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.

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.

Polytene Chromosomes - A Portrait of Functional Organization of the Drosophila Genome.

This mini-review is devoted to the problem genetic meaning of main polytene chromosome structures - bands and interbands. Generally, densely packed chromatin forms black bands, moderately condensed regions form grey loose bands, whereas decondensed regions of the genome appear as interbands. Recent progress in the annotation of the Drosophila genome and epigenome has made it possible to compare the banding pattern and the structural organization of genes, as well as their activity. This was greatly aided by our ability to establish the borders of bands and interbands on the physical map, which allowed to perform comprehensive side-by-side comparisons of cytology, genetic and epigenetic maps and to uncover the association between the morphological structures and the functional domains of the genome. These studies largely conclude that interbands 5'-ends of housekeeping genes that are active across all cell types. Interbands are enriched with proteins involved in transcription and nucleosome remodeling, as well as with active histone modifications. Notably, most of the replication origins map to interband regions. As for grey loose bands adjacent to interbands, they typically host the bodies of house-keeping genes. Thus, the bipartite structure composed of an interband and an adjacent grey band functions as a standalone genetic unit. Finally, black bands harbor tissue-specific genes with narrow temporal and tissue expression profiles. Thus, the uniform and permanent activity of interbands combined with the inactivity of genes in bands forms the basis of the universal banding pattern observed in various Drosophila tissues.

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.

Chromatin Heterogeneity and Distribution of Regulatory Elements in the Late-Replicating Intercalary Heterochromatin Domains of Drosophila melanogaster Chromosomes.

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.

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.

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.

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.

Banding patterns in Drosophila melanogaster polytene chromosomes correlate with DNA-binding protein occupancy.

The most enigmatic feature of polytene chromosomes is their banding pattern, the genetic organization of which has been a very attractive puzzle for many years. Recent genome-wide protein mapping efforts have produced a wealth of data for the chromosome proteins of Drosophila cells. Based on their specific protein composition, the chromosomes comprise two types of bands, as well as interbands. These differ in terms of time of replication and specific types of proteins. The interbands are characterized by their association with "active" chromatin proteins, nucleosome remodeling, and origin recognition complexes, and so they have three functions: acting as binding sites for RNA pol II, initiation of replication and nucleosome remodeling of short fragments of DNA. The borders and organization of the same band and interband regions are largely identical, irrespective of the cell type studied. This demonstrates that the banding pattern is a universal principle of the organization of interphase polytene and non-polytene chromosomes.

Late replication domains in polytene and non-polytene cells of Drosophila melanogaster.

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.

Protein composition of interband regions in polytene and cell line chromosomes of Drosophila melanogaster.

BACKGROUND: Despite many efforts, little is known about distribution and interactions of chromatin proteins which contribute to the specificity of chromomeric organization of interphase chromosomes. To address this issue, we used publicly available datasets from several recent Drosophila genome-wide mapping and annotation projects, in particular, those from modENCODE project, and compared molecular organization of 13 interband regions which were accurately mapped previously. RESULTS: Here we demonstrate that in interphase chromosomes of Drosophila cell lines, the interband regions are enriched for a specific set of proteins generally characteristic of the "open" chromatin (RNA polymerase II, CHRIZ (CHRO), BEAF-32, BRE1, dMI-2, GAF, NURF301, WDS and TRX). These regions also display reduced nucleosome density, histone H1 depletion and pronounced enrichment for ORC2, a pre-replication complex component. Within the 13 interband regions analyzed, most were around 3-4 kb long, particularly those where many of said protein features were present. We estimate there are about 3500 regions with similar properties in chromosomes of D. melanogaster cell lines, which fits quite well the number of cytologically observed interbands in salivary gland polytene chromosomes. CONCLUSIONS: Our observations suggest strikingly similar organization of interband chromatin in polytene chromosomes and in chromosomes from cell lines thereby reflecting the existence of a universal principle of interphase chromosome organization.

Identical functional organization of nonpolytene and polytene chromosomes in Drosophila melanogaster.

Salivary gland polytene chromosomes demonstrate banding pattern, genetic meaning of which is an enigma for decades. Till now it is not known how to mark the band/interband borders on physical map of DNA and structures of polytene chromosomes are not characterized in molecular and genetic terms. It is not known either similar banding pattern exists in chromosomes of regular diploid mitotically dividing nonpolytene cells. Using the newly developed approach permitting to identify the interband material and localization data of interband-specific proteins from modENCODE and other genome-wide projects, we identify physical limits of bands and interbands in small cytological region 9F13-10B3 of the X chromosome in D. melanogaster, as well as characterize their general molecular features. Our results suggests that the polytene and interphase cell line chromosomes have practically the same patterns of bands and interbands reflecting, probably, the basic principle of interphase chromosome organization. Two types of bands have been described in chromosomes, early and late-replicating, which differ in many aspects of their protein and genetic content. As appeared, origin recognition complexes are located almost totally in the interbands of chromosomes.

Induced decondensation of heterochromatin in Drosophila melanogaster polytene chromosomes under condition of ectopic expression of the Supressor of underreplication gene.

Overexpression of Suppressor of Underreplication protein (SUUR) induces giant reversible swellings in intercalary and pericentric heterochromatin of salivary gland polytene chromosomes. Here, we demonstrate that morphology and extent of swellings are highly dependent on the fixation conditions used: upon glutaraldehyde fixation, we observed moderate decondensation of heterochromatic regions, which was significantly more pronounced upon acetic-acid fixation. Swellings are formed in a PARP-independent fashion. Together with data on inactive transcription in them, this indicates that the swelling-forming regions fail to acquire any features of puffs, the regions typically forming locally decondensed chromatin. Large swellings display striking re-localization of histones and SUUR protein, which are now found at the periphery of the swellings, in contrast to the DNA that fills the entirety of the swelling. We show that swelling-embedded DNA is capable of undergoing replication, however SUUR overexpression drastically alters replication timing in salivary gland cells. We speculate that swelling formation results from SUUR tipping the balance against other proteins that contribute to the organization of repressed chromatin regions.

Gene density profile reveals the marking of late replicated domains in the Drosophila melanogaster genome.

Regulation of replication timing has been a focus of many studies. It has been shown that numerous chromosomal regions switch their replication timing on cell differentiation in Drosophila and mice. However, it is not clear which features of these regions are essential for such regulation. In this study, we examined the organization of late underreplicated regions (URs) of the Drosophila melanogaster genome. When compared with their flanks, these regions showed decreased gene density. A detailed view revealed that these regions originate from unusual combination of short genes and long intergenic spacers. Furthermore, gene expression study showed that this pattern is mostly contributed by short testis-specific genes abundant in the URs. Based on these observations, we developed a genome scanning algorithm and identified 110 regions possessing similar gene density and transcriptional profiles. According to the published data, replication of these regions has been significantly shifted towards late S-phase in two Drosophila cell lines and in polytene chromosomes. Our results suggest that genomic organization of the underreplicated areas of Drosophila polytene chromosomes may be associated with the regulation of their replication timing.

Paucity and preferential suppression of transgenes in late replication domains of the D. melanogaster genome.

BACKGROUND: Eukaryotic genomes are organized in extended domains with distinct features intimately linking genome structure, replication pattern and chromatin state. Recently we identified a set of long late replicating euchromatic regions that are underreplicated in salivary gland polytene chromosomes of D. melanogaster. RESULTS: Here we demonstrate that these underreplicated regions (URs) have a low density of P-element and piggyBac insertions compared to the genome average or neighboring regions. In contrast, Minos-based transposons show no paucity in URs but have a strong bias to testis-specific genes. We estimated the suppression level in 2,852 stocks carrying a single P-element by analysis of eye color determined by the mini-white marker gene and demonstrate that the proportion of suppressed transgenes in URs is more than three times higher than in the flanking regions or the genomic average. The suppressed transgenes reside in intergenic, genic or promoter regions of the annotated genes. We speculate that the low insertion frequency of P-elements and piggyBacs in URs partially results from suppression of transgenes that potentially could prevent identification of transgenes due to complete suppression of the marker gene. In a similar manner, the proportion of suppressed transgenes is higher in loci replicating late or very late in Kc cells and these loci have a lower density of P-elements and piggyBac insertions. In transgenes with two marker genes suppression of mini-white gene in eye coincides with suppression of yellow gene in bristles. CONCLUSIONS: Our results suggest that the late replication domains have a high inactivation potential apparently linked to the silenced or closed chromatin state in these regions, and that such inactivation potential is largely maintained in different tissues.

Localization and characteristics of DNA underreplication zone in the 75C region of intercalary heterochromatin in Drosophila melanogaster polytene chromosomes.

In Drosophila polytene chromosomes, regions of intercalary heterochromatin are scattered throughout the euchromatic arms. Here, we present data on the first fine analysis of the individual intercalary heterochromatin region, 75C1-2, located in the 3L chromosome. By using electron microscopy, we demonstrated that this region appears as three closely adjacent condensed bands. Mapping of the region on the physical map by means of the chromosomal rearrangements with known breakpoints showed that the length of the region is about 445 kb. Although it seems that the SUUR protein binds to the whole 75C1-2 region, the proximal part of the region is fully polytenized, so the DNA underreplication zone is asymmetric and located in the distal half of the region. Finally, we speculate that intercalary heterochromatin regions of Drosophila polytene chromosomes are organized into three different types with respect to the localization of the underreplication zone.

Conservation of domain structure in a fast-evolving heterochromatic SUUR protein in drosophilids.

Different genomic regions replicate at a distinct time during S-phase. The SuUR mutation alters replication timing and the polytenization level of intercalary and pericentric heterochromatin in Drosophila melanogaster salivary gland polytene chromosomes. We analyzed SuUR in different insects, identified conserved regions in the protein, substituted conserved amino acid residues, and studied effects of the mutations on SUUR function. SuUR orthologs were identified in all sequenced drosophilids, and a highly divergent ortholog was found in the mosquito genome. We demonstrated that SUUR evolves at very high rate comparable with that of Transformer. Remarkably, upstream ORF within 5' UTR of the gene is more conserved than SUUR in drosophilids, but it is absent in the mosquito. The domain structure and charge of SUUR are maintained in drosophilids despite the high divergence of the proteins. The N-terminal part of SUUR with similarity to the SNF2/SWI2 proteins displays the highest level of conservation. Mutation of two conserved amino acid residues in this region impairs binding of SUUR to polytene chromosomes and reduces the ability of the protein to cause DNA underreplication. The least conserved middle part of SUUR interacting with HP1 retains positively and negatively charged clusters and nuclear localization signals. The C terminus contains interlacing conserved and variable motifs. Our results suggest that SUUR domains evolve with different rates and patterns but maintain their features.

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.

SUUR joins separate subsets of PcG, HP1 and B-type lamin targets in Drosophila.

Drosophila melanogaster Suppressor of Under-Replication (SuUR) gene encodes a protein that modulates replicative properties of heterochromatin in endocycles of polytene cells. The SuUR mutation abolishes underreplication of intercalary heterochromatin and results in partial underreplication of pericentric heterochromatin. We performed a genome-wide mapping of SUUR target genes in non-polytenic Drosophila Kc cells by using the DamID approach. We show that SUUR preferentially binds genes that are transcriptionally silent and late-replicated. Distinct subsets of SUUR targets are associated with PcG proteins (Pc and Esc; Polycomb and Extra sexcombs), heterochromatic proteins [HP1 and SU(VAR)3-9] and B-type lamin. The SUUR binding profile negatively correlates with the DNA polytenization levels of salivary gland polytene chromosomes. Finally, SUUR target genes are repressed in Drosophila embryos and gradually activated later in development. Together these results suggest that SUUR is a ubiquitous marker of heterochromatin in different cell types.