The role of the activin cistrome in liver progenitor cell-mediated liver regeneration

激活素反应元件（Activin response element， ARE）定位于主分化基因的启动子中。该序列基序是SMAD4-SMAD2/3-FOXH1复合物的特异性结合位点，参与器官发育过程中胚胎干细胞（ESC）分化的调控。没有细胞外信号，整个定向基因表达，随后被称为“激活素池”，在ESC中被组蛋白H3复合物上的抑制染色质压缩因子hp1 -赖氨酸三甲基化阻断，从而使ESC处于沉默状态。当来自TGF-家族成员（如Nodal、activin和TGF-）的信号激活激活激活素池时，静止的ESC切换到分化模式。ESC暴露于这些细胞因子导致形成由TRIM33和磷酸化的SMAD2和SMAD3组成的复合物，取代HP1并揭示ARE。随后，TGF-诱导的转录因子复合物SMAD4-SMAD2/3-FoxH1与激活素应答元件结合，诱导主分化基因的表达，启动细胞分化。在人类中，肝祖细胞（LPC），成人肝干细胞样细胞，在严重肝损伤（如肝硬化、急性肝衰竭（ALF）和急性慢性肝衰竭（ACLF））中，在恢复肝脏质量和功能方面起着至关重要的作用。成功的LPC介导的肝再生需要LPC快速分化为肝细胞。目前尚不清楚LPC向肝细胞的分化是否受激活素池的控制。本项目的初步数据显示，阴性结果（即肝移植或死亡）的ALF或ACLF患者肝脏中p-SMAD2、TRIM33和FOXH1的表达较弱或检测不到。相反，代偿性肝硬化患者在LPC和LPC源性肝细胞中表达高水平的这三种转录因子。体外研究进一步表明，lpc向肝细胞分化需要SMAD2/3、TRIM33和FOXH1。因此，我们假设通过染色质重塑激活激活素是LPC向肝细胞分化的关键。本项目旨在阐明(i) are介导的LPC分化是否发生在肝硬化、ALF和ACLF等严重肝病患者中；（ii）在这些严重的情况下，TGF-或激活素或两者均诱导LPC激活激活素池；（iii）激活素池的激活如何影响体外LPC向肝细胞的分化；（iv）干扰激活素池的成分是否会影响动物中LPC介导的肝脏再生。该项目将确定激活素池在lpc介导的肝脏再生中的作用和分子机制，并使我们能够在肝硬化和ALF/ACLF事件中利用这些干细胞的再生能力。

Activin response element (ARE) are localized in the promoters of master differentiation genes. The sequence motif is a specific binding site for the SMAD4-SMAD2/3-FOXH1 complex, involved in the regulation of embryonic stem cell (ESC) differentiation during organ development. Without extracellular signals, the entirety of ARE-directed gene expression, subsequently called the "activin cistrome", is blocked in ESC by repressive chromatin compacting factor HP1-lysine trimethylation on histone H3 complexes, thus holding ESC in a silent state. Quiescent ESC switch into differentiation mode once the activin cistrome is activated by signals from TGF- family members e.g. Nodal, activin and TGF-. Exposure of ESC to these cytokines results in the formation of complexes comprising TRIM33 and phosphorylated SMAD2 and SMAD3, which replace HP1 and uncover the ARE. Subsequently, the TGF--induced transcription factor complex SMAD4-SMAD2/3-FoxH1 binds to the activin response elements, and induces expression of master differentiation genes, initiating cell differentiation. In humans, liver progenitor cells (LPC), adult liver stem-like cells, play a crucial role in restoring liver mass and function in severe liver damage, e.g. cirrhosis, acute liver failure (ALF) and acute-on-chronic liver failure (ACLF). Successful LPC-mediated liver regeneration requires rapid LPC differentiation into hepatocytes. It is unknown to date if LPC differentiation into hepatocytes is governed by the activin cistrome. Preliminary data for this project show that ALF or ACLF patients with a negative outcome, i.e. liver transplantation or death, showed weak or undetectable hepatic expression of p-SMAD2, TRIM33 and FOXH1. In contrast, compensated cirrhotic patients express high levels of the three transcription factors in LPC and LPC-derived hepatocytes. In vitro study further showed that LPC-to-hepatocyte differentiation required SMAD2/3, TRIM33 and FOXH1. Thus, we hypothesize that activation of the activin cistrome by chromatin remodelling is crucial for LPC differentiation into hepatocytes. This project aims to elucidate (i) whether ARE-mediated LPC differentiation occurs in patients suffering from severe liver disease, e.g. cirrhosis, ALF and ACLF; (ii) if TGF- or activin or both induce activation of the activin cistrome in LPC under these severe circumstances; (iii) how activation of the activin cistrome impacts LPC differentiation into hepatocytes in vitro and (iv) if interfering with components of the activin cistrome influences LPC-mediated liver regeneration in animals. The project will define the role and molecular mechanisms of the activin cistrome in LPC-mediated liver regeneration and enable us to harness the regenerative power of these stem-like cells in events of cirrhosis and ALF/ACLF.