Characterizing the Activity of TWIST1 Enhancers during Embryonic Development

Gene regulatory elements such as enhancers are crucial for executing spatiotemporal transcription programs that are required for the organism. Alteration of these regulatory elements can affect the transcription of their targeted gene and lead to human disorders, such as limb and craniofacial malformations. One of the known genes related to craniofacial and limb development is TWIST1 that plays a vital role in mesoderm development. Accordingly, haploinsufficiency of TWIST1 can cause premature fusion of the skull suture, coronal craniosynostosis and polydactyly. However, the molecular basis of TWIST1 transcriptional regulation during development has yet to be elucidated. Using zebrafish enhancer assay we characterized active enhancers in the TWIST1-HDAC9 locus that drive transcription in craniofacial tissues. Each enhancer has a discrete activity pattern, and together comprise a spatiotemporal transcription. The evolution of body structure is thought to be tightly coupled to changes in regulatory sequences, but specific molecular events associated with major morphological transitions in vertebrates have remained elusive. As TWIST1 expression pattern is conserved in vertebrates, I hypothesized that at least in part, its regulatory elements are also conserved through evolution. Therefore, I analyzed the positive enhancer sequences for their evolutionary conservation and found that several enhancers are evolutionarily conserved between vertebrates but highly diverged in snakes. Furthermore, I compared DNA samples from different organism classes including few species of snakes and preformed a zebrafish enhancer assay to identify the major differences in TWIST1 enhancers. This study associated between TWIST1 enhancer sequences and changes in the on the morphology through evolution.