H4K16ac is dispensable for mammalian transcriptional control but necessary for a faithful genome duplication program [ChIP-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: To unveil H4K16ac genome-wide deposition, we deployed ChIPseq in HME1 cells: pull down was performed with two antibodies (two replicates each) in WT cells (Control KO) and cells lacking the histone mark (MSL3 KO cells). ChIPseq in KO cells retrieves the non-specific signal and can be subtracted to obtain the real H4K16ac occupancy in the genome. As a control for gene-assiciated signal, H3K36me3 ChIPseq was performed in HME1 cells