Fig 1.
Predicted Egr binding sites and ChIP primer locations upstream of genes preferentially expressed during sustained ERK signaling in response to NGF.
Of the 69 genes that Mullenbrock et al. (2011) determined are preferentially expressed during sustained ERK signaling in response to NGF, 21 contained putative Egr binding sites within 5 kb upstream of their transcription start site (TSS). The locations of each Egr binding site are denoted by the vertical blue lines. The red horizontal lines denote the relative locations of primer sets used for real time PCR to detect Egr binding to nearby Egr binding site(s). For genes with multiple dispersed Egr binding sites, multiple primers sets were designed (denoted P1, P2, etc.) to detect Egr binding to the nearest predicted Egr binding site.
Fig 2.
Time course analysis of Egr family expression following NGF treatment.
PC12 cell cultures were treated with 50 ng/ml NGF for 0–4 h before harvest. (a) Total RNA was extracted and subjected to real-time RT-PCR to quantify changes in mRNA levels or (b) total cell lysates were prepared and subjected to SDS-PAGE and Western blot analysis to evaluate changes in protein levels. NS, nonspecific band.
Fig 3.
ChIP analysis to evaluate binding of Egr1 and Egr2 upstream of genes with predicted Egr binding sites.
PC12 cultures were treated with or without 50 ng/ml NGF for 1 h and subjected to ChIP assay using antibodies against Egr1, Egr2, or an IgG control. Real-time PCR was then conducted on the immunoprecipitated DNA using primers within 250 bp of each predicted Egr binding site (see Fig 1 and S2 Table for primer locations and sequences). For genes with multiple dispersed Egr binding sites, multiple primer sets (denoted P1, P2, etc.) were used; only data for the set that detected highest level of binding are included here (see S1 Fig for data from the remaining primer sets). Primers to amplify a region approximately 100 bp upstream of the Myog gene were used as a negative control for Egr1 binding. (a) Data from Egr1 ChIP are plotted as % input and are averages from three to four independent experiments ± S.E. *, One-tailed Student’s t tests were conducted comparing the % input value for Myog after Egr1 IP to the % input values for each predicted Egr target after Egr1 IP, which yielded p values ≤ 0.05 for 12 predicted Egr targets. (b) Data from Egr2 ChIP are plotted as % input and are averages from two independent experiments ± S.E.
Fig 4.
Sustained Egr1 expression and binding upstream of target genes in response to NGF versus EGF.
(a) PC12 cell cultures were treated with 50 ng/ml NGF or 25 ng/ml EGF for 0–4 h. Total cell lysates were harvested and subjected to SDS-PAGE and Western blot for Egr1. Egr1 levels were quantified by densitometry and converted to % maximum levels for ten independent experiments and plotted (average ± S.E.). (b) PC12 cultures were treated with or without 25 ng/ml EGF for 1 h and subjected to ChIP assay using antibodies against Egr1 or an IgG control. Real-time PCR was then conducted on the immunoprecipitated DNA using primers within 250 bp of predicted Egr binding sites (see Fig 1 and S2 Table for primer locations and sequences). Primers to amplify a region approximately 100 bp upstream of the Myog gene were used as a negative control for Egr1 binding. Data are plotted as % input and are averages from three to six independent experiments ± S.E. *, One-tailed Student’s t tests were conducted comparing the % input value for Myog after Egr1 IP to the % input values for each predicted Egr target after Egr1 IP, which yielded p values ≤ 0.05. (c) PC12 cell cultures were treated with 50 ng/ml NGF or 25 ng/ml EGF for 0–4 h and subjected to ChIP with anti-Egr1 antibody. Immunoprecipitated DNA was then subjected to real-time PCR with primers to detect Egr1 binding to a subset of its target genes, which was quantified as % input. Percent input values were then converted into % maximum values, which were plotted. *, One-tailed Student’s t tests were conducted comparing the % input values at each time point after NGF versus EGF treatment, which yielded p values ≤ 0.05 at the 3-hour time point for Arc, Kctd11, Trib1, Tph1, and Vgf.
Fig 5.
AP-1, CREB, and Egr1 cooperatively regulate 28 genes during their preferential expression in response to NGF and sustained ERK signaling.
The Venn diagram summarizes genes bound by AP-1, CREB, and/or Egr1 during their preferentially expression in response to NGF and sustained ERK signaling, as detected by Mullenbrock et al. (2011) and the present study.