It's not magic - Hsp90 and its effects on genetic and epigenetic variation

Abstract

Canalization, or phenotypic robustness in the face of environmental and genetic perturbation, is an emergent property of living systems. Although this phenomenon has long been recognized, its molecular underpinnings have remained enigmatic until recently. Here, we review the contributions of the molecular chaperone Hsp90, a protein that facilitates the folding of many key regulators of growth and development, to canalization of phenotype - and de-canalization in times of stress - drawing on studies in eukaryotes as diverse as baker's yeast, mouse ear cress, and blind Mexican cavefish. Hsp90 is a hub of hubs that interacts with many so-called 'client proteins,' which affect virtually every aspect of cell signaling and physiology. As Hsp90 facilitates client folding and stability, it can epistatically suppress or enable the expression of genetic variants in its clients and other proteins that acquire client status through mutation. Hsp90's vast interaction network explains the breadth of its phenotypic reach, including Hsp90-dependent de novo mutations and epigenetic effects on gene regulation. Intrinsic links between environmental stress and Hsp90 function thus endow living systems with phenotypic plasticity in fluctuating environments. As environmental perturbations alter Hsp90 function, they also alter Hsp90's interaction with its client proteins, thereby re-wiring networks that determine the genotype-to-phenotype map. Ensuing de-canalization of phenotype creates phenotypic diversity that is not simply stochastic, but often has an underlying genetic basis. Thus, extreme phenotypes can be selected, and assimilated so that they no longer require environmental stress to manifest. In addition to acting on standing genetic variation, Hsp90 perturbation has also been linked to increased frequency of de novo variation and several epigenetic phenomena, all with the potential to generate heritable phenotypic change. Here, we aim to clarify and discuss the multiple means by which Hsp90 can affect phenotype and possibly evolutionary change, and identify their underlying common feature: at its core, Hsp90 interacts epistatically through its chaperone function with many other genes and their gene products. Its influence on phenotypic diversification is thus not magic but rather a fundamental property of genetics.

Keywords: Buffering; Canalization; Capacitor; Hsp90; Potentiate; Robustness.

Figure 1. Canalization minimizes phenotypic variation

A. Quantitative traits exhibit some degree of variation, represented here by a distribution. A canalized trait shows tight distributions (blue) regardless of genetic or environmental perturbation. Some traits can be de-canalized by environmental or genetic perturbations which increases the degree of phenotypic variation (green). B. A possible mechanism of assimilation of a new phenotype occurs via de-canalization. Over several generations of selection for a crossveinless wing phenotype in Drosophila, the rare phenotype was assimilated to a large fraction of the population.

Figure 2. Phenotypic variability revealed by inhibition of Hsp90

A. Inhibition of Hsp90 in Drosophila reveals phenotypes including black facets in one eye, notched wings, and extraneous tissue (reprint from Rutherford and Lindquist, 1998). B. Inhibition of Hsp90 by geldanamycin in A. thaliana reveals phenotypes including disruption of typical symmetry and oval shaped, flat leaves (reprint from Queitsch et al., 2002). C. Inhibition of Hsp90 in Mexican cavefish, A. mexicanus, results in variable eye size of larval fish (reprint from Rohner et al., 2013). D. Human cells expressing the FANCA mutant allele R880Q exhibit increased sensitivity to Hsp90 inhibitor ganetespib but a wild-type allele or non-buffered alleles do not. In this case, the detrimental growth phenotype revealed upon Hsp90 inhibition is only observed in a specific FANCA mutant background (reprint from Karras et al., 2017).

Figure 3. Hsp90 structure and function

A. The N-terminus of Hsp90 contains a conserved ATP binding domain (lime green), a middle domain that may bind client proteins and co-chaperones (green), and a C-terminal domain responsible for dimerization (blue). B. The Hsp90 chaperone cycle begins with the binding of co-chaperones and clients. Here co-chaperones are colored blue. All other co-chaperones and ATP are drawn in gray. The progesterone receptor is one of the best understood clients. It binds one co-chaperone, then recruits another. After ATP binding, the Hsp90 dimer clamps together. This final hydrolysis step includes binding of another co-chaperone as well as ATP. Upon ATP hydrolysis, the clamp opens, releasing a mature client protein. C. Protein interaction studies have defined the vast network of Hsp90 interactors. Hsp90 interacts with hundreds of proteins of diverse functions including protein folding, signaling, cell cycle, translation and metabolism.

Figure 4. Mechanistic examples of buffering and potentiating

A. Activity of v-Src but not c-Src is dependent on Hsp90 (reprint from Xu and Lindquist, 1993). B. Likewise, malignant transformation with v-Src is also dependent on Hsp90. Upon treatment with the Hsp90 inhibitor geldanamycin, the normal contact inhibition of growth is restored (reprint from Whitesell et al., 1994). C. Graphic illustration of Hsp90 potentiating oncogenic v-Src constitutively active kinase activity. The original observed phenotype is graphed on a phenotypic scale in black. The phenotype dependent on Hsp90 is graphed in purple with a vector designating the phenotypic difference of buffered alleles. D. Ste12 contributes to both mating and invasion. A wild-type STE12 allele results in normal mating efficiency and invasion. A mutant ste12 K150I allele results in decreased mating efficiency only at high temperature or upon inhibition of Hsp90. The same allele results in increased invasion only at high temperature. E. In the RM background, the MEC1 allele is not dependent on Hsp90. In the BY background, the MEC1 allele is dependent on Hsp90; HU-resistance and UV-resistance decreases when Hsp90 in inhibited. F. Hsp90 potentiates BCR-ABL imatinib resistance. G. Hsp90 potentiates fluconazole resistance in several erg3 alleles and other gene deletions.

Figure 5. Environmental perturbations regulate Hsp90, a central node that integrates stress sensing with the manifestation and generation of de novo variants

Many environmental perturbations including heat, salinity, and drought have the potential to alter Hsp90 activity. This altered activity affects cryptic genetic variation, buffered and potentiated variants, de novo mutations, self-templating protein conformations, and epigenetic variation. All of these will in turn alter the relationship between genotype and phenotype.

Figure 6. Hsp90-responsive phenotypes in deeply mutagenized Col-0 seedlings often resemble those commonly arising in the wild-type Col-0 background but their frequency in the population and severity is significantly increased

This observation is consistent with mutations affecting genes encoding clients that are already susceptible to Hsp90 perturbation in the wild-type through standing variation. Novel Hsp90-dependent phenotypes are also observed; genetic variation in the genes underlying these phenotypes has likely been purged in the wild-type Col-0 population. A. Phenotype examples of EMS mutagenized seedlings in the Col-0 background. B. Response to Hsp90 perturbation (geldanamycin: GdA) in Col-0 and mutagenized lines (M₃ generation). Dark red color denotes 10% increase in frequency of phenotypes under Hsp90-reduced conditions. Dark blue color denotes 10% decrease in frequency of phenotypes under Hsp90-reduced conditions. Black lines represent phenotypic frequency in the Col-0 background. An asterisk (*) denotes a significant p-value for Fisher’s Exact test (p-adj < 0.05). C. Odds ratios of seedling phenotypes in M₃ lines derived from EMS mutagenized Col-0. Seedlings were assayed for 16 early-seedling phenotypes under geldanamycin (GdA) and mock (DMSO) treatment. Increasing red intensity reflects higher Odds Ratios, with grey color reflecting an infinite Odds Ratio for that comparison. An asterisk (*) denotes a significant p-value for Fisher’s Exact test (p-adj < 0.05).

Figure 7. Phenotypic neighborhoods are rewired by changes in environment

In this cartoon example, two phenotypes are graphed in two dimensional phenotypic spaces (axes). Each point represents a different genotype. The same genotype is graphed twice when it presents a different phenotype in a different environmental condition (colored points). Phenotypes dependent on the three different environmental perturbations are graphed (purple, orange, red) at the end of vectors representing the difference due to buffering or potentiation. Research illuminating examples such as these will greatly advance our understanding of how Hsp90 and environmental perturbations alter phenotypic landscapes.