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.
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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).
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Communicated by E.A. Nigg
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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
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DOI: https://doi.org/10.1007/s00412-009-0228-2