Elucidation of the Role of Setd8 and H4K20me1 in Erythropoiesis

染色质凝聚是许多造血细胞最终成熟的关键特征，但 在红细胞中尤其关键，它在准备过程中经历快速的核和染色质凝聚 做眼球摘除手术。人类平均每秒必须产生约250万个红细胞才能保持稳定 状态和避免贫血。从已确定的红血球生成去核红细胞的过程 祖细胞是极其复杂的，在细胞快速分裂期间需要显著的基因表达。 和染色质凝聚。染色质凝集缺陷在遗传性贫血中尤其常见。 和骨髓增生异常综合征，然而调节红系染色质的特定表观遗传机制 凝集，染色质凝集与基因表达的关系较差 明白了。这个项目的目标是描绘特定的表观遗传机制，以调节和协调， 红系终末成熟过程中基因表达和染色质结构的变化。在上一次资助期间 期间，我们证明了成熟的红系细胞积累组蛋白H4、赖氨酸20的单甲基化 (H4K20me1)。我们进一步证明，Setd8，唯一能够产生这种标记的酶，对于 红血球生成。Setd8的中断，以及随后H4K20me1的丢失，导致了 全球和特定位点的染色质缩合中的转录抑制和缺陷。整体而言 这项提案的目标是描述Setd8和H4K20me1调节红细胞生成的机制。 质谱学研究表明，H4K20me1在成熟红细胞中的积累是 伴随着组蛋白H4 K16Ac的丢失，这干扰了邻近核小体的结合 并促进组蛋白的解压，以及组蛋白H4赖氨酸20二甲基化的丢失(H4K20me2)，这是 降低组蛋白H4赖氨酸20与表观遗传阅读器相互作用的能力。我们假设这是 表观遗传转换是基因表达和高阶染色质结构的重要决定因素 成熟的红系细胞。在具体目标1中，我们将确定建立H4染色质的机制 成熟的红细胞中的景观。H4K20me1通过调节染色质结构和基因表达 招募表观遗传学读者。唯一与H4K20me1相互作用的蛋白在 红细胞是凝集素II复合体。我们证明了凝聚素II亚单位NCAPH2是必不可少的 用于红系造血。此外，NCAPH2/和SETD8/的转录本存在显著重叠 红细胞，支持凝聚素II与H4K20me1相互作用调节基因表达的模型 红系成熟过程中的染色质结构。在具体目标2中，我们将描述通过哪些机制 凝集素II复合体调节红系终末成熟。总而言之，这些研究将提供新的 对红系终末成熟调控的洞察。因为染色质凝聚是 由于许多细胞类型的终末成熟，这些研究可能具有超越红细胞生成的广泛意义。

Chromatin condensation is a key feature in the terminal maturation of many hematopoietic cell types, but is particularly critical in erythroblasts, which undergo rapid nuclear and chromatin condensation in preparation for enucleation. The average human must generate ~2.5 million red blood cells per second to maintain steady state and avoid anemia. The process of generating an enucleate red blood cell from a committed erythroid progenitor is extremely complex, and requires significant in gene expression during a time of rapid cell division and chromatin condensation. Defects in chromatin condensation are particularly common in inherited anemias and myelodysplastic syndromes, however the specific epigenetic mechanisms that regulate erythroid chromatin condensation, and the relationship between chromatin condensation and gene expression are poorly understood. The goal of this project is to delineate specific epigenetic mechanisms that regulate, and coordinate, changes in gene expression and chromatin structure during terminal erythroid maturation. During the last funding period, we demonstrated that maturing erythroid cells accumulate mono-methylation of histone H4, lysine 20 (H4K20me1). We further demonstrated Setd8, the sole enzyme capable of generating this mark, is essential for erythropoiesis. Disruption of Setd8, and subsequent loss of H4K20me1, resulted in a profound defect in transcriptional repression and defects in chromatin condensation, both globally and at specific loci. The overall goal of this proposal is to delineate mechanisms by which Setd8 and H4K20me1 regulate erythropoiesis. Mass spectrometry studies demonstrated that accumulation of H4K20me1 in maturing erythroblasts was accompanied by loss of histone H4 K16Ac, which interferes with the association of neighboring nucleosomes and promotes histone decompaction, as well as loss of histone H4 lysine 20 dimethylation (H4K20me2), which decreases the ability of histone H4 lysine 20 to interact with epigenetic readers. We hypothesize that this epigenetic transition is an important determinate of gene expression and higher order chromatin structure in maturing erythroblasts. In specific Aim 1, we will determine the mechanisms that establish the H4 chromatin landscape in maturing erythroblasts. H4K20me1 regulates chromatin structure and gene expression through recruitment of epigenetic readers. The only H4K20me1 interacting protein expressed at significant levels in erythroblasts is the Condensin II complex. We demonstrate that the Condensin II subunit NCAPH2 is essential for erythropoiesis. Further, there is significant overlap in the transcriptomes of NCAPH2 / and SETD8 / erythroblasts, supporting a model where Condensin II interacts with H4K20me1 to regulate gene expression and chromatin structure during erythroid maturation. In specific aim 2, we will delineate the mechanisms by which the Condensin II complex regulates terminal erythroid maturation. Together, these studies will provide novel insights into the regulation of terminal erythroid maturation. As chromatin condensation is a common feature of the terminal maturation of many cell types, these studies may have broad implications beyond erythropoiesis.