Generation and characterization of novel antibodies highly selective for phosphorylated linked histone H1 in Tetrahymena and HeLa cells

Abstract

Phosphorylated forms of Tetrahymena macronuclear histone H1 were separated from each other and from dephosphorylated H1 by cation-exchange HPLC. A homogeneous fraction of hyperphosphorylated macronuclear H1 was then used to generate novel polyclonal antibodies highly selective for phosphorylated H1 in Tetrahymena and in human cells. These antibodies fail to recognize dephosphorylated forms of H1 in both organisms and are not reactive with most other nuclear or cytoplasmic phosphoproteins including those induced during mitosis. The selectivity of these antibodies for phosphorylated forms of H1 in Tetrahymena and in HeLa argues strongly that these antibodies recognize highly conserved phosphorylated epitopes found in most H1s and from this standpoint Tetrahymena H1 is not atypical. Using these antibodies in indirect immunofluorescence analyses, we find that a significant fraction of interphase mammalian cells display a strikingly punctate pattern of nuclear fluorescence. As cells enter S-phase, nuclear staining becomes more diffuse, increases significantly, and continues to increase as cells enter mitosis. As cells exit from mitosis, staining with the anti-phosphorylated H1 antibodies is rapidly lost presumably owing to the dephosphorylation of H1. These immunofluorescent data document precisely the cell cycle changes in the level of H1 phosphorylation determined by earlier biochemical studies and suggest that these antibodies represent a powerful new tool to probe the function(s) of H1 phosphorylation in a wide variety of eukaryotic systems.

The Molecular Chaperone Hsp90 Is Required for Cell Cycle Exit in Drosophila melanogaster

“…However, we also wanted to determine if Hsp83 6-55 mutants experience ectopic Cyclin/Cdk activity after cell cycle exit. To test this, we utilized two reporters for Cyclin/Cdk activity: anti-phospho-histone H1 antibodies, which detect phospho-epitopes of multiple Cyclin/Cdk complexes in several systems, but which have been shown to specifically respond to Cyclin E/Cdk2 activity in Drosophila endoreplicating follicle cells [46], [47], and the MPM2 antibody, which detects a small focus of nuclear staining at the histone locus body (HLB) in response to Cyclin E/Cdk2 activity ([48], [49] and Figure 5A–A′). At both early time points after wild-type cells had exited the cell cycle (26–28 hr APF) and a later time point (44 hr APF), Hsp83 6-55 mutant cells exhibited labeling with both of these markers of Cyclin/Cdk activity, whereas the control cells surrounding the Hsp83 6-55 mutant clone did not (Figure 5B–F″).…”

Site-specific regulation of histone H1 phosphorylation in pluripotent cell differentiation

“…Inhibitors thought to be selective for CDK2 such as CVT-313 and roscovitine decreased global levels of phosphorylated H1 and its association with the MMTV promoter [29], and roscovitine and olomoucine diminished H1 phosphorylation at replication foci [87], but several issues affect these studies. Both employed antisera raised against phosphorylated Tetrahymena macronuclear H1, which lacks SPXZ motifs [88], to detect phosphorylated H1 even though the sites in metazoan H1 variants that are recognized by this antisera have not been established. Moreover, CVT-313, roscovitine and olomoucine are now known to inhibit additional CDKs with similar potency [89–92], and prolonged treatments with these drugs can arrest cell cycle progression [91, 93] and consequently affect H1 phosphorylation levels indirectly.…”

DNA Copy-Number Control through Inhibition of Replication Fork Progression

“…Recently, SNF2H has been shown to associate with replication forks, suggesting that chromatin remodeling activity could be important for replication fork progression (Lopez-Contreras et al, 2013; Sirbu et al, 2013). Histone H1 is phosphorylated throughout S phase, and this phosphorylation is thought to decondense histone H1-containing chromatin (Gurley et al, 1978; Lu et al, 1994). Cdc45, a key component of the replicative helicase, may function to recruit Cdk2 to replication forks to phosphorylate histone H1 and decondense chromatin, thereby facilitating replication of histone H1-containing regions (Alexandrow and Hamlin, 2005).…”