Figure 4

Validation of Twist1 ChIP-seq peak specificity ECC, limb, or PNST cells. ChIP was performed with anti-Twist1 in E12.5 ECCs (blue bars), E10.5 limb buds (green bars), and PNST cells (red bars) followed by sequence-specific qPCR to examine relative binding of Twist1 to the three binding regions with the highest fold enrichment, from MACS analysis, associated with expressed genes in each tissue. In E12.5 ECCs, Twist1 occupies binding regions associated with leukocyte cell derived chemotaxin 1 (Lect1), shroom family member 4 (Shroom4), and bromodomain and WD repeat domain containing 3 (Brwd3). In E10.5 limb buds, Twist1 occupies binding regions associated with son of sevenless homolog 2 (Sos2), transmembrane protein 18 (Tmem18), and abhydrolase domain containing 6 (Abhd6). In PNST cells, Twist1 occupies binding regions associated with CD44 antigen (Cd44) and calcium/calmodulin-dependent serine protein kinase (Cask), and transforming growth factor, beta 2 (Tgfb2). ChIP quantification was performed by qPCR, and fold-enrichment was calculated versus an anti-rabbit IgG control for each tissue. All analyses were performed in biological triplicate. * indicates p < 0.05 as determined by student’s t-test comparing IgG and Twist1 ChIP qPCR ΔΔCt values.