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.

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. [ABSTRACT FROM AUTHOR]