Elucidation of Twist1 transcriptional hierarchies regulating cell proliferation and migration will further the understanding of the molecular mechanisms by which Twist1 functions in heart development and cancer progression. We have identified Twist1-responsive ECRs, predicted to act as gene enhancers, associated with Tbx20, Cdh11, Sema3C, Gadd45a, and Rab39b genes that promote cell proliferation and migration. These enhancers are directly bound by Twist1 in developing heart valves, and conserved E-box consensus sequences were identified that are required for Twist1-responsive gene expression. Unlike other bHLH transcription factors, whose transcriptional activity requires paired E-box consensus sequences, Twist1 appears to only require one E-box consensus site to promote gene expression. With the exception of Cdh11, each of the ECRs identified in this study contains a single E-box consensus sequence. Conversely, Cdh11-Intron1 contains 2 E-box consensus sequences, however, Twist1 binding and gene induction was detected only for E-box1. rVista2.0, oPOSSUM, DiRE, and Trafac analysis for transcription NVP-BKM120 PI3K inhibitor factor binding sequences revealed that each identified enhancer has additional conserved transcription factor binding consensus sequences. Enhancer sequences identified in these studies are located in upstream genomic regions, proximal to the gene, in 39UTR, and intronic gene regions, consistent with locations of previously identified enhancers within the genome. Interestingly, regions within close proximity to the E-box consensus site are enriched for A/T sequences, relative to more distal flanking regions. However, no common binding sequences within close proximity to the E-box consensus site of the Twist1 responsive ECRs were identified. From these data, we predict that Twist1 does not require a specific co-factor protein to promote gene expression from its downstream target genes. Although an obligate Twist1 co-factor was not identified from these experiments, Twist1 binds to the E-box consensus sequence as either a homodimer or heterodimer with E-proteins. In other systems, bHLH dimer composition dictates target gene responsiveness, but dimer-specificity of Twist1 function in heart valve development has not yet been determined. Identified Twist1 target genes involved in cell migration include Y-27632 dihydrochloride Sema3C and Cdh11. Sema3C is the gene with the greatest decrease in expression resulting from in siTwist1 treatment of MC3T3-E1 cells. Previous studies have demonstrated that Sema3C promotes cell migration of axons, neural crest cells, and metastatic cancer cells. Sema3C null mice die within the first 24 hours of life from persistent truncus arteriosus and aortic arch malformations due to neural crest migration defects. Similar to Twist1, Sema3C is important for NCC contribution to OFT development, but a role in heart valve development has not previously been reported.