近日,華中農業大學動物科學技術學院、動物醫學院苗義良團隊首次揭示了圍著床期胚胎譜系分離中選擇性自噬與細胞命運决定的調控機制。研究成果以“ATG7-mediated autophagy facilitates embryonic stem cell exit from naive pluripotency and marks commitment to differentiation”為題在Autophagy發表。
胚胎著床由精密複雜的訊號調控和錶觀遺傳調控所决定,其分子機理研究是動物繁殖領域發展亟需解决的覈心問題之一。受精後,尚未植入子宮(pre-implantation)的胚胎中生成了數十個原始態(naive)胚胎幹細胞,這些細胞具有分化成動物體內所有細胞的潜能。這種naive態幹細胞的多能性是高度動態且連續發展的,當胚胎植入子宮後,naive幹細胞很快邁出向特定細胞類型分化的第一步,變為始發態(primed)胚胎幹細胞。naive-primed態過渡期間,naive態幹細胞的染色質結構以及錶觀遺傳發生了全域重塑以抑制naive態幹細胞多能性基因的表達,啟動早期分化基因,進而促進primed態胚胎的著床及發育。
作為一種重要的分解代謝過程,自噬以溶酶體降解和再迴圈途徑維持多能性相關蛋白的適當水准和幹細胞內穩態。現時的觀點認為,特异性自噬調節因數局限於調控線粒體重構以促進體細胞重程式設計,以及通過降解功能紊亂的線粒體,降低細胞氧化應激來維持naive態細胞的多能性。然而,現時尚無研究揭示細胞自噬能否從錶觀遺傳水准調控胚胎幹細胞的多能性轉換。
本研究構建了mCherry-GFP-LC3雙螢光報告細胞系,利用體外胚胎幹細胞naive-primed轉換模型,通過電子顯微觀察及活細胞監測等科技闡述了naive態胚胎幹細胞多能性轉變過程中自噬通量的變化,證實了自噬對幹細胞命運轉變的决定性作用(圖1)。
圖1:naive-primed轉化過程中自噬流的動態變化
基於RNA-seq和染色質轉座酶可及性測序(ATAC-seq)聯合生物資訊學分析,本研究描繪了胚胎幹細胞多能性轉換過程染色質可及性動態變化規律,並發現自噬阻斷條件下NANOG作為封锁多能性轉變的屏障,一方面抑制分化相關位點的開放,另一方面維持naive多能性基因位點的開放以抑制多能性退出。此外通過染色質免疫共沉澱科技(ChIP)證明NANOG通過競爭性結合OTX2調控的特异性神經外胚層發育的相關區域來抑制神經元分化。通過CRISPR/Cas9構建的ATG7敲除小鼠模型,評估了自噬喪失會破壞圍著床期胚胎的發育,進而導致新生小鼠神經發育异常及死亡。圍著床期胚胎的异常發育是導致早期妊娠流產的主要原因,針對選擇性自噬在圍著床期胚胎發育中調控機制開發相關科技,對改善動物妊娠和構建naive態大動物胚胎幹細胞具有重要意義(圖2)。
圖2:細胞命運轉換過程中選擇性自噬參與的蛋白穩態調控及染色質變化
華中農業大學動科動醫學院副研究員周吉隆和何海楠博士、張晶晶博士為論文的共同第一作者,華中農業大學苗義良教授為論文通訊作者。本研究受到了國家重點研發計畫和國家自然科學基金的資助。
【英文摘要】
Macroautophagy/autophagy is a conserved cellular mechanism to degrade unneeded cytoplasmic proteins and organelles to recycle their components,and it is critical for embryonic stem cell(ESC)self-renewal and somatic cell reprogramming.Whereas autophagy is essential for early development of embryos,no information exists regarding its functions during the transition from naive-to-primed pluripotency.Here,by using an in vitro transition model of ESCs to epiblast-like cells(EpiLCs),we find that dynamic changes in ATG7-dependent autophagy are critical for the naive-to-primed transition,and are also necessary for germline specification.RNA-seq and ATAC-seq profiling reveal that NANOG acts as a barrier to prevent pluripotency transition,and autophagy-dependent NANOG degradation is important for dismantling the naive pluripotency expression program through decommissioning of naive-associated active enhancers.Mechanistically,we found that autophagy receptor protein SQSTM1/p62 translocated into the nucleus during the pluripotency transition period and is preferentially associated with K63 ubiquitinated NANOG for selective protein degradation.In vivo,loss of autophagy by ATG7 depletion disrupts peri-implantation development and causes increased chromatin association of NANOG,which affects neuronal differentiation by competitively binding to OTX2-specific neuroectodermal development-associated regions.Taken together,our findings reveal that autophagy-dependent degradation of NANOG plays a critical role in regulating exit from the naive state and marks distinct cell fate allocation during lineage specification.
原文連結:https://www.tandfonline.com/doi/full/10.1080/15548627.2022.2055285