Factor binding and chromatin modification in the promoter of murine Egr1 gene upon induction

Abstract

The influence of chromatin on immediate-early gene expression has been studied in a model of Egr1 induction in intact mouse cells. ChIP analysis of factor and RNA polymerase binding reveals that the gene is constitutively poised for transcription in nonstimulated cells, but a repressing chromatin structure hampers productive transcription. Stimulation with phorbol esters results in a transient activation, which starts at 5 min and peaks at 30 min. Quantitative mapping of promoter occupancy by the different factors shows for the first time that no direct competition between SP1 and EGR1 occurs. The phosphorylation of ELK1 and CREB, which involves both the cascades of MEK1/2 and p38 kinases, is required for gene expression, which ceases following the binding of NAB1 and NAB2 to the promoter. The changes in histone acetylation and the differential recruitment of histone-modifying complexes further show the role of chromatin in the activation of this immediate-early gene.

Fig. 1a–e

TPA-induced expression of Egr1 gene in murine MLP29 progenitor cells. The steady state level of Egr1 mRNA was analyzed by RT-PCR (a, b) and the real-time transcription rate was determined by RNAPol-ChIP (c, d). Results of representative semiquantitative analyses (a, c) and of quantitative (b, d) PCRs are given. The latter represent the average ± SE of three (b) or six (d) determinations. In the quantitative analyses, Egr1 expression is normalized to the value in the absence of TPA and plotted as fold induction. e Results of a ChIP analysis of phosphoacetylation of histone H3

Fig. 2a–c

ChIP analysis of factor binding to Egr1 promoter after TPA stimulation. To facilitate the interpretation of the experiments, a map of the Egr1 promoter is depicted (a) in which the relevant elements of the promoter are represented. The bars below the map correspond to the different amplicons used in the PCR analyses (see Table 1 for details). Western blots of extracts from MLP29 progenitor cells, showing the specificity of antibodies used, are given in b. The results of a representative ChIP assay with amplicon P are given in c. As a negative control, c shows the results obtained with the same antibodies in the promoter of α-actin gene. NA Control with no antibody added

Fig. 3a–c

Quantitative mapping of the binding of several factors to the Egr1 promoter. a Plot of the relative binding of SRF in nonstimulated cells, determined as detailed in the experimental procedures, against the position of the centers of the analyzed amplicons. The bimodal distribution shows that both clusters of SREs (see Fig. 2a) are being used. b Relative binding of SP1 in nonstimulated cells. c Relative binding of EGR1 (upper panel), NAB1 (middle panel), and NAB2 (lower panel), plotted for 30 min (open symbols and solid line) and 60 min (filled symbols and dashed line) of TPA treatment. To estimate the relative binding of the factors, the DNA concentration obtained for each amplicon was normalized to the value obtained for the input (see the section “Materials and methods”)

Fig. 4a–d

Factor phosphorylation and Egr1 activity. a Western blots of extracts from MLP29 progenitor cells, showing the specificity of antibodies used. b Time-dependent phosphorylation of ELK1 and CREB in Egr1 promoter. ChIP analyses, similar to those of Fig. 2, were carried out with antibodies specific for the phosphorylated forms of ELK1 and CREB. c Effects of several kinase inhibitors on TPA-induced Egr1 expression. The average ± SE of three independent quantitative RT-PCR determinations at amplicon C (normalized relative to rRNA 18S gene) is represented for cells treated or not with the inhibitors (i) of the indicated kinases. A control determination (from untreated cells) was arbitrarily set to 1. d ChIP analysis of the effects of kinase inhibitors on the phosphorylation of CREB and ELK1, and on the binding of RNA polymerase II to the Egr1 promoter. The results of the PCR analyses of a and c were integrated and plotted

Fig. 5a–d

Binding of HDACs and HATs to Egr1 promoter. ChIP analysis (amplicon P) of some components of HDAC (b) and HAT (d) complexes to Egr1 promoter. The effects of kinase inhibitors on the presence of histone-modifying complexes at the Egr1 promoter of cells stimulated 15 min are shown in c. The results of the PCR analyses were integrated as in Fig. 4. Western blots of extracts from MLP29 progenitor cells, showing the specificity of antibodies used, are given in a

Fig. 6

Histone modifications at the promoter of the repressed and active Egr1 gene. The figure shows the results of a ChIP experiment carried out in nonstimulated (TPA 0 min) and TPA-treated cells for 30 min, in which the Egr1 gene is fully active (TPA 30 min) with the indicated antibodies. The immunoprecipitated DNA was PCR-amplified with the primers corresponding to amplicon P (see Table 1). The results obtained at the promoter of the α-actin gene were included as negative control. The appropriate primers (see Table 1) were used