H4K16ac is dispensable for mammalian transcriptional control but necessary for a faithful genome duplication program [RNA-seq]

Histone acetylation has widely been assumed to directly instruct gene activation. Among acetylated residues, H4K16ac is one of the most abundant modifications, conserved across all eukaryotes. Despite its established role in X-chromosome hyperactivation in Drosophila, its function in mammalian cells has remained elusive. Here, we show that in human somatic cells, H4K16ac does not regulate gene expression, but instead controls the spatiotemporal program of genome duplication. By combining a meta-analysis of public datasets and perturbation experiments free of confounding effects, we found that H4K16ac is neither associated with nor required for transcriptional activity. Rather, H4K16ac depletion resulted in premature replication of heterochromatic regions and widespread alterations in replication timing across the genome. These defects were driven by the aberrant activation of cryptic replication origins at long terminal repeats (LTRs)—repetitive elements typically marked by H4K16ac and whose sequence context resembles that of canonical origins in euchromatic regions. Our findings reveal an unexpected role for one of the most prevalent chromatin modifications and uncover a new regulatory mechanism that ensures accurate genome duplication. Overall design: For the study of H4K16ac impact on specific gene programs, the 3 complex-specific subunits of MSL complex were independently deleted using synthetic sgRNAs. The not-expressed TNP2 gene was used as negative control. Three days after transfection, cells were collected, and RNA was extracted using a RNeasy Plus Micro Kit (Qiagen, 74034). 3 replicates per condition were generated. For the study of H4K16ac impact on global levels of transcription, stable KO cells (either MSL3 or not expressed control TNP2) were used. cells were grown for 2 days in two identical replicate plates. Cells in one plate were fixed with 4% paraformaldehyde (PFA, Alfa Aesar, #43368) followed by permeabilization with 0.5% Triton X-100 in PBS and nuclei staining with SYTOX Green Nucleic Acid Stain (Thermo Fisher Scientific, #S7020). Imaging and quantification were performed using an Incucyte S3 Live-Cell Analysis System. Cell counts generated were then used to take the same number of cells (400.000) in all conditions in the other plate. Total RNA extraction was performed using a RNeasy Plus Micro kit (Qiagen, 74034). RNA concentration was checked with the nanodrop. 1ug of RNA was taken from the lowest concentrated sample and same volume was taken for all the other samples. 2 µL (1:100) ERCC Spike-in (Thermo Fisher Scientific, 4456740) was added to each sample. Two replicates per condition were generated.

组蛋白乙酰化（Histone acetylation）长期以来被广泛认为可直接指导基因激活。在乙酰化残基中，H4K16ac是丰度最高的修饰之一，且在所有真核生物中均保守存在。尽管已明确其在果蝇X染色体超激活中的功能，但其在哺乳动物细胞中的作用仍未明确。本研究显示，在人类体细胞中，H4K16ac并不调控基因表达，而是控制基因组复制的时空程序。本研究通过整合公共数据集的荟萃分析（meta-analysis）并开展无混杂效应的扰动实验，发现H4K16ac既不与转录活性相关，也并非转录活性所必需。相反，H4K16ac的缺失会导致异染色质区域复制提前，并引发全基因组范围内复制时序的广泛改变。上述缺陷源于长末端重复序列（long terminal repeats, LTRs）上隐蔽复制起始位点的异常激活：这类重复元件通常带有H4K16ac标记，其序列环境与常染色质区域的经典复制起始位点相似。本研究揭示了这一最普遍的染色质修饰之一的意外功能，并阐明了一种确保基因组精确复制的新型调控机制。

实验总体设计：
为研究H4K16ac对特定基因程序的影响，本研究利用合成的单向导RNA（single-guide RNA, sgRNA）分别敲除MSL复合物的3个特异性亚基。以不表达的TNP2基因作为阴性对照。转染3天后收集细胞，使用RNeasy Plus Micro试剂盒（Qiagen，74034）提取RNA，每个实验组设置3个生物学重复。
为研究H4K16ac对全局转录水平的影响，本研究使用稳定敲除细胞（分别敲除MSL3或作为对照的不表达基因TNP2）。将细胞接种于两块完全相同的培养板中培养2天。其中一块培养板的细胞用4%多聚甲醛（paraformaldehyde, PFA，Alfa Aesar，#43368）固定，随后用含0.5% Triton X-100的磷酸盐缓冲液（phosphate buffered saline, PBS）透化，再用SYTOX Green核酸染料（Thermo Fisher Scientific，#S7020）对细胞核进行染色。使用Incucyte S3活细胞分析系统完成成像与定量分析。根据得到的细胞计数，从另一块培养板的所有实验组中取等量细胞（400,000个）。使用RNeasy Plus Micro试剂盒（Qiagen，74034）提取总RNA，采用纳米紫外分光光度计检测RNA浓度。从浓度最低的样本中取1 μg RNA，其余样本均取相同体积。向每个样本中加入2 μL（1:100稀释）的ERCC外源转录本对照（ERCC Spike-in，Thermo Fisher Scientific，4456740），每个实验组设置2个生物学重复。