Distinct contributions of KAT2A histone acetyl-transferase complexes to human erythropoiesis [RNA-seq]. Distinct contributions of KAT2A histone acetyl-transferase complexes to human erythropoiesis [RNA-seq]

KAT2A is a histone acetyl-transferase involved in stabilization of transcriptional activity through acetylation of lysine residue 9 of Histone 3. Mouse knockout models suggest that Kat2a is dispensable for haematopoietic stem and progenitor cell activity, despite a central role in survival and maintenance of Acute Myekoid Leukaemia cells through block of differentiation. Herein, we investigate KAT2A activity in human cord blood haematopoiesis and identify a specific requirement in the establishment of the erythroid lineage. KAT2A is required for specification and survival of Erythroid-Megakaryocytic progenitors, with regulation of expression of the erythropoietin receptor (EPOR) gene, which participates in lineage commitment, as well as of later effector genes in the platelet and erythroid lineages. Early and late lineage roles can be distinctly attributed to the ATAC and SAGA complexes in which context KAT2A exerts its activity. ATAC is active earlier in erythroid lineage development and mediates MEP specification from HSC, whilst SAGA activity is required post-commitment, including in expression of haemoglobin genes. We thus position KAT2A as a novel regulator of human erythropoiesis and separate early and later effects in lineage development with complex specificity. This has implications for putative therapeutic targeting of KAT2A complexes in leukaemia. Overall design: 2 individual cord blood samples treated as independent biological replicates were enriched for CD34+ haematopoietic stem cells (HSC) and progenitors by magnetic selection using the StemCell Technologies EasySep system. CD34+ cells from each cord blood sample were transduced with lentiviral vectors (LV) encoding a control non-targeting shRNA or a shRNA directed at KAT2A, as well as reporter GFP gene. Four days after LV transduction, cells were sorted by flow cytometry on the basis of GFP expression, coupled with CD34+ CD38- CD45RA- staining to isolate HSC and multipotent progenitors expressing the shRNA of interest. KAT2A knockdown on the paired samples was confirmed by qRT-PCR prior to library preparation.

KAT2A是一种组蛋白乙酰转移酶（histone acetyl-transferase），可通过乙酰化组蛋白3（Histone 3）第9位赖氨酸残基稳定转录活性。小鼠基因敲除模型显示，尽管KAT2A在通过阻断分化维持急性髓系白血病（Acute Myeloid Leukaemia）细胞的存活与稳态中发挥核心作用，但它对于造血干细胞和祖细胞（haematopoietic stem and progenitor cell）的功能并非必需。本研究探究了KAT2A在人脐带血造血过程中的活性，并明确了其在红系谱系（erythroid lineage）建立过程中的特定必需功能。KAT2A对红系-巨核系祖细胞（Erythroid-Megakaryocytic progenitors）的特化与存活至关重要，可调控促红细胞生成素受体（erythropoietin receptor, EPOR）基因的表达——该基因参与谱系定型，同时还调控血小板与红系谱系后期效应基因的表达。KAT2A发挥功能的ATAC与SAGA复合物（ATAC and SAGA complexes）可分别介导其在谱系发育的早期与晚期作用：ATAC复合物在红系谱系发育早期更为活跃，可介导造血干细胞（haematopoietic stem cell, HSC）向巨核红系祖细胞（MEP）的特化；而SAGA复合物的活性则在谱系定型后不可或缺，包括调控血红蛋白基因的表达。综上，本研究将KAT2A界定为人类红细胞生成过程中的新型调控因子，并明确了其在谱系发育中兼具早期与晚期效应且具有复杂特异性。该发现对于以KAT2A复合物为潜在治疗靶点的白血病治疗策略具有重要参考价值。实验设计：本研究选取2份独立的脐带血样本作为生物学重复，通过StemCell Technologies公司的EasySep系统进行磁珠分选，以富集CD34阳性造血干细胞与祖细胞。将每份脐带血样本中的CD34阳性细胞用慢病毒载体（lentiviral vectors, LV）进行转导，该载体携带对照非靶向shRNA、靶向KAT2A的shRNA以及报告基因绿色荧光蛋白（GFP）。慢病毒转导4天后，通过流式细胞术根据绿色荧光蛋白表达情况，结合CD34+CD38-CD45RA-染色，分选获得携带目标shRNA的造血干细胞与多能祖细胞。在构建测序文库前，通过定量实时聚合酶链反应（qRT-PCR）验证了配对样本中KAT2A的敲低效率。