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. 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.