Unraveling the transcriptional regulation of TWIST1 in limb development

Abstract

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.

A GWAS in Latin Americans identifies novel face shape loci, implicating VPS13B and a Denisovan introgressed region in facial variation

“…Although HDAC9 does not appear to be expressed in developing mouse craniofacial tissues, many enhancers located within HDAC9 have been shown to form long-range interactions with the neighboring TWIST1 gene during the development of limbs and pharyngeal arches in mice (34). TWIST1 is a transcription factor important in limb and craniofacial development (34), and mutations of TWIST1 have been shown to result in Saethre-Chotzen syndrome, defined by its facial and cranial gestalt (OMIM #101400). These observations suggest that the effect of SNPs showing association with profile traits in the CANDELA individuals could be mediated through regulatory elements within HDAC9 controlling TWIST1 expression during craniofacial development.…”

Quis Custodiet Ipsos Custodes (Who Controls the Controllers)? Two Decades of Studies on HDAC9

“…In an elegant study using 4C-seq, it was demonstrated 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. CRISPR technology was used to prove the impact of these distal regulative elements on Twist1 expression [ 102 ]. Alongside the role played by HDAC9 in regulating fitness [ 93 ], survival [ 26 , 103 ] and the metabolic reprogramming [ 104 ] of cancer cells, recent evidence reports a role of the deacetylase in regulating the tumor microenvironment and anti-tumor immunity.…”

The regulatory landscape of early maize inflorescence development

“…Based on studies in animals and plants, CREs can reside within the core or proximal promoters of genes (i.e., at or near the transcriptional start site (TSS)), or within cis-regulatory modules distal to their target genes, such as enhancer sequences that can influence gene expression at long range [2,3]. Functional studies on distal enhancers, predominantly in animal systems, showed they can integrate various signals and fine-tune gene expression through combinatorial recruitment of TFs that are available in distinct spatiotemporal patterns [23][24][25]. Aside from a handful of functionally tested cases, the majority identified by Quantitative Trait Loci (QTL), our knowledge of distal regulatory sequences in plants remains poorly defined [26].…”