5 μL nuclease-free H2O)懸浮細(xì)胞核。37℃孵育30 min;使用Qiagen MinElute PCR Purification Kit純化DNA; 10 μL elution buffer洗脫產(chǎn)物;取轉(zhuǎn)座后DNA片段配制成PCR反應(yīng)體系(10 μL DNA、10 μL nuclease-free H2O、2.5 μL PCR Primer 1、2.5 μL Barcoded PCR Primer 2和25 μl NEBNext High-Fidelity 2× PCR Master Mix)。反應(yīng)條件:72℃延伸5 min; 98℃變性30 s (1循環(huán));98℃變性10 s, 63℃退火30 s, 72℃延伸1 min (共5循環(huán)); 72℃延伸5 min; 4℃冷卻。最后使用Qiagen MinElute PCR Purification Kit純化PCR產(chǎn)物[9]。庫(kù)檢合格后進(jìn)行Illumina HiSeq測(cè)序。
測(cè)序數(shù)據(jù)下機(jī)之后,獲得原始測(cè)序序列(raw data),在有相關(guān)物種參考序列或參考基因組的情況下,通過(guò)標(biāo)準(zhǔn)流程進(jìn)行生物信息分析。
首先使用FastQC軟件來(lái)查看測(cè)序數(shù)據(jù)的質(zhì)量情況,包括測(cè)序錯(cuò)誤率分布和堿基含量分布。下機(jī)的原始序列進(jìn)行去接頭處理,使用BWA軟件將得到的有效數(shù)據(jù)(clean data)比對(duì)到參考基因組hg38_genecode上,該軟件采用BWT算法建立索引的短序列快速比對(duì),常用于將短序列比對(duì)到大型基因組上[10]。隨后比對(duì)分析后的bam文件作為輸入文件,使用MACS2軟件進(jìn)行Call Peak(使用統(tǒng)計(jì)學(xué)方法計(jì)算出參考基因組中比對(duì)上的Reads顯著富集的區(qū)域),篩選閾值為q value<0.05[11]。對(duì)每個(gè)peak區(qū)域從上、下游兩個(gè)方向分別拓寬200 bp提取出DNA序列,使用HOMER軟件的findMotifsGenome.pl工具對(duì)DNA序列預(yù)測(cè)motif,然后將預(yù)測(cè)的motif與各大數(shù)據(jù)庫(kù)(HOMER和JASPAR)中已有的motif數(shù)據(jù)進(jìn)行匹配,找到已發(fā)表的ChIP-seq實(shí)驗(yàn)中的motif和相應(yīng)的轉(zhuǎn)錄因子[12]?;蚋浇盘?hào)分布圖的分析使用deeptools軟件,對(duì)所有基因根據(jù)基因長(zhǎng)度進(jìn)行歸一化處理,計(jì)算在轉(zhuǎn)錄起始位點(diǎn)(transcription start site, TSS)上游3 kb到轉(zhuǎn)錄終止位點(diǎn)(transcription termination site, TSS)下游3 kb范圍內(nèi)每個(gè)位點(diǎn)上所有基因的平均信號(hào)值,并繪制成曲線圖[13]。最后利用DAVID (Database for Annotation, Visualization and Integrated Discovery)對(duì)染色質(zhì)開(kāi)放區(qū)域所關(guān)聯(lián)的基因進(jìn)行基因本體論(Gene Ontology, GO)富集分析[14];基于KEGG數(shù)據(jù)庫(kù)對(duì)peak鄰近基因進(jìn)行信號(hào)通路富集分析[15]。
在成骨誘導(dǎo)劑作用下,體外培養(yǎng)的MSCs逐漸出現(xiàn)形態(tài)變化,至14 d,分化為能分泌骨基質(zhì)的成骨細(xì)胞(圖1)。我們使用ATAC-seq來(lái)檢測(cè)早期不同分化時(shí)點(diǎn)(0、3、5和7 d)染色質(zhì)開(kāi)放區(qū)域的動(dòng)態(tài)變化。測(cè)序下機(jī)的原始數(shù)據(jù)經(jīng)篩選后,采用BWA軟件比對(duì),各組Reads比對(duì)率均高于90%; Reads在基因區(qū)域的信號(hào)分布均富集在TSS附近(圖2A)。使用MACS軟件對(duì)各組Reads進(jìn)行Call Peak,在MSC-0d組篩選出110 369個(gè)Reads顯著富集的區(qū)域(peak),在OB-3d組篩選出113 785個(gè)peak,在OB-5d組篩選出110 267個(gè)peak,在OB-7d組篩選出68 216個(gè)peak,表明成骨分化的第7天,細(xì)胞轉(zhuǎn)錄活性區(qū)域顯著減少(圖2B)。功能性區(qū)域分布顯示,位于啟動(dòng)子與TSS之間的peak比例大約在10%~15%之間變動(dòng),但是各組之間也存在細(xì)微的區(qū)別。值得注意的是,在成骨誘導(dǎo)的第7天,位于啟動(dòng)子與TSS的染色質(zhì)開(kāi)放區(qū)域的比例在各組中最高(15.98%),盡管這一時(shí)期整個(gè)peaks數(shù)最少(圖2C)。
Figure 1. Alizarin red staining of MSCs at different time points of differentiation (scale bar=20 μm). A: 0 d; B: 7 d; C: 14 d.
Figure 2. Identification of the chromatin accessibility. A: signal distribution map near the gene (the horizontal axis represents the normalized gene range coordinates, and the vertical axis represents the average signal value of the site); B: the number of peaks in each group; C: peak distribution in functional areas between MSC-0d and OB-7d groups.
HOMER軟件對(duì)4組細(xì)胞染色質(zhì)開(kāi)放區(qū)域的DNA序列進(jìn)行motif分析,顯示各組細(xì)胞中結(jié)合最多的motif均是亮氨酸拉鏈(basic leucine zipper, bZIP)轉(zhuǎn)錄因子家族的成員,如、、、、、-等,但是每組之間的排列順序又略有差異(表1)。
表1 各組細(xì)胞染色質(zhì)開(kāi)放區(qū)內(nèi)motif排序表
隨后我們對(duì)各分化組與MSC-0d組之間的差異motif進(jìn)行分析,顯示在OB-3d組與MSC-0d組之間上調(diào)數(shù)量變化最顯著的motif是基因家族、基因家族和等;在OB-5d組與MSC-0d組之間新增數(shù)量變化最為顯著的motif是基因家族、基因家族和等;在OB-7d組與MSC-0d組之間, motif則發(fā)生了更為顯著的變化,數(shù)量增加最為明顯的是、、、、等(圖3A)。RUNX2作為調(diào)控成骨早期分化的關(guān)鍵轉(zhuǎn)錄因子,我們重點(diǎn)追蹤了其結(jié)合序列開(kāi)放性的動(dòng)態(tài)變化。結(jié)果顯示,在成骨誘導(dǎo)的第3天和第5天,結(jié)合motif的開(kāi)放數(shù)量在整個(gè)細(xì)胞中的排序中前移,但在第7天顯著下降(圖3B),表明的轉(zhuǎn)錄活性在第5天后逐漸弱化。
Figure 3. Motif analysis in each group. A: the main different motifs in each group; B: the dynamic ranks of RUNX2 motif during osteogenic differentiation.
GO富集分析顯示, MSC-0d、OB-3d、OB-5d和OB-7d組細(xì)胞染色質(zhì)開(kāi)放區(qū)域所關(guān)聯(lián)基因的主要生物學(xué)過(guò)程(biological process, BP)并無(wú)顯著差異,大多是蛋白磷酸化和代謝途徑等,但各組間細(xì)胞差異染色質(zhì)開(kāi)放區(qū)域所關(guān)聯(lián)的基因BP有所不同。在OB-3d組與MSC-0d組之間,上調(diào)最為顯著的BP有正性調(diào)節(jié)GTP酶活性(positive regulation of GTPase activity)、正性調(diào)控Rho GTP酶活性(positive regulation of Rho GTPase activity)、正性調(diào)控Rac GTP酶活性(positive regulation of Rac GTPase activity)、細(xì)胞外基質(zhì)組成(extracellular matrix organization)、正性調(diào)控Wnt信號(hào)通路(positive regulation of Wnt signaling pathway)等;在OB-5d組與MSC-0d組之間,上調(diào)最為顯著的BP與在OB-3d組與MSC-0d組之間相似;但在OB-7d組與MSC-0d組之間,差異BP發(fā)生顯著變化,上調(diào)最顯著的BP為細(xì)胞黏附(cell adhesion)、細(xì)胞外基質(zhì)組成、細(xì)胞-基質(zhì)黏附(cell-matrix adhesion)等(圖4)。
Figure 4. Differential GO enrichment analysis of peak adjacent genes in each group.
信號(hào)通路富集分析顯示, MSC-0d、OB-3d、OB-5d和OB-7d組細(xì)胞染色質(zhì)開(kāi)放區(qū)域所關(guān)聯(lián)的主要信號(hào)通路都包括磷酸化、轉(zhuǎn)錄調(diào)控和代謝過(guò)程等。但各組細(xì)胞之間差異信號(hào)通路有所不同。在OB-3d組與MSC-0d組之間,上調(diào)較為顯著的信號(hào)通路有Rap1信號(hào)通路、黏著斑通路、炎癥介質(zhì)對(duì)TRP通道的調(diào)節(jié)等。在OB-5d組與MSC-0d組之間,上調(diào)較為顯著的信號(hào)通路有Rap1信號(hào)通路、黏著連接和黏著斑通路等。在OB-7d組與MSC-0d組之間,上調(diào)較為顯著信號(hào)通路有PI3K-Akt信號(hào)通路、Rap1信號(hào)通路、黏著斑通路等(圖5)。
Figure 5. Differential pathway enrichment analysis of peak adjacent genes in each group.
骨髓MSCs是在骨髓中發(fā)現(xiàn)的非造血干細(xì)胞群,其顯著特征在于具有強(qiáng)大的自我更新能力與多向分化潛能[16]。在分化早期,骨髓MSCs定向分化為特異性祖細(xì)胞是決定譜系定向的關(guān)鍵環(huán)節(jié),因此,我們將觀察的染色質(zhì)開(kāi)放區(qū)的時(shí)點(diǎn)設(shè)定為成骨誘導(dǎo)后的第3天、第5天和第7天。
MSCs向成骨細(xì)胞分化過(guò)程中伴隨著眾多基因的轉(zhuǎn)錄激活或抑制。在調(diào)控基因轉(zhuǎn)錄的啟動(dòng)子區(qū)和增強(qiáng)子區(qū),“開(kāi)放”和“封閉”染色質(zhì)分別代表單個(gè)基因的轉(zhuǎn)錄激活和抑制狀態(tài)?!伴_(kāi)放”的染色質(zhì)區(qū)域顯示較低的核小體密度甚至無(wú)核小體,并與修飾的核心組蛋白的特定組合;相反,轉(zhuǎn)錄抑制的基因通常在“封閉”染色質(zhì)結(jié)構(gòu)域內(nèi)[17-18]。ATAC-seq技術(shù)檢測(cè)的內(nèi)容為基因組中的“開(kāi)放”區(qū)域,即與轉(zhuǎn)錄因子結(jié)合調(diào)控基因表達(dá)的序列,對(duì)于了解組織分化和分化的分子機(jī)制提供了新的手段。本研究顯示,來(lái)源于骨髓間充質(zhì)的第六代干細(xì)胞中有超過(guò)十萬(wàn)個(gè)染色質(zhì)開(kāi)放區(qū)域,并且主要富集在轉(zhuǎn)錄起始點(diǎn)附近,表明MSCs在體外培養(yǎng)條件下就具有較高的基因轉(zhuǎn)錄活性。但是由于體外培養(yǎng)環(huán)境與體內(nèi)生長(zhǎng)環(huán)境的差異,我們尚不能以此來(lái)推測(cè)MSCs在體內(nèi)生長(zhǎng)時(shí)的染色質(zhì)開(kāi)放狀態(tài)。在成骨誘導(dǎo)劑作用第3天和第5天,染色質(zhì)開(kāi)放區(qū)域的數(shù)量趨于穩(wěn)定,而到了第7天,顯著下降,表明具有轉(zhuǎn)錄活性的基因明顯減少。我們推測(cè)成骨細(xì)胞分化的第7天可能是細(xì)胞功能的分水嶺:在成骨誘導(dǎo)劑的作用的前7 d,MSCs以定向分化為主,轉(zhuǎn)錄因子活躍,參與的基因數(shù)量眾多;而7 d之后,細(xì)胞趨于成熟,功能上以分泌細(xì)胞外基質(zhì)和礦化為主,所以激活基因數(shù)量顯著下降。這一推測(cè)需要相應(yīng)的RNA-seq進(jìn)行驗(yàn)證。
除了染色質(zhì)開(kāi)放數(shù)量,各組間功能性區(qū)域peak分布也有所不同。在成骨誘導(dǎo)的第3天和第5天,功能性區(qū)域peak分布在啟動(dòng)子與轉(zhuǎn)錄起始點(diǎn)之間的染色質(zhì)開(kāi)放區(qū)域的比例微小變動(dòng),但在第7天,這一比例明顯升高,提示細(xì)胞功能發(fā)生了明顯變化。值得注意的是,雖然基因間檢測(cè)到的peak較多,但是由于基因間區(qū)在基因組上占比極大,所以基因間區(qū)上微弱的信號(hào)一般不是真的調(diào)控因子結(jié)合位點(diǎn)。
與染色質(zhì)開(kāi)放數(shù)量趨勢(shì)相似的是各組間motif變化。有意思的是,各組細(xì)胞中占主導(dǎo)地位的轉(zhuǎn)錄因子結(jié)合序列均是bZIP基因家族成員(如、、和-等)。bZIP是堿性亮氨酸拉鏈(basic leucine zipper)蛋白大家族,普遍存在于動(dòng)植物及微生物中,通過(guò)二聚化亮氨酸拉鏈區(qū)對(duì)基因轉(zhuǎn)錄發(fā)揮廣泛的調(diào)控作用[19]。顯著的motif變化發(fā)生在成骨誘導(dǎo)的第7天,排序靠前的motif中出現(xiàn)轉(zhuǎn)錄因子和,這兩種轉(zhuǎn)錄因子如何參與細(xì)胞功能的轉(zhuǎn)變尚不清楚。
比各組間motif比較更有實(shí)際意義的是組間差異motif。測(cè)序結(jié)果顯示,與未誘導(dǎo)的MSC相比,成骨誘導(dǎo)第3天和第5天顯著變動(dòng)的motif主要有、和bZIP家族中的等,提示其可能是調(diào)控成骨分化早期的重要轉(zhuǎn)錄因子。其中是為人們熟知的促進(jìn)成骨分化的關(guān)鍵轉(zhuǎn)錄因子。基因?qū)儆赥EA/ATTS結(jié)構(gòu)域家族的成員,也被證實(shí)與成骨分化有關(guān)[20]。在成骨誘導(dǎo)第7天,組間差異motif變化與前5 d相比更為顯著。和的motif數(shù)量下降,取而代之的則是基因家族和基因家族,表明主要參與成骨早期的細(xì)胞分化,同時(shí)也表明MSCs的成骨分化到了第7天后可能進(jìn)入新的轉(zhuǎn)錄調(diào)控階段。
GO富集分析的目的是對(duì)基因進(jìn)行注釋和分類(lèi)。在MSC-0d、OB-3d、OB-5d和OB-7d各組細(xì)胞,GO富集分析的主要BP并無(wú)顯著差異,大多是蛋白磷酸化、生物代謝等,說(shuō)明以上這些功能可能是各類(lèi)細(xì)胞基礎(chǔ)生理活動(dòng)所共有。而各組間差異peak鄰近基因GO的富集分析顯示不同的基因功能。在成骨誘導(dǎo)劑作用的第3天和第5天,上調(diào)的基因多與GTP酶活性有關(guān)。事實(shí)上,已有研究認(rèn)為GTP酶活性是MSCs成骨定向分化的重要參與者[21]。此外,細(xì)胞外基質(zhì)的形成、Wnt信號(hào)通路功能和骨骼發(fā)育相關(guān)功能也被激活,這與成骨方向的分化是密切相關(guān)的[22-24]。第7天,新增的基因功能轉(zhuǎn)變?yōu)榧?xì)胞黏附、細(xì)胞外基質(zhì)組織、細(xì)胞與基質(zhì)黏附等活動(dòng),表明細(xì)胞功能出現(xiàn)明顯變化。此外,我們?cè)趐eak鄰近基因的信號(hào)通路富集分析和差異peak鄰近基因信號(hào)通路富集分析也看到類(lèi)似的變化。
綜合分析,本項(xiàng)目通過(guò)追蹤骨髓MSCs成骨分化早期染色質(zhì)開(kāi)放區(qū)域的動(dòng)態(tài)變化,為研究成骨定向分化的調(diào)控機(jī)制及鑒定有效靶基因提供了新的切入點(diǎn),后期工作將聯(lián)合RNA-seq數(shù)據(jù)和ChIP-seq數(shù)據(jù)進(jìn)行深入挖掘。
[1] Johnell O, Kanis JA. An estimate of the worldwide prevalence and disability associated with osteoporotic fractures [J]. Osteoporos Int, 2006, 17 (12):1726-1733.
[2] Muruganandan S, Roman AA, Sinal CJ. Adipocyte differentiation of bone marrow-derived mesenchymal stem cells: cross talk with the osteoblastogenic program[J]. Cell Mol Life Sci, 2009, 66(2):236-253.
[3] Botolin S, McCabe LR. Bone loss and increased bone adiposity in spontaneous and pharmacologically induced diabetic mice [J]. Endocrinology, 2007, 148(1):198-205.
[4]杲麗,胡成虎,金巖. 骨髓間充質(zhì)干細(xì)胞在小鼠早衰性骨質(zhì)疏松中的細(xì)胞生物學(xué)功能研究[J]. 中國(guó)病理生理雜志, 2013, 29(4):707-712.
Gao L, Hu CH, Jin Y. Age-related changes of bone marrow mesenchymal stem cells in senile osteoporosis[J].Chin J Pathophysiol, 2013, 29(4):707-712.
[5] Fiorentini E, Granchi D, Leonardi E, et al. Effects of osteogenic differentiation inducers onexpanded adult mesenchymal stromal cells [J]. Int J Artif Organs, 2011, 34(10):998-1011.
[6] Arnsdorf EJ, Tummala P, Castillo AB, et al. The epigenetic mechanism of mechanically induced osteogenic differentiation[J]. J Biomech, 2010, 43(15):2881-2886.
[7] Scott-Browne JP, Lopez-Moyado IF, Trifari S, et al. Dynamic changes in chromatin accessibility occur in CD8+T cells responding to viral infection[J]. Immunity, 2016, 45(6):1327-1340.
[8] Zhao Y, Zheng D, Cvekl A. Profiling of chromatin accessibility and identification of general-regulatory mechanisms that control two ocular lens differentiation pathways[J]. Epigenetics Chromatin, 2019, 12(1):27.
[9] Buenrostro JD, Wu B, Chang HY, et al. ATAC-seq: a method for assaying chromatin accessibility genome-wide[J]. Curr Protoc Mol Biol, 2015, 109:21.29.1-21.29.9.
[10] Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2[J]. Nat Methods, 2012, 9(4):357-359.
[11] Zhang Y, Liu T, Meyer CA, et al. Model-based analysis of ChIP-Seq (MACS)[J]. Genome Biol, 2008, 9(9):R137.
[12] Heinz S, Benner C, Spann N, et al. Simple combinations of lineage-determining transcription factors prime-regulatory elements required for macrophage and B cell identities[J]. Mol Cell, 2010, 38(4):576-589.
[13] Ramirez F, Dundar F, Diehl S, et al. deepTools: a flexible platform for exploring deep-sequencing data[J]. Nucleic Acids Res, 2014, 42(Web Server issue):W187-W191.
[14] Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources[J]. Nat Protoc, 2009, 4(1):44-57.
[15] Kanehisa M, Goto S, Sato Y, et al. KEGG for integration and interpretation of large-scale molecular data sets[J]. Nucleic Acids Res, 2012, 40(Database issue):D109-D114.
[16] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells[J]. Science, 1999, 284(5411):143-147.
[17] Long HK, Prescott SL, Wysocka J. Ever-changing landscapes: transcriptional enhancers in development and evolution[J]. Cell, 2016, 167(5):1170-1187.
[18] Tsompana M, Buck MJ. Chromatin accessibility: a window into the genome[J]. Epigenetics Chromatin, 2014, 7(1):33.
[19] Hurst HC. Transcription factors 1: bZIP proteins[J]. Protein Profile, 1995, 2(2):101-168.
[20] Hakelien AM, Bryne JC, Harstad KG, et al. The regulatory landscape of osteogenic differentiation[J]. Stem Cells, 2014, 32(10):2780-2793.
[21] Strzelecka-Kiliszek A, Mebarek S, Roszkowska M, et al. Functions of Rho family of small GTPases and Rho-associated coiled-coil kinases in bone cells during differentiation and mineralization[J]. Biochim Biophys Acta Gen Subj, 2017, 1861(5 Pt A):1009-1023.
[22] Ward DF Jr, Salasznyk RM, Klees RF, et al. Mechanical strain enhances extracellular matrix-induced gene focusing and promotes osteogenic differentiation of human mesenchymal stem cells through an extracellular-related kinase-dependent pathway[J]. Stem Cells Dev, 2007, 16(3):467-480.
[23] Baksh D, Boland GM, Tuan RS. Cross-talk between Wnt signaling pathways in human mesenchymal stem cells leads to functional antagonism during osteogenic differentiation [J]. J Cell Biochem, 2007, 101(5):1109-1124.
[24] Deng ZL, Sharff KA, Tang N, et al. Regulation of osteogenic differentiation during skeletal development[J]. Front Biosci, 2008, 13:2001-2021.
Dynamic changes of chromatin accessibility in early osteogenic differentiation of human mesenchymal stem cells
LIU Jian-yun, ZHANG Jie, MA Bai-cheng, WU Ping, XIONG Jian-jun
(,,332000,)
To observe the genome-wide dynamic profiling of chromatin accessibility in early osteogenic differentiation of human bone marrow mesenchymal stem cells (MSCs).The 6th generation of primarily cultured human bone marrow MSCs were treated with osteogenic inducer for 0 d, 3 d, 5 d and 7 d, and then were harvested to receive the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). The numbers of open chromatin regions, the specific activated DNA motifs in open chromatin regions and the functional enrichment analysis of peak adjacent genes were analyzed by bioinformatics.At different time points in the MSCs at early stage of osteogenic differentiation, the number of open chromatin regions changed significantly, and functional peak distributions in each group were mainly enriched near the transcription start sites. Motif analysis revealed subtle alterations of activated DNA involved in transcriptional regulation between each group. Gene ontology analysis of peak's adjacent gene function showed that the biological processes of the newly opened chromatin regions were mainly concentrated on regulation of several GTPase activity, extracellular matrix organization, Wnt signaling pathway, and so on, while enrichment pathways in different genes included Rap1 signaling pathway, focal adhesion, adherens junction, and so on.During osteogenic differentiation of MSCs, the open chromatin regions change significantly at the early stage.
Mesenchymal stem cells; Osteogenic differentiation; ATAC-seq; Bioinformatics
R681; R363
A
10.3969/j.issn.1000-4718.2020.11.016
1000-4718(2020)11-2037-06
2020-03-14
2020-05-22
國(guó)家自然科學(xué)基金資助項(xiàng)目(No.81860165)
Tel: 0792-8577050; E-mail: jcyx_xiongjianjun@jju.edu.cn
(責(zé)任編輯:盧萍,羅森)