Justin Cotney, Ph.D.
Assistant Professor of Genetics and Molecular Biology, Department of Genetics and Genome Sciences
My lab is interested in understanding how gene expression is regulated by distantly located sequences during development. We aim to understand how such gene regulatory sequences arise and evolve, how they locate their target genes over long distances on the same chromosome, and how defective regulatory sequences contribute to human disease. We address these questions with a combination of genome-wide sequencing based experiments and complex computational pipelines.
Evolution of Mammalian Developmental Enhancers
For over 30 years researchers have hypothesized that the small number of differences in protein-coding genes between closely related mammalian species (i.e., human vs. chimp) are likely not sufficient to explain significant differences in physiology and morphology. Many have postulated that species differences in form and function likely arise not from differences in the functions of genes themselves but how their expression is regulated in both the adult and developing embryo. We were the first to address this question with functional genomics in mammalian development. Through comparative epigenetic analysis of human, rhesus monkey and mouse limb development we identified thousands of sequences that are potentially regulating gene expression in a human specific fashion. The human gains of gene regulatory activity are significantly associated with increased expression of approximately 300 genes in human limb tissue compared to similar stages in mouse suggesting bona fide regulatory change in embryonic limb development. This work demonstrated the first in vivo experimental evidence of a gain of enhancer function in human embryonic development and provided an initial estimate for the rate of gene regulatory change in embryonic development. However, it remains to be determined if this trend is the same across similar evolutionary time scales and tissues. My lab is employing functional genomics techniques in multiple rodent species to comprehensively identify enhancers active during developmental patterning of several structures (i.e., brain, lung, heart, and limb) and uncover the sequence changes that drive differential activation.
Long-distance Regulation of Gene Expression
To fully understand how developmental enhancers influence phenotype and cause disease their regulatory targets must be identified. Mammalian genomes consist of vast expanses of DNA that can be over one hundred million base pairs in length. Protein coding genes make up less than two percent of this DNA. The sequences that control these genes can be found in close proximity to their regulatory targets (less than 10,000 bp) or potentially operate over entire chromosomes (greater than 1 million base pairs). This indicates that the chromosomes in the nucleus form reproducible tertiary structures that allow regions very far apart from one another linearly to interact. Computational methods for predicting such gene-enhancer interactions have thus far not been very accurate. My lab is implementing cutting edge chromosome confirmation capture coupled with chromatin immunoprecipitation (Hi-ChIP) experiments to directly identify active regulatory loops with high resolution during development. These interaction maps will allow us to accurately identify the target gene for active enhancers and make specific hypotheses about the effects of enhancer mutations on gene regulation. We have already generated mice with large enhancer region deletions and currently characterizing their role in regulation of critical genes during craniofacial development. We aim to further expand these studies to identify the gene-enhancer network active during early heart patterning.
Enhancer Sequence Variation and Human Disease
The vast majority of commonly occurring variants identified by genome-wide association studies for a variety of diseases are located in non-coding portions of the genome suggesting improper regulation of gene expression is a major contributor to human disease. Genes required for developmental patterning, such as homeobox transcription factors or signaling molecules like Sonic hedgehog (SHH), have complex expression patterns in multiple embryonic tissues. Activation of such genes in a precise spatiotemporal fashion is facilitated by numerous enhancers found in the non-genic portions of the genome. Loss of function mutations in such genes involved in patterning are typically lethal or cause many abnormalities. In contrast disruptions of the enhancers that control those genes are much less dramatic and isolated to specific tissues. The tissue-specific nature of the few known enhancer defects make non-syndromic birth defects an area ripe for investigation of the general role of enhancer dysfunction and human disease. In particular clefts of the lip and/or palate are very common in humans and most individuals are considered non-syndromic due to no other tissues in the body being affected.
Using experimental and computational methods we have generated the first comprehensive annotation of early human craniofacial development spanning the first four to eight weeks of gestation. We have identified over 75,000 sequences in the human genome that likely contribute to the patterning of the human face, with over 6,000 of these never before indicated to be active in a human tissue. We have shown these craniofacial enhancer regions are strongly enriched for variants associated with increased risk for non-syndromic cleft lip and palate (NSCLP). My lab is now employing massively parallel enhancer assays to test the function of tens of thousands of sequences simultaneously to determine those that are likely to play a role in NSCLP. We then aim to functionally characterize the regulatory capacity of the identified enhancers and NSCLP associated alleles in mouse craniofacial embryonic development. These experiments will identify the stages of development that these enhancers exert regulatory control, will begin to reveal the genetic architecture of NSCLP, and provide targets for future genetic screening and therapies. The paradigm we have established here of profiling the early development of a tissue and intersecting with disease genetics will greatly improve our ability to interpret our genome and realize truly personalized genomic medicine in the future.