Polytene chromosomes reflect functional organization of the Drosophila genome

Polytene chromosomes of Drosophila melanogaster are a convenient model for studying interphase chromosomes of eukaryotes. They are giant in size in comparison with diploid cell chromosomes and have a pattern of cross stripes resulting from the ordered chromatid arrangement. Each region of polytene chromosomes has a unique banding pattern. Using the model of four chromatin types that reveals domains of varying compaction degrees, we were able to correlate the physical and cytological maps of some polytene chromosome regions and to show the main properties of genetic and molecular organization of bands and interbands, that we describe in this review. On the molecular map of the genome, the interbands correspond to decompacted aquamarine chromatin and 5’ ends of ubiquitously active genes. Gray bands contain lazurite and malachite chromatin, intermediate in the level of compaction, and, mainly, coding parts of genes. Dense black transcriptionally inactive bands are enriched in ruby chromatin. Localization of several dozens of interbands on the genome molecular map allowed us to study in detail their architecture according to the data of whole genome projects. The distribution of proteins and regulatory elements of the genome in the promoter regions of genes localized in the interbands shows that these parts of interbands are probably responsible for the formation of open chromatin that is visualized in polytene chromosomes as interbands. Thus, the permanent genetic activity of interbands and gray bands and the inactivity of genes in black bands are the basis of the universal banding pattern in the chromosomes of all Drosophila tissues. The smallest fourth chromosome of Drosophila with an atypical protein composition of chromatin is a special case.  Using the model of four chromatin states and fluorescent in situ hybridization, its cytological map was refined and the genomic coordinates of all bands and interbands were determined. It was shown that, in spite of the peculiarities of this chromosome, its band organization in general corresponds to the rest of the genome. Extremely long genes of different Drosophila chromosomes do not fit the common scheme, since they can occupy a series of alternating bands and interbands (up to nine chromosomal structures) formed by parts of these genes

Tethering of CHROMATOR and dCTCF proteins results in decompaction of condensed bands in the <i>Drosophila melanogaster</i> polytene chromosomes but does not affect their transcription and replication timing

<div><p>Instulator proteins are central to domain organization and gene regulation in the genome. We used ectopic tethering of CHROMATOR (CHRIZ/CHRO) and dCTCF to pre-defined regions of the genome to dissect the influence of these proteins on local chromatin organization, to analyze their interaction with other key chromatin proteins and to evaluate the effects on transcription and replication. Specifically, using UAS-GAL4DBD system, CHRO and dCTCF were artificially recruited into highly compacted polytene chromosome bands that share the features of silent chromatin type known as intercalary heterochromatin (IH). This led to local chromatin decondensation, formation of novel DHSes and recruitment of several “open chromatin” proteins. CHRO tethering resulted in the recruitment of CP190 and Z4 (PZG), whereas dCTCF tethering attracted CHRO, CP190, and Z4. Importantly, formation of a local stretch of open chromatin did not result in the reactivation of silent marker genes <i>yellow</i> and <i>mini</i>-<i>white</i> immediately adjacent to the targeting region (UAS), nor did RNA polII become recruited into this chromatin. The decompacted region retained late replicated, similarly to the wild-type untargeted region.</p></div

11A6-9 band splits upon tethering of CHRO^GAL4DBD (A-D) and dCTCF^GAL4DBD (E,F) into the regions of EY01976 (A,B) and EY00353 (C-F) insertions.

<p>Phase contrast (left column). Overlay of phase contrast and immunostaining (right column). Thin arrow indicates EY01976 insertion (A—control, B—CHRO^GAL4DBD expression and splitting of the band 11A6-9 in its distal part), thick arrow indicates EY00353 insertion in the middle of the band (C,E—control; D,F—tethering of CHRO^GAL4DBD and dCTCF^GAL4DBD, respectively).</p

Ectopic tethering of CHRO^GAL4DBD does not lead to transcription of the decompacted chromatin.

<p>(A)–anti-MYC signal (UAS) and anti-RNA PolII Ser5 signal do not co-localize in the decompacted interband-like regions formed in the bands 10A1-2 and 11A6-9. Upper row: phase contrast of the split-band morphology, bottom row: immunostaining signals for RNA PolII Ser5 (green) and MYC (red). Positions of UASes are denoted by arrows; (B)–wing bristle pigmentation in <i>Oregon</i> R (dark), EY00353; DBDGAL4 (brown) and EY00353; CHRO^GAL4DBD (brown) flies indicates that the reporter <i>yellow</i>⁺ gene present in EY elements is not induced upon CHRO^GAL4DBD tethering.</p

Replication timing in the band 10A1-2.

<p>Control 10A-UAS; GAL4DBD chromosomes (A,B), 10A-UAS; CHRO^GAL4DBD (C,D), CHRO (green), PCNA (red). Arrow indicates the decondensation site.</p

Summary of protein immunodetection data in the chromosomes from CHRO^GAL4DBD- and dCTCF^GAL4DBD-expressing animals.

<p>Summary of protein immunodetection data in the chromosomes from CHRO^GAL4DBD- and dCTCF^GAL4DBD-expressing animals.</p

11A6-9 band splits upon tethering of CHRO^GAL4DBD (A-D) and dCTCF^GAL4DBD (E,F) into the regions of EY01976 (A,B) and EY00353 (C-F) insertions.

<p>Phase contrast (left column). Overlay of phase contrast and immunostaining (right column). Thin arrow indicates EY01976 insertion (A—control, B—CHRO^GAL4DBD expression and splitting of the band 11A6-9 in its distal part), thick arrow indicates EY00353 insertion in the middle of the band (C,E—control; D,F—tethering of CHRO^GAL4DBD and dCTCF^GAL4DBD, respectively).</p

Late Replication Domains Are Evolutionary Conserved in the <i>Drosophila</i> Genome

<div><p><i>Drosophila</i> chromosomes are organized into distinct domains differing in their predominant chromatin composition, replication timing and evolutionary conservation. We show on a genome-wide level that genes whose order has remained unaltered across 9 <i>Drosophila</i> species display late replication timing and frequently map to the regions of repressive chromatin. This observation is consistent with the existence of extensive domains of repressive chromatin that replicate extremely late and have conserved gene order in the <i>Drosophila</i> genome. We suggest that such repressive chromatin domains correspond to a handful of regions that complete replication at the very end of S phase. We further demonstrate that the order of genes in these regions is rarely altered in evolution. Substantial proportion of such regions significantly coincide with large synteny blocks. This indicates that there are evolutionary mechanisms maintaining the integrity of these late-replicating chromatin domains. The synteny blocks corresponding to the extremely late-replicating regions in the <i>D. melanogaster</i> genome consistently display two-fold lower gene density across different <i>Drosophila</i> species.</p></div