Late-replicating domains (intercalary heterochromatin) in the Drosophila genome display a number of features suggesting their organization is quite unique. Typically, they are quite large and encompass clusters of functionally unrelated tissue-specific genes. They correspond to the topologically associating domains and conserved microsynteny blocks. Our study aims at exploring further details of molecular organization of intercalary heterochromatin and has uncovered surprising heterogeneity of chromatin composition in these regions. Using the 4HMM model developed in our group earlier, intercalary heterochromatin regions were found to host chromatin fragments with a particular epigenetic profile. Aquamarine chromatin fragments (spanning 0.67% of late-replicating regions) are characterized as a class of sequences that appear heterogeneous in terms of their decompactization. These fragments are enriched with enhancer sequences and binding sites for insulator proteins. They likely mark the chromatin state that is related to the binding of cis-regulatory proteins. Malachite chromatin fragments (11% of late-replicating regions) appear to function as universal transitional regions between two contrasting chromatin states. Namely, they invariably delimit intercalary heterochromatin regions from the adjacent active chromatin of interbands. Malachite fragments also flank aquamarine fragments embedded in the repressed chromatin of late-replicating regions. Significant enrichment of insulator proteins CP190, SU(HW), and MOD2.2 was observed in malachite chromatin. Neither aquamarine nor malachite chromatin types appear to correlate with the positions of highly conserved non-coding elements (HCNE) that are typically replete in intercalary heterochromatin. Malachite chromatin found on the flanks of intercalary heterochromatin regions tends to replicate earlier than the malachite chromatin embedded in intercalary heterochromatin. In other words, there exists a gradient of replication progressing from the flanks of intercalary heterochromatin regions center-wise. The peculiar organization and features of replication in large late-replicating regions are discussed as possible factors shaping the evolutionary stability of intercalary heterochromatin.

The Drosophila melanogaster polytene chromosomes are the best model for studying the genome organization during interphase. Despite of the long-term studies available on genetic organization of polytene chromosome bands and interbands, little is known regarding long gene location on chromosomes. To analyze it, we used bioinformatic approaches and characterized genome-wide distribution of introns in gene bodies and in different chromatin states, and using fluorescent in situ hybridization we juxtaposed them with the chromosome structures. Short introns up to 2 kb in length are located in the bodies of housekeeping genes (grey bands or lazurite chromatin). In the group of 70 longest genes in the Drosophila genome, 95% of total gene length accrues to introns. The mapping of the 15 long genes showed that they could occupy extended sections of polytene chromosomes containing band and interband series, with promoters located in the interband fragments (aquamarine chromatin). Introns (malachite and ruby chromatin) in polytene chromosomes form independent bands, which can contain either both introns and exons or intron material only. Thus, a novel type of the gene arrangement in polytene chromosomes was discovered; peculiarities of such genetic organization are discussed.

The precise genomic localization of the borders of 62 intercalary heterochromatin bands in Drosophilapolytene chromosomes was determined. A new type of bands containing chromatin of different states...

The molecular and chromosomal localization of the duncegene was studied. This gene (167.3 kb) consists almost entirely of introns, in which a cluster of seven short tissue-specific genes is...

The molecular and chromosomal localization of the dunce gene was studied. This gene (167.3 kb) consists almost entirely of introns, in which a cluster of seven short tissue-specific genes is located. On the basis of the results of FISH analysis of the gene fragments, we established that the dunce gene is located within nine chromosomal structures (four bands and five interbands), which contradicts the common idea that genes are located in only one structure (band or interband) or at the boundary of these structures. Our results are quite unexpected and original and greatly expand the current understanding of the genetic organization of interphase chromosomes.

A novel method for mapping the interphase genome architecture in Drosophila polytene chromosomes is proposed. The method is based on physical mapping of the boundaries between interbands, gray bands, and black bands. Mapping relies on three criteria: the 4HMM bioinformatic model that considers the associations between each of the four chromatin states and polytene chromosome structures and the findings of ChIP-seq analysis in salivary glands for two critical markers: the CHRIZ/CHRO protein localized in housekeeping gene promoters and H3K36me3 histone modification, a marker of transcriptional elongation. For mapping the 1AF region of the X chromosome, FISH probes were selected from AQUAMARINE chromatin (interbands) at the boundary with black bands, which provided precise coordinates of the edges of black bands (localization of the developmental genes). The localization of promoters and housekeeping gene bodies was then identified by ChIP-seq analysis for CHIRIZ and H3K36me3 in salivary glands (the interbands and gray bands, respectively). These maps will allow one to better understand the architecture of an actively functioning genome.