Binding of c-Myc to chromatin mediates mitogen-induced acetylation of histone H4 and gene activation

Abstract

The Myc protein binds DNA and activates transcription by mechanisms that are still unclear. We used chromatin immunoprecipitation (ChIP) to evaluate Myc-dependent changes in histone acetylation at seven target loci. Upon serum stimulation of Rat1 fibroblasts, Myc associated with chromatin, histone H4 became locally hyperacetylated, and gene expression was induced. These responses were lost or severely impaired in Myc-deficient cells, but were restored by adenoviral delivery of Myc simultaneous with mitogenic stimulation. When targeted to chromatin in the absence of mitogens, Myc directly induced H4 acetylation. In addition, Myc recruited TRRAP to chromatin, consistent with a role for this cofactor in histone acetylation. Finally, unlike serum, Myc alone was very inefficient in inducing expression of most target genes. Myc therefore governs a step, most likely H4 acetylation, that is required but not sufficient for transcriptional activation. We propose that Myc acts as a permissive factor, allowing additional signals to activate target promoters.

Figure 1

Schematic representation of the genes characterized in this study and of the amplicons used in ChIP analysis. PCR amplicons are numbered with the position of their 5′ end relative to the 5′ end of exon 1 (+1), as annotated in GenBank. For the bidirectional loci AIRC/GPAT and HSP10/60, we took as reference GPAT and HSP60, respectively. The accession numbers for all genomic sequences and the sequences of the PCR primers are given in Materials and Methods.

Figure 2

Quantification of chromatin immunoprecipitation by real-time PCR. Chromatin extracted from cross-linked Rat1 or myc^−/− cells was immunoprecipitated with anti-Myc antibodies. DNA was recovered and used as a template for real-time PCR in a Taqman 5700 (Perkin Elmer). (A) Representative PCR-amplification curves, as displayed by the Taqman 5700. In this example, we used primers amplifying the NUC E-box domain (amplicon +574, Fig. 1). Calculation of the amount of immunoprecipitated NUC E-box DNA relative to that present in total input chromatin is shown at the bottom. (CT) Cycle threshold, cycle number at which each PCR reaction reaches a predetermined fluorescence threshold, set within the linear range of all reactions. (B) Graphic representation of the data for NUC and control promoters analyzed in the same experiment (GLU, GLY, PCNA, and ACHR). In this experiment, cells were cross-linked and harvested 4 h after mitogenic stimulation.

Figure 3

Mitogens induce association of Myc with chromatin and acetylation of histone H4. At the indicated time points after serum stimulation, Rat1 cells (shaded bars) and myc^−/− cells (black bars) were analyzed by ChIP. Antibodies against (A) Myc, (B) acetylated histone H4, and (C) acetylated histone H3 were used in parallel immunoprecipitations. Precipitated DNA samples were amplified with primers recognizing the E-box domains of Myc-target genes (see corresponding maps and amplicons in Fig. 1) or the control ACHR promoter as indicated in A. % total: as defined in Figure 2.

Figure 4

(A) Kinetics of histone H4 acetylation at Myc-binding sites. The experiment shown in Figure 3B was repeated over a longer time course. (B) Myc rescues histone H4 acetylation in myc^−/− cells. Quiescent myc^−/− cells (open bar) were serum-stimulated and simultaneously infected with either AdGFP (black bars) or AdGFP-Myc (shaded bars). ChIP was used to measure H4 acetylation at the NUC E-box domain (amplicon +574, Fig. 1).

Figure 5

Localization of Myc and acetylated histones on Myc-target genes by ChIP. (A) Mapping of Myc-binding along its target genes, 4 h after mitogenic stimulation in Rat1 cells. (B) Distribution of acetylated histone H4 along Myc-target genes in quiescent Rat1 cells (black bars) and 4 h after serum stimulation (shaded bars). (C) Same experiment, in myc^−/− cells. The identity of each amplicon is indicated below the graphs (see maps in Fig. 1). (E) E-box sites.

Figure 6

Targeting of Myc to chromatin in quiescent cells promotes H4 acetylation. (A,B) Quiescent Rat1 cells expressing MycER were analyzed by ChIP before (black bars) or 1 h after OHT treatment (shaded bars). Data are shown for the same E-box and control amplicons as in Figure 3. (C,D) Rat1 cells harboring a control retrovirus (vector) or a Myc-expressing virus (Myc) were analyzed by ChIP during continuous growth (shaded bars) or following withdrawal from the cell cycle (black bars), as achieved by contact inhibition and serum starvation. ChIP data are shown for the NUC E-box domain (amplicon +574, Fig. 1). ChIP was performed with antibodies against Myc (A,C) or acetylated H4 (B,D). (E) Comparison of H4 acetylation in quiescent control and Myc-expressing cells (same as in D) with cells expressing the Myc mutants Δ7-91 and Δ106-145. In D and E, all data are expressed relative to the acetylation level in quiescent control cells (vector). (F) Immunoblot analysis of wild-type Myc, Δ7-91 and Δ106-145 in the same cells used in E, with the 9E10 monoclonal antibody (BabCO).

Figure 7

TRRAP is recruited to chromatin in a Myc-dependent manner. (A,B) Characterization of the TRRAP-CT antibody (see additional information in Materials and Methods). (A) TRRAP-CT was used in immunoprecipitation from a 293-cell lysate (500 μg), in the presence or absence of the cognate antigenic peptide, as indicated. Precipitates were analyzed by immunoblotting with TRRAP-CT itself. The first lane shows total cell lysate (50 μg). (B) TRRAP-CT was used for immunoblot analysis of the indicated cell lysates. (NHLF) normal human lung fibroblasts; (ML1) human myeloid leukemia cell line; (MEF) mouse embryonic fibroblasts. (C,D) TRRAP-binding to the NUC E-box domain (amplicon +574, Fig. 1) and control ACHR promoter was assayed by ChIP with TRRAP-CT, following (C) mitogenic stimulation of Rat1 (shaded bars) or myc^−/− cells (black bars) and (D) activation of MycER in quiescent cells.

Figure 8

Induction of Myc-target genes by mitogens is Myc-dependent. Relative mRNA levels for (A) Myc-target genes and (B) control genes, quantified by reverse transcription and real-time PCR. Rat1 cells (filled circles) and myc^−/− cells (filled triangles) were analyzed at the indicated time points following mitogenic stimulation. To restore Myc expression in myc^−/− cells, these cells were infected at the time of serum stimulation with the recombinant adenovirus AdGFP-Myc (open circles) or with the control virus AdGFP (open triangles). Expression of each gene is represented as the fold-induction relative to untreated quiescent cells (time 0).

Figure 9

Mitogenic stimuli and activation of Myc alone are not equivalent in inducing expression of Myc-target genes. Quiescent Rat1-MycER cells were stimulated with either OHT alone (open squares) or serum alone (filled circles). Serum-stimulated myc^−/− cells are shown as a reference (filled triangles). mRNA expression was quantified as in Figure 8.