EGR1 CHIP and RNA sequencing following EGR1 knockdown in CD34+ human hematopoeitic stem and progenitor cells

Therapy-related myeloid neoplasms (t-MN) share many similarities with AML de novo in the elderly. One common factor is that they arise in the setting of chronic inflammation, likely due to advanced age or chemotherapy-induced senescence. Here, we examined the impact of haploinsufficient loss of the del(5q) tumor suppressor gene, EGR1, commonly deleted in high-risk MNs. In mice, under the exogenous inflammatory stress of either serial transplant or successive doses of the alkylating agent ENU, Egr1-haploinsufficient hematopoietic stem cells (HSCs) exhibit a clonal advantage. Complete loss of EGR1 function is incompatible with transformation; mutations of EGR1 are rare and are not observed in the remaining allele in del(5q) patients and complete knockout of Egr1 in mice leads to HSC exhaustion. Using chromatin immunoprecipitation sequencing (ChIP-seq), we identify EGR1 binding sites in human CD34+ cord blood-derived stem and progenitor cells (HSPCs) and show that EGR1 binds genes critical for stem cell differentiation, inflammatory signaling, and the DNA damage response. Notably, in the chromosome 5 sequences frequently deleted in patients, there is a significant enrichment of innate and inflammatory genes, which may confer a fitness advantage in an inflammatory environment. Short hairpin RNA (shRNA) mediated silencing of EGR1 biases HSPCs towards a self-renewal transcriptional signature. In the absence of EGR1, cells upregulate MYC-driven proliferative signals, downregulate CDKN1A (p21), disrupt the DNA damage response, and downregulate inflammation - adaptations anticipated to confer a relative fitness advantage for stem cells especially in an environment of chronic inflammation. RNA sequencing following EGR1 knockdown in CD34+ human hematopoeitic stem and progenitor cells (HSPC) isolated from cord blood. EGR1 CHIP-seq from CD34+ HSPCs.

治疗相关髓系肿瘤（therapy-related myeloid neoplasms, t-MN）与老年原发性急性髓系白血病（de novo AML）存在诸多相似之处。二者的共同特征之一是均发生于慢性炎症环境中，该炎症环境可能由高龄或化疗诱导的细胞衰老所引发。本研究针对高危髓系肿瘤中常见缺失的、位于del(5q)区域的抑癌基因EGR1的单倍体功能缺失效应展开了探究。在小鼠模型中，当接受连续移植或烷化剂ENU连续给药所致的外源性炎症应激时，Egr1单倍体功能缺失的造血干细胞（hematopoietic stem cells, HSCs）会呈现克隆优势。而EGR1功能完全缺失则无法实现恶性转化；EGR1突变极为罕见，且在del(5q)患者的剩余等位基因中未检测到该突变；同时，小鼠体内Egr1完全敲除会导致造血干细胞耗竭。本研究通过染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq），在人脐带血来源CD34+造血干祖细胞（hematopoietic stem and progenitor cells, HSPCs）中鉴定出EGR1的结合位点，并证实EGR1可结合与干细胞分化、炎症信号通路及DNA损伤应答密切相关的基因。值得注意的是，在患者常见缺失的5号染色体序列中，先天性免疫及炎症相关基因存在显著富集，这或许可使细胞在慢性炎症环境中获得生存优势。短发夹RNA（short hairpin RNA, shRNA）介导的EGR1沉默可使造血干祖细胞偏向于呈现自我更新转录特征。在EGR1缺失的情况下，细胞会上调MYC介导的增殖信号、下调CDKN1A（p21）、破坏DNA损伤应答通路并抑制炎症反应——这些适应性改变有望使干细胞在慢性炎症环境中获得相对生存优势。本研究开展了两项组学实验：其一为对脐带血分离得到的CD34+人造血干祖细胞进行EGR1敲低后的RNA测序；其二为对CD34+造血干祖细胞进行EGR1染色质免疫共沉淀测序。