AP-2α and AP-2β cooperatively function in the craniofacial surface ectoderm to regulate chromatin and gene expression dynamics during facial development
Abstract
The facial surface ectoderm is essential for normal development of the underlying cranial neural crest cell populations, providing signals that direct appropriate growth, patterning, and morphogenesis. Despite the importance of the ectoderm as a signaling center, the molecular cues and genetic programs implemented within this tissue are understudied. Here we show that removal of two members of the AP-2 transcription factor family, AP-2α and AP-2ß, within the early embryonic ectoderm of the mouse leads to major alterations in the craniofacial complex. Significantly, there are clefts in both the upper face and mandible, accompanied by fusion of the upper and lower jaws in the hinge region. Comparison of ATAC-seq and RNA-seq analyses between controls and mutants revealed significant changes in chromatin accessibility and gene expression centered on multiple AP-2 binding motifs associated with enhancer elements within these ectodermal lineages. In particular, loss of these AP-2 proteins affects both skin differentiation as well as multiple signaling pathways, most notably the WNT pathway. We also determined that the mutant clefting phenotypes that correlated with reduced WNT signaling could be rescued by Wnt1 ligand overexpression in the ectoderm. Collectively, these findings highlight a conserved ancestral function for AP-2 transcription factors in ectodermal development and signaling, and provide a framework from which to understand the gene regulatory network operating within this tissue that directs vertebrate craniofacial development.
Data availability
Sequencing data has been deposited in the Gene Expression Omnibus under accession code GSE199342.
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ATAC-seq, histone-seq, and RNA-seq of mouse craniofacial tissuesNCBI Gene Expression Omnibus, GSE199342.
Article and author information
Author details
Funding
National Institute of Dental and Craniofacial Research (2R01 DE12728)
- Trevor Williams
National Institute of Dental and Craniofacial Research
- Eric Van Otterloo
University of Iowa
- Eric Van Otterloo
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All experiments were conducted in accordance with all applicable guidelines and regulations, following the 'Guide for the Care and Use of Laboratory Animals of the National Institutes of Health'. The animal protocol utilized was approved by the Institutional Animal Care and Use Committee of the University of Colorado - Anschutz Medical Campus (#14) and the Institutional Animal Care and Use Committee of the University of Iowa (#9012197).
Copyright
© 2022, Van Otterloo et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Further reading
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- Chromosomes and Gene Expression
- Evolutionary Biology
Gene regulation is essential for life and controlled by regulatory DNA. Mutations can modify the activity of regulatory DNA, and also create new regulatory DNA, a process called regulatory emergence. Non-regulatory and regulatory DNA contain motifs to which transcription factors may bind. In prokaryotes, gene expression requires a stretch of DNA called a promoter, which contains two motifs called –10 and –35 boxes. However, these motifs may occur in both promoters and non-promoter DNA in multiple copies. They have been implicated in some studies to improve promoter activity, and in others to repress it. Here, we ask whether the presence of such motifs in different genetic sequences influences promoter evolution and emergence. To understand whether and how promoter motifs influence promoter emergence and evolution, we start from 50 ‘promoter islands’, DNA sequences enriched with –10 and –35 boxes. We mutagenize these starting ‘parent’ sequences, and measure gene expression driven by 240,000 of the resulting mutants. We find that the probability that mutations create an active promoter varies more than 200-fold, and is not correlated with the number of promoter motifs. For parent sequences without promoter activity, mutations created over 1500 new –10 and –35 boxes at unique positions in the library, but only ~0.3% of these resulted in de-novo promoter activity. Only ~13% of all –10 and –35 boxes contribute to de-novo promoter activity. For parent sequences with promoter activity, mutations created new –10 and –35 boxes in 11 specific positions that partially overlap with preexisting ones to modulate expression. We also find that –10 and –35 boxes do not repress promoter activity. Overall, our work demonstrates how promoter motifs influence promoter emergence and evolution. It has implications for predicting and understanding regulatory evolution, de novo genes, and phenotypic evolution.
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- Chromosomes and Gene Expression
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