Principal Investigator: Martha Bulyk
The human genome is often referred to as a blueprint; the genome of each individual, however, varies slightly from the reference version of the blueprint. One of the biggest challenges in interpreting personal human genomes is predicting how variants affect the functions of genes, and how these variants may contribute to human disease.
In our project, we focus on understanding how variants affect the activities of homeodomain transcription factors. Homeodomain transcription factors are proteins within cells that bind to DNA, and they play important roles in the normal functions and development of humans. Numerous homeodomain mutations have been associated with an array of human diseases. By studying how the DNA-binding activities of homeodomains change in the presence of particular variants, we are able to better understand the consequences of variants in the human genome.
Our results suggest possible ways in which disease-associated variants contribute to human disease. Our data also help clarify variants in the human genome that have been annotated as being of uncertain significance; we find that several of these variants affect the DNA-binding properties of the homeodomain transcription factors. We envision our findings contributing towards a deeper understanding of homeodomain-DNA recognition rules, and improved human variant interpretation.
Homeodomains are transcription factors with important roles in development, patterning, and cellular differentiation. Since every single position within the homeodomain DNA-binding domain (DBD) has been reported in ClinVar to have pathogenic variants and variants of uncertain significance (VUS), a more comprehensive understanding of the effects of these variants is important for variant interpretation and insights into disease mechanisms.
We sought to elucidate the consequences of such missense variation on DNA binding by profiling >90 DBD variants across 30 human homeodomains using protein-binding microarrays. Most of the disease-associated variants we examined showed diminished binding affinity. Several variants demonstrated stark changes in DNA-binding specificity, with recognition of novel sets of sequences. More surprisingly, we identified variants that altered binding affinity specifically for sets of sequences that were recognised by the wild-type homeodomain at moderate affinity, which we interpreted as a subtler change in DNA-binding specificity. We identified 9 novel DNA-binding specificity-determining positions, and performed structural modelling to suggest mechanisms for alterations in DNA-binding specificity.
Our results suggest pathogenic mechanisms for several disease-associated variants and genes, and contribute towards clarification of VUS. We envision our findings contributing towards a deeper understanding of homeodomain-DNA recognition rules, and improved variant interpretation and target gene prediction.
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