Fig. 6

Fig. 6

Synergistic Binding Is Sequence Based and Associated with Hoxa2-Activated Genes

(A) Sequence logo of the top motifs identified using de novo motif discovery.

(B) Distribution of motifs in Meis peaks (entire ChIP-seq), top Meis peaks far from Hoxa2, and Meis peaks corresponding to Hoxa2-Meis synergistic binding (200 nt summit regions). Red, yellow, and blue columns represent the occurrence of TGACAG, GATNNAT, and TAAT, respectively. The occurrence of GATNNAT is significantly higher in synergistic binding events relative to Meis ChIP-seq (p values shown on the tops of columns).

(C) Percentage of Hoxa2-bound regions associated to a Hoxa2-regulated gene in the entire Hoxa2 ChIP-seq (Hoxa2_ALL), Hoxa2/Meis synergistic binding, top 1% Hoxa2 peaks (TOP_Hoxa2_ONLY), and top 1% Meis peaks (TOP_Meis_ONLY). For each category, the corresponding number of regions is indicated on the x axis.

(D) High enrichment of the histone mark H3K27Ac on Hoxa2/Meis synergistic binding regions in IIBA chromatin, relative to a negative control region using ChIP qPCR. Data are presented as the average of two independent experiments in duplicate and error bars indicate the SEM.

(E) Integration of a lacZ reporter gene in genomic regions containing Hoxa2/Meis synergistic binding events. The expression of the reporter (top) and the expression of Hoxa2-regulated genes associated to the integration sites (bottom) are shown.

(F) ChIP-seq tracks corresponding to the genomic region containing Zfp703. Meis binding in the IIBA overlapping a Hoxa2 peak (enclosed by the red rectangle) is enhanced relative to Meis binding in the IBA. Black arrow shows similar binding of Meis in the IBA and IIBA in regions not bound by Hoxa2.

(G–J) Whole-mount ISH on E11.5 wild-type (G and I) and Hoxa2 mutant (H and J) embryos, using Zfp703 (G and H) and Zfp503 (I and J) probes. Both Zfp703 and Zfp503 are specifically downregulated in the IIBA (black arrows) of Hoxa2 mutant embryos. See also Figure S5.