H1 linker histones regulate the balance of repressive and active chromatin domains via localized genomic compaction [CUT&Tag]

H1 linker histones are the most abundant chromatin binding proteins. Their association with chromatin determines the spacing between nucleosomes and enables arrays of nucleosomes to fold into more compact chromatin structures. Mammals express multiple H1 proteins and are able to compensate for the loss of one or even two members by increasing synthesis of other members to maintain a constant H1 to nucleosome stoichiometry. To study the role of H1 in mammalian development, we generated a conditional triple H1 knockout (H1cTKO) mouse strain that enables depletion of H1 in specific cell types. Here, we report on the effects of depleting H1 in adult hematopoietic cells. Deletion of the genes encoding three widely expressed H1 subtypes (H1c, H1d, and H1e) has particularly profound effects on B- and T- lymphocyte development. H1 depletion leads to de-repression of T-cell activation genes, and a shift in T-cells towards effector functions, a process that mimics normal T-cell activation. Comparison of chromatin structure in normal and H1-depleted CD8+ T-cells revealed that H1 binding produces localized chromatin compaction within spatially defined chromatin domains containing above average levels of H1. Reduction of H1 stoichiometry in these regions leads to decreases in H3K27 methylation and increases in H3K36 methylation. In vitro, H1 promotes PRC2-mediated H3K27 methylation and inhibits NSD2-mediated H3K36 methylation. Mechanistically, H1 mediates these opposite effects by promoting physical compaction of the chromatin substrate. These findings identify H1 as a critical regulator of the epigenetic landscape in mammalian cells. Overall design: CD8+ splenic T cells were isolated from adult C57BL/6 WT or H1 cTKO mice. CD8+ splenic T cells were stimulated ex vivo for 3 days using IL2 and CD3e/CD28 co-stimulatory beads. CUT&Tag was performed with the indicated antibody. 2 independent replicates were used for IGG and 4 independent replicates were used for H1 and H3 antibodies. H1 and H3 CUT&Tag was performed before and after ex vivo stimulation.

H1连接组蛋白（H1 linker histones）是丰度最高的染色质结合蛋白。其与染色质的结合决定了核小体之间的间距，并可使核小体阵列折叠为更为紧凑的染色质结构。哺乳动物可表达多种H1蛋白，能够通过上调其他成员的合成以补偿单个乃至两个H1亚型的缺失，从而维持稳定的H1与核小体化学计量比。
为研究H1在哺乳动物发育中的功能，我们构建了条件性三重H1敲除（conditional triple H1 knockout, H1cTKO）小鼠品系，该品系可实现特定细胞类型中H1的耗竭。本研究报道了成年造血细胞内H1耗竭所产生的效应。敲除三种广泛表达的H1亚型（H1c、H1d与H1e）的编码基因，对B淋巴细胞与T淋巴细胞的发育具有极为显著的影响。
H1耗竭会导致T细胞激活基因的去阻抑，并使T细胞向效应功能表型偏移，这一过程模拟了正常的T细胞激活事件。通过对比正常与H1耗竭的CD8+ T细胞的染色质结构，我们发现H1结合可在空间限定的染色质结构域内诱导局部染色质压缩，此类结构域中的H1水平高于整体平均水平。在这些区域中，H1化学计量比的降低会引发H3K27甲基化水平下降，同时伴随H3K36甲基化水平上升。
体外实验证实，H1可促进多梳抑制复合体2（PRC2）介导的H3K27甲基化，同时抑制核受体结合SET结构域蛋白2（NSD2）介导的H3K36甲基化。从分子机制来看，H1通过促进染色质底物的物理压缩来介导这两种相反的调控效应。上述研究结果确认H1是哺乳动物细胞表观遗传景观的关键调控因子。
整体实验设计：从成年C57BL/6野生型（WT）或H1cTKO小鼠体内分离CD8+脾脏T细胞；将分离得到的CD8+脾脏T细胞在体外经白细胞介素2（IL2）与CD3e/CD28共刺激磁珠刺激3天；使用指定抗体开展CUT&Tag（染色质靶向切割与标签测序）实验。IgG对照组设置2个独立生物学重复，针对H1与H3抗体的实验则设置4个独立生物学重复；分别在体外刺激前后完成H1与H3的CUT&Tag实验。