Chromosoma

, Volume 119, Issue 2, pp 177–194

Chromatin insulators specifically associate with different levels of higher-order chromatin organization in Drosophila

  • Heather A. Wallace
  • Maria P. Plata
  • Hyuck-Joon Kang
  • Misty Ross
  • Mariano Labrador
Research Article

DOI: 10.1007/s00412-009-0246-0

Cite this article as:
Wallace, H.A., Plata, M.P., Kang, HJ. et al. Chromosoma (2010) 119: 177. doi:10.1007/s00412-009-0246-0

Abstract

Chromatin insulators are required for proper temporal and spatial expression of genes in metazoans. Here, we have analyzed the distribution of insulator proteins on the 56F–58A region of chromosome 2R in Drosophila polytene chromosomes to assess the role of chromatin insulators in shaping genome architecture. Data show that the suppressor of Hairy-wing protein [Su(Hw)] is found in three structures differentially associated with insulator proteins: bands, interbands, and multi-gene domains of coexpressed genes. Results show that bands are generally formed by condensation of chromatin that belongs to genes containing one or more Su(Hw) binding sites, whereas, in interbands, Su(Hw) sites appear associated with open chromatin. In addition, clusters of coexpressed genes in this region form bands characterized by the lack of CP190 and BEAF-32 insulator proteins. This pattern correlates with the distribution of specific chromatin marks and is conserved in nurse cells, suggesting that this organization may not be limited to one cell type but represents the basic organization of interphasic chromosomes.

Supplementary material

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Supplementary Table 1Distribution of Su(Hw) binding sites in cytological subdivisions 56E–58A from polytene chromosomes. Su(Hw) binding sites in this region were named sequentially following Bridges cytological subdivisions. Cytological subdivisions are shown in alternative background colors. Sites from modENCODE and Bushey et al. (2009) are compared. Only 11 sites were found in data from modENCODE that were not found in Bushey et al. (2009) whereas four sites were found in Bushey et al. (2009) and were missing in modENCODE. The position of binding sites in relation to genes is also indicated. Red characters are used for sites localized within transcribed regions of genes. Black characters are used to indicate sites localized in intergenic DNA. Thirty-seven sites map in intergenic DNA whereas 41 sites map in transcribed DNA. Nucleotide positions are given for sites as well as for the probe used in in situ hybridizations. Sites selected for in situ hybridization that are indicated with a “plus” sign correspond to a visible immunostaining band, while those that do not are indicated with a “minus“ sign. Sites selected for ChIP are indicated with a “plus” sign if significantly enriched for the Su(Hw) protein or a “minus“ sign if the level of enrichment was not significant (DOC 140 kb)
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Supplementary Table 2Distribution of clusters of coexpressed and non-coexpressed genes found in cytological subdivisions 56F–58A. Clusters of coexpressed genes were obtained from Spellman and Rubin (2002) and merged with the Drosophila-annotated genome (release 5.5). Clusters of coexpressed genes are indicated by an orange background color whereas clusters of non-coexpressed genes are indicated by a gray color. Boundaries are indicated in green. Insulator sites are annotated in column H. Sites in red correspond to intragenic sites, and sites in black correspond to intergenic sites. Sites are named as in Supplementary Table 1 (XLS 91 kb)
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Supplementary Table 3Oligonucleotide sequences used in in situ hybridization and real-time PCR after chromatin immunoprecipitation (XLS 22 kb)
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Supplementary Fig. 1

In situ hybridization combined with immunostaining in cytological subdivisions 56F–58A from polytene chromosomes. A total of 29 probes containing Su(Hw) binding sites are shown. DAPI is shown in blue. In situ hybridization signal is shown in red, and Su(Hw) immunostaining is shown in green. a Sites 56F.1to 57B.29. (GIF 3746 kb)

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Supplementary Fig. 2

In situ hybridization combined with immunostaining in cytological subdivisions 56F–58A from polytene chromosomes. A total of 29 probes containing Su(Hw) binding sites are shown. DAPI is shown in blue. In situ hybridization signal is shown in red, and Su(Hw) immunostaining is shown in green. b Sites 57D.1 to 58A.8 (GIF 3596 kb)

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Supplementary Fig. 3

High-resolution images of Su(Hw) immunostaining in polytene chromosomes reveal independent binding sites at distances as low as 11 kb. Su(Hw) binding sites obtained by tiling microarray (a) are compared with the distribution of Su(Hw) immunostaining sites in polytene chromosomes (b). Sites separated by distances less than 10 kb, such as those Glycogenin (57D.1–57D.2) or CG18375 (57D.3–57D.4) never appear as independent sites. Sites 57D.5 and 57D.6 are separated by 30 kb and may appear as a single or as a double band. c Distances of 10 kb are clearly resolved between King tubby (57B.30) and CG4050 (57B.31) sites. Microarray data from modENCODE is shown using Integrated Genome Browser (GIF 805 kb)

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Supplementary Fig. 4

Chromatin immunoprecipitation results are highly variable when comparing individual Su(Hw) binding sites along the cytological subdivisions 56F–58A. a Percentage of input obtained from ChIP experiments using 13 independent Su(Hw) binding sites. An endogenous copy of the gypsy retrotransposon was used as positive control (gypsy) and the RpL32 gene (Rp49) was used as a negative control. b Polytene chromosome immunostaining, using an antibody against Su(Hw), spanning the same cytological region. Although there is a certain correlation between the intensity of polytene chromosome immunostaining and ChIP assays, results also show that relatively intense signals in polytene chromosomes may produce a low level of enrichment in ChIP assays using embryonic chromatin (56F.7 or 57E.1) whereas fainter signals in polytene chromosomes may yield a high enrichment in ChIP assays (58A.1 or 58A.8) (GIF 641 kb)

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Supplementary Fig. 5

Ectopic activation of Sdc by Gal4 disorganizes the flanking polytene chromosome structure across more than 380 kb. The banding pattern revealed by DAPI in polytene chromosomes in the Oregon R (OR) stock (a) is compared with the same pattern in the SdcEY04602 stock after ectopic activation of transcription at the Sdc promoter by Gal4 (b). The dense DAPI staining normally associated with Sdc disappears and is substituted by dark patches that span more than 300 kb in both directions. Su(Hw) and CP190 immunostaining is also altered in the sites flanking Sdc, and these changes map hundreds of kilobase pairs away from the Sdc gene (c) (GIF 2588 kb)

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Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Heather A. Wallace
    • 1
  • Maria P. Plata
    • 1
  • Hyuck-Joon Kang
    • 1
  • Misty Ross
    • 1
    • 2
  • Mariano Labrador
    • 1
  1. 1.Department of Biochemistry, Cellular and Molecular BiologyThe University of TennesseeKnoxvilleUSA
  2. 2.Quillen College of MedicineEast Tennessee State UniversityJohnson CityUSA