The SU(VAR)3-9/HP1 Complex Differentially Regulates the Compaction State and Degree of Underreplication of X Chromosome Pericentric Heterochromatin in Drosophila melanogaster

Abstract

In polytene chromosomes of Drosophila melanogaster, regions of pericentric heterochromatin coalesce to form a compact chromocenter and are highly underreplicated. Focusing on study of X chromosome heterochromatin, we demonstrate that loss of either SU(VAR)3-9 histone methyltransferase activity or HP1 protein differentially affects the compaction of different pericentric regions. Using a set of inversions breaking X chromosome heterochromatin in the background of the Su(var)3-9 mutations, we show that distal heterochromatin (blocks h26-h29) is the only one within the chromocenter to form a big ''puff''-like structure. The ''puffed'' heterochromatin has not only unique morphology but also very special protein composition as well: (i) it does not bind proteins specific for active chromatin and should therefore be referred to as a pseudopuff and (ii) it strongly associates with heterochromatin-specific proteins SU(VAR)3-7 and SUUR, despite the fact that HP1 and HP2 are depleted particularly from this polytene structure. The pseudopuff completes replication earlier than when it is compacted as heterochromatin, and underreplication of some DNA sequences within the pseudopuff is strongly suppressed. So, we show that pericentric heterochromatin is heterogeneous in its requirement for SU(VAR)3-9 with respect to the establishment of the condensed state, time of replication, and DNA polytenization.

Analysis of Drosophila Species Genome Size and Satellite DNA Content Reveals Significant Differences Among Strains as Well as Between Species

“…Structural features of heterochromatin satellite repeats, as opposed to specific sequences, have been proposed to act as replication barriers (Leach et al 2000). The fact that underreplication is conserved, despite great differences in satellite DNA content and species-specific repeat sequence motifs, implies that structural and possibly epigenetic factors act as fork barriers (Demakova et al 2007). The availability of additional Drosophila genome sequences will allow a more thorough analysis of underreplicated genomic regions and genetic elements such as fork barriers that may control this conserved process.…”

The Release 6 reference sequence of the Drosophila melanogaster genome

“…The diploid larval neuroblast mitotic chromosomes provide a complete and unbiased representation of the pericentric, centric, and Y-chromosome heterochromatin (Gatti and Pimpinelli 1992) and have been subdivided into a map of 61 regions with diverse cytological features, designated h1 to h61 (Supplemental Figure S1; Gatti et al 1994). The map of larval salivary gland polytene chromosomes in the w m4 , SuUR Su(var)3-9 06 strain, in which some domains in pericentric heterochromatin acquire fine banding patterns due to suppression of the normal underreplication of these regions Demakova et al 2007), provides higher spatial resolution but is limited to polytenized regions. BACs were labeled and hybridized to mitotic and polytene chromosomes in parallel experiments using the same BAC DNA preparations.…”

“…One very large contig on chromosome arm 2R was oriented by single-probe mitotic FISH experiments with probes, BACR27E03 and BACR24N15, separated by 1.6 Mb (Supplemental Table S1); polytene FISH experiments did not orient this contig. We performed polytene chromosome FISH mapping using the w m4 , SuUR Su(var)3-9 06 strain Demakova et al 2007). Of 68 BACs tested, 24 hybridized to unique locations, 26 hybridized to multiple locations but with one unambiguous primary location, three produced ambiguous results, and 15 failed to localize specifically (Supplemental Table S1).…”

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

“…Both SuUR and Su(var)3-9 06 mutations independently affect the polytenization level of different heterochromatic domains (32). However, SuUR Su(var)3-9 06 double mutants display a clear additive effect in terms of improved structure of pericentric regions.…”

“…The histone-methyltransferase (HMTase) SU(VAR)3-9, in concert with HP1 and SU(VAR)3-7 proteins, is believed to define the compacted state of heterochromatin (27)(28)(29)(30). Mutants for any of these genes show dramatically decompacted heterochromatin (31,32). As opposed to SuUR mutants, the polytene chromosomes from SuUR Su(var)3-9 06 double mutants show polytenization of additional blocks of deep pericentric heterochromatin.…”