Recycling of H2A-H2B provides short-term memory of chromatin states [ChOR-Seq]. Recycling of H2A-H2B provides short-term memory of chromatin states [ChOR-Seq]

Chromatin landscapes are disrupted during DNA replication and must be restored faithfully to maintain genome regulation and cell identity. The H3-H4 modification landscape is restored by parental histone recycling and post-replication modification of new histone H3-H4. How DNA replication impact on histone H2A-H2B is unknown. Here, we track H2A-H2B modifications and H2A.Z during DNA replication and across the cell cycle using quantitative genomics. We show that H2AK119ub, H2BK120ub, and H2A.Z are recycled quantitatively and accurately during DNA replication. H2A-H2B are recycled symmetrically to daughter strands largely independent of known H3-H4 recycling pathways. Post-replication, H2A-H2B modifications are rapidly restored, and the rapid wave of H2AK119ub supports accurate restoration of H3K27me3. This work reveals epigenetic transmission of H2A-H2B modification during DNA replication and identifies H3-H4 and H2A-H2B crosstalk in epigenome propagation. We propose that rapid short-term memory of recycled H2A-H2B modifications facilitates reestablishment of slow, long-term chromatin state memory. Overall design: ChIP-Seq and ChOR-seq measuring chromatin occupancy for H3K27me3, H2AK119ub, H2A.Z, H2BK120ub, pan-H2A, pan-H3 in wildtype (WT) or Ring1B-mAID BAP1dTAG Ring1AKO mouse embryonic stem cells with corresponding clickedInputs controls in two biological replicates. CHOR-seq involves a 10min EdU labelling step, followed by a chase in medium without EdU (T15-T720; in min) or immediate harvesting (T0). Optional treatments are indicated. ClickedInputs are the corresponding inputs from the ChOR-seq time course where the EdU-labelled fraction has been clicked to Biotin, Streptavidin-purified and libraries generated.

DNA复制过程中，染色质景观（chromatin landscapes）会遭到破坏，必须被精准恢复以维持基因组调控与细胞身份。组蛋白H3-H4（H3-H4）的修饰景观可通过亲本组蛋白回收与新合成组蛋白H3-H4的复制后修饰得以恢复。目前，DNA复制对组蛋白H2A-H2B的影响尚不明确。

本研究借助定量基因组学技术，追踪了DNA复制期间及全细胞周期中H2A-H2B修饰与组蛋白H2A.Z（H2A.Z）的动态变化。研究发现，组蛋白H2A赖氨酸119泛素化（H2AK119ub）、组蛋白H2B赖氨酸120泛素化（H2BK120ub）及H2A.Z在DNA复制过程中可被精准且定量地回收。H2A-H2B会对称地被转运至子代DNA链，且该过程在很大程度上不依赖于已知的H3-H4回收通路。复制完成后，H2A-H2B修饰会被快速恢复，而H2AK119ub的快速重塑有助于组蛋白H3赖氨酸27三甲基化（H3K27me3）的精准恢复。

本研究揭示了DNA复制过程中H2A-H2B修饰的表观遗传传递机制，并阐明了表观基因组传播过程中H3-H4与H2A-H2B的串扰调控。我们提出，回收的H2A-H2B修饰所形成的快速短期记忆，有助于重建缓慢形成的长期染色质状态记忆。

实验整体设计：本研究采用染色质免疫共沉淀测序（ChIP-Seq）与ChOR-seq技术，在野生型（WT）或Ring1B-mAID BAP1dTAG Ring1AKO小鼠胚胎干细胞中，检测H3K27me3、H2AK119ub、H2A.Z、H2BK120ub、泛H2A（pan-H2A）、泛H3（pan-H3）的染色质结合水平，并设置对应的clickedInputs对照，共设置两个生物学重复。

ChOR-seq实验流程包含10分钟的5-乙炔基-2'-脱氧尿苷（EdU）标记步骤，随后在不含EdU的培养基中进行追踪培养（时间范围T15-T720，单位：分钟），或直接收获细胞（T0）；实验可设置可选处理。clickedInputs样本来源于ChOR-seq时间序列实验中的对应输入样本：先将EdU标记的DNA片段与生物素进行点击化学反应，再通过链霉亲和素纯化并构建测序文库。