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

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Abstract

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.

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Acknowledgments

The authors are very grateful to Prof. K. McKim, Prof. E. Käs, and Prof. H. Saumweber for the antibodies; thank Bloomington Drosophila Stock Center and Prof. P.G. Georgiev for the flystocks; and thank Dr. A. Gorchakov for the discussion. This work was supported by the Program of Presidium of the Russian Academy of Sciences “Molecular and Cellular Biology” (grant no. 6.4) and by the grant of Russian Foundation of Basic Research 12-04-31128 (DAM).

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Authors and Affiliations

  1. Institute of Molecular and Cellular Biology SB RAS, Novosibirsk, 630090, Russia

    Dmitry E. Koryakov, Galina V. Pokholkova, Daniil A. Maksimov, Stepan N. Belyakin, Elena S. Belyaeva & Igor F. Zhimulev

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  1. Dmitry E. Koryakov
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  2. Galina V. Pokholkova
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  3. Daniil A. Maksimov
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  4. Stepan N. Belyakin
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  5. Elena S. Belyaeva
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  6. Igor F. Zhimulev
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Corresponding author

Correspondence to Elena S. Belyaeva.

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Responsible editor: Terry Orr-Weaver

Dmitry E. Koryakov and Galina V. Pokholkova contributed equally to this work.

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Supplementary Fig. 1

Changes in chromatin make-up upon induced transcription of EY01976. Shown is a fragment of X chromosome (regions 10-11) with inactive insertion (a), insertion activated by hsp70-Gal4 at 18°С (b) and by da-Gal4 (c). Phase contrast (a-c) and overlay of phase contrast and immunostaining (d-h). Lines connect the same chromosome regions. Asterisk denotes a puff at 10EF. Bar, 5 μm (JPEG 59 kb)

High resolution image (TIFF 7995 kb)

Supplementary Fig. 2

Normalized RNA quantification around EY01976 insertion, shown as a log scale. qRT-PCR probes used and their positions on molecular map are indicated below the columns. Genotypes and induction conditions are color-coded. Error bars represent standard deviation (GIF 6 kb)

High resolution image (EPS 96 kb)

Supplementary Fig. 3

Degree of polytenization within the region 11A6-9 harboring EY01976 transgene, activated with da-Gal4 driver. x-axis shows genomic coordinates, y-axis shows the level of polytenization. The y-axis was scaled so that Actin42A gene polytenization level was equal 1. Red-shaded curves (red, orange, pink) correspond to the lines with inactive insertions or with Gal4-drivers. Blue-shaded lines (dark blue, light blue, purple) depict the polytenization profiles in stocks where transgenes are Gal4-activated. Arrows indicate insertion and PCR probes positions. Error bars represent standard deviation (GIF 32 kb)

High resolution image (EPS 155 kb)

Supplementary Fig. 4

Examples of typical immunostaining results with antibodies against RNA pol II, H3K9me2, and H3K9ac in the polytene sections 10-11. Fluorescent signal and phase contrast images are shown separately. The images with fluorescence clearly demonstrate the fact that prior to activation the region 11A6-9 (indicated by arrows) was not associated with these proteins. Bar, 5 μm (JPEG 99 kb)

High resolution image (TIFF 12368 kb)

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Koryakov, D.E., Pokholkova, G.V., Maksimov, D.A. et al. Induced transcription results in local changes in chromatin structure, replication timing, and DNA polytenization in a site of intercalary heterochromatin. Chromosoma 121, 573–583 (2012). https://doi.org/10.1007/s00412-012-0382-9

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