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Unusual chromatin state in Rhynchosciara americana (Diptera: Sciaridae)

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Abstract

Euchromatin and heterochromatin are usually defined by the degree of DNA compaction, gene content and combinations of histone and non-histone proteins. More recent studies on protein location have been able to specify a variety of chromatin types thus adding chromatin configurations other than the two basic reference states. Chromatin research exploiting non-model organisms has the potential to provide novel information related to epigenetic modifications and their impact on chromosome structure and function. Polytene chromosomes of Rhynchosciara americana display a particular region within the A9 sub-section characterised by lack of DNA compaction as well as an usual polytene banding pattern. DNA content in the sub-section seems to be low as deduced by DAPI staining. Antibodies to H3K4me, a conserved epigenetic transcription marker, labelled the A9 sub-section strongly. In contrast, transcriptional activity in the region, if any, seems to be low as inferred by detection of RNA polymerase II and RNA. Histone markers related to heterochromatin formation such as H3K9me and H3K27me are underrepresented in the A9 sub-section. However, a chromodomain-containing sciarid protein was detected in the region, displaying levels of fluorescence very close to those observed in pericentric heterochromatin. A plasmid micro-library constructed with micro-dissected DNA from the A9 sub-section was screened for repetitive DNA. The proportion of inserts containing repeats was found to be similar to that contained in another micro-library made with DNA from a single chromosome end of this species. The data suggest an unusual “chromatin colour” indicating that high levels of histone markers related to transcription coexist with a significant presence of chromodomain-containing proteins and the virtual absence of histone modifications observed in heterochromatin formation.

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Abbreviations

CCD:

Charged coupled device

DAPI:

4′,6-diamidino-2-phenylindole

DOP-PCR:

Degenerate oligonucleotide primed PCR

dNTP:

Deoxy-ribonucleotide 5′-triphosphate

dUTP:

2′-deoxy-uridine 5′-triphosphate

FITC:

Fluorescein isothiocyanate

H3K27ac:

Histone H3 acetylated at lysine 27

H3K4me2:

Histone H3 dimethylated at lysine 4

H3K4me3:

Histone H3 trimethylated at lysine 4

H3K9me1:

Histone H3 monomethylated at lysine 9

H3K9me2:

Histone H3 dimethylated at lysine 9

H3K9me3:

Histone H3 trimethylated at lysine 9

H3K27me1:

Histone H3 monomethylated at lysine 27

H3K27me2:

Histone H3 dimethylated at lysine 27

H3K27me3:

Histone H3 trimethylated at lysine 27

IgG:

Immunoglobulin

Kbp:

Kilobase pairs

PCR:

Polymerase chain reaction

SDS:

Sodium dodecyl sulfate

SSC:

Sodium saline citrate

TBS:

Tris-buffered saline

TBST:

Tris buffered saline, 0.1 % Triton X-100

TRITC:

Tetramethyl rhodamine isothiocyanate

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Acknowledgments

The authors thank Dr. C. Goday for the gift of antibodies to Sciara proteins, Dr. G. J. Filion and an anonymous referee for valuable comments on the manuscript and Dr. A. J. Stocker for critical reading of the text. This work was supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo).

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Authors and Affiliations

  1. Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, Rua do Matão 277, Cidade Universitária, São Paulo, SP, CEP: 05508-090, Brazil

    Alejandra Badaracco & Eduardo Gorab

Authors
  1. Alejandra Badaracco
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  2. Eduardo Gorab
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Correspondence to Eduardo Gorab.

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Ethical standards

The experiments comply with Brazilian current laws.

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The authors declare that they have no competing interests.

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Responsible Editor: Wendy Bickmore

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ESM 1

Polytene chromosomes A and X of R. americana after tritiated uridine incorporation (modified from Santelli 1972). The vertical bar points to the extension of the A9 sub-section (GIF 197 kb)

High resolution image (TIFF 2471 kb)

ESM 2

Distribution of chromodomain-containing protein in R. americana polytene chromosomes. a Indirect immunofluorescence. b DAPI staining. c Superimposed images from a and b. Pericentric regions of the four chromosomes are indicated (A10-12, B15, C11, X12) (GIF 57 kb)

High resolution image (TIFF 877 kb)

ESM 3

(a) Plasmid DNAs (1–11) were cut with EcoRI and analysed in an agarose gel. 1: pRaA-37; 2; 3: pRaA-46; 9: pRa1-11. DNA from the gel was transferred and (b) hybridised to salivary gland DNA of R. americana. Hybridisation results of pRaA-7, pRaA-35 and pRaA-42 were obtained in another membrane (data not shown) (GIF 85 kb)

High resolution image (TIFF 1010 kb)

ESM 4

a Sequence alignment with the pRaA-11 and part of the pRa-43 (Rossato et al. 2007). b, c pRaA-7 and pRaA-35 insert sequences. d Protein alignments using conceptual translation from part of pRaA-7 and pRaA-35 insert sequences with GL22367 (Drosophila persimilis) and GF21664 (Drosophila ananassae) sequences. e Protein alignments using GL22367 and AJ278684.1 (D. melanogaster) sequences. f pRaA-37 insert sequence and the corresponding BLAST result. g BLAST alignment with pRaA-37 and H. melpomene sequences. h pRaA-42 insert sequence showing below the underlined nucleotides aligned with mariner sequences. i pRaA-46 insert sequence and the corresponding BLAST search result (PDF 22 kb)

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Badaracco, A., Gorab, E. Unusual chromatin state in Rhynchosciara americana (Diptera: Sciaridae). Chromosome Res 23, 781–790 (2015). https://doi.org/10.1007/s10577-015-9497-1

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