Chromosome Research

, 15:1015 | Cite as

Analysis of heterochromatic epigenetic markers in the holocentric chromosomes of the aphid Acyrthosiphon pisum

Article

Abstract

Monomethylated-K9 H3 histones (Me9H3) and heterochromatin protein 1 (HP1) are reported as heterochromatin markers in several eukaryotes possessing monocentric chromosomes. In order to confirm that these epigenetic markers are evolutionarily conserved, we sequenced the HP1 cDNA and verified the distribution of Me9H3 histones and HP1 in the holocentric chromosomes of the aphid Acyrthosiphon pisum. Sequencing indicates that A. pisum HP1 cDNA (called ApHP1) is 1623 bp long, including a 170 bp long 5′UTR and a 688 bp long 3′UTR. The ApHP1 protein consists of 254 amino acidic residues, has a predicted molecular mass of 28 kDa and a net negative charge. At the structural level, it shows an N terminal chromo domain and a chromo shadow domain at the C terminus linked by a short hinge region. At the cytogenetic level, ApHP1 is located exclusively in the heterochromatic regions of the chromosomes. The same heterochromatic regions were labelled after immuno-staining with antibodies against Me9H3 histones, confirming that Hp1 and Me9H3 co-localize at heterochromatic chromosomal areas. Surprisingly, aphid heterochromatin lacks DNA methylation and methylated cytosine residues were mainly spread at euchromatic regions. Finally, the absence of DNA methylation is observed also in aphid rDNA genes that have been repeatedly described as mosaic of methylated and unmethylated units in vertebrates.

Key words

Acyrthosiphon aphid chromatin heterochromatin histone HP1 methylation 

References

  1. Adams RLP (1996) DNA methylation. In: Adams RLP, ed. Principles of Medical Biology, vol. 5. New York: JAI Press Inc., pp. 33–66.Google Scholar
  2. Bizzaro D, Mandrioli M, Zanotti M, Giusti M, Manicardi GC (2000) Chromosome analysis and molecular characterization of highly repeated DNAs in the aphid Acyrthosiphon pisum (Aphididae, Hemiptera). Genetica 108: 197–02CrossRefPubMedGoogle Scholar
  3. Blackman RL (1987) Reproduction, cytogenetics and development. In: Minsk AK, Harrewijn P, eds. Aphids: Their Biology, Natural Enemies and Control, vol. A. Amsterdam: Elsevier, pp. 163–195.Google Scholar
  4. Bongiorni S, Prantera G (2003) Imprinted facultative heterochromatization in mealybugs. Genetica 117: 271–79.CrossRefPubMedGoogle Scholar
  5. Bongiorni S, Mazzuoli M, Masci S, Prantera G (2001) Facultative heterochromatization in parahaploid male mealybugs: involvement of a heterochromatin-associated protein. Development 128: 3809–817.PubMedGoogle Scholar
  6. Borsatti F, Mandrioli M (2005) Conservation of heterochromatic components and epigenetic markers in the holocentric chromosomes of the cabbage moth, Mamestra brassicae (Lepidoptera). Eur J Entomol 102: 625–32.Google Scholar
  7. Brock GJR, Bird AP (1997) Mosaic methylation of the repeat unit of the human ribosomal RNA genes. Hum Mol Genet 6: 451–56.CrossRefPubMedGoogle Scholar
  8. Cowell IG, Aucott R, Mahadevaiah SK et al. (2002) Heterochromatin, HP1 and methylation at lysine 9 of histone H3 in animals. Chromosoma 111: 22–6.CrossRefPubMedGoogle Scholar
  9. Donlon TA, Magenis RE (1983) Methyl green is a substitute for distamycin A in the formation of distamycin A/DAPI C-bands. Hum Genet 65: 144–46.CrossRefPubMedGoogle Scholar
  10. Eissenberg JC, Elgin SCR (2000) The HP1 protein family: getting a grip on chromatin. Curr Opin Genet Dev 10: 204–10.CrossRefPubMedGoogle Scholar
  11. Eissenberg JC, Ge YW, Hartnett T (1994) Increased phosphorylation of HP1, a heterochromatin-associated protein of Drosophila, is correlated with heterochromatin assembly. J Biol Chem 269: 21315–1321.PubMedGoogle Scholar
  12. Epstein H, James TC, Singh PB (1992) Cloning and expression of Drosophila HP1 homologues form a mealybug, Planococcus citri. J Cell Sci 101: 463–74.PubMedGoogle Scholar
  13. Eskeland R, Eberharter A, Imhof A (2007) HP1 binding to chromatin methylated at H3K9 is enhanced by auxiliary factors. Mol Cell Biol 27: 453–65.CrossRefPubMedGoogle Scholar
  14. Field LM, Lyko F, Mandrioli M, Prantera G (2004) DNA methylation in insects. Insect Mol Biol 13: 109–17.CrossRefPubMedGoogle Scholar
  15. Frohman MA (1990) RACE: Rapid amplification of cDNA ends. In: Innis DA, Gelfand DH, Snisky JJ, eds. PCR Protocols. San Diego: Academic Press, pp. 28–38.Google Scholar
  16. Huang DW, Fanti L, Pak DTS, Botchan MR, Pimpinelli S, Kellum R (1998) Distinct cytoplasmic and nuclear fractions of Drosophila heterochromatin protein 1: their phosphorylation levels and associations with origin recognition complex proteins. J Cell Biol 142: 307–18.CrossRefPubMedGoogle Scholar
  17. Hughes-Schrader S, Schrader F (1961) The kinetocore of the Hemiptera. Chromosoma 12: 327–50.CrossRefPubMedGoogle Scholar
  18. Jacobs SA, Taverna SD, Zhang Y et al. (2001) Specificity of the HP1 chromo domain for the methylated N-terminus of histone H3. EMBO J 20: 5232–241.CrossRefPubMedGoogle Scholar
  19. James TC, Elgin SCR (1986) Identification of non-histone chromosomal protein associated with heterochromatin in Drosophila and its gene. Mol Cell Biol 6: 3862–872.PubMedGoogle Scholar
  20. Jenuwein T, Allis CD (2001) Translating the histone code. Science 293: 1074–080.CrossRefPubMedGoogle Scholar
  21. Jones DO, Cowell IG, Singh PB (2000) Mammalian chromo domain proteins: their role in genome organization and expression. Bioessays 22: 124–37.CrossRefPubMedGoogle Scholar
  22. Kellum R (2003) HP1 complexes and heterochromatin assembly. Curr Top Microbiol Immunol 274: 53–7.PubMedGoogle Scholar
  23. Lachner M, O’Carroll D, Rea S, Mechtler K, Jenuwein T (2001) Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410: 116–20.CrossRefPubMedGoogle Scholar
  24. Mandrioli M (2004) Epigenetic tinkering and evolution: is there any continuity in the functional role of cytosine methylation from invertebrates to vertebrates? Cell Mol Life Sci 61: 2425–427.CrossRefPubMedGoogle Scholar
  25. Mandrioli M, Borsatti F (2006) DNA methylation of fly genes and transposons. Cell Mol Life Sci 63: 1963–966.CrossRefGoogle Scholar
  26. Mandrioli M, Volpi N (2003) The genome of the lepidopteran Mamestra brassicae has a vertebrate-like content of methyl-cytosine. Genetica 119: 187–91.CrossRefPubMedGoogle Scholar
  27. Mandrioli M, Bizzaro D, Gionghi D, Bassoli L, Manicardi GC, Bianchi U (1999) Molecular cytogenetic characterization of a highly repeated DNA sequence in the peach potato aphid Myzus persicae. Chromosoma 108: 436–42.CrossRefPubMedGoogle Scholar
  28. Manicardi GC, Mandrioli M, Bizzaro D, Bianchi U (2002) Cytogenetic and molecular analysis of heterochromatic areas in the holocentric chromosomes of different aphid species. In: Sobti RG, Obe G, Athwal RS, eds. Chromosome Structure and Function. Dordrecht, The Nethelands: Kluwer Academic Publishers, pp. 47–56.Google Scholar
  29. Pfarr W, Webersinke G, Paar C, Wechselberger C (2005) Immunodetection of 5′-methylcytosine on Giemsa-stained chromosomes. Biotechniques 38: 527–30.CrossRefPubMedGoogle Scholar
  30. Rea S, Eisenhaber F, O’Carroll D, Strahl BD et al. (2000) Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406: 593–99.CrossRefPubMedGoogle Scholar
  31. Sabater-Muñoz B, Legeai F, Rispe C et al. (2006) Large-scale gene discovery in the pea aphid Acyrthosiphon pisum (Hemiptera). Genome Biol 7: R21.CrossRefPubMedGoogle Scholar
  32. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning. A laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.Google Scholar
  33. Schweizer D (1976) Reverse fluorescent chromosome banding with chromomycin and DAPI. Chromosoma 58: 307–24.CrossRefPubMedGoogle Scholar
  34. Sumner AT (1972) A simple technique for demonstrating centromeric heterochromatin. Exp Cell Res 75: 304–06.CrossRefPubMedGoogle Scholar
  35. Sumner AT, Taggart MH, Mezzanotte R, Ferrucci L (1990) Patterns of digestion of human chromosomes by restriction endonucleases demonstrated by in situ nick translation. Histochem J 22: 639–52.CrossRefPubMedGoogle Scholar
  36. Tweedie S, Charlton J, Clark V, Bird A (1997) Methylation of genomes and genes at the invertebrate-vertebrate boundary. Mol Cell Biol 17: 1469–475.PubMedGoogle Scholar
  37. Wang G, Ma A, Chow CM et al. (2000) Conservation of heterochromatin protein 1 function. Mol Cell Biol 20: 6970–983.CrossRefPubMedGoogle Scholar

Copyright information

© Springer 2007

Authors and Affiliations

  1. 1.Dipartimento di Biologia AnimaleUniversità di Modena e Reggio EmiliaModenaItaly

Personalised recommendations