Title: Unique features of metazoan gene regulation provide robust cell fate decisions from stochastic components.
Alistair N Boettiger, Jacques Bothma, Michael Perry, Michael Levine
Abstract:
The developmental patterning of multicellular organisms requires precise control on the scale of microns and chemical sensitivity on the scale of tens of molecules. The molecular system which regulates this careful construction however operates in an environment of substantial stochastic variation. We quantify this variation in the muscle fate-determination pathway in the early Drosophila embryo and study two evolutionary innovations by which the effects of such variation is minimized to allow very precise tissue specification.
The substantial degree of stochastic variation in the muscle differentiation process within clonal populations of embryos, can readily be unmasked by complimenting mild genetic and environmental stress. This results in a substantial array phenotypic differences in developmental patterning, from fully viable to lethal ones (despite identical genetic and environmental conditions). We have developed a collection of single-molecule imaging techniques with the ability to simultaneously detect hundreds of thousands of molecules to study the molecular sources of this variation. We apply these techniques to measure the contribution from independent regulatory sequences involved in regulation the key muscle determination factor, snail. We show that while individual sequences can be removed without destroying downstream developmental processes, such modifications do substantially increase variation in on the cellular and molecular level. Moreover, we find that this molecular scale variation in a random subset of embryos are such as to produce developmental defects. This work demonstrates that gene-regulatory mechanisms which appear redundant on scale of individual embryos in fact play a critical role for the fitness of the overall population. The regulatory features of the genome which allow multiple independent, spatially distributed regulatory sequences to control a common promoter of the same gene, critical for these effects we observe, is unique to metazoan genomes.
These measurements also provide some novel insights into the molecular mechanisms of regulating gene expression. In particular they suggest that for the response to muscle determination transcription factors, it is not the site occupancy of transcription factor at the regulatory site that is limiting for transcription, but the rate of interaction between the regulatory site and its cognate promoter. Using a mathematical abstraction of the gene regulation, we predict the maximum contribution from combining multiple regulatory sequences based on measurements from only the independent components. This work suggests that chromatin dynamics are a substantial source of noise in gene expression and molecular mechanisms which modulate chromatin interaction rates may function to substantially reduce the resulting variation.
We have also observed that the snail gene employs a recently characterized type of promoter common to multicellular organisms and rarely if ever found in single-cell species. This promoter binds polymerase in a non-tissue specific fashion, but only releases the polymerase to a productive elongation state in the presumptive muscle tissue. We use a combination of modeling and high resolution imaging techniques to show that this regulation of mRNA elongation instead of regulation of Pol II recruitment is a mechanism that further dampens the noise produced by molecular scale fluctuations.