Wild-type phenotypes are remarkably robust to environmental and genetic variation, yet evolutionary novelty continues to arise. What are the molecular underpinnings of phenotypic robustness? How do novel shapes and functions evolve from a robust wild-type phenotype? Natural selection draws upon phenotypic variation among individuals. Although selection can only fix traits with an underlying genetic basis, phenotypic variance results from a complex interplay of genetic, epigenetic, and environmental factors. We seek to identify and understand molecular mechanisms that have the potential to rapidly generate selectable phenotypic variation. Our current projects focus on Hsp90- and Pol V-mediated evolutionary capacitance, the interactions of microRNA-mediated regulation with HSP90, the phenotypic consequences of length variability polymorphisms in microsatellites, and the dynamics of the plant chromatin landscape across development and different environments.

Phenotypic capacitance

In eukaryotes, the environmentally responsive chaperone Hsp90 assists the maturation of many key regulatory proteins and ensures correct protein folding. As an unexpected consequence, genetic variation can accumulate and remain phenotypically silent. Challenging HSP90 function uncovers such cryptic genetic variation and can therefore produce altered phenotypes. Moderate environmental change alone can reveal similar selectable cryptic genetic variants in plants and flies in the laboratory. The presence of HSP90-dependent cryptic genetic variation with a plausible natural release mechanism has wide-ranging implications for phenotypic variation and possibly evolutionary change. We have recently shown that HSP90-buffered genetic variants are common in the plant Arabidopsis thaliana, suggesting that the chaperone plays an important role in genotype-phenotype translation. Our current efforts focus on identifying genomic footprints of HSP90's hypothesized evolutionary role, for example by mapping HSP90- dependent loci in natural A. thaliana populations. We have started to explore interactions of the microRNA pathway with HSP90 in plants and the role of HSP90 in bacteria. Our group is also interested in identifying novel, functionally distinct capacitors to address unique and shared features compared to HSP90. In preliminary studies, RNA Polymerase Pol V has emerged as a promising candidate.

Microsatellites in local adaptation and population genetics

Microsatellites are fast evolving, repetitive genetic elements. They frequently reside in regulatory and coding regions, where repeat copy number mutations can alter gene regulation or protein function with significant phenotypic consequences. For example, expansion of polyglutamine tracts in several human genes is associated with neurodegenerative disorders. We hypothesize that microsatellite variation contributes critically to local adaptation in A. thaliana. In proof-of principle experiments, we use classical genetics to assess the phenotypic consequences of microsatellite variation in key developmental genes. Unlike SNPs, microsatellites have entirely escaped genome-wide assessment due to technical barriers. We aim to develop a high- throughput, cost-effective method to genotype microsatellite variation genome- wide across many individuals.

The Plant Chromatin landscape

Gene regulation occurs at the level of chromatin accessibility; here, myriad proteins interact with regulatory DNA elements to orchestrate the activation and repression of genes ultimately determining the form and function of cells. Accessible regulatory DNA elements such as promoters, enhancers and insulators can be detected by their characteristic sensitivity to the endonuclease DNaseI. We have developed methods for genome-wide DNaseI-seq in A. thaliana. Our protocols utilize either the INTACT method for biotin-based capture of nuclei from specific cell types or a gradient-based method of isolating non-labeled nuclei from whole seedlings. This project is conducted in collaboration with John Stamatoyannopoulos and Jennifer Nemhauser and is funded by NSF.

Phenotypic Robustness and the Missing Heritability of Disease

The missing heritability in complex human diseases has been explained with the failure to identify the numerous genetic modifiers and environmental exposures leading to disease. Prompted by recent findings on increased mutation burden in patients with complex disease and our model organism studies, we propose an alternative explanation: genetic predispositions will translate into disease in individuals with generally decreased organismal robustness. Employing A. thaliana, we are developing molecular markers for organismal robustness that are applicable in large human populations and will test their predictive power in proof-of-principle experiments. This project received a 2011 NIH Innovator Award.