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. Author manuscript; available in PMC: 2021 May 10.
Published in final edited form as: Cell Rep. 2016 Jul 28;16(6):1642–1652. doi: 10.1016/j.celrep.2016.06.100

Figure 6. Chx10 and the Isl1-Lhx3-complex bind common genomic regions linked MN genes.

Figure 6.

(A) Strategy to analyze ChIPseq datasets for Chx10 and Isl1-Lhx3. DREME and MEME algorithms identified a nearly identical DNA motif that is enriched in the 927 common genomic regions recruiting both Chx10 and Isl1-Lhx3. TOMTOM algorithm demonstrated that this motif resembles Chx10-binding motif in the database.

(B) Graphical representation of the frequency of the motif from the 927 common ChIPseq peaks in relation to the central position (0 in X-axis) of the peaks.

(C) Fold change of expression for 12 genes associated with Chx10/Isl1-Lhx3 common ChIPseq peaks, which are downregulated by Chx10 (blue) in Chx10-ESCs while being upregulated by Isl1-Lhx3 (red) in Isl1-Lhx3-ESCs, as obtained with RNAseq analyses. Y-axis shows log10 values of gene expression levels in Dox-treated ESCs relative to vehicle treated controls (-Dox).

(D) Overlapping peaks for Isl1-Lhx3 (purple) and Chx10 (green) from ChIPseq datasets, associated with MN genes Hb9, Lmo1 and Slit3. The peaks are located upstream of Hb9 and Lmo1 coding regions and intron of the Slit3 gene.

(E) ChIP-qPCR analyses in E12.5 mouse spinal cords show that Isl1, Lhx3 and Chx10 are recruited to the ChIPseq peak regions associated with Hb9, Lmo1 and Slit3, but not to the Untr6 gene, a negative control genomic region. The error bars represent the standard deviation. ***, p<0.005; *, p<0.05, compared to IgG.

(F) In situ hybridization analyses in chick spinal cords electroporated (+ side) with Isl1-Lhx3, Chx10, EnR-Chx10 or VP16-Chx10. Isl1-Lhx3 promotes the expression of Lmo1 and Slit3 in the dorsal spinal cord (bracket), whereas Chx10, EnR-Chx10 and VP16-Chx10 inhibit the expression of LMO1 and Slit3 in MNs.

(G,H) Luciferase assays in P19 cells using luciferase reporters linked to a Chx10/Isl1-Lhx3-bound genomic element upstream of the Lmo1 (G) or Hb9 (H) genes. Each reporter was co-transfected with constructs shown below the graph. Chx10 WT, but not Chx10 DNA-binding mutant (Chx10 mt, Chx10-N51A), represses the transcriptional activation of the reporter gene. The error bars represent the standard deviation. ***: p<0.005, * p<0.05, ns: non-significant.