Skip to main content
SpringerLink
Log in

Site-specifically phosphorylated forms of H1.5 and H1.2 localized at distinct regions of the nucleus are related to different processes during the cell cycle

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

Abstract

The cell cycle-associated phosphorylation of histone H1.5 is manifested as three discrete phosphorylated forms, occurring exclusively on Ser17, Ser172, and Ser188 during interphase. During late G2 and mitosis the up-phosphorylation occurs exclusively on threonine at either Thr137 or Thr154 to build the tetraphosphorylated forms of H1.5, whereas the pentaphosphorylated forms result from phosphorylation at Thr10. To determine the kinetic and spatial distribution of histone H1 phosphorylation within the nucleus of synchronized Hela cells we localized three distinct phosphorylation sites of histone subtype H1.5 using affinity-purified polyclonal antibodies generated against phosphorylated Ser17, Ser172, and Thr10. Immunofluorescence labeling of synchronized HeLa cells using the specific antibodies revealed that phosphorylation of H1.5 Ser17 appeared early in G1 at discrete speckles followed by phosphorylation of Ser172. Thr10 phosphorylation started during prophase, showed highest phosphorylation levels during metaphase, and disappeared clearly before chromatin decondensation occurred. Experiments using the kinase inhibitor staurosporine indicate the involvement of different kinases at the various phospho-sites. Colocalization studies revealed that Ser172 phosphorylation of H1.5 and H1.2 does colocalize to DNA replication and transcription sites. These results favor the idea that the various site-specifically phosphorylated forms of H1.5 and H1.2 localized at distinct regions of the nucleus are related to different functions during the cell cycle.

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
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Albig W, Meergans T, Doenecke D (1997) Characterization of the H1.5 gene completes the set of human H1 subtype genes. Gene 184:141–148

    Article  PubMed  CAS  Google Scholar 

  • Alexandrow MG, Hamlin JL (2005) Chromatin decondensation in S-phase involves recruitment of Cdk2 by Cdc45 and histone H1 phosphorylation. J Cell Biol 168:875–886

    Article  PubMed  CAS  Google Scholar 

  • Banks GC, Deterding LJ, Tomer KB, Archer TK (2001) Hormone-mediated dephosphorylation of specific histone H1 isoforms. J Biol Chem 276:36467–36473

    Article  PubMed  CAS  Google Scholar 

  • Bhan S, May W, Warren SL, Sittman DB (2008) Global gene expression analysis reveals specific and redundant roles for H1 variants, H1c and H1(0), in gene expression regulation. Gene 414:10–18

    Article  PubMed  CAS  Google Scholar 

  • Bleher R, Martin R (1999) Nucleo-cytoplasmic translocation of histone H1 during the HeLa cell cycle. Chromosoma 108:308–316

    Article  PubMed  CAS  Google Scholar 

  • Breneman JW, Yau P, Teplitz RL, Bradbury EM (1993) A light microscope study of linker histone distribution in rat metaphase chromosomes and interphase nuclei. Exp Cell Res 206:16–26

    Article  PubMed  CAS  Google Scholar 

  • Buck SB, Bradford J, Gee KR, Agnew BJ, Clarke ST, Salic A (2008) Detection of S-phase cell cycle progression using 5-ethynyl-2'-deoxyuridine incorporation with click chemistry, an alternative to using 5-bromo-2'-deoxyuridine antibodies. Biotechniques 44:927–929

    Article  PubMed  CAS  Google Scholar 

  • Bustin M, Catez F, Lim JH (2005) The dynamics of histone H1 function in chromatin. Mol Cell 17:617–620

    Article  PubMed  CAS  Google Scholar 

  • Cao G, Liu LM, Cleary SF (1991) Modified method of mammalian cell synchronization improves yield and degree of synchronization. Exp Cell Res 193:405–410

    Article  PubMed  CAS  Google Scholar 

  • Chadee DN, Taylor WR, Hurta RA, Allis CD, Wright JA, Davie JR (1995) Increased phosphorylation of histone H1 in mouse fibroblasts transformed with oncogenes or constitutively active mitogen-activated protein kinase kinase. J Biol Chem 270:20098–20105

    Article  PubMed  CAS  Google Scholar 

  • Chen D, Dundr M, Wang C, Leung A, Lamond A, Misteli T, Huang S (2005) Condensed mitotic chromatin is accessible to transcription factors and chromatin structural proteins. J Cell Biol 168:41–54

    Article  PubMed  CAS  Google Scholar 

  • Dimitrova DS, Berezney R (2002) The spatio-temporal organization of DNA replication sites is identical in primary, immortalized and transformed mammalian cells. J Cell Sci 115:4037–4051

    Article  PubMed  CAS  Google Scholar 

  • Dou Y, Gorovsky MA (2000) Phosphorylation of linker histone H1 regulates gene expression in vivo by creating a charge patch. Mol Cell 6:225–231

    Article  PubMed  CAS  Google Scholar 

  • Dou Y, Mizzen CA, Abrams M, Allis CD, Gorovsky MA (1999) Phosphorylation of linker histone H1 regulates gene expression in vivo by mimicking H1 removal. Mol Cell 4:641–647

    Article  PubMed  CAS  Google Scholar 

  • Gorka C, Fakan S, Lawrence JJ (1993) Light and electron microscope immunocytochemical analyses of histone H1(0) distribution in the nucleus of Friend erythroleukemia cells. Exp Cell Res 205:152–158

    Article  PubMed  CAS  Google Scholar 

  • Halmer L, Gruss C (1995) Influence of histone H1 on the in vitro replication of DNA and chromatin. Nucleic Acids Res 23:773–778

    Article  PubMed  CAS  Google Scholar 

  • Happel N, Stoldt S, Schmidt B, Doenecke D (2008) M-phase-specific phosphorylation of histone H1.5 at threonine 10 by GSK-3. J Mol Biol 386:339–350

    Article  PubMed  CAS  Google Scholar 

  • Hassan AB, Errington RJ, White NS, Jackson DA, Cook PR (1994) Replication and transcription sites are colocalized in human cells. J Cell Sci 107:425–434

    PubMed  CAS  Google Scholar 

  • Hellauer K, Sirard E, Turcotte B (2001) Decreased expression of specific genes in yeast cells lacking histone H1. J Biol Chem 276:13587–13592

    PubMed  CAS  Google Scholar 

  • Hendzel MJ, Wei Y, Mancini MA, Van Hooser A, Ranalli T, Brinkley BR, Bazett-Jones DP, Allis CD (1997) Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106:348–360

    Article  PubMed  CAS  Google Scholar 

  • Jackson DA, Hassan AB, Errington RJ, Cook PR (1993) Visualization of focal sites of transcription within human nuclei. EMBO J 12:1059–1065

    PubMed  CAS  Google Scholar 

  • Koop R, Di Croce L, Beato M (2003) Histone H1 enhances synergistic activation of the MMTV promoter in chromatin. EMBO J 22:588–599

    Article  PubMed  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Lennox RW, Cohen LH (1988) The production of tissue-specific histone complements during development. Biochem Cell Biol 66:636–649

    Article  PubMed  CAS  Google Scholar 

  • Lever MA, Th'ng JP, Sun X, Hendzel MJ (2000) Rapid exchange of histone H1.1 on chromatin in living human cells. Nature 408:873–876

    Article  PubMed  CAS  Google Scholar 

  • Lindner H, Sarg B, Helliger W (1997) Application of hydrophilic-interaction liquid chromatography to the separation of phosphorylated H1 histones. J Chromatogr A 782:55–62

    Article  PubMed  CAS  Google Scholar 

  • Lu MJ, Dadd CA, Mizzen CA, Perry CA, McLachlan DR, Annunziato AT, Allis CD (1994) Generation and characterization of novel antibodies highly selective for phosphorylated linker histone H1 in Tetrahymena and HeLa cells. Chromosoma 103:111–121

    PubMed  CAS  Google Scholar 

  • Lu MJ, Mpoke SS, Dadd CA, Allis CD (1995) Phosphorylated and dephosphorylated linker histone H1 reside in distinct chromatin domains in Tetrahymena macronuclei. Mol Biol Cell 6:1077–1087

    PubMed  CAS  Google Scholar 

  • Misteli T, Gunjan A, Hock R, Bustin M, Brown DT (2000) Dynamic binding of histone H1 to chromatin in living cells. Nature 408:877–881

    Article  PubMed  CAS  Google Scholar 

  • Nakamura H, Morita T, Sato C (1986) Structural organizations of replicon domains during DNA synthetic phase in the mammalian nucleus. Exp Cell Res 165:291–297

    Article  PubMed  CAS  Google Scholar 

  • Nakayasu H, Berezney R (1989) Mapping replicational sites in the eucaryotic cell nucleus. J Cell Biol 108:1–11

    Article  PubMed  CAS  Google Scholar 

  • Parseghian MH, Hamkalo BA (2001) A compendium of the histone H1 family of somatic subtypes: an elusive cast of characters and their characteristics. Biochem Cell Biol 79:289–304

    Article  PubMed  CAS  Google Scholar 

  • Parseghian MH, Harris DA, Rishwain DR, Hamkalo BA (1994) Characterization of a set of antibodies specific for three human histone H1 subtypes. Chromosoma 103:198–208

    Article  PubMed  CAS  Google Scholar 

  • Parseghian MH, Newcomb RL, Winokur ST, Hamkalo BA (2000) The distribution of somatic H1 subtypes is non-random on active vs. inactive chromatin: distribution in human fetal fibroblasts. Chromosome Res 8:405–424

    Article  PubMed  CAS  Google Scholar 

  • Parseghian MH, Newcomb RL, Hamkalo BA (2001) Distribution of somatic H1 subtypes is non-random on active vs. inactive chromatin II: distribution in human adult fibroblasts. J Cell Biochem 83:643–659

    Article  PubMed  CAS  Google Scholar 

  • Roque A, Ponte I, Arrondo JL, Suau P (2008) Phosphorylation of the carboxy-terminal domain of histone H1: effects on secondary structure and DNA condensation. Nucleic Acids Res 36:4719–4726

    Article  PubMed  CAS  Google Scholar 

  • Roth SY, Allis CD (1992) Chromatin condensation: does histone H1 dephosphorylation play a role? Trends Biochem Sci 17:93–98

    Article  PubMed  CAS  Google Scholar 

  • Salic A, Mitchison TJ (2008) A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci U S A 105:2415–2420

    Article  PubMed  Google Scholar 

  • Sancho M, Diani E, Beato M, Jordan A (2008) Depletion of human histone H1 variants uncovers specific roles in gene expression and cell growth. PLoS Genet 4:e1000227

    Article  PubMed  CAS  Google Scholar 

  • Sarg B, Helliger W, Talasz H, Forg B, Lindner HH (2006) Histone H1 phosphorylation occurs site-specifically during interphase and mitosis: identification of a novel phosphorylation site on histone H1. J Biol Chem 281:6573–6580

    Article  PubMed  CAS  Google Scholar 

  • Sarg B, Chwatal S, Talasz H, Lindner HH (2009) Testis-specific linker histone H1t is multiply phosphorylated during spermatogenesis: identification of phosphorylation sites. J Biol Chem 284:3610–3618

    Article  PubMed  CAS  Google Scholar 

  • Shen X, Gorovsky MA (1996) Linker histone H1 regulates specific gene expression but not global transcription in vivo. Cell 86:475–483

    Article  PubMed  CAS  Google Scholar 

  • Sweet MT, Jones K, Allis CD (1996) Phosphorylation of linker histone is associated with transcriptional activation in a normally silent nucleus. J Cell Biol 135:1219–1228

    Article  PubMed  CAS  Google Scholar 

  • Takami Y, Nishi R, Nakayama T (2000) Histone H1 variants play individual roles in transcription regulation in the DT40 chicken B cell line. Biochem Biophys Res Commun 268:501–508

    Article  PubMed  CAS  Google Scholar 

  • Talasz H, Helliger W, Puschendorf B, Lindner H (1996) In vivo phosphorylation of histone H1 variants during the cell cycle. Biochemistry 35:1761–1767

    Article  PubMed  CAS  Google Scholar 

  • Talasz H, Sapojnikova N, Helliger W, Lindner H, Puschendorf B (1998) In vitro binding of H1 histone subtypes to nucleosomal organized mouse mammary tumor virus long terminal repeat promotor. J Biol Chem 273:32236–32243

    Article  PubMed  CAS  Google Scholar 

  • Thiriet C, Hayes JJ (2008) Linker histone phosphorylation regulates global timing of replication origin firing. J Biol Chem 284:2823–2829

    Article  PubMed  CAS  Google Scholar 

  • Vermaak D, Steinbach OC, Dimitrov S, Rupp RA, Wolffe AP (1998) The globular domain of histone H1 is sufficient to direct specific gene repression in early Xenopus embryos. Curr Biol 8:533–536

    Article  PubMed  CAS  Google Scholar 

  • Wei X, Somanathan S, Samarabandu J, Berezney R (1999) Three-dimensional visualization of transcription sites and their association with splicing factor-rich nuclear speckles. J Cell Biol 146:543–558

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgment

We thank L. Sattler and Dr. M. Rittinger for their excellent technical assistance. This work, as part of the European Science Foundation EUROCORES Program EuroDYNA, was supported by funds from the Austrian Science Foundation (project I23-B03).

Author information

Authors and Affiliations

  1. Division of Clinical Biochemistry, Biocenter, Innsbruck Medical University, Fritz-Pregl-Str. 3, 6020, Innsbruck, Austria

    Heribert Talasz, Bettina Sarg & Herbert H. Lindner

Authors
  1. Heribert Talasz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bettina Sarg
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Herbert H. Lindner
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Herbert H. Lindner.

Additional information

Communicated by E.A. Nigg

Rights and permissions

Reprints and permissions

About this article

Cite this article

Talasz, H., Sarg, B. & Lindner, H.H. Site-specifically phosphorylated forms of H1.5 and H1.2 localized at distinct regions of the nucleus are related to different processes during the cell cycle. Chromosoma 118, 693–709 (2009). https://doi.org/10.1007/s00412-009-0228-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00412-009-0228-2

Keywords

Search

Navigation