Article Figures & Data
Figures
- FIG. 1.
EGR2 and CITED1 are overexpressed in KI mammary tumors and show nuclear localization. (A) EGR2 and CITED1 transcript levels were analyzed in KI and MMTV-activated ErbB2 mammary tumors by real-time RT-PCR. EGR2 and CITED1 transcript levels were normalized to GAPDH by the ΔΔCT method. Each relative transcript level represents the mean value of three independent amplification reactions (± the standard deviation). (B) Lysates prepared from primary mammary tumor tissues were subjected to SDS-PAGE and blotted with an EGR2 (top)- or CITED1 (middle)-specific antibody. Grb2 (bottom) was used as a control for equal loading. EGR2 (C) and CITED1 (D) expression (brown) in tissue excised from primary KI breast tumors by immunohistochemical (IHC) staining with antibodies specific to EGR2 and CITED1 is also shown. AU, arbitrary units.
- FIG. 2.
EGR2 binds to a probe corresponding to a region of the erbB2 promoter and associates with the erbB2 promoter in vivo. (A) Lysates of 293T cells transfected with V5-tagged EGR2 were incubated with a radiolabeled probe corresponding to positions −706 to −675 of the erbB2 promoter (lanes 2 to 6). Anti-V5 antibody (Ab) (lane 3), an unlabeled specific competitor (lanes 4 and 5), or a nonspecific competitor (lane 6) was added. (B) A probe with a single base pair substitution (lanes 4 to 6) was used. Black, gray, and white arrowheads represent free probe, shift, and supershift, respectively. WT, wild type. (C) Diagrammatic representation of the probes used. The EGR2 binding site is in bold, and the mutated oligonucleotide is underlined. (D) Chromatin from a KI mammary tumor-derived cell line expressing V5-EGR2 was immunoprecipitated (IP) with a V5-specific antibody, a nonspecific antibody, or a no-antibody control. Chromatin was amplified with primers to the EGR2 binding site within the erbB2 gene (positions −723 to −612) or an upstream site (positions −2284 to −2136). (E) Endogenous EGR2 was immunoprecipitated from KI mammary tumor cells with an antibody directed to mouse EGR2 (Covance) and the associated chromatin amplified with the primers described for panel D. (F) Q-PCR of EGR2-associated chromatin. n-Fold enrichment represents the amount of chromatin corresponding to the amplified region with respect to the product obtained with the no-antibody control following normalization for input levels. Each result shown is the mean value obtained in three amplification reactions performed for two independent immunoprecipitations (± the standard deviation). (G) Diagrammatic representation of the primers used.
- FIG. 3.
EGR2 activates transcription from the erbB2 promoter. (A) C6 cells were cotransfected with 200 ng of a reporter construct containing no promoter (pgL3-basic), the −1225 to −182 region of the erbB2 promoter (pGL3-wt promoter), or the same region containing a mutated EGR2 binding site (pGL3-mut. promoter) and 750 ng of empty vector or EGR2 expression plasmid as indicated. Cytoplasmic extracts were prepared, and luciferase activity was measured. (B) HS578T cells were cotransfected with 200 ng of the pGL3-wt promoter construct and empty vector or EGR2 expression vector in the amounts indicated. Cytoplasmic extracts were assayed for luciferase activity. Luciferase activity is shown as relative light units normalized to β-galactosidase activity and is the mean result of three separate experiments with triplicate wells (± the standard deviation). AU, arbitrary units. (C) Diagrammatic representation of the reporter constructs used with the EGR2 binding site mutation indicated.
- FIG. 4.
CITED1 interacts with EGR2 and enhances transcription from the erbB2 promoter. (A) Extracts of 293T cells cotransfected with V5-EGR2 and HA-CITED1 were immunoprecipitated (IP) with an anti-HA antibody (Ab) or a control antibody, and the proteins were separated by SDS-PAGE. Membranes were probed with an anti-V5 antibody to detect EGR2 (top). The same lysates were precipitated with an anti-V5 antibody, and the isolated proteins were immunoblotted (IB) for CITED1 (bottom). The amount of input lysate shown is 5% of the total protein used in the immunoprecipitations. (B) HS578T cells were cotransfected with 200 ng of the wild-type erbB2 promoter-reporter construct described in the legend to Fig. 3 and an empty vector or an EGR2 expression vector (250 ng) and increasing amounts of a CITED1 vector (0, 250, 500, or 750 ng). Cytoplasmic extracts were assayed for luciferase activity. (C) ChIP with an HA-tagged CITED1-infected KI mammary tumor-derived cell line. Eluted chromatin was amplified with primers to the EGR2 binding site within the erbB2 promoter or to an upstream control site. (D) Chromatin associated with endogenous CITED1 in the KI tumor-derived cell line was isolated by immunoprecipitation with an antibody raised against full-length human CITED1 (2H11) and amplified as described for panel C. (E) Q-PCR of chromatin associated with endogenous CITED1. Fold enrichment represents the amount of chromatin corresponding to the −723 to −612 region of the erbB2 promoter compared to the no-antibody control following normalization for input levels. Each result shown is the mean value obtained in three separate amplification reactions for two independent immunoprecipitations (± the standard deviation). (F) Diagrammatic representation of the primers used.
- FIG. 5.
14-3-3σ overexpression leads to a decrease in ErbB2 levels and results in relocalization of EGR2 to the cytoplasm. (A) 14-3-3σ was immunoprecipitated (IP) from lysates of TM15 cells stably expressing 14-3-3σ, and the precipitated proteins were resolved by SDS-PAGE and immunoblotted (IB) for EGR2. WCL, whole-cell lysate. (B) Lysates of 293T cells cotransfected with EGR2-S376A-V5 and 14-3-3σ were immunoprecipitated with V5- and 14-3-3σ-specific antibodies (Ab). The isolated complexes were blotted for EGR2 (top) or 14-3-3σ (bottom). (C) 14-3-3σ (red) and EGR2 (green). Note the nucleus-localized EGR2 in cells expressing low levels of 14-3-3σ (green arrowheads) and the cytoplasmic EGR2 in the high-14-3-3σ-expressing cells (red arrowheads). (D) 14-3-3σ (green) and ErbB2 (red). Note the low ErbB2 levels in cells expressing high levels of 14-3-3σ (green arrowheads), whereas cells with low 14-3-3σ levels have high ErbB2 expression (red arrowheads). (E) Lysates of 293T cells cotransfected with EGR2-V5 and HA-14-3-3ζ were immunoprecipitated with V5- and HA-specific antibodies. The isolated complexes were blotted for EGR2 (top) or HA (bottom). (F) TM15 cells stably expressing HA-14-3-3ζ were stained with antibodies to HA (red) and EGR2 (green). Note that EGR2 remains nucleus localized in the presence of high levels of 14-3-3ζ.
