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. 2015 Aug 18;43(14):6827-46.
doi: 10.1093/nar/gkv589. Epub 2015 Jul 2.

Identification of in vivo DNA-binding mechanisms of Pax6 and reconstruction of Pax6-dependent gene regulatory networks during forebrain and lens development

Jian Sun  1 , Shira Rockowitz  1 , Qing Xie  1 , Ruth Ashery-Padan  2 , Deyou Zheng  3 , Ales Cvekl  4
Affiliations

Identification of in vivo DNA-binding mechanisms of Pax6 and reconstruction of Pax6-dependent gene regulatory networks during forebrain and lens development

Jian Sun et al. Nucleic Acids Res. .

Abstract

The transcription factor Pax6 is comprised of the paired domain (PD) and homeodomain (HD). In the developing forebrain, Pax6 is expressed in ventricular zone precursor cells and in specific subpopulations of neurons; absence of Pax6 results in disrupted cell proliferation and cell fate specification. Pax6 also regulates the entire lens developmental program. To reconstruct Pax6-dependent gene regulatory networks (GRNs), ChIP-seq studies were performed using forebrain and lens chromatin from mice. A total of 3514 (forebrain) and 3723 (lens) Pax6-containing peaks were identified, with ∼70% of them found in both tissues and thereafter called 'common' peaks. Analysis of Pax6-bound peaks identified motifs that closely resemble Pax6-PD, Pax6-PD/HD and Pax6-HD established binding sequences. Mapping of H3K4me1, H3K4me3, H3K27ac, H3K27me3 and RNA polymerase II revealed distinct types of tissue-specific enhancers bound by Pax6. Pax6 directly regulates cortical neurogenesis through activation (e.g. Dmrta1 and Ngn2) and repression (e.g. Ascl1, Fezf2, and Gsx2) of transcription factors. In lens, Pax6 directly regulates cell cycle exit via components of FGF (Fgfr2, Prox1 and Ccnd1) and Wnt (Dkk3, Wnt7a, Lrp6, Bcl9l, and Ccnd1) signaling pathways. Collectively, these studies provide genome-wide analysis of Pax6-dependent GRNs in lens and forebrain and establish novel roles of Pax6 in organogenesis.

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Figures

Figure 1.
Figure 1.
Identification, initial characterization and genomic distribution of Pax6 peaks in embryonic forebrain and newborn lens chromatin. (A) Venn diagram showing a large overlap of Pax6 peaks in lens and forebrain chromatin. (B) Genomic location of Pax6 peaks. A bar diagram indicates the percentages of Pax6-binding sites in the promoter (±2 kb relative to TSS), gene body (exons and introns), distal regions (±50 kb relative to TSS/TES) and intergenic regions.
Figure 2.
Figure 2.
Pax6 binding in forebrain and lens chromatin at Pax6, Mab21l1, Fezf2, Ngn2, Cryaa, and Crybb3 loci. A representative panel of six loci occupied by Pax6: Pax6, and Mab21l1 (common peaks), Fezf2, and Ngn2 (forebrain-specific binding), and Cryaa and Crybb3 (lens-specific peaks). The Pax6 peaks (labeled by roman numbers) were independently validated by qChIPs as shown in Figure 5A. The previously identified Pax6 ectodermal enhancer (EE) corresponds to Pax6 peak IV. The non-bound regions (NR) examined by qChIPs are also indicated.
Figure 3.
Figure 3.
De novo enriched motifs within the Pax6 peaks. (A) A comparison between top motifs identified in lens and forebrain using all peaks (centered ± 100 bp from the peak summits) with a consensus Pax6 binding site determined by in vitro SELEX studies (JASPAR database (36)). The graphs show distribution of the de novo identified motifs within the 200 bp region examined. (B) A comparison between the second most frequent motifs and Pax6-binding sequences and identification of the common 5′-ATTA-3′ motif (boxed).
Figure 4.
Figure 4.
Identification of directly regulated Pax6 genes in forebrain and lens. (A) Venn diagram of genes identified by Pax6 ChIP-seq in E12.5 forebrain compared to differentially expressed genes in E12.5 wild type and Pax6 null embryos (22). (B) Venn diagram of genes identified by Pax6 ChIP-seq in newborn lens compared to differentially expressed genes in E14.5 wild type and conditionally inactivated Pax6 in lens (41). Note that 12 genes were differentially expressed and bound by Pax6 in both tissues. The direct target genes are shown within larger functional groups. Genes up-regulated (down-regulated) by Pax6 are shown in red (blue), respectively.
Figure 5.
Figure 5.
Validation of Pax6 directly regulated genes in forebrain and lens by qChIPs and qRT-PCR. (A) ChIP analysis of Pax6, Ephb1, Ngn2, Dmrta1, Ascl1, Fezf2, Ccnd1, Emx1, and Emx2 in E12.5 forebrain, and Pax6, Mab21l1, Prox1 and Fgfr2 in newborn lens chromatin. Position of the peaks and non-bound regions (NRs) is shown in Figure 1B and Supplementary Figure S4. The NRs were arbitrarily selected in nearby regions to the Pax6-bound peaks within the same loci. (B) RT-PCR analysis of Pax6, Ngn2, Dmrta1, Ascl1, Fezf2, Gsx2, Ephb1, Emx2 and Ccnd1 in Pax6−/- E12.5 forebrain, and Pax6, Prox1, and c-Maf in conditionally deleted E9.5 prospective lens ectoderm.
Figure 6.
Figure 6.
Heatmaps showing the co-occurrence of Pax6, core histone PTMs and RNA polymerase II. (A) Heatmap of maximal read coverage in 50 bp bins from −5 kb to +5 kb of the peak summits at forebrain Pax6 peaks (n = 3514). ChIP-seq data from H3K4me1, H3K4me3, H3K27ac, H3K27me3 and RNA polymerase II in ES cell and forebrain chromatin shown as labeled. The numbers of Pax6 peaks from clusters I to VIII are shown in table format. (B) Heatmap of maximal read coverage in 50 bp bins from −5 kb to +5 kb of the peak summits at lens Pax6 peaks (n = 3723). ChIP-seq data from H3K4me1, H3K4me3, H3K27ac, H3K27me3 and RNA polymerase II in ES cell and lens chromatin shown as labeled. The numbers of Pax6 peaks from clusters I to VII are shown in table format. (C–F) Profiles of H3K4me1, H3K4me3, H3K27ac, H3K27me3 and RNA polymerase II at forebrain Pax6 peaks in ES cell, forebrain and lens chromatin at individual clusters. Y-axis shows the read density per 50 bp averaged over Pax6-bound peaks in each tissue from −5 kb to +5 kb of the peak summits. Data were normalized to a read depth of 10 million mapped reads. Formation of forebrain-specific class II enhancers compared to their status in ES cells in clusters V and VI marked by H3K27me3 in both cell types (panels C,D). Activation of poised and inactive enhancers in ES cells in lens identified by clusters IV and VI (panels E,F), respectively.
Figure 7.
Figure 7.
Pax6-binding, RNA abundance and extended H3K27ac regions in Ccnd1, Hes5, Msi1 and Pax6 loci. (A) A comparison of genes highly expressed in forebrain (red dots) or lens (green dots) determined by RNA-seq and their enriched gene ontology (GO) functions. (B) Comparison of transcripts levels (box plots) linked to ‘Pax6-occupied’ and ‘Pax6-free enhancers’. TE, typical enhancer (i.e. marked by H3K27ac). (C) Examples of extended regions marked by abundant H3K27ac identified in Ccnd1, Hes5, Msi1, and Pax6 loci.
Figure 8.
Figure 8.
Pax6-dependent gene regulatory networks and analysis of enhancers of key regulatory genes in forebrain. (A) GRN of early stages of cortical neurogenesis. (B) GRN of cell cycle exit control in radial glial cells. (C) Dual roles of Pax6 in a subcircuit that indirectly controls expression of Tbr1 and Bhlhb5. (D) Direct repression of Ascl1 and Grx2 in the boundary region. Pax6 direct targets identified (validated) here are shown in blue (green), respectively. Pax6-binding in both forebrain and lens, and distribution of histone PTMs and RNA polymerase II is shown in Supplementary Figure S5.
Figure 9.
Figure 9.
Pax6-dependent gene regulatory networks and analysis of enhancers of key regulatory genes in lens. (A) GRN of crystallin gene expression. (B) GRN of cell cycle exit control via FGF signaling in lens cells. (C) Pax6 and Wnt signaling in lens differentiation. (D) Pax6 directly regulates genes implicated in filopodia, Wnt signaling and cytoskeleton. (E) GRN of extracellular matrix (ECM) gene expression. Pax6 direct targets identified (validated) here are shown in blue (green), respectively. Pax6-binding in both forebrain and lens, and distribution of histone PTMs and RNA polymerase II is shown in Supplementary Figure S6.
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