The histone methyltransferase Setd8 alters the chromatin landscape and regulates the expression of key transcription factorsduring erythroid differentiation. The histone methyltransferase Setd8 alters the chromatin landscape and regulates the expression of key transcription factorsduring erythroid differentiation

SETD8 is the sole methyltransferase capable of mono-methylating histone H4, lysine 20. SETD8 is highly expressed in erythroid cells and erythroid deletion of Setd8 is embryonic lethal by embryonic day 11.5 (E11.5) due to profound anemia, suggesting it has an erythroid-specific function. To gain insights into the function of SETD8 during erythroid differentiation, we performed ATAC-seq on sorted populations of E10.5 Setd8 null and control erythroblasts. Accessibility profiles were integrated with expression changes and a mark of heterochromatin (H3K27me3) performed in wild type E10.5 erythroblasts to further understand the role of SETD8 in erythropoiesis. Data integration identified regions of greater chromatin accessibility in Setd8 null cells that co-located with H3K27me3 in wild type E10.5 erythroblasts suggesting that these regions, and their associated genes, are repressed during normal erythropoiesis. Pathway analysis of genes identified through data integration revealed stemness-related pathways. Among those genes were multiple transcriptional regulators active in multipotent progenitors but repressed during erythroid differentiation including Hhex, Hlx, and Gata2. Consistent with a role for SETD8 in erythroid specification, SETD8 expression is upregulated upon erythroid commitment, and Setd8 disruption impairs erythroid colony forming ability. Taken together, our results suggest that Setd8 is important for the establishment of appropriate patterns of gene expression during erythroid differentiation. Overall design: Biological replicates were generated for setd8-null(2) and setd8-control(3) embryos. Biological replicates were aggregated, sequencing depth normalized, and a continuous log2 ratio was computed to identify regions of differential accessiblity in the setd8-null compared to control.

SETD8是唯一可对组蛋白H4的赖氨酸20位点进行单甲基化的甲基转移酶。SETD8在红系细胞中高表达，红系特异性敲除Setd8会导致胚胎于胚胎第11.5天（E11.5）因重度贫血发生胚胎致死，提示其具有红系特异性功能。为探究SETD8在红系分化过程中的功能，本研究对分选获得的E10.5期Setd8敲除型与对照型成红细胞开展了转座酶可及性测序（ATAC-seq）。将染色质可及性图谱与基因表达变化，以及在野生型E10.5成红细胞中检测的异染色质标记H3K27me3进行整合分析，以进一步阐明SETD8在红细胞生成中的作用。通过数据整合，本研究鉴定出Setd8敲除细胞中染色质可及性更高的区域，这些区域在野生型E10.5成红细胞中与H3K27me3共定位，提示此类区域及其关联基因在正常红细胞生成过程中处于沉默抑制状态。对数据整合得到的基因进行通路分析后发现，其富集于干性相关通路。其中包含多个在多能祖细胞中活跃、但在红系分化过程中被抑制的转录调控因子，包括Hhex、Hlx与Gata2。与SETD8在红系定向分化中的功能相符，SETD8的表达在红系定向发生时会上调，而Setd8敲除会损害红系集落形成能力。综上，本研究结果表明Setd8对于红系分化过程中恰当基因表达模式的建立具有重要作用。实验整体设计：本研究为Setd8敲除组（2个生物学重复）与对照组（3个生物学重复）的胚胎设置了生物学重复样本。将生物学重复样本合并后进行测序深度标准化，并计算连续型log₂比值，以鉴定Setd8敲除组相较于对照组的差异染色质可及性区域。