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

Abstract

Chromatin structure has emerged as a key contributor to spatial and temporal control over the initiation of DNA replication. However, despite genome-wide correlations between early replication of gene-rich, accessible euchromatin and late replication of gene-poor, inaccessible heterochromatin, a causal relationship between chromatin structure and replication initiation remains elusive. Here, we combined histone gene engineering and whole-genome sequencing in Drosophila to determine how perturbing chromatin structure affects replication initiation. We found that most pericentric heterochromatin remains late replicating in H3K9R mutants, even though H3K9R pericentric heterochromatin is depleted of HP1a, more accessible, and transcriptionally active. These data indicate that HP1a loss, increased chromatin accessibility, and elevated transcription do not result in early replication of heterochromatin. Nevertheless, a small amount of pericentric heterochromatin with increased accessibility replicates earlier in H3K9R mutants. Transcription is de-repressed in these regions of advanced replication but not in those regions of the H3K9R mutant genome that replicate later, suggesting that transcriptional repression may contribute to late replication. We also explored relationships among chromatin, transcription, and replication in euchromatin by analyzing H4K16R mutants. In Drosophila, the X Chromosome gene expression is up-regulated twofold and replicates earlier in XY males than it does in XX females. We found that H4K16R mutation prevents normal male development and abrogates hyperexpression and earlier replication of the male X, consistent with previously established genome-wide correlations between transcription and early replication. In contrast, H4K16R females are viable and fertile, indicating that H4K16 modification is dispensable for genome replication and gene expression.

Rif1 functions in a tissue-specific manner to control replication timing through its PP1-binding motif

“…Cells of the wing disc are derived from the embryonic mesoderm while ovarian follicle cells are derived from the embryonic ectoderm. To generate RT profiles, we used fluorescence-activated cell sorting (FACS) to isolate and subsequently sequence the genomes of S phase nuclei from each tissue and compared these data to those obtained from G1 phase nuclei from wing discs (Figure 1A; (Armstrong et al 2018)). The premise of this method is that early-replicating DNA sequences are over-represented relative to late-replicating sequences within the S phase population.…”

“…To determine how lineage contributes to RT, we generated RT values at 100kb windows tiled at 10kb intervals across the genome for both wing discs and follicle cells and used a stringent significance threshold to identify differential RT between each tissue (Materials and Methods; (Armstrong et al 2018)). RT profiles generated from individual replicates of wildtype wing discs and follicle cells were strongly correlated (Pearson’s correlations = 0.95 and 0.95, respectively; Figure S1A), whereas RT values between the two lineages were significantly more divergent (Pearson’s correlation = 0.39; Figure 1B).…”

“…We found that ~30% of the genome had different RT in the two tissue types we examined, and that transcriptional changes do not account for these changes. Studies in other systems also have failed to establish a direct relationship between changes in RT and changes in transcriptional activity (MacAlpine et al 2004; Lubelsky et al 2014; Siefert et al 2017; Almeida et al 2018; Armstrong et al 2018). While transcriptional activity has long been correlated with RT, there are clearly mechanisms that control RT independently of transcription.…”

New, easy, quick and efficient DNA replication timing analysis by high-throughput approaches

“…We wanted to know if START-R suite can run the correct analyses with this type of data and also with other organisms than mouse and human. We performed exactly the same pipeline used for early-late Repli-seq data described above for Drosophila, zebrafish and human S/G1 data (Armstrong et al, 2018;Siefert et al, 2017;Massey et al, 2019), in order to be sure that the integration into the START-R pipeline was correct. Then and as expected,…”

“…Different laboratories analyze variations of DNA copy number between G1 and S phase cells (S/G1 ratio) to study the replication timing program with Repli-seq. We used data obtained from different organisms such as Drosophila, zebrafish and human (Armstrong et al, 2018;Siefert et al, 2017;Massey et al, 2019), to validate the START-R suite (GEO accession numbers: GSM3154888 and GSM3154890 for HWT Drosophila melanogaster female larvae wing disc cells in S and G1 phase, respectively; GSM2282090 for 28hpf Danio rerio embryos; SRX3413939-40 for HEK293T human cells in S and G1 phase, respectively). As previously, reads from G1 and S fractions are mapped with Bowtie2, then PCR duplicates are removed by RmDUP tool, and Bamcoverage is used to obtain the coverage with RPKM.…”