巨核细胞（MK）是血小板前体细胞，起源于造血干细胞（HSCs）。虽然MK系列承诺和分化伴随着基因表达的改变，但仍有很多调节巨核造血的因素有待揭示。复制启动决定蛋白（RepID）具有包括溴结构域、隐秘图多域和WD40结构域的多种组蛋白编码阅读器，以及由RepID介导的染色质上的Cullin 4-RING E3泛素连接酶复合物（CRL4）。我们的目的是调查RepID-CRL4是否参与MK分化所需的转录变化。使用来源于RepID合格或RepID缺失的K562红细胞白血病细胞系的cDNA进行PCR阵列分析。通过在癌细胞线系百科全书（CCLE）上使用CellMinerCDB门户检查RepID和DAB2表达之间的相关性。通过估计细胞大小以及计数已知为MK表型的多核细胞，并通过qRT-PCR分析利用丙酸激酯12-肉毒素13-乙酸酯（PMA）介导的MK分化条件下的MK标记转录本来确定RepID缺失K562细胞中MK分化的加速程度。使用BioGRID数据库、免疫沉淀和近距离连接测定研究CRL4与组蛋白甲基化修饰酶之间的相互作用。使用亚细胞分离后的免疫印迹研究RepID、CRL4和组蛋白甲基化修饰酶的表达和染色质结合亲和力的改变。通过染色质免疫沉淀和qPCR分析来分析基于RepID-CRL4-JARID1A的DAB2启动子上的表观遗传变化。表达MK标记的RepID缺失K562细胞显示出加速的MK分化，细胞大小增加、多裂核以及比RepID合格的K562细胞更早达到MK标记表达的最大水平。在RepID缺失背景中恢复含有WD40结构域的RepID构建抑制了DAB2的表达。CRL4A形成与组蛋白H3K4脱甲基酶JARID1A的复合物在可溶性细胞核中，并以RepID依赖的方式加载到DAB2启动子上，在增殖状态下。在MK分化过程中，RepID、CRL4A和JARID1A从染色质中解离，导致DAB2启动子的松刺染色质化。本研究揭示了RepID-CRL4A-JARID1A通路在调节MK分化的基因表达中的作用，为诱导血小板生产的新治疗方法奠定了基础。视频摘要。© 2023年。BioMed Central有限公司，施普林格自然出版集团的一部分。

Megakaryocytes (MKs) are platelet precursors, which arise from hematopoietic stem cells (HSCs). While MK lineage commitment and differentiation are accompanied by changes in gene expression, many factors that modulate megakaryopoiesis remain to be uncovered. Replication initiation determinant protein (RepID) which has multiple histone-code reader including bromodomain, cryptic Tudor domain and WD40 domains and Cullin 4-RING E3 ubiquitin ligase complex (CRL4) recruited to chromatin mediated by RepID have potential roles in gene expression changes via epigenetic regulations. We aimed to investigate whether RepID-CRL4 participates in transcriptional changes required for MK differentiation.The PCR array was performed using cDNAs derived from RepID-proficient or RepID-deficient K562 erythroleukemia cell lines. Correlation between RepID and DAB2 expression was examined in the Cancer Cell Line Encyclopedia (CCLE) through the CellMinerCDB portal. The acceleration of MK differentiation in RepID-deficient K562 cells was determined by estimating cell sizes as well as counting multinucleated cells known as MK phenotypes, and by qRT-PCR analysis to validate transcripts of MK markers using phorbol 12-myristate 13-acetate (PMA)-mediated MK differentiation condition. Interaction between CRL4 and histone methylation modifying enzymes were investigated using BioGRID database, immunoprecipitation and proximity ligation assay. Alterations of expression and chromatin binding affinities of RepID, CRL4 and histone methylation modifying enzymes were investigated using subcellular fractionation followed by immunoblotting. RepID-CRL4-JARID1A-based epigenetic changes on DAB2 promoter were analyzed by chromatin-immunoprecipitation and qPCR analysis.RepID-deficient K562 cells highly expressing MK markers showed accelerated MKs differentiation exhibiting increases in cell size, lobulated nuclei together with reaching maximum levels of MK marker expression earlier than RepID-proficient K562 cells. Recovery of WD40 domain-containing RepID constructs in RepID-deficient background repressed DAB2 expression. CRL4A formed complex with histone H3K4 demethylase JARID1A in soluble nucleus and loaded to the DAB2 promoter in a RepID-dependent manner during proliferation condition. RepID, CRL4A, and JARID1A were dissociated from the chromatin during MK differentiation, leading to euchromatinization of the DAB2 promoter.This study uncovered a role for the RepID-CRL4A-JARID1A pathway in the regulation of gene expression for MK differentiation, which can form the basis for the new therapeutic approaches to induce platelet production. Video Abstract.© 2023. BioMed Central Ltd., part of Springer Nature.