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. 2019 May 29;10(1):2361.
doi: 10.1038/s41467-019-10287-w.

Ep400 Deficiency in Schwann Cells Causes Persistent Expression of Early Developmental Regulators and Peripheral Neuropathy

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Free PMC article

Ep400 Deficiency in Schwann Cells Causes Persistent Expression of Early Developmental Regulators and Peripheral Neuropathy

Franziska Fröb et al. Nat Commun. .
Free PMC article

Abstract

Schwann cells ensure efficient nerve impulse conduction in the peripheral nervous system. Their development is accompanied by defined chromatin changes, including variant histone deposition and redistribution. To study the importance of variant histones for Schwann cell development, we altered their genomic distribution by conditionally deleting Ep400, the central subunit of the Tip60/Ep400 complex. Ep400 absence causes peripheral neuropathy in mice, characterized by terminal differentiation defects in myelinating and non-myelinating Schwann cells and immune cell activation. Variant histone H2A.Z is differently distributed throughout the genome and remains at promoters of Tfap2a, Pax3 and other transcriptional regulator genes with transient function at earlier developmental stages. Tfap2a deletion in Ep400-deficient Schwann cells causes a partial rescue arguing that continued expression of early regulators mediates the phenotypic defects. Our results show that proper genomic distribution of variant histones is essential for Schwann cell differentiation, and assign importance to Ep400-containing chromatin remodelers in the process.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Peripheral neuropathy resulting from Ep400 deletion in Schwann cells (SCs). ad Occurrence of Ep400 in SCs of sciatic nerves from control (a, b) and Ep400ΔPNS (c, d) mice at P21 as determined by co-immunofluorescence studies with antibodies against Ep400 (red) and Sox10 (green) to prove efficient SC-specific deletion. Sox10-negative cells in the nerve retained Ep400 and may represent endoneurial fibroblasts, pericytes, endothelial cells, or immune cells. Scale bar: 25 µm. eh Hindlimb clasping phenotype (e, g) and sciatic nerve hypomyelination (f, h) in Ep400ΔPNS (g, h) as compared to control (e, f) mice at P21. ip, s, t, w, x Representative electron microscopic pictures of sciatic nerve sections from control (i, j, m, n) and Ep400ΔPNS (k, l, o, p, s, t, w, x) mice at P21 (il, s, t) and 2 months (2 mo) (mp, w, x) in overview (ip) and at higher resolution (s, t, w, x). Magnifications depict an activated macrophage (s) and various myelin abnormalities (t, w, x). Arrow, unmyelinated axon; arrowhead, hypomyelinated axon; asterisk, myelin debris. Scale bars: 2.5 µm. q, r, u, v Determination of the mean g ratio (q, u) and the number of myelinated axons as percentage of total axons with a diameter ≥1 µm (r, v) in ultrathin sciatic nerve sections of control (black bars) and Ep400ΔPNS (white bars) mice at P21 (q, r) and 2 mo (u, v). All large caliber axons were myelinated in control mice. Statistical significance was determined by unpaired, two-tailed Student’s t test (*P ≤ 0.05; ***P ≤ 0.001). Exact values are listed in Supplementary Tables 1 and 2 and source data are provided as a Source Data file
Fig. 2
Fig. 2
Sciatic nerve features in Ep400ΔPNS mice. a Relative distribution of internodal length in stained teased fibers from sciatic nerves of control (black bars) and Ep400ΔPNS (white bars) mice at P21 (n = 3, approximately 50 internodes measured per nerve). b Representative confocal images of paranode (green, for Caspr) and node (red, for Nav1.6) stainings in teased fibers obtained from sciatic nerves of control and Ep400ΔPNS mice at P21 by immunohistochemistry. Scale bar: 5 µm. c, d Length determination of node and paranode in sciatic nerves of control and Ep400ΔPNS mice at P21 from confocal images of teased fibers after immunohistochemistry (n = 3; mean values ± SEM). ek Comparison of the total number of Sox10-positive Schwann cells (SCs) (e), Oct6-positive pro-myelinating SCs (f), Krox20-positive myelinating SCs (g), Ki67-positive proliferating SCs (h), cleaved Caspase 3-positive apoptotic SCs (i), total TUNEL-labeled cells (j), and Iba1-positive macrophages (k) in sciatic nerve sections of control and Ep400ΔPNS mice at P0, P9, P15, P21, and at 2 months (2mo) of age (n = 3; mean values ± SEM). l, m Determination of transcript levels for Ep400, ErbB3, Fabp7, Cdh19, L1Cam, S100b, Dhh, Sox10, Oct6, Krox20, Connexin-32 (Cx32), Mbp, Mpz, Pmp22, Periaxin, Neurofascin (Nfasc), and Ankyrin 2 (Ank2) in sciatic nerve of Ep400ΔPNS mice at increasing age (P9, P21, and 2mo from left to right, indicated by triangle below the bars) by quantitative reverse transcriptase PCR as compared to transcript levels in age-matched control tissue, which were arbitrarily set to 1 (marked by line) (n = 3; mean values ± SEM). Statistical significance was determined for a particular parameter or gene at a particular age between control and Ep400ΔPNS mice by two-tailed Student’s t test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). Exact values are listed in Supplementary Tables 3–6 and source data are provided as a Source Data file
Fig. 3
Fig. 3
Expression and H2A.Z-binding profiles in nerves of Ep400ΔPNS mice. a Pie chart of expression data from RNA-Seq studies on sciatic nerves at P9 depicting genes with ≥2-fold upregulated (red), ≥2-fold downregulated (blue), and unchanged (dark gray) expression in Ep400ΔPNS mice relative to controls. b, c Gene ontology (GO) analysis (GORILLA) for biological processes enriched among genes upregulated (b) or downregulated (c) in Ep400ΔPNS nerves. Processes are sorted by statistical significance. d, e Heatmaps of H3K27ac (d) and H2A.Z (e) chromatin immunoprecipitation–sequencing (ChIP-Seq) signals ±2.5 kb around the transcriptional start site (TSS) in control and Ep400ΔPNS nerves at P9. The order of genes from top to bottom is according to the strength of the signal for the respective histone modification in the control. f, g Pie chart showing the fraction of promoters (f) and enhancers (g) that are active in early postnatal Schwann cells and at the same time occupied by H2A.Z in control nerves (dark blue/red), Ep400ΔPNS nerves (medium blue/red), or both (light blue/red). h Pie chart depicting H2A.Z peaks selectively detected only in control (black) or Ep400ΔPNS (light gray) mice or shared between the samples (rose colored) according to ChIP-Seq studies on sciatic nerves at P9. i, j GO analysis (GORILLA) for biological processes enriched among genes exhibiting H2A.Z peaks only in control (i) or Ep400ΔPNS (j) nerves. k, l Selected tracks for genes with H2A.Z peaks only in control (Bpifc, k) or Ep400ΔPNS (Cxcr3, l) nerves. Shown are H2A.Z precipitates and input for both genotypes. Peaks are marked by red (ctrl) and orange (Ep400ΔPNS) lines below the respective equally colored tracks
Fig. 4
Fig. 4
Aberrant H2A.Z occupancy and expression of developmental transcription factor genes in Ep400ΔPNS nerves. a Pie chart of shared H2A.Z peaks grouped as unchanged (dark gray), broader and higher (ochre) or narrower and lower (green) in Ep400ΔPNS nerves at P9. b, c Gene ontology terms (GORILLA) enriched among genes with broader and higher (b) or narrower and lower (c) H2A.Z peaks in Ep400ΔPNS nerves, sorted by statistical significance. d Venn diagram showing overlap between upregulated genes and transcription-related genes with broader and higher H2A.Z peaks in Ep400ΔPNS nerves. e Biological processes linked to upregulated transcription factors (blue, further analyzed) with broader and higher H2A.Z peaks in Ep400ΔPNS nerves. f, g H2A.Z (red), H3K27ac (green), and input (blue) tracks for Tfap2a (f) and Pax3 (g) genes in control (dark colors) and Ep400ΔPNS nerves (light colors) with peaks marked below the tracks. h Immunofluorescent visualization and quantification of Sox2 expressing (red) Krox20-positive (green) Schwann cells in control and Ep400ΔPNS sciatic nerve sections at P21 (n = 3; mean values ± SEM). i Reverse transcriptase PCR quantification of Tfap2a, Pou3f3, Pax3, Sox1, Sox2, and Sox3 transcripts in Ep400ΔPNS sciatic nerves at increasing age (P9, P21, and 2 months from left to right, indicated by triangle) as compared to age-matched control (arbitrarily set to 1 and marked by red line; n = 3; mean values ± SEM; statistics performed between control and Ep400ΔPNS mice for each gene and age). j In situ hybridization for Tfap2a, Pax3, and Pou3f3 on control and Ep400ΔPNS sciatic nerve sections at P21. km Activation of luciferase reporters under control of Krox20 (k), Pmp22 (l), Periaxin (Prx), Mag, Connexin32 (Cx32), and Mpz (m) regulatory regions in transiently transfected Neuro2a cells by Sox10, Krox20, Pax3, Sox2, Sox3, Tfap2a, and combinations (n = 3; presented as fold inductions ± SEM, transfections without added transcription factors set to 1 for each regulatory region). Scale bars: 5 µm (h), 50 µm (j). Statistical significance was determined by unpaired two-tailed Student’s t test (h, i) or analysis of variance (km) (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). Exact values are listed in Supplementary Tables 6–8 and source data are provided as a Source Data file
Fig. 5
Fig. 5
Partial rescue of Schwann cell (SC) defects in Ep400ΔPNS mice by Tfap2a deletion. ae Determination of Sox10-positive SCs (a) and the number of Sox2- (b), Oct6- (c), Krox20- (d), and Mbp-positive (e) subpopulations in sciatic nerve sections of control (black bars), Tfap2aΔPNS (dark gray bars), Ep400ΔPNS (white bars), and double knockout (dko, light gray bars) mice at P0, P9, P21, and 2 months (n = 3; mean values ± SEM). fm Immunohistochemistry with antibodies against Mbp (fi) and in situ hybridization with a Mpz-specific probe (jm) on sciatic nerve sections from control, Tfap2aΔPNS, Ep400ΔPNS, and dko mice at P21. nr Determination of mean g ratio (n), myelin debris (o), myelin outfoldings (p), nodal (q), and paranodal (r) length from ultrathin sciatic nerve sections of control (blue bars), Ep400ΔPNS (red bars), and dko (light gray bars) mice at P21 (mean values ± SEM, n = 3 in or and 100–200 axons in n). Values for control and Ep400ΔPNS mice are those shown in Fig. 1q and Supplementary Fig. 3s–v. s–u Comparison of Ki67-positive proliferating SCs (s), cleaved Caspase 3-positive apoptotic SCs (t), and Iba1-positive macrophages (u) in sciatic nerve sections of control (black bars), Ep400ΔPNS (white bars), and dko (light gray bars) mice at P0, P9, P21, and 2 months (n = 3; mean values ± SEM). Statistical significance was determined by analysis of variance (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). Exact values are listed in Supplementary Tables 1, 4, 5, 9, and 11 and source data are provided as a Source Data file. v Model summarizing the proposed action of Ep400. In normal SCs, Ep400 helps to turn off expression of transcriptional regulatory genes active in the precursor (Tfap2a) and immature (Pax3, Sox2) SC stage by replacing H2A.Z-containing by H2A-containing nucleosomes at their transcriptional start site. In Ep400-deficient SCs, genes keep their H2A.Z-containing nucleosomes, remain aberrantly expressed, and interfere with the expression of Krox20 and the myelin genes

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