Sarov, M. | bioRxiv Science

bioRxiv2020

Recapitulating evolutionary divergence in a single regulatory element causes expression changes of the lens gene Tdrd7

Mutations in cis-regulatory elements play important roles for phenotypic changes during evolution. Eye degeneration in the blind mole rat (BMR) and other subterranean mammals is significantly associated with widespread divergence of eye regulatory elements, but the effect of these regulatory mutations on eye development and function has not been explored. Here, we investigate the effect of mutations observed in the BMR sequence of a conserved non-coding element upstream of Tdrd7, a pleiotropic gene required for lens development and spermatogenesis. We first show that this conserved element is a transcriptional repressor in lens cells and that the BMR sequence partially lost repressor activity. Next, we recapitulated the evolutionary changes by precisely replacing the endogenous regulatory element in a mouse line by the orthologous BMR sequence with CRISPR-Cas9. Strikingly, this repressor element has a large effect, causing a more than two-fold up-regulation of Tdrd7 in developing lens. Interestingly, the increased mRNA level does not result in a corresponding increase in TDRD7 protein nor an obvious lens phenotype, likely explained by buffering at the posttranscriptional level. Our results are consistent with eye degeneration in subterranean mammals having a polygenic basis where many small-effect mutations in different eye-regulatory elements collectively contribute to phenotypic differences.

evolutionary biology↗

bioRxiv2020

Cooperative molecular networks drive a mammalian cell state transition

In the mammalian embryo, epiblast cells must exit their naive state and acquire formative pluripotency. This cell state transition is recapitulated by mouse embryonic stem cells (ESCs), which undergo pluripotency progression in defined conditions in vitro. However, our understanding of the molecular cascades and gene-networks involved in the exit from naive pluripotency remains fragmented. Here we employed a combination of genetic screens in haploid ESCs, CRISPR/Cas9 gene disruption, large-scale transcriptomics and computational systems-biology to delineate the regulatory circuits governing naive state exit. Transcriptome profiles for 73 knockout ESC lines predominantly manifest delays on the trajectory from naive to formative epiblast. We find that gene networks operative in ESCs are active during transition from pre- to post-implantation epiblast in utero. We identified 374 naive-associated genes tightly connected to epiblast state and largely conserved in human ESCs and primate embryos. Integrated analysis of mutant transcriptomes revealed funneling of multiple gene activities into discrete regulatory modules. Finally, we delineate how intersections with signaling pathways direct this pivotal mammalian cell state transition.

systems biology↗