Chromatin conformation and transcriptional activity are permissive regulators of DNA replication initiation in Drosophila

We replaced the endogenous histones of Drosophila melanogaster with either histones containing an H3K9R mutation or histones containing an H4K16R mutation to interrogate established genome-wide correlations between chromatin state, transcription, and DNA replication timing. We performed total RNA-seq in H4K16R males and females to investigate the role of H4K16 in dosage compensation of the male X chromosome. We found that H4K16 directly promotes hyper-expression of the male X chromosome in Drosophila. To generate replication timing profiles, we performed Repli-seq in HWT males and females, H4K16R males and females, and H3K9R females. We found that H3K9 promotes late replication of the pericentromeric heterochromatin and H4K16 promotes early replication of the male X chromosome. Experiments were performed on endogenous histone mutants rescued with a wild type (HWT), H4K16R mutant, or H3K9R mutant transgenic arrays. RNA-seq experiments were carried out in triplicate and Repli-seq experiments were carried out in duplicate. RNA-seq and Repli-seq were performed in wing imaginal discs from 3rd instar Drosophila larvae. S phase and G1 phase samples for Repli-seq were obtained using FACS on nuclei isolated from wing imaginal discs and replication timing values were calculated as S/G1 ratio.