Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.

Drosophila melanogaster polytene chromosomes display specific banding pattern; the underlying genetic organization of this pattern has remained elusive for many years. In the present paper, we analyze 32 cytology-mapped polytene chromosome interbands. We estimated molecular locations of these interbands, described their molecular and genetic organization and demonstrate that polytene chromosome interbands contain the 5′ ends of housekeeping genes. As a rule, interbands display preferential “head-to-head” orientation of genes. They are enriched for “broad” class promoters characteristic of housekeeping genes and associate with open chromatin proteins and Origin Recognition Complex (ORC) components. In two regions, 10A and 100B, coding sequences of genes whose 5′-ends reside in interbands map to constantly loosely compacted, early-replicating, so-called “grey” bands. Comparison of expression patterns of genes mapping to late-replicating dense bands vs genes whose promoter regions map to interbands shows that the former are generally tissue-specific, whereas the latter are represented by ubiquitously active genes. Analysis of RNA-seq data (modENCODE-FlyBase) indicates that transcripts from interband-mapping genes are present in most tissues and cell lines studied, across most developmental stages and upon various treatment conditions. We developed a special algorithm to computationally process protein localization data generated by the modENCODE project and show that Drosophila genome has about 5700 sites that demonstrate all the features shared by the interbands cytologically mapped to date.

Salivary gland polytene chromosomes of Drosophila melanogaster have a reproducible set of intercalary heterochromatin (IH) sites, characterized by late DNA replication, underreplicated DNA, breaks and frequent ectopic contacts. The SuUR mutation has been shown to suppress underreplication, and wild-type SuUR protein is found at late-replicating IH sites and in pericentric heterochromatin. Here we show that the SuUR gene influences all four IH features. The SuUR mutation leads to earlier completion of DNA replication. Using transgenic strains with two, four or six additional SuUR(+) doses (4-8xSuUR(+)) we show that wild-type SuUR is an enhancer of DNA underreplication, causing many late-replicating sites to become underreplicated. We map the underreplication sites and show that their number increases from 58 in normal strains (2xSuUR(+)) to 161 in 4-8xSuUR(+) strains. In one of these new sites (1AB) DNA polytenization decreases from 100% in the wild type to 51%-85% in the 4xSuUR (+) strain. In the 4xSuUR(+) strain, 60% of the weak points coincide with the localization of Polycomb group (PcG) proteins. At the IH region 89E1-4 (the Bithorax complex), a typical underreplication site, the degree of underreplication increases with four doses of SuUR(+) but the extent of the underreplicated region is the same as in wild type and corresponds to the region containing PcG binding sites. We conclude that the polytene chromosome regions known as IH are binding sites for SuUR protein and in many cases PcG silencing proteins. We propose that these stable silenced regions are late replicated and, in the presence of SuUR protein, become underreplicated.

In D. melanogaster polytene chromosomes, intercalary heterochromatin (IH) appears as large dense bands scattered in euchromatin and comprises clusters of repressed genes. IH displays distinctly low gene density, indicative of their particular regulation. Genes embedded in IH replicate late in the S phase and become underreplicated. We asked whether localization and organization of these late-replicating domains is conserved in a distinct cell type. Using published comprehensive genome-wide chromatin annotation datasets (modENCODE and others), we compared IH organization in salivary gland cells and in a Kc cell line. We first established the borders of 60 IH regions on a molecular map, these regions containing underreplicated material and encompassing ∼12% of Drosophila genome. We showed that in Kc cells repressed chromatin constituted 97% of the sequences that corresponded to IH bands. This chromatin is depleted for ORC-2 binding and largely replicates late. Differences in replication timing between the cell types analyzed are local and affect only sub-regions but never whole IH bands. As a rule such differentially replicating sub-regions display open chromatin organization, which apparently results from cell-type specific gene expression of underlying genes. We conclude that repressed chromatin organization of IH is generally conserved in polytene and non-polytene cells. Yet, IH domains do not function as transcription- and replication-regulatory units, because differences in transcription and replication between cell types are not domain-wide, rather they are restricted to small “islands” embedded in these domains. IH regions can thus be defined as a special class of domains with low gene density, which have narrow temporal expression patterns, and so displaying relatively conserved organization.

Background: Recently, we analyzed genome-wide protein binding data for the Drosophila cell lines S2, Kc, BG3 and Cl.8 (modENCODE Consortium) and identified a set of 12 proteins enriched in the regions corresponding to interbands of salivary gland polytene chromosomes. Using these data, we developed a bioinformatic pipeline that partitioned the Drosophila genome into four chromatin types that we hereby refer to as aquamarine, lazurite, malachite and ruby.Results: Here, we describe the properties of these chromatin types across different cell lines. We show that aquamarine chromatin tends to harbor transcription start sites (TSSs) and 5’ untranslated regions (5’UTRs) of the genes, is enriched in diverse “open” chromatin proteins, histone modifications, nucleosome remodeling complexes and transcription factors. It encompasses most of the tRNA genes and shows enrichment for non-coding RNAs and miRNA genes. Lazurite chromatin typically encompasses gene bodies. It is rich in proteins involved in transcription elongation. Frequency of both point mutations and natural deletion breakpoints is elevated within lazurite chromatin. Malachite chromatin shows higher frequency of insertions of natural transposons. Finally, ruby chromatin is enriched for proteins and histone modifications typical for the “closed” chromatin. Ruby chromatin has a relatively low frequency of point mutations and is essentially devoid of miRNA and tRNA genes. Aquamarine and ruby chromatin types are highly stable across cell lines and have contrasting properties. Lazurite and malachite chromatin types also display characteristic protein composition, as well as enrichment for specific genomic features. We found that two types of chromatin, aquamarine and ruby, retain their complementary protein patterns in four Drosophila cell lines.