Super-resolution microscopy reveals stochastic initiation of replication in Drosophila polytene chromosomes

Studying the probability distribution of replication initiation along a chromosome is a huge challenge. Drosophila polytene chromosomes in combination with super-resolution microscopy provide a unique opportunity for analyzing the probabilistic nature of replication initiation at the ultrastructural level. Here, we developed a method for synchronizing S-phase induction among salivary gland cells. An analysis of the replication label distribution in the first minutes of S phase and in the following hours after the induction revealed the dynamics of replication initiation. Spatial super-resolution structured illumination microscopy allowed identifying multiple discrete replication signals and to investigate the behavior of replication signals in the first minutes of the S phase at the ultrastructural level. We identified replication initiation zones where initiation occurs stochastically. These zones differ significantly in the probability of replication initiation per time unit. There are zones in which initiation occurs on most strands of the polytene chromosome in a few minutes. In other zones, the initiation on all strands takes several hours. Compact bands are free of replication initiation events, and the replication runs from outer edges to the middle, where band shapes may alter

Effects of Mutations in the Drosophila melanogaster Rif1 Gene on the Replication and Underreplication of Pericentromeric Heterochromatin in Salivary Gland Polytene Chromosomes

In Drosophila salivary gland polytene chromosomes, a substantial portion of heterochromatin is underreplicated. The combination of mutations SuURES and Su(var)3-906 results in the polytenization of a substantial fraction of unique and moderately repeated sequences but has almost no effect on satellite DNA replication. The Rap1 interacting factor 1 (Rif) protein is a conserved regulator of replication timing, and in Drosophila, it affects underreplication in polytene chromosomes. We compared the morphology of pericentromeric regions and labeling patterns of in situ hybridization of heterochromatin-specific DNA probes between wild-type salivary gland polytene chromosomes and the chromosomes of Rif1 mutants and SuUR Su(var)3-906 double mutants. We show that, despite general similarities, heterochromatin zones exist that are polytenized only in the Rif1 mutants, and that there are zones that are under specific control of Su(var)3-9. In the Rif1 mutants, we found additional polytenization of the largest blocks of satellite DNA (in particular, satellite 1.688 of chromosome X and simple satellites in chromosomes X and 4) as well as partial polytenization of chromosome Y. Data on pulsed incorporation of 5-ethynyl-2′-deoxyuridine (EdU) into polytene chromosomes indicated that in the Rif1 mutants, just as in the wild type, most of the heterochromatin becomes replicated during the late S phase. Nevertheless, a significantly increased number of heterochromatin replicons was noted. These results suggest that Rif1 regulates the activation probability of heterochromatic origins in the satellite DNA region

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

A Spontaneous Inversion of the X Chromosome Heterochromatin Provides a Tool for Studying the Structure and Activity of the Nucleolus in <i>Drosophila melanogaster</i>

The pericentromeric heterochromatin is largely composed of repetitive sequences, making it difficult to analyze with standard molecular biological methods. At the same time, it carries many functional elements with poorly understood mechanisms of action. The search for new experimental models for the analysis of heterochromatin is an urgent task. In this work, we used the Rif1 mutation, which suppresses the underreplication of all types of repeated sequences, to analyze heterochromatin regions in polytene chromosomes of Drosophila melanogaster. In the Rif1 background, we discovered and described in detail a new inversion, In(1)19EHet, which arose on a chromosome already carrying the In(1)sc8 inversion and transferred a large part of X chromosome heterochromatin, including the nucleolar organizer to a new euchromatic environment. Using nanopore sequencing and FISH, we have identified the eu- and heterochromatin breakpoints of In(1)19EHet. The combination of the new inversion and the Rif1 mutation provides a promising tool for studies of X chromosome heterochromatin structure, nucleolar organization, and the nucleolar dominance phenomenon. In particular, we found that, with the complete polytenization of rDNA repeats, the nucleolus consists of a cloud-like structure corresponding to the classical nucleolus of polytene chromosomes, as well as an unusual intrachromosomal structure containing alternating transcriptionally active and inactive regions

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