Tead1 regulates the expression of Peripheral Myelin Protein 22 during Schwann cell development

Abstract

Schwann cells are myelinating glia in the peripheral nervous system that form the myelin sheath. A major cause of peripheral neuropathy is a copy number variant involving the Peripheral Myelin Protein 22 (PMP22) gene, which is located within a 1.4-Mb duplication on chromosome 17 associated with the most common form of Charcot-Marie-Tooth Disease (CMT1A). Rodent models of CMT1A have been used to show that reducing Pmp22 overexpression mitigates several aspects of a CMT1A-related phenotype. Mechanistic studies of Pmp22 regulation identified enhancers regulated by the Sox10 (SRY sex determining region Y-box 10) and Egr2/Krox20 (Early growth response protein 2) transcription factors in myelinated nerves. However, relatively little is known regarding how other transcription factors induce Pmp22 expression during Schwann cell development and myelination. Here, we examined Pmp22 enhancers as a function of cell type-specificity, nerve injury and development. While Pmp22 enhancers marked by active histone modifications were lost or remodeled after injury, we found that these enhancers were permissive in early development prior to Pmp22 upregulation. Pmp22 enhancers contain binding motifs for TEA domain (Tead) transcription factors of the Hippo signaling pathway. We discovered that Tead1 and co-activators Yap and Taz are required for Pmp22 expression, as well as for the expression of Egr2 Tead1 directly binds Pmp22 and Egr2 enhancers early in development and Tead1 binding is induced during myelination, correlating with Pmp22 expression. The data identify Tead1 as a novel regulator of Pmp22 expression during development in concert with Sox10 and Egr2.

Figure 1.

H3K27ac-marked enhancers correlate with Pmp22 expression. ChIP-Seq analysis depicts genomic regions enriched with transcriptional regulators and enhancers in P15 rat (A) sciatic nerve and (B) spinal cord. Included are binding profiles of the Schwann cell and oligodendrocyte master regulator Sox10, as well as the Schwann cell-specific regulator Egr2. Profiles of H3K27ac and the oligodendrocyte-specific regulator Olig2 in cultured oligodendrocytes (OL) were obtained from published datasets (44). H3K27ac-enriched enhancers specific to sciatic nerve are indicated by grey boxes. (C) ChIP-Seq analysis depicts enrichment of histone H3K27 acetylation in P25 rat sham versus cut sciatic nerve (3 days after transection). Enhancers lost upon nerve injury are indicated by grey boxes. (D) ChIP-Seq analysis of H3K4me3 enrichment in adult sciatic nerve identifies active promoters of expressed genes. The overlapping segments of two duplicated regions (hg36 chr17:15,143,663–15,311,619; rn5 chr10:49,146,632-49,288,636) found in patients with a mild form of CMT (18,19) is shown as a red thick line. Previously characterized Pmp22 enhancers are labeled in (A–C), which includes the upstream sites A, B and C (16), as well as sites at −7 kb and +11 kb (15,17). H3K27ac-enrichment also reveals an enhancer at − 18 kb, as well as additional upstream enhancers (−162, −134, −128, −108, −104 kb) within the duplicated region. Marked genomic regions are listed with respect to the Pmp22 translation start site. Note that Cdrt4 and Tekt3 genes located within the duplicated region are not expressed in peripheral nerve (16).

Figure 2.

Pmp22 enhancers are permissive for activation early in development. (A) Dense ChIP-Seq profiles from sciatic nerve highlight the location of Pmp22 enhancers assayed by ChIP-qPCR. ChIP-qPCR analysis identifies (B) Sox10 binding and (C) H3K27ac-enhancer mark enrichment in P1 and P15 rat sciatic nerve. Genomic regions assayed are listed with respect to the Pmp22 translation start site. Included in the analysis is a negative control site (neg.) within Tekt3, a testes-specific gene that is not expressed in Schwann cells. Levels in (B) are shown relative to control IP (goat IgG) (grey line is set at 1), whereas in (C) levels are shown relative to total histone H3 ChIP (grey line demarcates the negative control site). Error bars indicate the standard deviation of three independent experiments (*P < 0.05). (D) Representative Tead binding motifs at H3K27ac-marked enhancers are shown.

Figure 3.

Tead1 is expressed in Schwann cells. (A) ChIP-Seq analysis in adult rat sciatic nerve reveals H3K4me3 enrichment at Tead1 locus, with the promoter region highlighted in a grey box. (B) Western blot analysis compares Tead1 and Pmp22 expression in P1 and P15 rat sciatic nerve. A representative blot from two biological replicates is shown. Quantitative RT-PCR was used to determine relative mRNA expression levels in (C) primary rat Schwann cells and (D) S16 rat Schwann cells treated with siRNA targeting Sox10. Gene expression levels of the housekeeping gene Ubce7 are included as a negative control. Levels are shown relative to the sample treated with a non-targeting control siRNA, which was set as 1. Error bars indicate the standard deviation of three independent experiments (*P < 0.05).

Figure 4.

Tead1 is required for Pmp22 gene activation. Quantitative RT-PCR was used to determine relative mRNA expression levels in (A) primary rat Schwann cells or (B) S16 rat Schwann cells treated with siRNA for Tead1. Levels are shown relative to the sample treated with a non-targeting control siRNA, which was set as 1 (grey line). Error bars indicate the standard deviation of three independent experiments (*P < 0.05).

Figure 5.

The Tead co-activators Taz and Yap are required for myelin gene expression in vivo. (A) Semithin cross-sections of sciatic nerves stained with Toluidine blue from control Mpz-Cre, Taz cKO and Taz cHet; Yap cKO mice at P3 and P20 (Bar, 10 µM). Quantitative RT-PCR was used to determine relative mRNA expression levels at (B) P3 and (C) P20 in Taz cKO and Taz cHet; Yap cKO sciatic nerves. Levels are shown relative to control sciatic nerves from Mpz-Cre transgenic mice, which was set at 1 (grey line). Error bars indicate the mean of at least three independent experiments as indicated, and statistical analysis was performed by one-way ANOVA (*P < 0.05).

Figure 6.

Tead1 binds the MSE enhancer driving Egr2 expression. ChIP-qPCR analysis identifies Sox10 and Tead1 binding in (A) S16 rat Schwann cells and in (B) P1 and (C) P15 rat sciatic nerve. Binding was assayed at a negative control site (Tekt3) as well as at Sox10-bound enhancers at the ErbB3, Sox10 and Egr2 loci. Levels are shown relative to control IP (goat IgG), thus background levels are indicated by grey line at 1. Error bars indicate the standard deviation of three independent experiments (*P < 0.05).

Figure 7.

Tead1 directly regulates Pmp22 expression through specific enhancer usage. ChIP-qPCR analysis identifies Tead1 binding in (A) S16 rat Schwann cells and in (B) P1 and P15 rat sciatic nerve. Genomic regions assayed are listed with respect to the Pmp22 translation start site. Shown in (A) are percent recoveries in a control IP (goat IgG) indicated by grey line at ∼0.05% recovery, whereas in (B) levels are shown relative to control IP (grey line is set at 1). (C) Reporter analysis was used to assess Pmp22 enhancer activity. Pmp22 enhancer regions were placed upstream of a luciferase reporter gene containing a minimal promoter. (i) Reporters were transiently transfected into RT4 rat Schwann cells, and exhibited different levels of activity relative to empty vector, which is depicted by logarithmic scale. (ii) Tead1 requirement for Pmp22 enhancer activation was assayed in RT4 cells treated with Tead1 siRNA. Levels are shown relative to the control sample, which was set as 1 (grey line). The control sample was treated with a non-targeting control siRNA and transfected with an empty luciferase reporter construct. Error bars in (A–B) and (Cii) indicate the standard deviation of three independent experiments (*P < 0.05).

Figure 8.

Tead1 binding motifs are required for Pmp22 enhancer activity. (A–C) Pmp22 enhancers were examined for conserved Tead1 consensus motif sequences. Shown in the schematics are the positions of previously described Sox10 (grey ovals) and Egr2 (white triangles) binding sites (15,16), newly identified Tead1 motifs (black squares), and the distances between the sites are labeled along the bottom. Shown below the schematics are conserved sequences at the Tead1 motifs (bolded). (D–F) Luciferase assays were performed in S16 Schwann cells with constructs containing wildtype (WT; white bars) or mutant (ΔTead1; black bars) genomic segments. Levels are shown relative to the wildtype construct activity, which was set as 1. Error bars indicate the standard deviation of eight technical replicate experiments (*P < 0.05).