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

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-fos and 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 ras expression results in a rapid increase in H3 phosphorylation. H3 phosphatase, identified as PP1, activities in ras-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.Histone H1 and the N-terminal tail of H3 have key roles in the folding and inter-association of the chromatin fiber (1-5). Modification of the N-and C-terminal tails of H1 by phosphorylation or the N-terminal tail of H3 by acetylation and/or phosphorylation could destabilize higher order chromatin structure (6, 7). Myc-and ras-transformed and Rb-deficient fibroblasts have a more decondensed chromatin structure than parental cells (8 -10). A general feature of these oncogenetransformed and Rb-deficient cells is increased H1 phosphorylation. H1 phosphorylation may relax chromatin by interfering with its action in chromatin folding and intermolecular fiberfiber interactions (3).Continuous stimulation of the Ras mitogen-activated protein kinase (MAPK) 1 signaling pathway in mouse fibroblasts transformed with oncogenes ras, fes, mos, and c-myc elevates the level of phosphorylated H1 (9, 11). Activation of the Ras-MAPK signaling pathway of quiescent fibroblasts treated with growth factors or phorbol esters results in the phosphorylation of H3 (12-14). Thus, persistent activation of the Ras-MAPK pathway in oncogene-transformed cells may also increase the level of phosphorylated H3, contributing to the destabilization of the higher order compaction of chromatin.The N-terminal domain of H3 can be phosphorylated on Ser-10 and/or Ser-28 (15, 16); however, the site of mitogeninduced phosphorylation remains unknown. Phosphorylation of H3 happens concurrently with the transcriptional activation of the immediate early response genes, e.g. c-fos (12, 13). However, inhibition of transcription does not prevent mitogen-activated H3 phosph...

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