Highly Conserved Dark Matter Genes May Play a Role in Brain Development

There are 481 genetic sequences within the human genome that share perfect homology with both mice and rats. Since these loci have been conserved for over 20,000 years, researchers were originally under the impression that they all must code for essential proteins for mammalian life, however, this does not seem to be the case. In fact, over half of these ultra conserved loci have absolutely no protein coding function at all, leaving researchers speculating as too why they were conserved. In order to answer this questions, a previous study analyzed the effect deleting many of these loci would have on the development of mice. Interestingly, the study revealed that the deletions did not have any affect on mouse viability, fitness, or overall functionality. However, researchers at the Broad Institute of MIT and Harvard were not convinced that such highly conserved genetic sequences had no effect on mammalian development, and must affect natural selection and other evolutionary trends. In order to solve this conundrum, they decided to knock out ultra conserved loci within regions of the Aristaless-related homeobox (Arx) gene on the X chromosome, a region which contains 4/5 of the longest ultra conserved regions within the human genome. First, they wanted to identify what cells expressed Arx, as well as confirming the enhancer activity of the ultra conserved regions. Through a combination of RNA seq, drop seq, and fluorescent reporter expression, they were able to reveal that Arx was primarily expressed within radial glia and immature neurons. They also showed that all four ultra conserved regions did have strong enhancer activity. To analyze the phenotypic function of the ultra conserved regions, CRISPR was used to knockout each region either individually or in pairs within developing mouse embryos. As expected, as the mice developed, there were no obvious affects on viability, fitness, or function. However, after thorough analysis of the brains of the genetically modified mice, it was evident that the ultra conserved loci did have a significant effect on neurodevelopment. Specifically, mice with a pair of deleted ultra conserved regions presented with a significant reduction in Arx expression in brain regions previously defined with strong enhancer activity. Single deletions lead to decreased body weight, a significant decrease in neuronal count, and a reduction in the length of the dentate gyrus, a brain region important for memory and seizure development. Interestingly, every phenotype was exacerbated in paired deletions, indicating that each ultra conserved region worked with each other to effectively enhance Arx expression and neuronal development. From this analysis, the authors are confident that many other “dark matter” genes do in fact have important functions in mammalian development and therefore are conserved for a reason.

Reference

• Dickel, Diane E., et al. Ultraconserved Enhancers Are Required for Normal Development. Cell (2018).

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