自噬调控造血干细胞多向分化中Notch-Sirt7-H3轴的作用

We have recently reported that autophagy contributes to the maintenance of balance between the quiescence and selfrenwal of hematopoietic stem cells (HSC) by limiting the protein level of cyclin D3 in collaboration with ubiquitination-proteasome system, and autophagy is able to selectively degrade intracellular Notch to impede Notch signaling, leading to release of Notch repression on HSC differentiation and reduction of leukemogenesis from abnomally activated Notch signaling in adult mice. However, the downstream cascades of Notch in the context of autophagic regulation of HSC differentiation remain unresolved. Interestingly, results in our ongoing experiments show that the upregulation of Notch signaling from loss of autophagy is selectively associated with the downregulation of sirtuin 7 (Sirt7), a member of class III histone deacetylases but upregulation of acetylation of histone 3 (H3). Moreover, artificial blokade in Notch signaling by inhibiting the cleavage of Notch receptor restores the level of Sirt7 protein, reduces the abnormally elevated level of H3 acetylation, and ultimately improves HSC multilineage differentiation and hematopoiesis. These preliminary data suggest that Notch-Sirt7-H3 axis may serve as a molecular platform downstream of autophagy in the regulation of HSC multilineage differetntion and hematopoiesis. To address this futher, we have generated a unique mouse model with autophagy essential gene atg7 exclusively deleted in HSCs and set to determine the role of Notch-Sirt7-H3 axis and its underlying mechanism in autophagic regulation of HSC multilineage differentiation in adult mice.

我们最近报道，自噬通过限制细胞周期素D3水平控制G1/S期转换以维护造血干细胞静息态与自我更新之间的平衡，并通过选择性降解Notch以下调Notch信号途径从而促进造血干细胞多向分化，降低白血病发生。但是，自噬调控造血干细胞多向分化过程中，其通过下调Notch信号后，如何继而促进造血干细胞多向分化的机制未明。我们的预实验显示：造血干细胞自噬缺陷引起Notch上调时，伴随去乙酰化蛋白酶Sirt7的表达选择性降低，组蛋白H3乙酰化水平升高，而且，在造血干细胞自噬缺陷小鼠中，阻断Notch信号可以逆转造血干细胞中Sirt7的表达水平和组蛋白H3乙酰化水平，并改善造血干细胞多向分化和小鼠造血功能，因此，提示Notch-Sirt7-H3轴可能是自噬调控造血干细胞多向分化的分子途径。本项目拟运用造血干细胞特异性自噬基因敲除小鼠模型探讨Notch-Sirt7-H3轴在自噬调控造血干细胞多向分化中的作用。

我们前期报道，自噬通过限制细胞周期素D3水平调控G1/S期转换以维护造血干细胞静息态与自我更新之间的平衡，并调控造血干细胞的多向分化，降低白血病发生。但是，自噬调控造血干细胞多向分化的机制未明。我们推测自噬调控造血干细胞的多向分化，可能涉及对组蛋白乙酰化水平的调控。.本项目研究显示，血液系统细胞中，条件性自噬缺陷导致核小体稳态破坏，体现于组蛋白H3.1在细胞核内显著减少而在细胞质内积累显著增加，而组蛋白H3.3在细胞核内水平显著减少，而并不发生细胞质内积累。血液系统自噬缺陷时，CD11b+髓系细胞的H3.1蛋白在细胞质堆积比较明显，而细胞核内H3.1蛋白水平显著减少。这一现象尤其发生于CD11b+Gr1low粒细胞中。自噬缺陷还导致组蛋白H3的乙酰化水平增加（K9,K18,K56）。所以，自噬缺陷导致造血干细胞多向分化异常，即髓系分化异常增强而淋系分化异常削弱，可能与核小体稳态被破坏和组蛋白修饰异常有关。.而且，我们发现自噬缺陷导致去乙酰化蛋白酶成员SIRT7蛋白水平下降。有趣的是，敲低SIRT7后，也发生以H3.1积累异常、H3.3显著减少为特征的核小体稳态破坏。相反，在自噬缺陷的血细胞中过表达SIRT7，可以挽救核小体稳态的破坏。.转录组测序及定量PCR验证显示，自噬缺陷不影响Sirt7基因的转录水平。用Co-IP技术研究发现，自噬缺陷时，SIRT7与泛素结合增加。进而发现，在自噬缺陷的血细胞中用特异性抑制剂阻断泛素化-蛋白酶体途径，可以遏制SIRT7蛋白的降解。说明，自噬缺陷导致SIRT7的泛素化降解是SIRT7水平降低的原因。因此，推测自噬通过维持SIRT7蛋白稳定性，以维持核小体稳态。.这一发现为进一步阐明自噬调控造血干细胞的多向分化奠定了重要基础。目前正在鉴定自噬调控SIRT7稳定性的直接靶分子。