Inhibition of DEK contributes to the tolerance of replication stress and restores HSC function in Fanconi anemia (2-ATAC). Inhibition of DEK contributes to the tolerance of replication stress and restores HSC function in Fanconi anemia (2-ATAC)

Replication stress drives functional decline in HSCs and is a major driver of BM failure in Fanconi anemia (FA). At present, how HSCs respond and counteract replication stress remains largely unknown. Using integrated multi-omics, we demonstrate that global chromatin relaxation is a prerequisite for the activation of stress-responsive genes and replication fork stabilization/progression in HSCs under replication stress. The reduced DEK (a chromatin architectural protein) contributes to the chromatin relaxation in HSCs, whereas DEK-overexpressed HSCs are confronted with a strong replication challenge, resulting in impaired HSC maintenance and hematopoiesis. Fancd2 deletion induces DEK expression and causes replication stress in HSCs, while haploinsufficiency of DEK promotes chromatin opening in Fancd2-deficient HSCs and substantially recovers HSC function. Notably, DEK expression is abnormally up-regulated in bone marrow (BM) CD34+ cells from FA patients. Inhibition of DEK significantly restores the proliferation capacity in vitro and engraftment in vivo of BM CD34+ cells from FA patients. At the molecular level, we identify that the transcriptional factor ATF2 directly promotes DEK transcription, mainly relying on the phosphorylated ATF2 (Thr69/71). Wild-type HSCs reduces DEK expression to counteract replication stress through the ATR kinase to phosphorylate ATF2 at Ser490/498. However, Fancd2 deficiency induces hyper-phosphorylation of p38 that further phosphorylates ATF2 at Thr69/71, causing DEK accumulation in HSCs. Collectively, our findings provide the first evidence of a functional link between chromatin relaxation and replication stress tolerance in HSCs, and highlight DEK as a potential molecular target for FA. Overall design: HSCs (CD48-CD150+LSK) from wild-type C57BL/6J mice were sorted and cultured with DMSO, Gemcitabine (Gem, 0.5 µM), or Hydroxyurea (HU, 100 µM) for 48 h, followed by ATAC-seq

复制应激（replication stress）可驱动造血干细胞（Hematopoietic Stem Cells, HSCs）功能衰退，亦是范可尼贫血（Fanconi Anemia, FA）患者骨髓（Bone Marrow, BM）衰竭的主要致病因素。目前，造血干细胞应答与拮抗复制应激的分子机制仍未完全阐明。本研究通过整合多组学分析，证实全基因组染色质松弛是复制应激状态下，造血干细胞激活应激应答基因、稳定并推进复制叉的必要前提。表达下调的DEK（一种染色质架构蛋白）可促进造血干细胞的染色质松弛；而DEK过表达的造血干细胞则会面临严重的复制应激挑战，进而导致造血干细胞维持功能受损及造血功能异常。敲除Fancd2可诱导造血干细胞中DEK的表达，并引发复制应激；而DEK单倍体剂量不足则可促进Fancd2缺陷型造血干细胞的染色质开放，显著恢复造血干细胞的功能。值得注意的是，范可尼贫血患者的骨髓CD34+细胞中，DEK的表达存在异常上调。抑制DEK可显著恢复范可尼贫血患者骨髓CD34+细胞的体外增殖能力与体内植入潜能。在分子层面，本研究证实转录因子ATF2可直接促进DEK的转录，这一过程主要依赖于磷酸化位点为Thr69/71的ATF2。野生型造血干细胞可通过ATR激酶磷酸化ATF2的Ser490/498位点，下调DEK的表达，以此拮抗复制应激。然而，Fancd2缺陷会诱导p38发生过度磷酸化，进而使ATF2的Thr69/71位点被磷酸化，最终导致造血干细胞中DEK的积累。综上，本研究首次证实了造血干细胞中染色质松弛与复制应激耐受之间的功能关联，并揭示DEK可作为范可尼贫血的潜在分子治疗靶点。实验整体设计：从野生型C57BL/6J小鼠中分选得到造血干细胞（CD48⁻CD150⁺LSK），分别用二甲基亚砜（DMSO）、吉西他滨（Gem, 0.5 μM）或羟基脲（HU, 100 μM）培养48小时，随后进行ATAC-seq测序。