Increased Ser-10 Phosphorylation of Histone H3 in Mitogen-stimulated and Oncogene-transformed Mouse Fibroblasts

doi:10.1074/jbc.274.35.24914

Increased Ser-10 Phosphorylation of Histone H3 in Mitogen-stimulated and Oncogene-transformed Mouse Fibroblasts*

From the ^‡Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, R3E 0V9 Canada, the ^§Department of Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, Manitoba, R3E 0W3 Canada, the ^**Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908, and the ^¶Department of Cell Biology and Anatomy, Faculty of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1 Canada

Abstract

When the Ras mitogen-activated protein kinase (MAPK) signaling pathway of quiescent cells is stimulated with growth factors or phorbol esters, the early response genes c-fosand c-myc are rapidly induced, and concurrently there is a rapid phosphorylation of histone H3. Using an antibody specific for phosphorylated Ser-10 of H3, we show that Ser-10 of H3 is phosphorylated, and we provide direct evidence that phosphorylated H3 is associated with c-fos and c-myc genes in stimulated cells. H3 phosphorylation may contribute to proto-oncogene induction by modulating chromatin structure and releasing blocks in elongation. Previously we reported that persistent stimulation of the Ras-MAPK signaling pathway in oncogene-transformed cells resulted in increased amounts of phosphorylated histone H1. Here we show that phosphorylated H3 is elevated in the oncogene-transformed mouse fibroblasts. Further we show that induction of rasexpression results in a rapid increase in H3 phosphorylation. H3 phosphatase, identified as PP1, activities inras-transformed and parental fibroblast cells were similar, suggesting that elevated H3 kinase activity was responsible for the increased level of phosphorylated H3 in the oncogene-transformed cells. Elevated levels of phosphorylated H1 and H3 may be responsible for the less condensed chromatin structure and aberrant gene expression observed in the oncogene-transformed cells.

Footnotes

↵* This work was supported by the Cancer Research Society, Inc. (to J. A. W., J .R. D., and D. P. B.-J.), by a Medical Research Council operating grant (to D. P. B.-J.), and by Public Health Service Grant GM 40922 from the National Institutes of Health (to C. D. A.). The awards of a Manitoba Health Research Council Studentship (to D. N. C.), a Medical Research Council of Canada Senior Scientist (to J. R. D.), and a National Cancer Institute of Canada Terry Fox Senior Scientist (to J. A. W.) are also gratefully acknowledged.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‖ Supported by a Postdoctoral Fellowship from the Medical Research Council of Canada and the Alberta Heritage Foundation for Medical Research.
↵‡ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Faculty of Medicine, University of Manitoba, Winnipeg, MB, Canada R3E 0W3. Tel.: 204-787-2391; Fax: 204-787-2190; E-mail: Davie@cc.umanitoba.ca.
↵2 M. J. Hendzel, unpublished observations.
Abbreviations:

MAPK

mitogen-activated protein kinase

pH3

phosphorylated H3

EGF

epidermal growth factor

TPA

12-O-tetradecanoylphorbol 13-acetate

PMSF

phenylmethylsulfonyl fluoride

IGκ MAR

immunoglobulin κ matrix attachment region

kb

kilobase

DAPI

diamidinophenolindole
- Received December 16, 1998.
- Revision received June 21, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.