Effect of the Suppressor of Underreplication (SuUR) gene on position-effect variegation silencing in Drosophila melanogaster

It has been previously shown that the SuUR gene encodes a protein located in intercalary and pericentromeric heterochromatin in Drosophila melanogaster polytene chromosomes. The SuUR mutation suppresses the formation of ectopic contacts and DNA underreplication in polytene chromosomes; SuUR+ in extra doses enhances the expression of these characters. This study demonstrates that heterochromatin-dependent PEV silencing is also influenced by SuUR. The SuUR protein localizes to chromosome regions compacted as a result of PEV; the SuUR mutation suppresses DNA underreplication arising in regions of polytene chromosomes undergoing PEV. The SuUR mutation also suppresses variegation of both adult morphological characters and chromatin compaction observed in rearranged chromosomes. In contrast, SuUR+ in extra doses and its overexpression enhance variegation. Thus, SuUR affects PEV silencing in a dose-dependent manner. However, its effect is expressed weaker than that of the strong modifier Su(var)2-5.     

Position-effect variegation and intercalary heterochromatin: a comparative study

The behaviour of IH (intercalary heterochromatin) regions of Drosophila melanogaster polytene chromosomes was compared with that of euchromatin condensed as a result of position-effect variegation. Normally replicating regions, when subject to such an effect, were found to become among the last regions in the genome to replicate. It is shown that the factors which enhance position effect (low temperature, the removal of the Y chromosome, genetic enhancers of position effect) increase the weak point frequency in the IH, i.e. enhance DNA underreplication in these regions. We suggest that the similarity in the properties of IH, CH (centromeric heterochromatin) and the dense blocks induced by position effect is due to strong genetic inactivation and supercondensation caused by specific proteins in early development. The primary DNA structure is not likely to play a key role in this process.     

Influence of the SuUR gene on intercalary heterochromatin in Drosophila melanogaster polytene chromosomes

Salivary gland polytene chromosomes of Drosophila melanogaster have a reproducible set of intercalary heterochromatin (IH) sites, characterized by late DNA replication, underreplicated DNA, breaks and frequent ectopic contacts. The SuUR mutation has been shown to suppress underreplication, and wild-type SuUR protein is found at late-replicating IH sites and in pericentric heterochromatin. Here we show that the SuUR gene influences all four IH features. The SuUR mutation leads to earlier completion of DNA replication. Using transgenic strains with two, four or six additional SuUR(+) doses (4-8xSuUR(+)) we show that wild-type SuUR is an enhancer of DNA underreplication, causing many late-replicating sites to become underreplicated. We map the underreplication sites and show that their number increases from 58 in normal strains (2xSuUR(+)) to 161 in 4-8xSuUR(+) strains. In one of these new sites (1AB) DNA polytenization decreases from 100% in the wild type to 51%-85% in the 4xSuUR (+) strain. In the 4xSuUR(+) strain, 60% of the weak points coincide with the localization of Polycomb group (PcG) proteins. At the IH region 89E1-4 (the Bithorax complex), a typical underreplication site, the degree of underreplication increases with four doses of SuUR(+) but the extent of the underreplicated region is the same as in wild type and corresponds to the region containing PcG binding sites. We conclude that the polytene chromosome regions known as IH are binding sites for SuUR protein and in many cases PcG silencing proteins. We propose that these stable silenced regions are late replicated and, in the presence of SuUR protein, become underreplicated.     

Intercalary heterochromatin in polytene chromosomes of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Intercalary heterochromatin consists of extended chromosomal domains which are interspersed throughout the euchromatin and contain silent genetic material. These domains comprise either clusters of functionally unrelated genes or tandem gene duplications and possibly stretches of noncoding sequences. Strong repression of genetic activity means that intercalary heterochromatin displays properties that are normally attributable to classic pericentric heterochromatin: high compaction, late replication and underreplication in polytene chromosomes, and the presence of heterochromatin-specific proteins. Late replication and underreplication occurs when the suppressor of underreplication protein is present in intercalary heterochromatic regions. Intercalary heterochromatin underreplication in polytene chromosomes results in free double-stranded ends of DNA molecules; ligation of these free ends is the most likely mechanism for ectopic pairing between intercalary heterochromatic and pericentric heterochromatic regions. No support has been found for the view that the frequency of chromosome aberrations is elevated in intercalary heterochromatin.     

Functional dissection of the Suppressor of UnderReplication protein of Drosophila melanogaster: identification of domains influencing chromosome binding and DNA replication

The Suppressor of UnderReplication (SuUR) gene controls the DNA underreplication in intercalary and pericentric heterochromatin of Drosophila melanogaster salivary gland polytene chromosomes. In the present work, we investigate the functional importance of different regions of the SUUR protein by expressing truncations of the protein in an UAS–GAL4 system. We find that SUUR has at least two separate chromosome-binding regions that are able to recognize intercalary and pericentric heterochromatin specifically. The C-terminal part controls DNA underreplication in intercalary heterochromatin and partially in pericentric heterochromatin regions. The C-terminal half of SUUR suppresses endoreplication when ectopically expressed in the salivary gland. Ectopic expression of the N-terminal fragments of SUUR depletes endogenous SUUR from polytene chromosomes, causes the SuUR ⁻ phenotype and induces specific swellings in heterochromatin.     

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.     

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.     

Localization and characteristics of DNA underreplication zone in the 75C region of intercalary heterochromatin in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> 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.     

Intercalary heterochromatin in Drosophila

Sites of intercalary heterochromatin (IH) in the complete set of Drosophila melanogaster polytene chromosomes were localized and studied according to the following criteria: tendency to break (weak points), ectopic pairing and late replication, the existence of repeats (in X and 2R) including those enriched with A-T bases. Correlation between these features investigated, the highest correlation coefficients found between weak point behavior, late replication, and ectopic pairing. The frequency of breaks in weak points in some IH bands was shown to be different in different tissues, strains and closely related Drosophila species. Sexual differences in morphology and manifestation of IH features were found in bands of the X chromosome: weak point behavior and participation in ectopic pairing of IH bands are an order of magnitude less frequent in male X chromosomes than in female X chromosomes. In autosomes such differences have not been observed. IH bands in male X chromosomes look more massive than the homologous ones in female X chromosomes: the DNA content of the 11A6-9 region is four times less in females than in males. The hypothesis is proposed that the specific features of intercalary heterochromatin bands are determined by tandem repetitiveness and late replication. The latter, if it occurs in a cluster of repetitions, could cause incomplete polytenization of the region and, as a consequence, breaks (or weak points) and the appearance of adhesive ends which may take part either in realization of ectopic contacts or in fixation of those occurring previously. Breaks caused by chromosome aberrations in regions with repeats may not result in a sharp decline of viability, so that break points of chromosome rearrangements in intercalary heterochromatin may be more frequent than in other regions.     

Distorted heterochromatin replication in Drosophila melanogaster polytene chromosomes as a result of euchromatin-heterochromatin rearrangements

Studies of the position effect resulting from chromosome rearrangements in Drosophila melanogaster have shown that replication distortions in polytene chromosomes correlate with heritable gene silencing in mitotic cells. Earlier studies mostly focused on the effects of euchromatin--heterochromatin rearrangements on replication and silencing of euchromatic regions adjacent to the heterochromatin breakpoint. This review is based on published original data and considers the effect of rearrangements on heterochromatin: heterochromatin blocks that are normally underrepresented or underreplicated in polytene chromosomes are restored. Euchromatin proved to affect heterochromatin, preventing its underreplication. The effect is opposite to the known inactivation effect, which extends from heterochromatin to euchromatin. The trans-action of heterochromatin blocks on replication of heterochromatin placed within euchromatin is discussed. Distortions of heterochromatin replication in polytene chromosomes are considered to be an important characteristic associated with the functional role of the corresponding genome regions.     

Distorted heterochromatin replication in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> polytene chromosomes as a result of euchromatin-heterochromatin rearrangements

Studies of the position effect resulting from chromosome rearrangements in Drosophila melanogaster have shown that replication distortions in polytene chromosomes correlate with heritable gene silencing in mitotic cells. Earlier studies mostly focused on the effects of euchromatin-heterochromatin rearrangements on replication and silencing of euchromatic regions adjacent to the heterochromatin breakpoint. This review is based on published original data and considers the effect of rearrangements on heterochromatin: heterochromatin blocks that are normally underrepresented or underreplicated in polytene chromosomes are restored. Euchromatin proved to affect heterochromatin, preventing its underreplication. The effect is opposite to the known inactivation effect, which extends from heterochromatin to euchromatin. The trans-action of heterochromatin blocks on replication of heterochromatin placed within euchromatin is discussed. Distortions of heterochromatin replication in polytene chromosomes are considered to be an important characteristic associated with the functional role of the corresponding genome regions.     

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.     

Replication of heterochromatin and structure of polytene chromosomes

Heterochromatin is characteristically the last portion of the genome to be replicated. In polytene cells, heterochromatic sequences are underreplicated because S phase ends before replication of heterochromatin is completed. Truncated heterochromatic DNAs have been identified in polytene cells of Drosophila and may be the discontinuous molecules that form between fully replicated euchromatic and underreplicated heterochromatic regions of the chromosome. In this report, we characterize the temporal pattern of heterochromatic DNA truncation during development of polytene cells. Underreplication occurred during the first polytene S phase, yet DNA truncation, which was found within heterochromatic sequences of all four Drosophila chromosomes, did not occur until the second polytene S phase. DNA truncation was correlated with underreplication, since increasing the replication of satellite sequences with the cycE(1672) mutation caused decreased production of truncated DNAs. Finally, truncation of heterochromatic DNAs was neither quantitatively nor qualitatively affected by modifiers of position effect variegation including the Y chromosome, Su(var)205(2), parental origin, or temperature. We propose that heterochromatic satellite sequences present a barrier to DNA replication and that replication forks that transiently stall at such barriers in late S phase of diploid cells are left unresolved in the shortened S phase of polytene cells. DNA truncation then occurs in the second polytene S phase, when new replication forks extend to the position of forks left unresolved in the first polytene S phase.     

Thoracic underreplication in Drosophila species estimates a minimum genome size and the dynamics of added DNA

Many cells in the thorax of Drosophila were found to stall during replication, a phenomenon known as underreplication. Unlike underreplication in nuclei of salivary and follicle cells, this stall occurs with less than one complete round of replication. This stall point allows precise estimations of early-replicating euchromatin and late-replicating heterochromatin regions, providing a powerful tool to investigate the dynamics of structural change across the genome. We measure underreplication in 132 species across the Drosophila genus and leverage these data to propose a model for estimating the rate at which additional DNA is accumulated as heterochromatin and euchromatin and also predict the minimum genome size for Drosophila. According to comparative phylogenetic approaches, the rates of change of heterochromatin differ strikingly between Drosophila subgenera. Although these subgenera differ in karyotype, there were no differences by chromosome number, suggesting other structural changes may influence accumulation of heterochromatin. Measurements were taken for both sexes, allowing the visualization of genome size and heterochromatin changes for the hypothetical path of XY sex chromosome differentiation. Additionally, the model presented here estimates a minimum genome size in Sophophora remarkably close to the smallest insect genome measured to date, in a species over 200 million years diverged from Drosophila.