Researchers in the lab of Justin Cotney, PhD, and the Center for Craniofacial Innovation at Children’s Hospital of Philadelphia (CHOP) discovered new genetic elements that play key roles in the development of the human face. The team focused on clusters of active genetic elements, known as superenhancers, which have been thought to be critical for other diseases like cancer. The team showed that too many or too few copies of one of these superenhancers can result in craniofacial birth defects in mice and humans. These findings shed new light onto the genetics of how the human face forms.
The human face forms through precise activation of genes in both location and timing. This activation occurs when genetic elements, known as enhancers, help control the expression of target genes. This is achieved through binding of combinations of proteins known as transcription factors that recognize the specific DNA sequence of the enhancer. Due to the flexible nature of DNA and the length of chromosomes that make up our genome, long loops or folds can form to allow the transcription factors bound to specific enhancers to interact with a specific gene and activate or “enhance” its expression. Most enhancers are relatively short in length. However, some locations of the genome harbor dozens of enhancers that become activated at the same time. These “superenhancers” have been shown to control genes known to be expressed in only a handful of tissue or cell types and are frequently hijacked by cancers. However, the role of such superenhancers during development has been largely unexplored.
Cotney Lab researchers sequenced human embryonic craniofacial tissue and discovered over 100,000 active enhancers. Through computational analysis, they were able to cluster these enhancers into around 4,000 superenhancers. They were then able to identify approximately 1,500 superenhancers unique to the developing human face.
The team focused on the largest superenhancer with the highest number of individual craniofacial enhancers. This region was located in a large gene “desert” between two genes, neither of which have been shown to be important for craniofacial development. Through specialized experiments they found that this superenhancer formed a long loop, able to interact with a cluster of genes called HOXA in both mouse and human craniofacial tissues. HOXA genes play critical roles in embryonic development and help make sure that structures form in the correct location. In collaboration with two other pediatric hospitals in the U.S. and in Belgium, they found copy number variants of the superenhancer in patients with severe craniofacial conditions but no clear genetic diagnosis. “Copy number” refers to the number of times a sequence or gene repeats in an individual’s genome.
To determine if the changes in the superenhancer are the likely cause of the defects observed in humans, the Cotney team used CRISPR technology to delete the entire region from the mouse genome. They found mice lacking the superenhancer displayed severe craniofacial defects, including cleft palate. Examination of gene expression in the tissues of these mice revealed that indeed HOXA genes were strongly affected by absence of the superenhancer, and none of 10 genes located more closely to this region. Importantly, these superenhancer copy number changes have never been previously described in patients. These findings represent a new connection between superenhancers and facial development.
This study highlights how superenhancers directly influence important developmental genes and how copy number changes can result in craniofacial abnormalities in humans. The Cotney Lab plans to investigate the superenhancer identified in this study further as well as other superenhancers specific for facial development. These efforts will broaden our understanding of the genetics underlying human facial development and can help inform future development of better genetic diagnostics.
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Researchers in the lab of Justin Cotney, PhD, and the Center for Craniofacial Innovation at Children’s Hospital of Philadelphia (CHOP) discovered new genetic elements that play key roles in the development of the human face. The team focused on clusters of active genetic elements, known as superenhancers, which have been thought to be critical for other diseases like cancer. The team showed that too many or too few copies of one of these superenhancers can result in craniofacial birth defects in mice and humans. These findings shed new light onto the genetics of how the human face forms.
The human face forms through precise activation of genes in both location and timing. This activation occurs when genetic elements, known as enhancers, help control the expression of target genes. This is achieved through binding of combinations of proteins known as transcription factors that recognize the specific DNA sequence of the enhancer. Due to the flexible nature of DNA and the length of chromosomes that make up our genome, long loops or folds can form to allow the transcription factors bound to specific enhancers to interact with a specific gene and activate or “enhance” its expression. Most enhancers are relatively short in length. However, some locations of the genome harbor dozens of enhancers that become activated at the same time. These “superenhancers” have been shown to control genes known to be expressed in only a handful of tissue or cell types and are frequently hijacked by cancers. However, the role of such superenhancers during development has been largely unexplored.
Cotney Lab researchers sequenced human embryonic craniofacial tissue and discovered over 100,000 active enhancers. Through computational analysis, they were able to cluster these enhancers into around 4,000 superenhancers. They were then able to identify approximately 1,500 superenhancers unique to the developing human face.
The team focused on the largest superenhancer with the highest number of individual craniofacial enhancers. This region was located in a large gene “desert” between two genes, neither of which have been shown to be important for craniofacial development. Through specialized experiments they found that this superenhancer formed a long loop, able to interact with a cluster of genes called HOXA in both mouse and human craniofacial tissues. HOXA genes play critical roles in embryonic development and help make sure that structures form in the correct location. In collaboration with two other pediatric hospitals in the U.S. and in Belgium, they found copy number variants of the superenhancer in patients with severe craniofacial conditions but no clear genetic diagnosis. “Copy number” refers to the number of times a sequence or gene repeats in an individual’s genome.
To determine if the changes in the superenhancer are the likely cause of the defects observed in humans, the Cotney team used CRISPR technology to delete the entire region from the mouse genome. They found mice lacking the superenhancer displayed severe craniofacial defects, including cleft palate. Examination of gene expression in the tissues of these mice revealed that indeed HOXA genes were strongly affected by absence of the superenhancer, and none of 10 genes located more closely to this region. Importantly, these superenhancer copy number changes have never been previously described in patients. These findings represent a new connection between superenhancers and facial development.
This study highlights how superenhancers directly influence important developmental genes and how copy number changes can result in craniofacial abnormalities in humans. The Cotney Lab plans to investigate the superenhancer identified in this study further as well as other superenhancers specific for facial development. These efforts will broaden our understanding of the genetics underlying human facial development and can help inform future development of better genetic diagnostics.
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