Linker Histone Phosphorylation Regulates Global Timing of Replication Origin Firing

Christophe Thiriet,Jeffrey J. Hayes

doi:10.1074/jbc.m805617200

Abstract

Despite the presence of linker histone in all eukaryotes, the primary function(s) of this histone have been difficult to clarify. Knock-out experiments indicate that H1s play a role in regulation of only a small subset of genes but are an essential component in mouse development. Here, we show that linker histone (H1) is involved in the global regulation of DNA replication in Physarum polycephalum. We find that genomic DNA of H1 knock-down cells is more rapidly replicated, an effect due at least in part to disruption of the native timing of replication fork firing. Immunoprecipitation experiments demonstrate that H1 is transiently lost from replicating chromatin via a process facilitated by phosphorylation. Our results suggest that linker histones generate a chromatin environment refractory to replication and that their transient removal via protein phosphorylation during S phase is a critical step in the epigenetic regulation of replication timing.

Highlights

Chromatin plays an active role in the control of gene expression and other nuclear processes that require access to DNA
We knocked down Physarum linker histone levels by direct incorporation of enzymatically generated siRNA into macroplasmodia in a manner similar to methods we developed for incorporation of exogenous proteins [16, 17]
Our results demonstrate that linker histone plays an essential role in proper regulation of replication timing, We have found that upon H1 knock-down, the majority of Physarum genomic DNA replication is achieved within 1.5 h instead of the normal 3 h, indicating that linker histone exerts a global influence on DNA replication, in contrast to the relatively minor global effect of linker histone depletion on transcription (4 – 6, 11)

Summary

EXPERIMENTAL PROCEDURES

Cultures of Physarum—P. polycephalum, strain TU291, was maintained in liquid cultures. Protein Analyses and Immunoprecipitation—Efficiency of RNA interference was assayed by Western blotting using an anti-PpH1 antiserum This antiserum recognizes biochemically purified PpH1 and H1 in whole cell extracts (Fig. 1, A and B) and is competed by a PpH1 peptide (supplemental Fig. S1). Immunoprecipitation of BrdUrd-incorporated DNA was used to determine the timing of replication of specific genes and was performed to Ref. 18. Cation of immunoprecipitated DNA was carried as described in Ref. 20 with the primers specific to Lav2.1. For experiments of phosphorylation of H1, detection of phospho-H1 was carrying out by Western blotting using anti-phospho-H1 (Abcam) with a dilution of 1:250 and 1:1000 for the secondary antibodies

RESULTS

We then examined the effects of

DISCUSSION