XB-IMG-169575
Xenbase Image ID: 169575
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Figure 6. Chd7 interacts with Sox10 and selectively activates myelination-related genes during OL differentiation(a) ChIP-seq density heatmaps for Chd7, H3K27Ac and Olig2 within ± 5 kb around the Chd7 peak center.(b) Histogram showing the distribution of ChIP-seq peaks relative to the TSS.(c,d) Average H3K27Ac and Olig2 ChIP-seq enrichment profiles around the central position of Chd7 binding regions.(e) Pie chart showing relative percentage of Chd7 target genes that are significantly upregulated or downregulated in Chd7cKO.(f) Box plots for mRNA levels of Chd7 targeted genes that are differentially expressed in Chd7cKO mutants.(g) Representative GO functional categories for Chd7 targeted genes.(h) ChIP-seq showing Chd7 enrichment at selected gene loci (Sox10, Myrf, Mbp and Ugt8). Tracks represent sequence tag enrichments for Chd7, H3K27Ac, Olig2, Brg1, H3K4me3 and IgG. Genome scale bars: 5 kb.(i) MEME analysis of the most highly represented de novo motif in the Chd7 cistrome in OLs. Sox10 binding motif was identified as the most significant matching binding site. Letter size indicates nucleotide frequency (presented as ‘bits’) at each position (horizontal axis).(j,k) Co-immunoprecipitation of HA-Sox10 with Chd7 from transiently transfected 293T cells (j) or endogenous Sox10 with Chd7 in differentiating Oli-Neu cells (k). Full-length blots are presented in Supplemental Figure 13.(l) Venn diagrams depicting overlap between Chd7 and Sox10 occupancy in OLs.(m,n) ChIP reads density plots for levels of H3K27Ac and Sox10 at loci targeted by Sox10 only and co-targeted by Sox10/Chd7.(o) Luciferase activity of Cnp (left) and Plp1 (right) promoter driven reporters in 293T cells co-transfected with Chd7, Sox10 or both. (The data are presented as mean ± s.e.m. For Cnp-Luc, n = 3 independent experiments, * p = 0.0138; for Plp1-Luc, n = 4 independent experiments, *p = 0.038; pairwise comparison for Sox10 compared with Sox10 + Chd7).(p) qRT-PCR analyses of differentiating markers Mbp, Plp1, Cnp and Myrf in Oli-Neu cells transduced with Sox10, Chd7 or both under proliferation condition (The data are presented as mean ± s.e.m. n = 3 biological replicates; For Mbp, ANOVA F = 1661; multiple comparisons with t-test, ** pChd7
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pCIG = 0.008, tChd7
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pCIG = 6.397, *** pChd7+Sox10
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Sox10 = 0.0002, tChd7+Sox10
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Sox10 = 9.876. For Plp1, ANOVA F = 137.6; multiple comparisons with t-test, * pChd7
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pCIG = 0.017, tChd7
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pCIG = 4.792, * pChd7+Sox10
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Sox10 = 0.01, tChd7+Sox10
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Sox10 = 5.836. For Cnp, ANOVA F = 119.6; multiple comparisons with t-test, * pChd7
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pCIG = 0.013, tChd7
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pCIG = 5.344, * pChd7+Sox10
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Sox10 = 0.01, tChd7+Sox10
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Sox10 = 5.794. For Myrf, ANOVA F = 57.61; multiple comparisons with t-test, * pChd7
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pCIG = 0.028, tChd7
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pCIG = 4.042, * pChd7+Sox10
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Sox10 = 0.02, tChd7+Sox10
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Sox10 = 5.43).(q) Left: qPCR validation of knockdown efficiency of Sox10 and Chd7 in primary rat OPCs under differentiation conditions (The data are presented as mean ± s.e.m. n = 5 biological replicates; for Sox10 *** p = 0.0003, t = 6.017; for Chd7 * p = 0.016, t = 3.041; Two-tailed unpaired Student’s t test). Right: qRT-PCR analyses of OL-differentiation associated genes following treatments with scrambled (n = 3), Sox10 (n = 5), Chd7 (n = 5), and both Sox10 and Chd7 siRNAs (n = 5), respectively. (mean ± s.e.m. n, numbers of biological replicates; for Mbp, ANOVA F (3, 14) = 36.26; multiple comparisons with t-test, * pChd7
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scrambled = 0.03, tChd7
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scrambled = 2.82, *** pSox10
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scrambled = 0.0004, tSox10
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scrambled = 6.84, ** pSox10+Chd7
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Sox10 = 0.006, tSox10+Chd7
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Sox10 = 3.7. For Plp1, ANOVA F (3, 14) = 38.12; multiple comparisons with t-test, * pChd7
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scrambled = 0.015, tChd7
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scrambled = 3.34, *** pSox10
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scrambled = 0.0005, tSox10
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scrambled = 6.68, ** pSox10+Chd7
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Sox10 = 0.004, tSox10+Chd7
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Sox10 = 3.86. For Cnp, ANOVA F (3, 14) = 37.99; multiple comparisons with t-test, * pChd7
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scrambled = 0.033, tChd7
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scrambled = 2.74, *** pSox10
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scrambled = 0.0002, tSox10
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scrambled = 7.55, * pSox10+Chd7
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Sox10 = 0.01, tSox10+Chd7
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Sox10 = 3.15. For Myrf, ANOVA F (3, 14) = 21.45; multiple comparisons with t-test, ** pSox10
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scrambled = 0.002, tSox10
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scrambled = 5.74, * pSox10+Chd7
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Sox10 = 0.03, tSox10+Chd7
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Sox10 = 2.69).(r) qRT-PCR analyses of differentiated OL markers Mbp, Cnp and Myrf in Oli-Neu cells transduced with Sox10, Chd7 K998R mutant form or both under proliferation condition (The data are presented as mean ± s.e.m. n = 4 independent experiments; for Mbp, ANOVA F (3, 12) = 8.803; multiple comparisons with t-test, * pSox10 vs pCIG = 0.022, tSox10 vs pCIG = 3.062, * pSox10+Chd7 K998R vs Sox10 =0.025, tSox10+Chd7 K998R vs Sox10 =2.97. For Cnp, ANOVA F (3, 12) = 4.802; multiple comparisons with t-test, * pSox10 vs pCIG = 0.035, tSox10 vs pCIG = 2.706, * pSox10+Chd7 K998R vs Sox10 =0.035, tSox10+Chd7 K998R vs Sox10 =2.71. For Myrf, ANOVA F (3, 12) = 12.76; multiple comparisons with t-test, * pChd7 K998R vs pCIG = 0.031, tChd7 K998R vs pCIG = 2.79, * pSox10 vs pCIG = 0.014, tSox10 vs pCIG = 3.447, * pSox10+Chd7 K998R vs Sox10 =0.015, tSox10+Chd7 K998R vs Sox10 =3.41). Image published in: He D et al. (2016) Image downloaded from an Open Access article in PubMed Central. Image reproduced on Xenbase with permission of the publisher and the copyright holder. Larger Image Printer Friendly View |