Stabilizing heterochromatin by ZKSCAN3 safeguards hMSCs from senescence

Zinc finger protein with KRAB and SCAN domain 3 (ZKSCAN3), a transcriptional repressor, involves in multiple cellular functions. However, the functions of ZKSCAN3 in the homeostatic maintenance of human stem cells remains elusive. Here, we demonstrated that ZKSCAN3 was crucial for preventing human mesenchymal stem cells (hMSCs) from senescence in an autophagy-independent manner. Downregulation of ZKSCAN3 was observed in senescent hMSCs, and depletion of ZKSCAN3 led to premature aging in hMSCs. Further study uncovered that ZKSCAN3 maintained heterochromatin (HC) stability by interacting with heterochromatin associated proteins KAP1 and HP1? as well as nuclear envelope proteins Lamin B1, LBR and Emerin. Deficiency of ZKSCAN3 resulted in detachment of Lamina-associated domains (LADs) from the lamina, loss of heterochromatin and more accessible chromatin status within the heterochromatin and aberrant expression of repetitive sequences. Overexpression of ZKSCAN3, KAP1 or HP1?? respectively rescued the senescent phenotypes in ZKSCAN3-/- hMSCs. Notably, reintroduction of ZKSCAN3 protein also retarded the cellular senescence in the replicative senescent hMSCs, as well as the pathological or physiological aging hMSCs. Together, our study reveals a novel, autophagy-independent role of ZKSCAN3 in the maintenance of heterochromatin stability and attenuation of hMSC senescence. Thus, ZKSCAN3 may be a candidate for alleviating human aging-related disorders. Overall design: Multi-omics sequencing of ZKSCAN3+/+ and ZKSCAN3-/- hMSCs.

含KRAB与SCAN结构域的锌指蛋白3（Zinc finger protein with KRAB and SCAN domain 3，ZKSCAN3）是一类转录抑制因子，参与诸多细胞生物学功能。然而，ZKSCAN3在人类干细胞稳态维持中的功能仍尚不明确。本研究证实，ZKSCAN3以不依赖自噬的方式，在防止人类间充质干细胞（hMSCs）衰老方面发挥关键作用。研究人员在衰老的hMSCs中观察到ZKSCAN3的表达下调，而ZKSCAN3缺失会导致hMSCs过早衰老。进一步研究发现，ZKSCAN3可通过与异染色质相关蛋白KAP1、HP1？以及核膜蛋白Lamin B1、LBR及Emerin结合，维持异染色质（HC）的稳定性。ZKSCAN3缺失会导致核纤层关联结构域（Lamina-associated domains，LADs）与核纤层脱离，引发异染色质丢失、异染色质区域内染色质可及性升高，以及重复序列的异常表达。分别过表达ZKSCAN3、KAP1或HP1？？，均可挽救ZKSCAN3敲除型（ZKSCAN3-/-）hMSCs的衰老表型。值得注意的是，重新导入ZKSCAN3蛋白同样能够延缓复制性衰老hMSCs，以及病理性或生理性衰老hMSCs的细胞衰老进程。综上，本研究揭示了ZKSCAN3在维持异染色质稳定性、延缓hMSCs衰老方面的全新且不依赖自噬的作用机制。因此，ZKSCAN3有望成为缓解人类衰老相关疾病的潜在治疗靶点。实验整体设计：对ZKSCAN3野生型（ZKSCAN3+/+）与ZKSCAN3敲除型（ZKSCAN3-/-）hMSCs进行多组学测序。