Neuropathy-associated Egr2 mutants disrupt cooperative activation of myelin protein zero by Egr2 and Sox10

Abstract

Dominant mutations in the early growth response 2 (Egr2/Krox20) transactivator, a critical regulator of peripheral myelin development, have been associated with peripheral myelinopathies. These dominant mutants interfere with the expression of genes required for myelination by Schwann cells, including that for the most abundant peripheral myelin protein, Myelin protein zero (Mpz). In this study, we show that Egr2 mutants specifically affect an Egr2-responsive element within the Mpz first intron that also contains binding sites for the transcription factor Sox10. Furthermore, Egr2 activation through this element is impaired by mutation of the Sox10 binding sites. Using chromatin immunoprecipitation assays, we found that Egr2 and Sox10 bind to this element in myelinating sciatic nerve and that a dominant Egr2 mutant does not perturb Egr2 binding but rather attenuates binding of Sox10 to the Mpz intron element. Sox10 binding at other sites of Egr2/Sox10 synergy, including a novel site in the Myelin-associated glycoprotein (Mag) gene, is also reduced by the dominant Egr2 mutant. These results provide the first demonstration of binding of Egr2/Sox10 to adjacent sites in vivo and also demonstrate that neuropathy-associated Egr2 mutants antagonize binding of Sox10 at specific sites, thereby disrupting genetic control of the myelination program.

FIG. 1.

Identification of a conserved region within the Mpz first intron that is sensitive to dominant mutants of Egr2. (A) The diagram of the Mpz locus indicates the position of the conserved intron element of Mpz. The putative Sox10 and Egr2 binding sites are indicated with ovals and squares, respectively. (B) Rat Schwann cells were cotransfected with a luciferase reporter construct containing the conserved region of the Mpz first intron (+984/+1749) and expression plasmids for wild-type (WT) Egr2 (50 ng) and the indicated Egr2 mutants (25, 50, and 100 ng). Similar assays were performed with luciferase reporters containing four consensus Egr binding sites upstream of a minimal promoter (4xEgr.syn) (C) or the Nab2 promoter (D). Induction is calculated relative to the activity of the reporter alone. EX, exons of the Mpz gene.

FIG. 2.

Dominant-negative Egr2 mutants selectively inhibit in a cell type-specific manner. HeLa cells (A) or B16/F10 melanocytes (B) were transfected as described for Fig. 1 with the Mpz intron element reporter and the indicated amounts of expression plasmids for Egr2 and the dominant S382R/D383Y (SR/DY) mutant.

FIG. 3.

Sox10 binds to the Mpz intron element in the S16 Schwann cell line. Formaldehyde cross-linked chromatin was prepared from S16 cells and immunoprecipitated with antibodies for Sox10, Egr2, or rabbit IgG as a negative control. Purified DNA fragments were analyzed by quantitative PCR using primers specific to the sites listed beneath each panel. Egr2 and Sox10 occupancy is expressed as the amount of DNA recovered relative to that in the input sample. (A) Amplicon positions are indicated on the diagram of the Mpz locus. Primer designations: −2.3 kb, 2.3 kb upstream from the transcription start site; PRO, Mpz promoter; IN1, Mpz first intron; IN5, Mpz intron 5. (B) Primers for the IgG2a promoter, Nab2 promoter, Cx32 promoter, Mag intron, and Egr2 MSE were used to detect binding of Sox10 and Egr2 to these sites. These data are representative of the Egr2 and Sox10 binding enrichments observed in triplicate quantitative PCR assays from two independent experiments.

FIG. 4.

Sox10 binds to the Mpz intron element in myelinating sciatic nerve. ChIP assays were performed by immunoprecipitating formaldehyde cross-linked chromatin prepared from sciatic nerves pooled from ∼10 rat pups at postnatal day 10, using antibodies for Sox10, Egr2, or rabbit IgG as a negative control, as described for Fig. 3. (A) Amplicon positions are indicated on the diagram of the Mpz locus. Primer designations: −2.3 kb, 2.3 kb upstream from the transcription start site; PRO, Mpz promoter; IN1, Mpz first intron; IN5, Mpz intron 5. (B) Primers for the IgG2a promoter, Cx32 promoter, Mag intron, and Egr2 MSE were used to detect binding for Sox10, Egr2, or the IgG control antibodies. These data are representative of quantitative PCR assays performed in triplicate with two independent sets of pooled rat sciatic nerves at P10.

FIG. 5.

Mutagenesis of Sox10 binding sites disrupts synergistic activation of the Mpz intron element by Egr2/Sox10. The two Sox10 sites in the Mpz intron element reporter were mutated by site-directed mutagenesis. The Mpz intron reporters (wild type and Sox10 mutant) were cotransfected in HeLa cells with expression plasmids for Egr2 (50 ng) and/or Sox10 (100 ng). The putative Sox10 and Egr2 binding sites are indicated with ovals and squares, respectively. The X in the diagram represents mutated Sox10 sites. The sequence alignment shows conservation of the Sox10 binding sites in mouse, human, and rat, and mutated bases are indicated by lines over the sequence. Induction (n-fold) is calculated relative to the activity of each reporter alone. (B) Mobility shift assays for Egr2 and Sox10 binding were performed using a fragment from the Mpz intron containing the two Sox10 sites and the second Egr2 site. These fragments either were wild type or had mutations in the Sox10 or Egr2 sites (indicated by X). Recombinant Egr2 and FLAG affinity-purified Sox10 were used. A faster-migrating, nonspecific band (asterisk) copurified with recombinant Egr2 but was unaffected by mutation of the Egr2 site. (C) Increasing amounts of purified Sox10 (8-fold range) were incubated in the presence or absence of Egr2 with the wild-type Mpz intron probe.

FIG. 6.

Mutation of Sox10 binding sites abrogates the dominant-negative effect of a dominant Egr2 mutant. Primary rat Schwann cells were cotransfected with the Mpz intron reporters (wild type and Sox10 mutant) along with expression plasmids for Egr2 (50 ng), the Egr2 (SR/DY) mutant (50 ng), and Sox10 (100 ng). Induction is calculated relative to the luciferase activity of each reporter alone. Means and standard errors are representative of two independent assays performed in duplicate.

FIG. 7.

Overexpression of Sox10 rescues the dominant-negative effect of a dominant Egr2 mutant. Primary rat Schwann cells were cotransfected with the wild-type Mpz intron reporter along with Egr2 (50 ng), Egr2 (SR/DY) mutant (50 ng), Sox10 (100 ng), and Sox11 (100 ng) expression plasmids. Induction is calculated relative to the luciferase activity of the reporter alone. Means and standard errors are representative of two independent assays performed in duplicate.

FIG. 8.

A dominant-negative Egr2 mutant downregulates expression of myelin genes. S16 Schwann cells were infected with Ad expressing either GFP or the dominant Egr2 mutant (SR/DY) and harvested 48 h postinfection. Relative levels of gene expression for Mpz, Mag, and Cx32 (A) and Egr2, Sox10, and Nab2 (B) were determined by quantitative PCR and normalized to the level of 18S rRNA. Induction (n-fold) is indicated relative to the level found in the untreated sample, which was set as 1 for each gene. Quantitative PCR experiments were performed in triplicate, and the standard errors are indicated. The results are representative of two independent experiments. (C) The immunoblot shows lysates of S16 cells infected with Ads expressing the indicated proteins. Blots were probed with antibodies directed against Sox10, Egr2, and α-tubulin as a loading control.

FIG. 9.

A dominant-negative Egr2 mutant attenuates Sox10 recruitment to the Mpz intron element. S16 cells were infected with Ad expressing either the Egr2 (SR/DY) mutant or a GFP control virus as described for Fig. 8. Formaldehyde cross-linked chromatin was immunoprecipitated using antibodies for Sox10, Egr2, or rabbit IgG as a negative control. Egr2 and Sox10 occupancy is expressed as the percent recovery relative to the input sample level, which was determined by quantitative PCR using primers targeted to the IgG2a promoter, Mpz intron, and Mpz promoter (A) and the Egr2 MSE, Cx32 promoter, and Mag intron (B). These data are representative of two independent experiments with quantitative PCR assays performed in triplicate. (C) Mobility shift assays for Egr2 and Sox10 binding were performed using a fragment from the Mpz intron as described for Fig. 5, with inclusion of the dominant Egr2 (SR/DY) mutant protein. All proteins were produced by in vitro transcription/translation and purified using the six-His tag (wild type and mutant Egr2) or the Flag tag (Sox10). An immunoblot using an anti-Egr2 antibody (lower panel) shows the relative levels of wild-type Egr2 and mutant Egr2 (SR/DY) proteins (in the mobility shift assay using the lowest level of mutant protein [lane 3]).

FIG. 10.

The N terminus of Egr2 is required to facilitate binding of Sox10 to the Mpz intron element. (A) Mobility shift assays for binding of Sox10 to the Mpz intron fragment were performed in the presence of either wild-type Egr2 or Egr2Δ1-180 (lacking the N-terminal domain of Egr2). (B) Purified Egr2 and Egr2Δ1-180, each containing an N-terminal HA epitope, were mixed and incubated with FLAG-Sox10. Sox10 was immunoprecipitated (IP) using an anti-FLAG affinity resin, and pulldown of Egr2 was detected using an anti-HA (α-HA) antibody. The input lane represents 5% of the amount of lysate used for the binding assay. IB, immunoblot.