Major Egr3 isoforms are generated via alternate translation start sites and differ in their abilities to activate transcription

Abstract

In previous studies, we detected a major, unidentified Egr response element (ERE) binding complex in brain extracts. We now report that this complex contains a truncated isoform of Egr3 generated by use of an alternate translation start site at methionine 106. Furthermore, the ERE binding complex previously thought to contain full-length Egr3 includes several isoforms generated by initiation at other internal methionines. Full-length and truncated (missing residues 1 to 105) Egr3 isoforms differ in the ability to stimulate transcription directed by a tandem repeat of two EREs but not by a single ERE. Taken together, our results indicate that alternative translation start sites are used to generate Egr3 isoforms with distinct transcriptional properties.

FIG. 1

Identification of a truncated Egr3 isoform. (A) αEgr3-INT detects two Egr3 isoforms in a gel shift analysis of recombinant Egr3 incubated either without antibody (−) or with αEgr3-NT (αNT) or αEgr3-INT (αINT). α and β refer to the slower- and faster-migrating Egr3-containing complexes, respectively. (B) Immunoblots of recombinant Egr3 run adjacent to control (Con) and 4-h post-MECS rat hippocampal extracts (HC) probed with αEgr3-NT (1:1,000 dilution) and labeled αNT (left) and of similar extracts probed with αEgr3-INT (1:500 dilution) and labeled αINT (right). The band labeled Egr3 is recognized by αEgr3-NT and αEgr3-INT. In contrast, the band labeled Egr3ΔNT is detected only by αEgr3-INT, indicating that it is truncated at the N terminus. (C) αEgr3-INT abolishes both delayed ERE binding complexes in vivo in ERE gel shift assays using ³²P-labeled ERE oligonucleotide probe on control and MECS-treated hippocampal (left) or cortical (right) extracts. α and β refer to Egr3-containing complexes. The slowest-migrating band in the lane labeled αNT (left) is a supershifted Egr3α complex. The slowest-migrating band in all other lanes is Egr1. The asterisk refers to a nonspecific band that does not display sequence-specific ERE binding activity. (D) Schematic representation of rat Egr3 (rEgr3) and the segments used to generate the αEgr3-NT (amino acids 1 to 100) and αEgr3-INT (amino acids 101 to 189) antibodies. The zinc finger DNA binding domain is highlighted in gray.

FIG. 2

Egr3 knockout mice lack α and β complexes. (A) Pattern of ERE binding complexes detected in 4-h postseizure forebrain (FB) extracts from wild-type (+/+) and Egr3 knockout (−/−) mice. In the right lane, labeled αINT, wild-type extracts were incubated with αEgr3-INT. α and β refer to specific Egr3-containing complexes in panels A and B. We eliminated binding of the nonspecific band in mouse brain extracts by inclusion of 100-fold excess of unlabeled mutant ERE oligonucleotide. (B) Mutation of Met 106 abolishes expression of truncated Egr3 isoform, determined by a gel shift assay of recombinant Egr3 synthesized in vitro from wild-type (wt) and mutated (M106L) Egr3 cDNA templates. (C) Immunoblot of recombinant extracts containing wild-type or mutated (M106L) Egr3 probed with αEgr3-INT (1:500 dilution). The asterisks refer to cross-reacting proteins that are present in the rabbit reticulocyte preparations. (D) Schematic representation of rat Egr3 protein depicting Met residues 1, 13, 49, and 55 within the NT epitope and Met residues 106, 129, and 140 within the INT epitope. The zinc finger DNA binding domain is shown in gray.

FIG. 3

Mutation of specific Met codons abolishes expression of Egr3 isoforms in vitro. (A) Pattern of ERE binding complexes detected in wild-type mouse forebrain (FB), included to highlight the close similarity between Egr3 complexes expressed in vivo and in vitro. (B and C) Gel shift assays of recombinant Egr3 synthesized in vitro from the mutated versions of the Egr3 cDNA indicated above the lanes. All Egr3 cDNAs shown in panels B and C are in the Egr3ΔM1 vector backbone and thus do not express Egr3α1.

FIG. 4

Mutagenesis studies of Egr3 in intact cells. (A) ERE binding complexes detected in hEK-293 cell extracts that were untransfected (UT) or transfected with CMVEgr3wt (wt [wild-type]) or ΔM1 cDNA. (B) Autoradiograph of a similar gel shift experiment performed on hEK-293 cell extracts transfected with CMVEgr3M13L, M49L, M49A/M55A, and M106L cDNAs. The mutated constructs in panel B are in the CMVEgr3ΔM1 backbone and thus do not express Egr3α1. The γ complex appears when M106 is mutated to Leu. (C) Immunoblot analysis using αEgr3-INT (1:500 dilution) performed on the nuclear extracts used for gel shift assays. αEgr3-INT detects two immunoreactive bands in CMVEgr3wt-transfected cells that correspond to the α (upper) and β (lower) gel shift complexes. The M106L mutation abolishes expression of the lower band and leads to the generation of a faster-migrating band that corresponds to that complex. (D) Schematic representation of the translation start sites of the Egr3 isoforms detected in vivo, Egr3α1 (M1), α2 (M13), α3 (M49/55), β (M106), and γ (M129). The zinc finger DNA binding domain is indicated in gray.

FIG. 5

Transcriptional activity of Egr3 isoforms. (A) Schematic representation of the expression and reporter vectors used in the reporter assays. The 1X ERE and 2X ERE reporter constructs contained either one or two copies of an ERE (GCG GGG GCG) upstream of the firefly luciferase gene. (B) Bar graph showing fold induction in luciferase activity induced by transfection with either CMVEgr3M49A/M106A (encoding Egr3α1/α2) or CMVEgr3Δ1-105/M129L (encoding Egr3β) over background reporter activity (taken from extracts of hEK-293 cell transfected with an empty CMV vector) for the 2X (left) and 1X (right) EREs. Error bars represent standard errors of the means. Statistical analysis of these data demonstrate that the Egr3 constructs tested differ significantly in their effects on the 2X ERE but not the 1X ERE (P < 0.0005 and P > 0.5, respectively; Student’s t test). (C) Immunoblot of untransfected hEK-293 cells run adjacent to hEK-293 cells transfected with 5 μg of CMVEgr3M49A/M106A (encoding Egr3α1/α2) or CMVEgr3Δ1-105/M129L (encoding Egr3β) probed with αEgr3-INT (1:500 dilution). (D) Representative gel shift experiment using a ³²P-labeled ERE oligonucleotide probe on extracts of hEK-293 cells untransfected (UT) or transfected with 5 μg of CMVEgr3wt (wt [wild type]) or CMVEgr3M49A/M106A (α1/α2).