High-resolution analysis of Drosophila heterochromatin organization using SuUR Su(var)3-9 double mutants

Abstract

The structural and functional analyses of heterochromatin are essential to understanding how heterochromatic genes are regulated and how centromeric chromatin is formed. Because the repetitive nature of heterochromatin hampers its genome analysis, new approaches need to be developed. Here, we describe how, in double mutants for Su(var)3-9 and SuUR genes encoding two structural proteins of heterochromatin, new banded heterochromatic segments appear in all polytene chromosomes due to the strong suppression of under-replication in pericentric regions. FISH on salivary gland polytene chromosomes from these double mutant larvae allows high resolution of heterochromatin mapping. In addition, immunostaining experiments with a set of antibodies against euchromatic and heterochromatic proteins reveal their unusual combinations in the newly appeared segments: binding patterns for HP1 and HP2 are coincident, but both are distinct from H3diMetK9 and H4triMetK20. In several regions, partial overlapping staining is observed for the proteins characteristic of active chromatin RNA Pol II, H3triMetK4, Z4, and JIL1, the boundary protein BEAF, and the heterochromatin-enriched proteins HP1, HP2, and SU(VAR)3-7. The exact cytological position of the centromere of chromosome 3 was visualized on salivary gland polytene chromosomes by using the centromeric dodeca satellite and the centromeric protein CID. This region is enriched in H3diMetK9 and H4triMetK20 but is devoid of other proteins analyzed.Drosophila melanogaster ͉ genome analysis

Regulatory functions and chromatin loading dynamics of linker histone H1 during endoreplication in Drosophila

“…Our results also implicate UR of TE sequences in polytenized cells as yet another putative mechanism that contributes to regulation of their expression. Interestingly, it was shown previously that double mutants encompassing both the Su(var)3-9 and SuUR mutant alleles exhibit a synthetically increased predominance of novel bandinterband structures at PH compared with the mutation of SuUR alone (Andreyeva et al 2007). While the evidence suggests a relationship between UR and transcriptionally repressive epigenetic states, such as H3K9 methylation, the nature of this relationship remains largely speculative (Posukh et al 2015).…”

“…(B) Close-up view of a representative genomic region (proximal 3L). (Open rectangles) Polytene cytological bands; (dotted line) the region corresponding to "Plato Atlantis" (Belyaeva et al 1998;Andreyeva et al 2007). (C) Suppression of UR in H1-depleted salivary gland cells.…”

“…In the SuUR ES mutant, UR of some regions of PH, such as the "Plato Atlantis" of the third chromosome, is suppressed, and they form band-interband patterns similar to those of euchromatin (Belyaeva et al 1998;Semeshin et al 2001;Andreyeva et al 2007). Whole-genome sequencing indicates that some of the mapped pericentric regions are severely underreplicated in the control, and the ploidy is partially increased in the absence of H1 (Fig.…”

The Release 6 reference sequence of the Drosophila melanogaster genome

“…These two BACs and the eight WGS3 scaffolds were identified because they contain dodeca satellite sequences found in heterochromatin region h53 (Supplemental Fig. S1; Abad et al 1992;Losada et al 2000;Andreyeva et al 2007). The order and orientation of these eight scaffolds have been determined using a physical map of the region (A Villasante, unpubl.…”

Heterochromatin: Not Just for Silencing AnymoreReport of the Eighth International Conference on Drosophila Heterochromatin

“…In this double mutant background, banded regions were generated from the amorphous material, allowing for major advances in ordering DNA sequences and localizing proteins within heterochromatin. 7 Genomic sequence Release 5.1 contained information from clones mapping to centric heterochromatin of the X chromosome and autosomes, but information regarding the Y chromosome remained limited. Even so, the Y-chromosome received much attention at the meeting.…”