The transcription factor TWIST1 plays a vital role in mesoderm development, particularly in limb and craniofacial formation. Accordingly, haploinsufficiency of TWIST1 can cause limb and craniofacial malformations as part of Saethre-Chotzen syndrome. However, the molecular basis of TWIST1 transcriptional regulation during development has yet to be elucidated. Here, we characterized active enhancers in the TWIST1-HDAC9 locus that drive transcription in the developing limb and branchial arches. Using available p300 and H3K27ac ChIP-seq data, we identified 12 enhancer candidates, located both within and outside the coding sequences of the neighboring gene, Histone deacetyase 9 (HDAC9). Using zebrafish and mouse enhancer assays, we showed that eight of these candidates have limb/fin and branchial arch enhancer activity that resemble Twist1 expression. Using 4C-seq, we showed that the Twist1 promoter region interacts with three enhancers (eTw-5, 6, 7) in the limb bud and branchial arch of mouse embryos at day 11.5. Furthermore, we found that two transcription factors, LMX1B and TFAP2, bind these enhancers and modulate their enhancer activity. Finally, using CRISPR/Cas9 genome editing, we showed that homozygous deletion of eTw5-7 enhancers reduced Twist1 expression in the limb bud and caused pre-axial polydactyly, a phenotype observed in Twist1+/- mice. Taken together, our findings reveal that each enhancer has a discrete activity pattern, and together comprise a spatiotemporal regulatory network of Twist1 transcription in the developing limbs/fins and branchial arches. Our study suggests that mutations in TWIST1 enhancers could lead to reduced TWIST1 expression, resulting in phenotypic outcome as seen with TWIST1 coding mutations.

Zinc finger E-box–binding homeobox 2 ( ZEB2 ) is a key developmental regulator of the central nervous system (CNS). Although the transcriptional regulation of ZEB2 is essential for CNS development, the elements that regulate ZEB2 expression have yet to be identified. Here, we identified a proximal regulatory region of ZEB2 and characterized transcriptional enhancers during neuronal development. Using chromatin immunoprecipitation sequencing for active (H3K27ac) and repressed (H3K27me3) chromatin regions in human neuronal progenitors, combined with an in vivo zebrafish enhancer assay, we functionally characterized 18 candidate enhancers in the ZEB2 locus. Eight enhancers drove expression patterns that were specific to distinct mid/hindbrain regions ( ZEB2#e3 and 5 ), trigeminal-like ganglia ( ZEB2#e6 and 7 ), notochord ( ZEB2#e2 , 4 and 12 ) and whole brain ( ZEB2#e14 ). We further dissected the minimal sequences that drive enhancer-specific activity in the mid/hindbrain and notochord. Using a reporter assay in human cells, we showed an increased activity of the minimal notochord enhancer ZEB2#e2 in response to AP-1 and DLX1 / 2 expressions, while repressed activity of this enhancer was seen in response to ZEB2 and TFAP2 expressions. We showed that Dlx1 but not Zeb2 and Tfap2 occupies Zeb2#e2 enhancer sequence in the mouse notochord at embryonic day 11.5. Using CRISPR/Cas9 genome editing, we deleted the ZEB2#e2 region, leading to reduction of ZEB2 expression in human cells. We thus characterized distal transcriptional enhancers and trans-acting elements that govern regulation of ZEB2 expression during neuronal development. These findings pave the path toward future analysis of the role of ZEB2 regulatory elements in neurodevelopmental disorders, such as Mowat–Wilson syndrome.