Concomitant Increase of Histone Acetyltransferase Activity and Degradation of p300 during Retinoic Acid-induced Differentiation of F9 Cells

Franck Brouillard,Chantal E Cremisi

doi:10.1074/jbc.m307123200

Abstract

The p300 and closely related CBP histone acetyltransferases (HAT) function as global transcriptional co-activators that play roles in many cell differentiation and signal transduction pathways. Despite their similarities, p300 and CBP have distinct functions during retinoic acid-induced differentiation of mouse F9 embryonal carcinoma cells. F9 cells constitute a well established model system for investigating the first steps of early development and retinoic acid signaling ex vivo. p300, but not CBP, was shown to be essential for F9 differentiation. In this study we have investigated the regulation of p300 during F9 differentiation. We report a dramatic decrease of p300, but not CBP protein levels, after 48 h of retinoic acid treatment. p300 is degraded via the ubiquitin-proteasome pathway. Although the large majority of p300 is degraded, its global HAT activity stays constant during F9 differentiation, which means that its specific HAT activity increases considerably. p300 is strongly phosphorylated in both undifferentiated and differentiated F9 cells; its HAT activity, however, is independent of phosphorylation before differentiation and becomes dependent on phosphorylation during differentiation. Furthermore, we show that protein kinase A affects p300 HAT activity both in vivo and in vitro as well as p300 phosphorylation in differentiated cells. Thus, we show that p300 is differentially phosphorylated in undifferentiated versus differentiated cells and that the changes in phosphorylation affect its HAT activity. Moreover, our study suggests an explanation for the functional switch of p300-mediated repression versus activation during F9 differentiation.

Highlights

Mouse embryonal carcinoma (EC)1 cell lines, derived from the stem cells of teratocarcinomas, provide an attractive ex vivo model system for studying the regulation of gene expression during mouse early embryogenesis [1]
The large majority of p300 is degraded, its global histone acetyltransferases (HAT) activity stays constant during F9 differentiation, which means that its specific HAT activity increases considerably. p300 is strongly phosphorylated in both undifferentiated and differentiated F9 cells; its HAT activity, is independent of phosphorylation before differentiation and becomes dependent on phosphorylation during differentiation
In this study we investigated the regulation of p300 expression and of its HAT activity during retinoic acid (RA)-induced F9 differentiation keeping in mind that the differentiation of EC cells mimics the early steps of development

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Mouse F9 EC cells were grown in high glucose Dulbecco’s modified Eagle’s medium (Invitrogen) containing 12.5% fetal calf serum supplemented with 100 units/ml penicillin and 100 ␮g/ml streptomycin in 0.1% gelatin-coated dishes. After incubation with the RW128 antibody, p300 immune complexes were divided in two equal portions; each of them was incubated with the same volume of protein A/G beads, washed 4 times with complete lysis buffer, once with lysis buffer without phosphatase inhibitors, and once with the ␭ phosphatase reaction buffer 1ϫ (50 mM Tris-HCl, pH 7.5, 0.1 mM Na2EDTA, 5 mM dithiothreitol, 0.01% Brij). P300 immune complexes were washed once with HAT buffer (50 mM Tris pH 7.6, 1 mM EDTA, 10 mM sodium butyrate, 20 mM NaF, 0.1 mM Na3VO4, and protease inhibitors) and mixed with the H4 biotinylated peptide (30 mM final concentration) in 30 ␮l of HAT buffer containing 100 nCi of [14C]acetyl-CoA (2.3 GBq/mmol, ICN). The immune complexes were visualized with the ECL detection system (PerkinElmer Life Sciences) or by autoradiography

RESULTS

Findings

DISCUSSION