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.

The N-Terminal Part of Drosophila CP190 Is a Platform for Interaction with Multiple Architectural Proteins.

CP190 is a co-factor in many Drosophila architectural proteins, being involved in the formation of active promoters and insulators. CP190 contains the N-terminal BTB/POZ (Broad-Complex, Tramtrack and Bric a brac/POxvirus and Zinc finger) domain and adjacent conserved regions involved in protein interactions. Here, we examined the functional roles of these domains of CP190 in vivo. The best-characterized architectural proteins with insulator functions, Pita, Su(Hw), and dCTCF, interacted predominantly with the BTB domain of CP190. Due to the difficulty of mutating the BTB domain, we obtained a transgenic line expressing a chimeric CP190 with the BTB domain of the human protein Kaiso. Another group of architectural proteins, M1BP, Opbp, and ZIPIC, interacted with one or both of the highly conserved regions in the N-terminal part of CP190. Transgenic lines of D. melanogaster expressing CP190 mutants with a deletion of each of these domains were obtained. The results showed that these mutant proteins only partially compensated for the functions of CP190, weakly binding to selective chromatin sites. Further analysis confirmed the essential role of these domains in recruitment to regulatory regions associated with architectural proteins. We also found that the N-terminal of CP190 was sufficient for recruiting Z4 and Chromator proteins and successfully achieving chromatin opening. Taken together, our results and the results of previous studies showed that the N-terminal region of CP190 is a platform for simultaneous interaction with various DNA-binding architectural proteins and transcription complexes.

Faint gray bands in Drosophila melanogaster polytene chromosomes are formed by coding sequences of housekeeping genes.

In Drosophila melanogaster, the chromatin of interphase polytene chromosomes appears as alternating decondensed interbands and dense black or thin gray bands. Recently, we uncovered four principle chromatin states (4НММ model) in the fruit fly, and these were matched to the structures observed in polytene chromosomes. Ruby/malachite chromatin states form black bands containing developmental genes, whereas aquamarine chromatin corresponds to interbands enriched with 5' regions of ubiquitously expressed genes. Lazurite chromatin supposedly forms faint gray bands and encompasses the bodies of housekeeping genes. In this report, we test this idea using the X chromosome as the model and MSL1 as a protein marker of the lazurite chromatin. Our bioinformatic analysis indicates that in the X chromosome, it is only the lazurite chromatin that is simultaneously enriched for the proteins and histone marks associated with exons, transcription elongation, and dosage compensation. As a result of FISH and EM mapping of a dosage compensation complex subunit, MSL1, we for the first time provide direct evidence that lazurite chromatin forms faint gray bands. Our analysis proves that overall most of housekeeping genes typically span from the interbands (5' region of the gene) to the gray band (gene body). More rarely, active lazurite chromatin and inactive malachite/ruby chromatin may be found within a common band, where both the housekeeping and the developmental genes reside together.

Tethering of SUUR and HP1 proteins results in delayed replication of euchromatic regions in Drosophila melanogaster polytene chromosomes.

We analyze how artificial targeting of Suppressor of Under-Replication (SUUR) and HP1 proteins affects DNA replication in the "open," euchromatic regions. Normally these regions replicate early in the S phase and display no binding of either SUUR or HP1. These proteins were expressed as fusions with DNA-binding domain of GAL4 and recruited to multimerized UAS integrated in three euchromatic sites of the polytene X chromosome: 3B, 8D, and 18B. Using PCNA staining as a marker of ongoing replication, we showed that targeting of SUUR(GAL4DBD) and HP1(GAL4DBD) results in delayed replication of appropriate euchromatic regions. Specifically, replication at these regions starts early, much like in the absence of the fusion proteins; however, replication completion is significantly delayed. Notably, delayed replication was insufficient to induce underreplication. Recruitment of SUUR(GAL4DBD) and HP1(GAL4DBD) had distinct effects on expression of a mini-white reporter, found near UAS. Whereas SUUR(GAL4DBD) had no measurable influence on mini-white expression, HP1(GAL4DBD) targeting silenced mini-white, even in the absence of functional SU(VAR)3-9. Furthermore, recruitment of SUUR(GAL4DBD) and HP1(GAL4DBD) had distinct effects on the protein composition of target regions. HP1(GAL4DBD) but not SUUR(GAL4DBD) could displace an open chromatin marker, CHRIZ, from the tethering sites.

Induced transcription results in local changes in chromatin structure, replication timing, and DNA polytenization in a site of intercalary heterochromatin.

In salivary gland polytene chromosomes of Drosophila melanogaster, the regions of intercalary heterochromatin are characterized by late replication, under-replication, and genetic silencing. Using Gal4/UAS system, we induced transcription of sequences adjacent to transgene insertions in the band 11A6-9. This activation resulted in a loss of "silent" and appearance of "active" epigenetic marks, recruitment of RNA polymerase II, and formation of a puff. The activated region is now early replicating and shows increased level of DNA polytenization. Notably, all these changes are restricted to the area around the inserts, whereas the rest of the band remains inactive and late replicating. Although only a short area near the insertion site is transcribed, it results in an "open" chromatin conformation in a much broader region. We conclude that regions of intercalary heterochromatin do not form stand-alone units of late replication and under-replication. Every part of such regions can be activated and polytenized independently of other parts.

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

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.

Mechanism and functional role of the interaction between CP190 and the architectural protein Pita in Drosophila melanogaster.

BACKGROUND: Pita is required for Drosophila development and binds specifically to a long motif in active promoters and insulators. Pita belongs to the Drosophila family of zinc-finger architectural proteins, which also includes Su(Hw) and the conserved among higher eukaryotes CTCF. The architectural proteins maintain the active state of regulatory elements and the long-distance interactions between them. In particular, Pita is involved in the formation of several boundaries between regulatory domains that controlled the expression of three hox genes in the Bithorax complex (BX-C). The CP190 protein is recruited to chromatin through interaction with the architectural proteins. RESULTS: Using in vitro pull-down analysis, we precisely mapped two unstructured regions of Pita that interact with the BTB domain of CP190. Then we constructed transgenic lines expressing the Pita protein of the wild-type and mutant variants lacking CP190-interacting regions. We have demonstrated that CP190-interacting region of the Pita can maintain nucleosome-free open chromatin and is critical for Pita-mediated enhancer blocking activity in BX-C. At the same time, interaction with CP190 is not required for the in vivo function of the mutant Pita protein, which binds to the same regions of the genome as the wild-type protein. Unexpectedly, we found that CP190 was still associated with the most of genome regions bound by the mutant Pita protein, which suggested that other architectural proteins were continuing to recruit CP190 to these regions. CONCLUSIONS: The results directly demonstrate role of CP190 in insulation and support a model in which the regulatory elements are composed of combinations of binding sites that interact with several architectural proteins with similar functions.

Genes Containing Long Introns Occupy Series of Bands and Interbands In Drosophila melanogaster polytene Chromosomes.

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.

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.

The SU(VAR)3-9/HP1 complex differentially regulates the compaction state and degree of underreplication of X chromosome pericentric heterochromatin in Drosophila melanogaster.

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.

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

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 yellow and mini-white 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.

Genetic organization of interphase chromosome bands and interbands in Drosophila melanogaster.

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.

Late replication domains are evolutionary conserved in the Drosophila genome.

Drosophila 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 Drosophila 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 Drosophila 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 D. melanogaster genome consistently display two-fold lower gene density across different Drosophila species.

Interaction between the Drosophila heterochromatin proteins SUUR and HP1.

SUUR (Suppressor of Under-Replication) protein is responsible for late replication and, as a consequence, for DNA underreplication of intercalary and pericentric heterochromatin in Drosophila melanogaster polytene chromosomes. However, the mechanism by which SUUR slows down the replication process is not clear. To identify possible partners for SUUR we performed a yeast two-hybrid screen using full-length SUUR as bait. This identified HP1, the well-studied heterochromatin protein, as a strong SUUR interactor. Furthermore, we have determined that the central region of SUUR is necessary and sufficient for interaction with the C-terminal part of HP1, which contains the hinge and chromoshadow domains. In addition, recruitment of SUUR to ectopic HP1 sites on chromosomes provides evidence for their association in vivo. Indeed, we found that the distributions of SUUR and HP1 on polytene chromosomes are interdependent: both absence and overexpression of HP1 prevent SUUR from chromosomal binding, whereas SUUR overexpression causes redistribution of HP1 to numerous sites occupied by SUUR. Finally, HP1 binds to intercalary heterochromatin when histone methyltransferase activity of SU(VAR)3-9 is increased. We propose that interaction with HP1 is crucial for the association of SUUR with chromatin.

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

The structural and functional analyses of heterochromatin are essential to understanding how heterochromatic genes are regulated and how centromeric chromatin is formed. Because the repetitive nature of heterochromatin hampers its genome analysis, new approaches need to be developed. Here, we describe how, in double mutants for Su(var)3-9 and SuUR genes encoding two structural proteins of heterochromatin, new banded heterochromatic segments appear in all polytene chromosomes due to the strong suppression of under-replication in pericentric regions. FISH on salivary gland polytene chromosomes from these double mutant larvae allows high resolution of heterochromatin mapping. In addition, immunostaining experiments with a set of antibodies against euchromatic and heterochromatic proteins reveal their unusual combinations in the newly appeared segments: binding patterns for HP1 and HP2 are coincident, but both are distinct from H3diMetK9 and H4triMetK20. In several regions, partial overlapping staining is observed for the proteins characteristic of active chromatin RNA Pol II, H3triMetK4, Z4, and JIL1, the boundary protein BEAF, and the heterochromatin-enriched proteins HP1, HP2, and SU(VAR)3-7. The exact cytological position of the centromere of chromosome 3 was visualized on salivary gland polytene chromosomes by using the centromeric dodeca satellite and the centromeric protein CID. This region is enriched in H3diMetK9 and H4triMetK20 but is devoid of other proteins analyzed.

DNA underreplication in intercalary heterochromatin regions in polytene chromosomes of Drosophila melanogaster correlates with the formation of partial chromosomal aberrations and ectopic pairing.

We studied the influence of the Suppressor of Underreplication (SuUR) gene expression on the intercalary heterochromatin (IH) regions of Drosophila melanogaster polytene chromosomes. We observed a strong positive correlation between increased SuUR expression, underreplication extent, amount of DNA truncation, and formation of ectopic contacts in IH regions. SuUR overexpression from heat shock-driven transgene results in the formation of partial chromosomal aberrations whose breakpoints map exclusively to the regions of intercalary and pericentric heterochromatin. It is important to note that all these effects are seen only if SuUR overexpression is induced during early stages of chromosome polytenization. Therefore, we developed the idea that ectopic pairing results from the joining of free DNA ends, which are formed as a consequence of underreplication.

Three distinct chromatin domains in telomere ends of polytene chromosomes in Drosophila melanogaster Tel mutants.

Drosophila melanogaster telomeric DNA is known to comprise two domains: the terminal tract of retrotransposons (HeT-A, TART and TAHRE) and telomere-associated sequences (TAS). Chromosome tips are capped by a protein complex, which is assembled on the chromosome ends independently of the underlying terminal DNA sequences. To investigate the properties of these domains in salivary gland polytene chromosomes, we made use of Tel mutants. Telomeres in this background are elongated owing to the amplification of a block of terminal retroelements. Supercompact heterochromatin is absent from the telomeres of polytene chromosomes: electron microscopy analysis identifies the telomeric cap and the tract of retroelements as a reticular material, having no discernible banding pattern, whereas TAS repeats appear as faint bands. According to the pattern of bound proteins, the cap, tract of retroelements and TAS constitute distinct and non-overlapping domains in telomeres. SUUR, HP2, SU(VAR)3-7 and H3Me3K27 localize to the cap region, as has been demonstrated for HP1. All these proteins are also found in pericentric heterochromatin. The tract of retroelements is associated with proteins characteristic for both heterochromatin (H3Me3K9) and euchromatin (H3Me3K4, JIL-1, Z4). The TAS region is enriched for H3Me3K27. PC and E(Z) are detected both in TAS and many intercalary heterochromatin regions. Telomeres complete replication earlier than heterochromatic regions. The frequency of telomeric associations in salivary gland polytene chromosomes does not depend on the SuUR gene dosage, rather it appears to be defined by the telomere length.