Chromosome Research

, Volume 24, Issue 3, pp 309–323 | Cite as

LINE-related component of mouse heterochromatin and complex chromocenters’ composition

  • Inna S. Kuznetsova
  • Dmitrii I. Ostromyshenskii
  • Alexei S. Komissarov
  • Andrei N. Prusov
  • Irina S. Waisertreiger
  • Anna V. Gorbunova
  • Vladimir A. Trifonov
  • Malcolm A. Ferguson-Smith
  • Olga I. Podgornaya


Chromocenters are interphase nuclear landmark structures of constitutive heterochromatin. The tandem repeat (TR)-enriched parts of different chromosomes cluster together in chromocenters. There has been progress in recent years in determining the protein content of chromocenters, although it is not clear which DNA sequences underly constitutive heterochromatin apart from the TRs. The aim of the current work was to find out which DNA sequences besides TRs are involved in chromocenters’ formation. Biochemically isolated chromocenters and microdissected centromeric regions were amplified by DOP-PCR, then cloned and sequenced. Alignment to Repbase, the mouse reference genome and WGS databases separated the sequences from both libraries into three groups: (1) sequences with similarity to pericentromere mouse major satellite; (2) sequences without similarity to any repetitive sequences; (3) sequences with similarity to long interspersed nuclear elements (LINEs). LINE-related sequences have a disperse pattern distribution on chromosomes predicted in silico. Selected clones were used for fluorescent in situ hybridization (FISH). The 10 clones tested hybridized to chromocenters and centromeric regions of metaphase chromosomes. These clones were used for double FISH with four known cloned TRs (satDNA, satellite DNA) and a probe specific for the sex chromosomes. The probes bind various chromocenters’ regions without overlapping; so, FISH results reveal a complex chromocenter composition. We mapped 18 LINE-derived clones to the RepBase L1 records. Most of them grouped in a ∼2-kb region at the end of the second ORF and 3′ untranslated region (UTR). So, even the limited number of the clones allows us to determine the region of the L1 element that is specific for heterochromatic regions. Although the L1 full-length probe did not hybridize at detectable levels to the heterochromatic region on any chromosome, the 2-kb fragment found is definitely a part of these regions. The precise LINE ∼2-kb fragment is the component of mouse and human constitutive heterochromatin enriched with TRs. The method used for amplification of the probes from two sources of the heterochromatic material uncovered the enrichment of a precise fragment of LINE within chromocenters.


Mouse genome Heterochromatin Tandem repeat LINE Bioinformatics analysis Fluorescent in situ hybridization (FISH) 





Chromocenters isolated by the biochemical approach


4′, 6-diami-dino-2-phenylindole


PCR with DOP primer described in the Material and methods section


Fluorescent in situ hybridization


Golden Path Gap, 3 Mb empty region around each centromere in assembled genome


Microdissected centromeric DNA


Mouse embryo fibroblast from C3H line

MiSat and MaSat

Centromeric minor and pericentromeric major satellites

MS3 and MS4

Mouse satellite 3 and 4, respectively


Long interspersed nuclear element


Pericentromeric heterochromatin


Satellite DNA


Short interspersed nuclear element


Transposable elements


Tandem repeat



The authors are entirely grateful to the anonymous reviewers for the very professional and helpful comments. This work was supported by the Russian Foundation for Basic Research (grant nos. 05-04-49156-а, 11-04-01700), the Russian Science Foundation (grant no.15-15-20026), Saint-Petersburg State University (grant no. and the granting program of “Molecular and Cell Biology” of the Presidium of Russian Academy of Sciences (no. 01.2.014571). We would like to thank Prof. Eugene D. Ponomarev (The Chinese University of Hong Kong) for the help with English corrections. Editing and publishing costs have been paid for by a grant from the Russian Science Foundation (grant no.15-15-20026).

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Ethical standards

Mice were housed and maintained according to the approved standards in the Laboratory Animal Resources facility at Institute of Cytology RAS (St Petersburg, Russia).

Supplementary material

10577_2016_9525_MOESM1_ESM.doc (2 mb)
Fig. S1 (DOC 2012 kb)
10577_2016_9525_MOESM2_ESM.doc (102 kb)
Table S1 (DOC 102 kb)


  1. Abdurashitov MA, Chernukhin VA, Gonchar DA, Degtyarev SK (2009) GlaI digestion of mouse γ-satellite DNA: study of primary structure and ACGT sites methylation. BMC Genomics 10(1):322CrossRefPubMedPubMedCentralGoogle Scholar
  2. Ahmed M, Liang P (2012) Transposable elements are a significant contributor to tandem repeats in the human genome. Comp Funct Genomics 2012Google Scholar
  3. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410CrossRefPubMedGoogle Scholar
  4. Andrey P, Kiêu K, Kress C et al (2010) Statistical analysis of 3D images detects regular spatial distributions of centromeres and chromocenters in animal and plant nuclei. PLoS Comput Biol 6(7):e1000853CrossRefPubMedPubMedCentralGoogle Scholar
  5. Arneson N, Hughes S, Houlston R, Done S (2008) Whole-genome amplification by degenerate oligonucleotide primed PCR (DOP-PCR). Cold Spring Harb Protoc 2008(1):pdb-prot4919Google Scholar
  6. Benson G (1999) Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res 27(2):573CrossRefPubMedPubMedCentralGoogle Scholar
  7. Boyle AL, Ballard SG, Ward DC (1990) Differential distribution of long and short interspersed element sequences in the mouse genome: chromosome karyotyping by fluorescence in situ hybridization. Proc Natl Acad Sci 87(19):7757–7761CrossRefPubMedPubMedCentralGoogle Scholar
  8. Broccoli D, Miller OJ, Miller DA (1990) Relationship of mouse minor satellite DNA to centromere activity. Cytogenet Genome Res 54(3-4):182–186CrossRefGoogle Scholar
  9. Broccoli D, Trevor KT, Miller OJ, Miller DA (1991) Isolation of a variant family of mouse minor satellite DNA that hybridizes preferentially to chromosome 4. Genomics 10(1):68–74CrossRefPubMedGoogle Scholar
  10. Carter NP, Bebb CE, Nordenskjo M, Ponder BA, Tunnacliffe A (1992) Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer. Genomics 13(3):718–725CrossRefPubMedGoogle Scholar
  11. Chinwalla AT, Cook LL, Delehaunty KD et al (2002) Initial sequencing and comparative analysis of the mouse genome. Nature 420(6915):520–562CrossRefPubMedGoogle Scholar
  12. Cooke HJ, Brown WAR (1984) Closely related sequences on human X and Y chromosomes outside the pairing region. Nature 311:259–261CrossRefPubMedGoogle Scholar
  13. Cooke HJ, Brown WR, Rappold GA (1985) Hypervariable telomeric sequences from the human sex chromosomes are pseudoautosomal. Nature 317:687–692CrossRefPubMedGoogle Scholar
  14. de Koning AP, Gu W, Castoe TA, Batzer MA, Pollock DD (2011) Repetitive elements may comprise over two-thirds of the human genome. PLoS Genet 7(12):e1002384CrossRefPubMedPubMedCentralGoogle Scholar
  15. Dernburg AF (2012) DOP-PCR amplification of probe DNA for whole-mount FISH in drosophila. Cold Spring Harb Protoc 2012(3):pdb-prot067306PubMedGoogle Scholar
  16. Elgin SC, Reuter G (2013) Position-effect variegation, heterochromatin formation, and gene silencing in Drosophila. Cold Spring Harb Perspect Biol 5(8):a017780CrossRefPubMedPubMedCentralGoogle Scholar
  17. Galaktionov NK, Solovyeva AI, Fedorov AV, Podgornaya OI (2014) Trematode Himasthla elongata mariner element (Hemar): structure and applications. J Exp Zool B Mol Dev Evol 322(3):142–155CrossRefPubMedGoogle Scholar
  18. Garagna S, Redi CA, Capanna E, Andayani N, Alfano RM, Viale G (1993) Genome distribution, chromosomal allocation, and organization of the major and minor satellite DNAs in 11 species and subspecies of the genus Mus. Cytogenet Genome Res 64(3-4):247–255CrossRefGoogle Scholar
  19. Guenatri M, Bailly D, Maison C, Almouzni G (2004) Mouse centric and pericentric satellite repeats form distinct functional heterochromatin. J Cell Biol 166(4):493–505CrossRefPubMedPubMedCentralGoogle Scholar
  20. Guo W, Wu H (2008) Metaphase preparation from murine bone. Exchange. doi: 10.1038/nprot.2008.16 Google Scholar
  21. Heitz E (1929) Heterochromatin, chromocentren. Chromomeren Berichte der Deutschen Botanischen Gesellschaft 47:274–284Google Scholar
  22. Helgason CD, Miller CL (eds) (2005) Basic cell culture protocols (Vol. 1). Humana PressGoogle Scholar
  23. Kipling D, Ackford HE, Taylor BA, Cooke HJ (1991) Mouse minor satellite DNA genetically maps to the centromere and is physically linked to the proximal telomere. Genomics 11(2):235–241CrossRefPubMedGoogle Scholar
  24. Kipling D, Wilson HE, Mitchell AR, Taylor BA, Cooke HJ (1994) Mouse centromere mapping using oligonucleotide probes that detect variants of the minor satellite. Chromosoma 103(1):46–55CrossRefPubMedGoogle Scholar
  25. Kipling D, Mitchell AR, Masumoto H, Wilson HE, Nicol L, Cooke HJ (1995) CENP-B binds a novel centromeric sequence in the Asian mouse Mus caroli. Mol Cell Biol 15(8):4009–4020CrossRefPubMedPubMedCentralGoogle Scholar
  26. Kohany O, Gentles AJ, Hankus L, Jurka J (2006) Annotation, submission and screening of repetitive elements in RepBase: RepBase submitter and censor. BMC Bioinf 7(1):474CrossRefGoogle Scholar
  27. Komissarov AS, Gavrilova EV, Demin SJ, Ishov AM, Podgornaya OI (2011) Tandemly repeated DNA families in the mouse genome. BMC Genomics 12(1):531CrossRefPubMedPubMedCentralGoogle Scholar
  28. Kuznetsova IS, Prusov AN, Enukashvily NI, Podgornaya OI (2005) New types of mouse centromeric satellite DNAs. Chromosom Res 13(1):9–25CrossRefGoogle Scholar
  29. Kuznetsova I, Podgornaya O, Ferguson-Smith MA (2006) High-resolution organization of mouse centromeric and pericentromeric DNA. Cytogenet Genome Res 112(3-4):248–255CrossRefPubMedGoogle Scholar
  30. Kuznetsova IS, Enukashvily NI, Noniashvili EM et al (2007) Evidence for the existence of satellite DNA‐containing connection between metaphase chromosomes. J Cell Biochem 101(4):1046–1061CrossRefPubMedGoogle Scholar
  31. Miga KH, Newton Y, Jain M, Altemose N, Willard HF, Kent WJ (2014) Centromere reference models for human chromosomes X and Y satellite arrays. Genome Res 24(4):697–707CrossRefPubMedPubMedCentralGoogle Scholar
  32. Moens PB, Pearlman RE (1990) Telomere and centromere DNA are associated with the cores of meiotic prophase chromosomes. Chromosoma 100(1):8–14CrossRefPubMedGoogle Scholar
  33. Morris CA, Moazed D (2007) Centromere assembly and propagation. Cell 128(4):647–650CrossRefPubMedGoogle Scholar
  34. Namekawa SH, Payer B, Huynh KD, Jaenisch R, Lee JT (2010) Two-step imprinted X inactivation: repeat versus genic silencing in the mouse. Mol Cell Biol 30(13):3187–3205CrossRefPubMedPubMedCentralGoogle Scholar
  35. Papait R, Pistore C, Grazini U et al (2008) The PHD domain of Np95 (mUHRF1) is involved in large-scale reorganization of pericentromeric heterochromatin. Mol Biol Cell 19(8):3554–3563CrossRefPubMedPubMedCentralGoogle Scholar
  36. Pertile MD, Graham AN, Choo KA, Kalitsis P (2009) Rapid evolution of mouse Y centromere repeat DNA belies recent sequence stability. Genome Res 19(12):2202–2213CrossRefPubMedPubMedCentralGoogle Scholar
  37. Probst AV, Almouzni G (2011) Heterochromatin establishment in the context of genome-wide epigenetic reprogramming. Trends Genet 27(5):177–185CrossRefPubMedGoogle Scholar
  38. Prusov AN, Zatsepina OV (2002) Isolation of the chromocenter fraction from mouse liver nuclei. Biochem Mosc 67(4):423–431CrossRefGoogle Scholar
  39. Radic MZ, Lundgren K, Hamkalo BA (1987) Curvature of mouse satellite DNA and condensation of heterochromatin. Cell 50(7):1101–1108CrossRefPubMedGoogle Scholar
  40. Saksouk N, Simboeck E, Déjardin J (2015) Constitutive heterochromatin formation and transcription in mammals. Epigenetics Chromatin 8(1):3CrossRefPubMedPubMedCentralGoogle Scholar
  41. Sambrook J, Russell DW (2001) Molecular cloning. A laboratory manual. Third. Cold pring Harbor Laboratory Press, New YorkGoogle Scholar
  42. Shatskikh AS, Gvozdev VA (2013) Heterochromatin formation and transcription in relation to trans-inactivation of genes and their spatial organization in the nucleus. Biochem Mosc 78(6):603–612CrossRefGoogle Scholar
  43. Snapp RR, Goveia E, Peet L, Bouffard NA, Badger GJ, Langevin HM (2013) Spatial organization of fibroblast nuclear chromocenters: component tree analysis. J Anat 223(3):255–261CrossRefPubMedPubMedCentralGoogle Scholar
  44. Solovei I, Kreysing M, Lanctôt C et al (2009) Nuclear architecture of rod photoreceptor cells adapts to vision in mammalian evolution. Cell 137(2):356–368CrossRefPubMedGoogle Scholar
  45. Telenius H, Ponder BA, Tunnacliffe A et al (1992) Cytogenetic analysis by chromosome painting using dop‐pcr amplified flow‐sorted chromosomes. Genes Chromosom Cancer 4(3):257–263CrossRefPubMedGoogle Scholar
  46. Vissel B, Choo KH (1989) Mouse major (γ) satellite DNA is highly conserved and organized into extremely long tandem arrays: implications for recombination between nonhomologous chromosomes. Genomics 5(3):407–414CrossRefPubMedGoogle Scholar
  47. Wijchers PJ, Geeven G, Eyres M, et al (2015) Characterization and dynamics of pericentromere-associated domains in mice. Genome Res gr-186643Google Scholar
  48. Wong AKC, Rattner JB (1988) Sequence organization and cytological localization of the minor satellite of mouse. Nucleic Acids Res 16(24):11645–11661CrossRefPubMedPubMedCentralGoogle Scholar
  49. Yang F, Trifonov V, Ng BL, Kosyakova N, Carter NP (2009) Generation of paint probes by flow-sorted and microdissected chromosomes. In Fluorescence In Situ Hybridization (FISH)—Application Guide. Springer Berlin Heidelberg, p 35–52Google Scholar
  50. Yunis JJ, Yasmineh WG (1971) Heterochromatin, satellite DNA, and cell function. Science 174(4015):1200–1209CrossRefPubMedGoogle Scholar
  51. Zatsepina OV, Zharskaya OO, Prusov AN (2008) Isolation of the constitutive heterochromatin from mouse liver nuclei. In The Nucleus. Humana Press, p 169-180Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Inna S. Kuznetsova
    • 1
    • 2
    • 3
  • Dmitrii I. Ostromyshenskii
    • 1
  • Alexei S. Komissarov
    • 1
    • 2
  • Andrei N. Prusov
    • 4
  • Irina S. Waisertreiger
    • 1
  • Anna V. Gorbunova
    • 1
  • Vladimir A. Trifonov
    • 5
  • Malcolm A. Ferguson-Smith
    • 6
  • Olga I. Podgornaya
    • 1
    • 2
    • 7
  1. 1.Institute of Cytology RASSt PetersburgRussia
  2. 2.St. Petersburg State UniversitySt PetersburgRussia
  3. 3.School of Biomedical SciencesThe Chinese University of Hong KongShatinHong Kong
  4. 4.A.N. Belozersky Institute of Physico-Chemical BiologyLomonosov Moscow State UniversityMoscowRussia
  5. 5.Institute of Molecular and Cellular Biology SB RAS, NovosibirskRussia; Novosibirsk State UniversityNovosibirskRussia
  6. 6.Cambridge Resource Centre for Comparative GenomicsUniversity of CambridgeCambridgeUK
  7. 7.Far Eastern Federal UniversityVladivostokRussia

Personalised recommendations