Figure 5

Twist1 and candidate cofactors occupy binding regions shared among ECC, limb bud, and PNST cells. A. Binding of Twist1 to regions associated with the genes midline 1 (Mid1) and Cdc42 binding protein kinase alpha (Cdc42bpa) detected in E12.5 ECCs and E10.5 limb ChIP-seq was validated by ChIP with anti-Twist1 in E12.5 ECCs and E10.5 limb buds followed by sequence-specific qPCR. B. Twist1 binding regions containing an E-box sequence, as detected by ChIP-seq in E12.5 ECCs, E10.5 limb buds, and PNST, are associated with receptor tyrosine kinase-like orphan receptor 2 (Ror2-a), carbohydrate sulfotransferase 11 (Chst11), spermatogenesis associated 5 (Spata5) and LPS-induced TN factor (Litaf). Twist1 ChIP followed by qPCR indicates that Twist1 binds each of these sequences in E12.5 ECCs, E10.5 limb buds, and PNST cells. C. Genomatix RegionMiner was utilized to identify RXR as a potential cofactor with Twist1 in E10.5 limb bud. Twist1 binding regions containing an E-box within 40 bp of an RXR binding consensus associated with Ror2-a and Wnt5b genes involved in limb development were chosen for validation. ChIP was performed with anti-RXR in E10.5 limb buds followed qPCR amplification of Twist1 binding region sequences. D. RREB1 consensus binding sites were identified within 40 base pairs of an E-box in Twist1 binding peak sequences of Chst11, Litaf, Ror2-a, and Spata5 detected in ECC, limb bud and PNST cells. ChIP was performed with anti-RREB1 followed by qPCR amplification of Twist1 binding region sequences in ECCs, limb buds, and PNST cells. E. Motif of RXR consensus site. F. Motif of RREB consensus site. Fold-enrichment was calculated relative to normal rabbit IgG control. All analyses were performed in biological triplicate. * indicates p < 0.05 as determined by student’s t-test comparing IgG and Twist1, IgG and RXR, or IgG and RREB1 ChIP qPCR ΔΔCt values.