[show abstract][hide abstract] ABSTRACT: 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.
PLoS ONE 01/2012; 7(1):e30035. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
PLoS ONE 01/2011; 6(10):e25960. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Proceedings of the National Academy of Sciences 08/2007; 104(31):12819-24. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: In Drosophila, dosage compensation requires assembly of the Male Specific Lethal (MSL) protein complex for doubling transcription of most X-linked genes in males. The recognition of the X chromosome by the MSL complex has been suggested to include initial assembly at approximately 35 chromatin entry sites and subsequent spreading of mature complexes in cis to numerous additional sites along the chromosome. To understand this process further we examined MSL patterns in a range of wild-type and mutant backgrounds producing different amounts of MSL components. Our data support a model in which MSL complex binding to the X is directed by a hierarchy of target sites that display different affinities for the MSL proteins. Chromatin entry sites differ in their ability to provide local intensive binding of complexes to adjacent regions, and need high MSL complex titers to achieve this. We also mapped a set of definite autosomal regions (approximately 70) competent to associate with the functional MSL complex in wild-type males. Overexpression of both MSL1 and MSL2 stabilizes this binding and results in inappropriate MSL binding to the chromocenter and the 4th chromosome. Thus, wild-type MSL complex titers are critical for correct targeting to the X chromosome.
[show abstract][hide abstract] ABSTRACT: The morphological characteristics of intercalary heterochromatin (IH) are compared with those of other types of silenced chromatin in the Drosophila melanogaster genome: pericentric heterochromatin (PH) and regions subject to position effect variegation (PEV). We conclude that IH regions in polytene chromosomes are binding sites of silencing complexes such as PcG complexes and of SuUR protein. Binding of these proteins results in the appearance of condensed chromatin and late replication of DNA, which in turn may result in DNA underreplication. IH and PH as well as regions subject to PEV have in common the condensed chromatin appearance, the localization of specific proteins, late replication, underreplication in polytene chromosomes, and ectopic pairing.
[show abstract][hide abstract] ABSTRACT: Regions of intercalary heterochromatin (IH) are dispersed in the euchromatic arms of polytene chromosomes and share the main properties of heterochromatin, namely chromosome constrictions resulting from DNA underreplication. These constrictions are frequent on the paired X chromosomes of females, but are practically absent from the single X chromosome of males. These sex-specific differences have been proposed to reflect the different levels of transcription and chromosome compaction due to dosage compensation, which in turn may affect the degree of underreplication in IH regions. To test this hypothesis, we induced dosage compensation in females by ectopic expression of MSL-2 protein. We then measured the extent of underreplication in IH regions by determining frequencies of constrictions, or by Southern blot analysis using a fragment of the ten (a) gene which is located in IH region 11A6-9. Females transheterozygous for Sxl (fhv1)/ Sxl (f1) or carrying a constitutive msl-2 transgene are known to hypertranscribe their X chromosomes. In such females, both the frequency of constrictions and DNA underreplication were reduced. Suppression of underreplication occurs only when a complete functional MSL complex assembles on the X chromosomes. We also used three strains that carried constitutive transgenes of msl-2 with mutations in the 5' untranslated regions. These strains produced normal levels of SXL protein, but variable levels of MSL-2 protein. The SXL protein did not prevent the formation of an MSL complex in these transgenic females. We found that the extent of underreplication of ten (a) DNA in IH region 11A6-9 negatively correlates with the amount of MSL complex.