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The ecdysone receptor (ScEcR-A) binds DNA puffs at the start of DNA amplification in Sciara coprophila

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Abstract

The steroid hormone ecdysone induces DNA amplification and subsequent DNA puff formation in late fourth larval instar salivary gland polytene chromosomes of the fungus fly, Sciara coprophila. Previous in vitro studies on DNA puff II/9A in Sciara demonstrated that the ecdysone receptor (ScEcR-A) efficiently binds an ecdysone response element adjacent to the origin recognition complex binding site within the II/9A amplification origin, implying a role for ScEcR-A in amplification. Here, we extrapolate the molecular details from locus II/9A to the rest of the genome using immunofluorescence with a ScEcR-A-specific antibody. ScEcR-A binds all DNA puff sites just as amplification begins and persists throughout the processes of amplification, transcription, and puffing. Ecdysone injections into pre-amplification stage larvae prematurely induce both DNA amplification and ScEcR-A binding to DNA puff sites. These data are consistent with a direct role for ScEcR-A in DNA amplification.

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Abbreviations

DAPI:

4′,6-Diamidino-2-phenylindole

EcR:

Ecdysone receptor

EcRE:

Ecdysone response element

ORC:

Origin recognition complex

ORI:

Origin of replication

Pol II:

RNA polymerase II

USP:

Ultraspiracle

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Acknowledgments

G.M.L. was supported by a fellowship from the Agency for Science, Technology and Research (A*STAR), Singapore.

Ethical Standards

All experiments presented herein were performed in compliance with the current laws of the USA.

Conflict of Interest

Gerald M. Liew, Michael S. Foulk, and Susan A. Gerbi declare that they have no conflict of interest. This article does not contain any studies with human or vertebrate animal subjects performed by any of the authors.

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Authors and Affiliations

  1. Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, 02912, USA

    Gerald M. Liew, Michael S. Foulk & Susan A. Gerbi

  2. Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, 94305, USA

    Gerald M. Liew

Authors
  1. Gerald M. Liew
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  2. Michael S. Foulk
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  3. Susan A. Gerbi
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Corresponding author

Correspondence to Susan A. Gerbi.

Additional information

Responsible Editor: Wendy Bickmore.

This paper is dedicated to Natalia Gabrusewycz-Garcia whose beautiful work identified the DNA puffs in Sciara polytene chromosomes.

Electronic supplementary material

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Online Resource 1

A long exposure of the ScEcR-A developmental Western blot. Labeled the same as in Fig. 1a (JPEG 14 kb)

High resolution image (TIFF 1,275 kb)

Online Resource 2

Control experiments demonstrate the specificity of the ScEcR-A antibody. a, b, c Chromosome squash from 14 × 7 eyespot stage larvae incubated with pre-immune serum and Alexa Fluor 488 dye-conjugated goat anti-rabbit IgG. Arrows point to fully expanded DNA puffs on chromosome III that are characteristic of the 14 × 7 stage but are not stained in this control experiment. d, e, f Chromosome squash from 14 × 7 eyespot stage larvae incubated only with secondary antibody (1:1,000 in PBT) shows no chromosomal staining. Arrows point to fully expanded DNA puffs. g, h, i Chromosome squash from early eyespot stage larvae incubated with a polyclonal antibody to ScEcR-A (primary antibody; 1:2,500 in PBT), secondary antibody; 1:1,000 in PBT. Arrow indicates a ScEcR-A signal at IV/19A, the only weak signal to appear on any chromosome if any signal was detected at all at the early eyespot stage. j, k, l Chromosome squash prepared from pre-amplification stage larvae mock-injected with 32 nl 50 % ethanol (containing 25 mg/ml of blue dextran as a tracer) at 18 h post-injection. Parentheses around a label indicate that no ScEcR-A signal was observed at that locus during a specific stage. Bar represents 50 μm (JPEG 1.92 mb)

High resolution image (TIFF 7.45 mb)

Online Resource 3

Exposure time required to detect ScEcR-A signal at stage 8 × 4 is 4.5 times longer than other stages examined. Average exposure time required for detection of ScEcR-A signal across different developmental stages examined. Reacted preparations were examined under a Zeiss Axiovert 200M microscope and photographed with a 40X objective. The accompanying AxioVision software was used to optimize exposure times automatically according to the immunostaining intensity of each chromosome preparation. These exposure times were then tabulated and summarized for each developmental stage. (JPEG 35 kb)

High resolution image (TIFF 148 kb)

Online Resource 4, 5 and 6

Little developmental variability in ScEcR-A immunofluorescence experiments. Examination of many immunofluorescence preparations from different larvae to assay for developmental variability demonstrated the reproducibility of the ScEcR-A signals. Each figure depicts chromosomes representative of the entire range seen during the eyespot stage indicated (Online Resource 4: 10 × 5; Online Resource 5: 12 × 6; Online Resource 6: 14 × 7). For brevity, only chromosome II is shown. a, d, g, j DAPI. b, e, h, k ScEcR-A. c, f, i, l Merged DAPI and ScEcR-A. Yellow arrows point to DNA puffs, red arrows point to RNA puffs, and white arrows point to prominent examples of “orphan signals” (see text). Bar represents 50 μm. (JPEG 1.90 mb)

High resolution image (TIFF 5.60 mb)

Online Resource 5

(JPEG 1.91 mb)

High resolution image (TIFF 5.79 mb)

Online Resource 6

(JPEG 1.87 mb)

High resolution image (TIFF 3.97 mb)

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Liew, G.M., Foulk, M.S. & Gerbi, S.A. The ecdysone receptor (ScEcR-A) binds DNA puffs at the start of DNA amplification in Sciara coprophila . Chromosome Res 21, 345–360 (2013). https://doi.org/10.1007/s10577-013-9360-1

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