Unraveling the transcriptional regulation of TWIST1 in limb development.

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

Noncoding structural variants disrupt the regulatory architecture of Rett genes

Rett syndrome is a progressive neurodevelopmental disorder, characterized by a severe developmental delay, absence of speech, seizures, hypotonia and stereotypic movements. It is typically caused by mutations in the MECP2 gene, but several other genes, including the transcription factors MEF2C and FOXG1, have been associated with a Rett-like phenotype as well. Recently, we and others identified several noncoding structural variants (SVs) in patients with Rett-like characteristics. All SVs are located proximal to the coding sequence of MEF2C or FOXG1, suggesting disruption of the regulatory structure governing these genes. Using Circularized Chromosome Conformation Capture (4C) sequencing in a neuronal cell line, we identified a complex regulatory interaction network in the MEF2C region. We found that the MEF2C promoter physically contacts multiple distal enhancer regions upstream of its coding sequence. Based on epigenetic enhancer marks and sequence conservation, we delineated 16 putative enhancer elements, of which 14 were active in in vitro luciferase assays and 8 displayed in vivo neuronal activity during zebrafish development. For FOXG1 as well, 4C-seq experiments have shown that the promoter interacts with at least three in vivo validated brain enhancers, all situated in a region affected by deletions or translocations in multiple Rett-like patients. In summary, Rett genes MEF2C and FOXG1 are part of complex regulatory networks involving multiple distal enhancers. Disruption of these regulatory structures by noncoding SVs could form the genetic basis of the Rett-like phenotype observed in some patients