H3K9 Promotes Under-Replication of Pericentromeric Heterochromatin in Drosophila Salivary Gland Polytene Chromosomes

Abstract

Chromatin structure and its organization contributes to the proper regulation and timing of DNA replication. Yet, the precise mechanism by which chromatin contributes to DNA replication remains incompletely understood. This is particularly true for cell types that rely on polyploidization as a developmental strategy for growth and high biosynthetic capacity. During Drosophila larval development, cells of the salivary gland undergo endoreplication, repetitive rounds of DNA synthesis without intervening cell division, resulting in ploidy values of ~1350C. S phase of these endocycles displays a reproducible pattern of early and late replicating regions of the genome resulting from the activity of the same replication initiation factors that are used in diploid cells. However, unlike diploid cells, the latest replicating regions of polyploid salivary gland genomes, composed primarily of pericentric heterochromatic enriched in H3K9 methylation, are not replicated each endocycle, resulting in under-replicated domains with reduced ploidy. Here, we employ a histone gene replacement strategy in Drosophila to demonstrate that mutation of a histone residue important for heterochromatin organization and function (H3K9) but not mutation of a histone residue important for euchromatin function (H4K16), disrupts proper endoreplication in Drosophila salivary gland polyploid genomes thereby leading to DNA copy gain in pericentric heterochromatin. These findings reveal that H3K9 is necessary for normal levels of under-replication of pericentric heterochromatin and suggest that under-replication at pericentric heterochromatin is mediated through H3K9 methylation.

Replication timing analysis in polyploid cells reveals Rif1 uses multiple mechanisms to promote underreplication in Drosophila

“…SUUR still localizes to these PH regions, but is unable to inhibit fork progression and promote UR because Rif1 is not present and SUUR is unable to inhibit fork progression in the absence of Rif1 (Munden et al, 2018). This model is in agreement with multiple observations indicating that SUUR binding to euchromatic and pericentric heterochromatin is different and that the genetic requirements for UR in euchromatin and pericentric heterochromatin are unique (Armstrong et al, 2019;Guarner et al, 2017).…”

Effects of Mutations in the Drosophila melanogaster Rif1 Gene on the Replication and Underreplication of Pericentromeric Heterochromatin in Salivary Gland Polytene Chromosomes

“…The intercalary heterochromatin regions correspond to extended domains of tissue-specific genes, that is, they are represented by unique rather than repeated sequences [ 14 , 26 ]. This finding made it possible to obtain detailed underreplication profiles of these areas [ 6 , 26 , 27 , 32 , 33 ]. The presence of intercalary heterochromatin regions leads to approximately ≤19% DNA underrepresentation in euchromatic polytene chromosome arms [ 6 ].…”

“…When histone H1 is knocked down, underreplication suppression is also observed in some pericentromeric heterochromatin areas [ 32 ]. H3K9 di-/tri-methylation and histone methyltransferase Su(VAR)3-9 affect underreplication of some heterochromatin sequences [ 33 , 60 , 62 ]. None of these factors has a significant effect on satellite DNA polytenization.…”

Flying High—Muscle-Specific Underreplication in Drosophila

“…Typically found in specialty tissues of Diptera, such as the well-studied polyploid salivary gland and ovarian follicle cells, UR results from a coupling of protein products from Rif1 and SuUR hindering progression of the DNA replication fork [3,4]. This stall usually occurs upon encountering heterochromatic DNA, which is tightly wound into nucleosomes, preventing this heterochromatic DNA from fully replicating [5,6]. While recent work has investigated the machinery involved in this stalling of replication, the mechanisms behind this process are not fully understood [2,7,8].…”