Phosphorylation of the ancestral histone variant H3.3 amplifies stimulation-induced transcription

Complex organisms are able to rapidly induce select genes among thousands in response to diverse environmental cues. This occurs in the context of large genomes condensed with histone proteins into chromatin. The macrophage response to pathogen sensing, for example, rapidly engages highly conserved signaling pathways and transcription factors (TF) for coordination of inflammatory gene induction. Enriched integration of histone H3.3, the ancestral histone H3 variant, is a feature of inflammatory genes and, in general, dynamically regulated chromatin and transcription. However, little is known of how chromatin is regulated at rapidly induced genes and what features of H3.3, conserved from yeast to human, might enable rapid and high-level transcription. The amino-terminus of H3.3 contains a unique serine residue as compared with alanine residues found in “canonical” H3.1/2. We find that this H3.3-specific serine residue, H3.3S31, is phosphorylated (H3.3S31ph) in a stimulation-dependent manner along the gene-bodies of rapidly induced response genes in mouse macrophages responding to pathogen sensing. Further, this selective mark of stimulation-responsive genes directly engages histone methyltransferase (HMT) Setd2, a component of the active transcription machinery. Our structure-function studies reveal that a conserved positively charged cleft in Setd2 contacts H3.3S31ph and specifies preferential methylation of H3.3S31ph nucleosomes. We propose that features of H3.3, including H3.3S31ph, engage delegated mechanisms to afford selective and rapid transcription. Overall design: Primary mouse bone marrow derived macrophages (BMDM) were used for NGS experiments. Briefly, BMDM were used unstimulated or following stimulation with S.typhosa LPS (100ng/mL) for the indicated period of time. Included in this submission are RNA-seq data for control DMSO treated BMDM in triplicate (60' and 120' LPS stimulation) as well as wild type, hypomorphic macrophage-like RAW264.7 cell lines, and CRISPR-generated H3f3a/H3f3b double knockout lines in duplicate (60' and 120' LPS stimulation). Included is also ChIP-seq data for time course in LPS stimulated BMDM (0', 60', 120') for H3S31ph, H3S28ph, H3K36me3, H3K36me2, H3K27ac, and H3.3.

复杂生物可响应多样环境信号，快速诱导数千个基因中的特定基因表达。该过程发生在由组蛋白组装形成染色质的大型基因组环境中。例如，巨噬细胞对病原体感知的应答，会快速激活高度保守的信号通路与转录因子（Transcription Factor, TF），以协调炎症基因的诱导表达。组蛋白H3.3——祖先型组蛋白H3变体——的富集整合是炎症基因的特征，通常也与动态调控的染色质及转录过程相关。然而，目前对于快速诱导基因处的染色质调控机制，以及从酵母到人类均保守的H3.3具有哪些特征可支持快速高水平转录，仍知之甚少。
相较于“经典”H3.1/2中的丙氨酸残基，H3.3的氨基末端拥有一个独特的丝氨酸残基。我们发现，这一H3.3特异性丝氨酸残基H3.3S31会在小鼠巨噬细胞响应病原体感知的快速诱导应答基因的基因体区域，以刺激依赖的方式被磷酸化（形成H3.3S31ph）。进一步研究表明，这种应答刺激的选择性修饰可直接结合组蛋白甲基转移酶（Histone Methyltransferase, HMT）Setd2——一种活跃转录机器的组成成分。我们的结构-功能研究揭示，Setd2中一个保守的带正电荷裂隙可与H3.3S31ph结合，并特异性偏好甲基化H3.3S31ph修饰的核小体。我们提出，包括H3.3S31ph在内的H3.3相关特征，可通过委派的机制实现选择性快速转录。
整体实验设计：本研究采用原代小鼠骨髓来源巨噬细胞（Bone Marrow Derived Macrophage, BMDM）进行下一代测序（Next Generation Sequencing, NGS）实验。简言之，将BMDM分为未刺激组，以及以S.typhosa脂多糖（Lipopolysaccharide, LPS，100ng/mL）刺激指定时长的组别。本数据集包含以下数据：经二甲基亚砜（DMSO）对照处理的BMDM的RNA测序（RNA-seq）数据（生物学重复3次，对应60分钟、120分钟LPS刺激组）；野生型、功能减退型巨噬细胞样RAW264.7细胞系，以及CRISPR编辑获得的H3f3a/H3f3b双敲除细胞系的RNA-seq数据（生物学重复2次，对应60分钟、120分钟LPS刺激组）。此外还包含LPS刺激的BMDM的时间进程染色质免疫沉淀测序（ChIP-seq）数据，对应靶点包括H3S31ph、H3S28ph、H3K36me3、H3K36me2、H3K27ac及H3.3，采样时间点为0分钟、60分钟、120分钟。