Skip to main content
SpringerLink
Log in

Ubiquitinated proteins including uH2A on the human and mouse inactive X chromosome: enrichment in gene rich bands

  • Research Article
  • Published:
Chromosoma Aims and scope Submit manuscript

Abstract

The inactive X chromosome (Xi) forms a heterochromatic structure in the nucleus that is known to have several modifications to specific histones involving acetylation or methylation. Using three different antibodies in four different cell lines, we demonstrate that the Xi in human and mouse cells is highly enriched in ubiquitinated protein(s), much of which is polyubiquitinated. This ubiquitination appears specific for the Xi as it was not observed for centromeres or other regions of heterochromatin. Results using an antibody specific to ubiquitinated H2A provide a clear link between H2A ubiquitination and gene repression, as visualized across an entire inactive chromosome. Interestingly, the ubiquitination of the chromosome persists into mitosis and can be seen in a reproducible banded pattern. This pattern matches that of Xist RNA which forms bands as it detaches from the mitotic X chromosome. Both ubiquitination and Xist RNA appear enriched in gene dense regions and depleted in gene poor bands, but do not correlate with L1 LINE elements which have been suggested as key to X-inactivation. These results provide evidence that ubiquitination along with Xist RNA plays an important role in the formation of facultative heterochromatin during X-inactivation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Aguilar RC, Wendland B (2003) Ubiquitin: not just for proteasomes anymore. Curr Opin Cell Biol 15:184–190

    Article  CAS  PubMed  Google Scholar 

  • Bach I, Ostendorff HP (2003) Orchestrating nuclear functions: ubiquitin sets the rhythm. Trends Biochem Sci 28:189–195

    Article  CAS  PubMed  Google Scholar 

  • Bailey JA, Carrel L, Chakravarti A, Eichler EE (2000) Molecular evidence for a relationship between LINE-1 elements and X chromosome inactivation: the Lyon repeat hypothesis. Proc Natl Acad Sci USA 97:6634–6639

    Article  CAS  PubMed  Google Scholar 

  • 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 USA 87:7757–7761

    CAS  PubMed  Google Scholar 

  • Chadwick BP, Willard HF (2001) Histone H2A variants and the inactive X chromosome: identification of a second macroH2A variant. Hum Mol Genet 10:1101–1113

    Article  CAS  PubMed  Google Scholar 

  • Chadwick BP, Willard HF (2002) Cell cycle-dependent localization of macroH2A in chromatin of the inactive X chromosome. J Cell Biol 157:1113–1123

    Article  CAS  PubMed  Google Scholar 

  • Chadwick BP, Willard HF (2003) Barring gene expression after XIST: maintaining facultative heterochromatin on the inactive X. Semin Cell Dev Biol 14:359–367

    Article  CAS  PubMed  Google Scholar 

  • Chen HY, Sun JM, Zhang Y, Davie JR, Meistrich ML (1998) Ubiquitination of histone H3 in elongating spermatids of rat testes. J Biol Chem 273:13165–13169

    Article  CAS  PubMed  Google Scholar 

  • Chow JC, Brown CJ (2003) Forming facultative heterochromatin: silencing of an X chromosome in mammalian females. Cell Mol Life Sci 60:2586–2603

    Article  CAS  PubMed  Google Scholar 

  • Chureau C, Prissette M, Bourdet A, Barbe V, Cattolico L, Jones L, Eggen A, Avner P, Duret L (2002) Comparative sequence analysis of the X-inactivation center region in mouse, human, and bovine. Genome Res 12:894–908

    CAS  PubMed  Google Scholar 

  • Clemson CM, McNeil JA, Willard HF, Lawrence JB (1996) XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure. J Cell Biol 132:259–275

    Article  CAS  PubMed  Google Scholar 

  • Costanzi C, Pehrson JR (1998) Histone macroH2A1 is concentrated in the inactive X chromosome of female mammals. Nature 393:599–601

    Article  CAS  PubMed  Google Scholar 

  • Davie JR, Delcuve GP, Nickel BE, Moirier R, Bailey G (1987) Reduced levels of histones H1o and H1b, and unaltered content of methylated DNA in rainbow trout hepatocellular carcinoma chromatin. Cancer Res 47:5407–5410

    CAS  PubMed  Google Scholar 

  • Duthie SM, Nesterova TB, Formstone EJ, Keohane AM, Turner BM, Zakian SM, Brockdorff N (1999) Xist RNA exhibits a banded localization on the inactive X chromosome and is excluded from autosomal material in cis. Hum Mol Genet 8:195–204

    Article  CAS  PubMed  Google Scholar 

  • Fey EG, Ornelles DA, Penman S (1986) Association of RNA with the cytoskeleton and the nuclear matrix. J Cell Sci Suppl 5:99–119

    CAS  PubMed  Google Scholar 

  • Fujimuro M, Sawada H, Yokosawa H (1994) Production and characterization of monoclonal antibodies specific to multi-ubiquitin chains of polyubiquitinated proteins. FEBS Lett 349:173–180

    Article  CAS  PubMed  Google Scholar 

  • Ganesan S, Silver DP, Greenberg RA, Avni D, Drapkin R, Miron A, Mok SC, Randrianarison V, Brodie S, Salstrom J, Rasmussen TP, Klimke A, Marrese C, Marahrens Y, Deng CX, Feunteun J, Livingston DM (2002) BRCA1 supports XIST RNA concentration on the inactive X chromosome. Cell 111:393–405

    Article  CAS  PubMed  Google Scholar 

  • Goldknopf IL, Taylor CW, Baum RM, Yeoman LC, Olson MO, Prestayko AW, Busch H (1975) Isolation and characterization of protein A24, a histone-like non-histone chromosomal protein. J Biol Chem 250:7182–7187

    CAS  PubMed  Google Scholar 

  • Hall LL, Lawrence JB (2003) The cell biology of a novel chromosomal RNA: chromosome painting by XIST/Xist RNA initiates a remodeling cascade. Semin Cell Dev Biol 14:369–378

    Article  CAS  PubMed  Google Scholar 

  • Hall LL, Byron M, Sakai K, Carrel L, Willard HF, Lawrence JB (2002a) An ectopic human XIST gene can induce chromosome inactivation in postdifferentiation human HT-1080 cells. Proc Natl Acad Sci USA 99:8677–8682

    Article  CAS  PubMed  Google Scholar 

  • Hall LL, Clemson CM, Byron M, Wydner K, Lawrence JB (2002b) Unbalanced X;autosome translocations provide evidence for sequence specificity in the association of XIST RNA with chromatin. Hum Mol Genet 11:3157–3165

    Article  CAS  PubMed  Google Scholar 

  • Hashizume R, Fukuda M, Maeda I, Nishikawa H, Oyake D, Yabuki Y, Ogata H, Ohta T (2001) The RING heterodimer BRCA1-BARD1 is a ubiquitin ligase inactivated by a breast cancer-derived mutation. J Biol Chem 276:14537–14540

    Article  CAS  PubMed  Google Scholar 

  • Heard E, Rougeulle C, Arnaud D, Avner P, Allis CD, Spector DL (2001) Methylation of Histone H3 at Lys-9 Is an Early Mark on the X Chromosome during X Inactivation. Cell 107:727–738

    Article  CAS  PubMed  Google Scholar 

  • Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67:425–479

    Article  CAS  PubMed  Google Scholar 

  • Hicke L (2001) Protein regulation by monoubiquitin. Nat Rev Mol Cell Biol 2:195–201

    Article  CAS  PubMed  Google Scholar 

  • Jason LJ, Moore SC, Lewis JD, Lindsey G, Ausio J (2002) Histone ubiquitination: a tagging tail unfolds? Bioessays 24:166–174

    Article  CAS  PubMed  Google Scholar 

  • Jenuwein T, Allis CD (2001) Translating the histone code. Science 293:1074–1080

    Article  CAS  PubMed  Google Scholar 

  • Johnson CV, Singer RH, Lawrence JB (1991) Fluorescent detection of nuclear RNA and DNA: implication for genome organization. Methods Cell Biol 35:73–99

    CAS  PubMed  Google Scholar 

  • Keohane AM, O’Neill LP, Belyaev ND, Lavender JS, Turner BM (1996) X-inactivation and histone H4 acetylation in embryonic stem cells. Dev Biol 180:618–630

    Article  CAS  PubMed  Google Scholar 

  • Lorick KL, Jensen JP, Fang S, Ong AM, Hatakeyama S, Weissman AM (1999) RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Proc Natl Acad Sci USA 96:11364–11369

    Article  CAS  PubMed  Google Scholar 

  • Lyon MF (1998) X-chromosome inactivation: a repeat hypothesis. Cytogenet Cell Genet 80:133–137

    Article  CAS  PubMed  Google Scholar 

  • Nickel BE, Davie JR (1989) Structure of polyubiquitinated histone H2A. Biochemistry 28:964–968

    CAS  PubMed  Google Scholar 

  • Panning B, Jaenisch R (1998) RNA and the epigenetic regulation of X chromosome inactivation. Cell 93:305–308

    Article  CAS  PubMed  Google Scholar 

  • Pham AD, Sauer F (2000) Ubiquitin-activating/conjugating activity of TAFII250, a mediator of activation of gene expression in Drosophila. Science 289:2357–2360

    Article  CAS  PubMed  Google Scholar 

  • Pickart CM (2001) Mechanisms underlying ubiquitination. Annu Rev Biochem 70:503–533

    Article  CAS  PubMed  Google Scholar 

  • Riggs S, J S-S, Keith DH (1985) Methylation of the PGK promoter region and an enhancer way-station model for X-chromosome inactivation. In Sandberg AA (ed) Biochemistry and biology of DNA methylation, Alan R. Liss, New York, pp 211–222

    Google Scholar 

  • Rougeulle C, Chaumeil J, Sarma K, Allis CD, Reinberg D, Avner P, Heard E (2004) Differential histone H3 Lys-9 and Lys-27 methylation profiles on the X chromosome. Mol Cell Biol 24:5475–5484

    Article  CAS  PubMed  Google Scholar 

  • Ruffner H, Joazeiro CA, Hemmati D, Hunter T, Verma IM (2001) Cancer-predisposing mutations within the RING domain of BRCA1: loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity. Proc Natl Acad Sci USA 98:5134–5139

    Article  CAS  PubMed  Google Scholar 

  • Schnell JD, Hicke L (2003) Non-traditional functions of ubiquitin and ubiquitin-binding proteins. J Biol Chem 278:35857–35860

    Article  CAS  PubMed  Google Scholar 

  • Tam R, Johnson C, Shopland L, McNeil J, Lawrence JB (2002) Applications of RNA FISH for visualizing gene expression and nuclear architecture. In: Beatty BG, Mai S, Squire JA (eds) FISH. Oxford University Press, Oxford, pp 93–118

    Google Scholar 

  • Vassilev AP, Rasmussen HH, Christensen EI, Nielsen S, Celis JE (1995) The levels of ubiquitinated histone H2A are highly upregulated in transformed human cells: partial colocalization of uH2A clusters and PCNA/cyclin foci in a fraction of cells in S-phase. J Cell Sci 108(Pt 3):1205–1215

    CAS  PubMed  Google Scholar 

  • Wang H, Wang L, Erdjument-Bromage H, Vidal M, Tempst P, Jones RS, Zhang Y (2004) Role of histone H2A ubiquitination in Polycomb silencing. Nature 431:873–878

    Article  CAS  PubMed  Google Scholar 

  • West MH, Bonner WM (1980) Histone 2B can be modified by the attachment of ubiquitin. Nucleic Acids Res 8:4671–4680

    CAS  PubMed  Google Scholar 

  • Wu-Baer F, Lagrazon K, Yuan W, Baer R (2003) The BRCA1/BARD1 heterodimer assembles polyubiquitin chains through an unconventional linkage involving lysine residue K6 of ubiquitin. J Biol Chem 278:34743–34746

    Article  CAS  PubMed  Google Scholar 

  • Zhang Y (2003) Transcriptional regulation by histone ubiquitination and deubiquitination. Genes Dev 17:2733–2740

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank John McNeil for assistance with the bioinformatic analysis and Barbara Panning and Hunt Willard for reagents used in this study. This work was made possible by NIH Public Health Service grant R01 GM-053234 to J.B.L. The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

Author information

Authors and Affiliations

  1. Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA, 01655, USA

    Kelly P. Smith, Meg Byron, Christine M. Clemson & Jeanne B. Lawrence

Authors
  1. Kelly P. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Meg Byron
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christine M. Clemson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jeanne B. Lawrence
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jeanne B. Lawrence.

Additional information

Communicated by S.A. Gerbi

Rights and permissions

Reprints and permissions

About this article

Cite this article

Smith, K.P., Byron, M., Clemson, C.M. et al. Ubiquitinated proteins including uH2A on the human and mouse inactive X chromosome: enrichment in gene rich bands. Chromosoma 113, 324–335 (2004). https://doi.org/10.1007/s00412-004-0325-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00412-004-0325-1

Keywords

Search

Navigation