Project description: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.
Project description:Transcription profiling was performed of second branchial arches of E11.5 embryos from Hoxa2+/- intercrosses. After genotyping the embryos, wild type and Hoxa2-/- were profiled by microarray.
Project description:We have undertaken a screen of mouse limb tendon cells in order to identify molecular pathways involved in tendon development. Mouse limb tendon cells were isolated based on Scleraxis (Scx) expression at different stages of development: E11.5, E12.5 and E14.5 Microarray comparisons were carried out between tendon progenitor and differentiated stages. Forelimbs from E11.5, E12.5 and E14.5 Scx-GFP embryos were collected and dissociated with trypsin to obtain cell suspensions. Scx-positive tendon cells were isolated by FACS. RNA was extracted and Fragmented biotin-labelled cRNA samples were hybridized on Affymetrix Gene Chip Mouse Genome 430 2.0 arrays.
Project description:We have undertaken a screen of mouse limb tendon cells in order to identify molecular pathways involved in tendon development. Mouse limb tendon cells were isolated based on Scleraxis (Scx) expression at different stages of development: E11.5, E12.5 and E14.5 Microarray comparisons were carried out between tendon progenitor and differentiated stages.
Project description:Pbx ChIP-seq on mouse second branchial arches, and posterior branchial arches connected to outflow tract of the heart (PBA/OFT) at embryonic day (E) 11.5.
Project description:Spatiotemporal gene expression programs are orchestrated by transcriptional enhancers which interact with target-gene promoters to regulate gene expression. The BMP antagonist Gremlin1 (Grem1) is an important node in the gene regulatory network that controls vertebrate limb development. In this study, we used a combination of open chromatin profiling (ATAC-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) in mouse and chick to identify putative cis-regulatory modules (CRMs) that regulate Grem1 in limb buds. Using CRISPR/Cas genome editing we generated different Grem1 regulatory alleles lacking either individual CRMs, combinations or entire enhancer clusters. To study potential interactions of these CRMs with the Grem1 promoter, we generated 4C-seq profiles of specific regulatory alleles. Taken together our results revealed that the spatio-temporal changes in Grem1 expression are caused by cis-regulatory alterations due to the deletions of enhancer clusters rather than global changes in chromatin architecture.
