ABSTRACT
Organismal development is remarkably robust, tolerating stochastic errors to produce consistent, so-called canalized adult phenotypes. The mechanistic underpinnings of developmental robustness are poorly understood, but recent studies implicate certain features of genetic networks such as functional redundancy, connectivity, and feedback. Here, we examine the BRZ/BEH gene family, whose function is crucial for embryonic stem development in the plant Arabidopsis thaliana, to test current assumptions on functional redundancy and trait robustness. Our analyses of BRZ/BEH gene mutants and mutant combinations revealed that functional redundancy among gene family members does not contribute to trait robustness. Connectivity is another commonly cited determinant of robustness; however, we found no correlation between connectivity among gene family members or their connectivity with other transcription factors and effects on robustness. Instead, we found that only BEH4, the most ancient family member, modulated developmental robustness. We present evidence that regulatory cross-talk among gene family members is integrated by BEH4 and promotes wild-type levels of developmental robustness. Further, the chaperone HSP90, a known determinant of developmental robustness, appears to act via BEH4 in maintaining robustness of embryonic stem length. In summary, we demonstrate that even among closely related transcription factors, trait robustness can arise through the activity of a single gene family member, challenging common assumptions about the molecular underpinnings of robustness.
INTRODUCTION
Development relies on the coordinated action of low concentrations of regulatory factors diffusing within and between cells, which inevitably results in random developmental errors. Typically, organisms tolerate developmental errors, resulting in canalized, wild-type-like individuals (Waddington 1942; Masel and Siegal 2009; Lempe et al. 2012; Whitacre 2012; Félix and Barkoulas 2015). Robustness to developmental errors is an intrinsic property of all organisms and is genetically controlled (Hall et al. 2007; Ansel et al. 2008; Sangster et al. 2008a; Rinott et al. 2011; Jimenez-Gomez et al. 2011; Metzger et al. 2015; Ayroles et al. 2015). However, the molecular mechanisms that regulate developmental robustness are poorly understood, which is largely due to the technical obstacles of studying this phenomenon in complex, multicellular organisms.
Regulation of developmental robustness has been attributed to a handful of molecular mechanisms and features of gene regulatory networks (reviewed in (Masel and Siegal 2009; Lempe et al. 2012; Whitacre 2012; Félix and Barkoulas 2015; Lachowiec et al. 2015b)). In Caenorhabditis elegans, large-scale double mutant analysis identified several highly connected chromatin modifiers as positive regulators of developmental robustness (Lehner et al. 2006). In Arabidopsis thaliana, QTL mapping for regulators of developmental robustness found evidence that the pleiotropic genes ERECTA and ELF3 regulate developmental robustness (Hall et al. 2007; Jimenez-Gomez et al. 2011); both genes are also highly connected in genetic networks. Nevertheless, these and other plant studies suggest that robustness modulators act in a trait-specific rather than global manner, presumably through epistasis with several specific partner genes.
The protein chaperone HSP90, known for its role in promoting genetic robustness (Rutherford and Lindquist 1998; Queitsch et al. 2002; Yeyati et al. 2007; Jarosz and Lindquist 2010; Rohner et al. 2013; Lachowiec et al. 2013, 2015a) also maintains developmental robustness in many organisms. For example, HSP90 perturbation across many isogenic plants results in vastly increased phenotypic diversity (Queitsch et al. 2002; Sangster et al. 2007, 2008a). Similarly, naturally low levels of HSP90 correlate with greater penetrance of mutations in isogenic worms (Burga et al. 2011; Casanueva et al. 2012). HSP90’s apparently global role in developmental robustness of plants and animals is consistent with the chaperone’s exceedingly high connectivity in genetic networks (i.e. epistasis with many different partner genes), particularly with many genes encoding kinases and transcription factors important for growth and development (Taipale et al. 2010; Lachowiec et al. 2015a).
Theoretical and empirical studies suggest that developmental robustness emerges from the circuitry of genetic networks. For example, highly connected nodes in genetic networks may be of particular importance in regulating robustness to noise due to their many interactions (Levy and Siegal 2008; Masel and Siegal 2009; Whitacre 2012). Another feature of genetic networks commonly associated with developmental robustness is functional redundancy among genes (Gutiérrez and Maere 2014). Functional redundancy will compensate for stochastic losses of function in specific gene family members or paralogs (DeLuna et al. 2008, 2010).
Gene duplication is one obvious source of network redundancy, and thereby developmental robustness. In Arabidopsis thaliana, one-third of genes belong to multi-member gene families (Swarbreck et al. 2008), which have arisen through three well-supported whole genome duplications (Simillion et al. 2002; Bowers et al. 2003), in addition to segmental and tandem duplication events (The Arabidopsis Initiative 2000). Duplication of transcription factor genes provides a plausible but potentially complex form of robustness regulation. Transcription factor family members recognize highly similar DNA motifs (Franco-Zorrilla et al. 2014) and often regulate one another (Phillips and Hoopes 2008), showing functional redundancy as well as feedback regulation (Wang et al. 2012; Sullivan et al. 2014; Lachowiec et al. 2015b). At the same time, transcription factors are particularly vulnerable nodes for developmental robustness due to their often low cellular concentrations and positions as both master regulators (Chan and Kyba 2013) and endpoints of signaling cascades (Li et al. 2014). It is unclear how these different features of transcription factors and their gene families converge to regulate developmental robustness.
The BESI/BZRI HOMOLOG(BEH) transcription factors belong to a small gene family exclusive to plants. With only six members (Wang et al. 2002), this family is tractable for studying the role of redundancy, connectivity, and feedback on developmental robustness. The well-studied founding members of the BEH family, BRII-EMS-SUPRESSOR1 (BES1) and BRASSINAZOLE-RESISTANT1 (BZR1) result from the most recent whole genome duplication in the A. thaliana lineage and are highly similar in sequence (Blanc et al. 2003). They are thought to be the primary transcription factors in brassinosteroid signaling; studies of phenotypic effects are largely restricted to dominant mutants (Zhao et al. 2002; Yin et al. 2002; Wang et al. 2002). Brassinosteroid signaling regulates a large number of physiological processes in plants, ranging from seed maturation to senescence (Clouse 2002). Brassinosteroids are recognized by the membrane-associated receptor BRI1 that then represses the activity of the GSK3 kinase BIN2. In the absence of brassinosteroids, BIN2 phosphorylates and inhibits BES1 and BZR1 (Zhao et al. 2002). In this phosphorylated state, BES1 and BZR1 are prohibited from entering the nucleus (Gampala et al. 2007). In the presence of brassinosteroids, BES1 and BZR1 are dephosphorylated (Tang et al. 2011) and localize to the nucleus, where they activate and repress different sets of targets genes (Yin et al. 2005; He et al. 2005; Sun et al. 2010; Yu et al. 2011). BES1 and BZR1 are known to interact with several other proteins to regulate transcription. For example, BES1 dimerizes with BIM family proteins (Yin et al. 2005) to increase DNA binding affinity in vitro, interacts with its target gene MYBL2 (Ye et al. 2012), and works with ISW1 (Li et al. 2010a), ELF6, and REF6 (Yu et al. 2008) to alter chromatin accessibility. Some studies have revealed differences in BES1 and BZR1 protein interactions. For example, BES1, but not BZR1, interacts with the known robustness regulator HSP90 (Shigeta et al. 2013; Lachowiec et al. 2013).
In contrast, the other family members BEH1-4 are little studied, largely due to the lack of well-characterized loss-of function or dominant mutants. As BES1 and BZR1, BEH1, BEH2, BEH3, and BEH4 are thought to act as transcription factors (Wang et al. 2002; He et al. 2005). Moreover, BEH1, BEH2, BEH3, and BEH4 are phosphorylated in a manner similar to BES1 and BZR1 (Yin et al. 2005), and yeast two-hybrid analyses show that BEH2, in addition to BES1 and BZR1, interacts with a GSK3 kinase (Rozhon et al. 2010). In sum, previous studies support that BEH1, BEH2, BEH3, and BEH4 act redundantly with the well-studied transcription factors BES1 and BZR1 (Krizek 2009; Ye et al. 2012).
Here, we systematically examined the entire BEH family for effects on developmental robustness through the lenses of redundancy, connectivity, and feedback. Contrary to commonly held assumptions about the importance of redundancy and connectivity in robustness, we observe that robustness in hypocotyl growth arises largely due to the function of a single gene, BEH4, which appears to maintain proper cross-talk among BEH family members. Further, we trace HSP90’s role in maintaining robustness of hypocotyl length to the function of BEH4, thereby elucidating how this well-known regulator of global developmental robustness specifically affects this trait.
METHODS
Plant materials and growth conditions
bes1-2 (Lachowiec et al. 2013), bzr1-2 (GABI_857E04), beh3-1 (SALK_017577), and beh4-1 (SAIL_750_F08) are in the Col-0 background. beh1-1 (SAIL_40_D04) and beh2-1 (SAIL_76_B06) are in the Col-3 background. Using qPCR (see below), we confirmed that none of the mutants produced full-length transcripts; most produced no transcript at all.
For hypocotyl length assays, seeds were sterilized for 10 minutes in 70% ethanol, 0.01% Triton X-100, followed by 5 minutes of 95% ethanol. After sterilization, seeds were suspended in 0.1% agarose and spotted on plates containing 0.5x Murashige Minimal Organics Medium and 0.8% bactoagar. Seeds on plates were then stratified in the dark at 4°C for 3 days and then transferred to an incubator cycling between 22° for 16 hours and 20° for 8 hours to imitate long days. Plate position was changed every 24 h to minimize position effect for light grown seedlings. Racks of plates containing dark-grown seedlings were wrapped in foil. For HSP90-inhibitor assays, 1μM geldanamycin (Sigma) was suspended in the medium. Equivalent amounts of the solvent DMSO were used for control treatment.
Phenotyping
For estimates of hypocotyl CV, three replicates of n > 50 were measured. Assays of mean hypocotyl length were completed in triplicate with n > 15. Photos were taken of each plate, and individual hypocotyls were manually measured using NIH ImageJ1.46r.
qPCR
Three biological replicates of sixty pooled 5-day dark grown seedlings were harvested. Tissue was frozen in liquid nitrogen and ground by hand with a pestle. RNA was extracted using the SV Total RNA Isolation kit (Promega). To remove contaminating DNA, a second DNase treatment was completed according to the Turbo DNase protocol (Ambion). Poly-A tail cDNA was produced using LightCycler kit with oligo-dT primers (Life Technologies). qPCR primers are listed in Table S3. In both the bzr1-2 and beh2-1 mutants these qPCR primers amplified products. The absence of the full-length transcripts was confirmed using primers that target the full-length transcript.
RESULTS
BEH family members share function in regulating hypocotyl elongation in the dark
To dissect the individual functions of different members of the BEH family, equivalent mutants, ideally recessive, complete loss-of-function (lof) mutants, are required for genetic analysis. For studies of BES1 and BZR, researchers have largely relied on the dominant mutants bes1-D and bzr1-1D, which introduce the same nucleotide change in their respective PEST domains (Yin et al. 2002; Wang et al. 2002). This mutation appears to stabilize PEST interaction with a de-phosphatase PP2A (Tang et al. 2011), thereby creating dominant mutants that are constitutively active. Not all members of the BEH family are predicted to contain homologous PEST domains (Rogers et al. 1986) (Figure S1), so comparable dominant mutants cannot be created. To generate comparable lof mutants, we acquired T-DNA insertion mutants for each gene family member (bes1-2, bzr1-2, beh1-1, beh2-1, beh3-1, and beh4-1) (Lamesch et al. 2012) (Figure S1). Based on expression analysis, we are confident that we have generated complete lof mutants for each family member, thereby enabling unbiased phenotype comparisons.
The phenotypes of bes1-D and bzr1-1D included hyper-elongation of hypocotyls when grown in the dark (Yin et al. 2002; Wang et al. 2002), suggesting that BEH1, BEH2, BEH3, and BEH4 may function in promoting hypocotyl growth. Indeed, the bes1-2, bzr1-2, beh3-1, and beh4-1 recessive lof mutants produced significantly shorter hypocotyls than wild-type in the dark (Figure 1a, p < 0.0001, linear mixed effects model, n = 70), demonstrating that these four family members, but not BEH1 and BEH2, are positive regulators of dark growth. Our results are consistent with previous findings in which RNAi targeting BES1 reduces hypocotyl length (Yin et al. 2005; Wang et al. 2013), and the bes1-1 T-DNA insertion mutant exhibits reduced hypocotyl length (He et al. 2005). Curiously, the recessive lof mutants of the founding and best-studied members of the BEH family, BES1 and BZR1, were not the most affected in dark growth; the lof mutants of the ancestral members BEH3 and BEH4 showed larger effects on dark growth, with beh4-1 exhibiting the strongest defect (Figure 1a), though the effect size was still small. The small but significant effects in these four mutants suggest that these gene family members share function but are not fully redundant in regulation hypocotyl growth in the dark.
There was no significant difference in dark growth between bes1-2 and bzr1-2 mutant seedlings, suggesting that BES1 and BZR1 contribute to dark growth to the same degree (Figure 1a). This finding is consistent with the similar phenotypes of the dominant bes1-D and bzr1-1D mutants (Lachowiec et al. 2013); it is also consistent with the high sequence identity between BES1 and BZR1 (Wang et al. 2002), and their overlapping patterns of expression (Yin et al. 2002; Wang et al. 2002). To determine whether BES1 and BZR1 independently (i.e. additively) regulate dark growth, we examined the bes1-2;bzr1-2 double mutant. The double mutant tended to be shorter than either single mutant, but was only significantly shorter than bes1-2 (Figure 1b), suggesting that BZR1 is epistatic to BES1 in promoting hypocotyl growth in the dark. Thus, although BZR1 and BES1 do not act fully redundantly in hypocotyl elongation, they appear to have overlapping rather than independent functions in its regulation. We speculate that these degenerate functions of BES1 and BZR1 may arise from different interacting protein partners.
Among the six family members, BEH1 and BEH2 did not affect dark growth (Figure 1a). Both genes are highly similar in sequence. To explore potential functional redundancy between BEH1 and BEH2, we created the respective double mutant and assessed hypocotyl growth. The double mutant beh1-1;beh2-1 exhibited no significant growth defect compared to wild-type, or the single mutants beh1-1, or beh2-1 (Figure 1c). This result indicates either that BEH1 and BEH2 do not regulate hypocotyl elongation or that they act redundantly with other family members or other, unrelated genes in regulating dark growth. Taken together, BES1, BZR1, BEH3, and BEH4 share the function of regulating hypocotyl growth during growth in the dark. Notably, the beh4-1 single mutant was significantly shorter than the bes1-2;bzr1-2 double mutant (p < 0.0001, linear mixed effects model, n = 70), demonstrating BEH4’s dominant role in controlling dark growth.
In addition to skotomorphogenesis, BES1 and BZR1 are also important for photomorphogenesis and flowering (Li et al. 2010b) based on bes1-D and bzr1-1D phenotypes. BES1 is known to interact with the flowering time regulating proteins, ELF6 and REF6 (Yu et al. 2008). We detected no significant defects for the BEH family recessive lof mutants for flowering time (Figure S2), which agrees with earlier findings for a BES1 T-DNA insertion line, bes1-1 (He et al. 2005).
When grown in the light, bes1-D and bzr1-1D exhibit opposing effects on hypocotyl growth, with bzr1-1D showing shortened hypocotyls (He et al. 2005; Gampala et al. 2007). In previous work, the recessive line bes1-1 showed reduced growth in the light (He et al. 2005). Therefore, we examined all of our recessive mutants for light growth. As light-grown seedlings have very short hypocotyls, at least 70 seedlings per genotype were required to detect significant differences for an effect size of 0.5mm (power analysis, power = 0.8). We hypothesized that the bzr1-2 would show longer hypocotyls than wild-type in the light, based on the shortened bzr1-1D phenotype. Indeed, bzr1-2 showed significantly longer hypocotyls than wild-type (p = 0.0087, linear mixed effects model, n = 70, Figure S3). In summary, our results reveal extensive, yet not complete, functional redundancy among these closely related transcription factors and emphasize the importance of using recessive, lof mutants for genetic analysis to elucidate function and primacy of individual genes in gene families
BEH4 is a determinant of robustness
We hypothesized that the observed extensive functional redundancy among BES1, BZR1, BEH3, and BEH4 may contribute to developmental robustness of dark grown hypocotyls (Wagner 2000; Gu et al. 2003; Lachowiec et al. 2015b). Measuring developmental robustness is straightforward in isogenic lines. By growing isogenic lines randomized in the same controlled environment, any variation in phenotype is attributed to errors in development and used as a measure of developmental robustness (Waddington 1942; Queitsch et al. 2002). Developmental robustness is often expressed as the coefficient of variation or CV (s2/u) (Lempe et al. 2012; Geiler-Samerotte et al. 2013; Gutiérrez and Maere 2014). We measured hypocotyl length with high replication in the BEH family single mutants using a randomized design to control for micro-environmental differences. Mutants in the founding members of the BEH family, bes1-2 and bzr1-1 did not significantly affect developmental robustness in hypocotyl length. Instead, beh4-1 showed a highly replicable and significant decrease in developmental robustness (Figure 2a, p = 3.145* 10−7, Levene’s test, n = 210). No other single mutant significantly affected robustness. We conclude that robustness in dark grown hypocotyls was most affected by BEH4 activity, which also affected trait mean the most (Figure 1a). This result recalls the results of a prior study, in which we found that HSP90-dependent loci for developmental robustness of dark grown hypocotyls often coincide with those for trait means upon HSP90 perturbation (Sangster et al. 2008a).
We hypothesized that we may observe a further loss of robustness by removing additional functional BEH family members. We examined the bes1-2;beh4-1 double mutant because both single mutants affected mean hypocotyl length in the dark. Surprisingly, we found that loss of BES1 activity partially rescued developmental robustness in the double mutant (Figure 2b). Similarly, loss of BES1 activity partially rescues mean trait value in the bes1-2;beh4-1 compared to the single beh4-1 mutant (Figure 2c). We conclude that BEH4 and BES1 do not act redundantly in generating hypocotyl developmental robustness and trait means. Rather, we suggest that developmental robustness arises through the integrated activity of various family members. Note that bes1-2 alone did not affect developmental robustness. It is only through its interaction with BEH4 that we observed its apparently stabilizing effect. Indeed, others have argued that BES1 directly or indirectly regulates BEH4 as shown by ChIP-analysis, at least in aerial tissues (Yu et al. 2011).
Expression feedback among members of the BEH family in the light and dark
We wanted to further explore the putative functional integration among BEH family members that may underlie BEH4-dependent developmental robustness. Specifically, we hypothesized that BEH4 acts as hub gene among BEH family members. Highly connected hub genes such as the well-characterized HSP90 are thought to affect robustness through their interaction with many other loci; hub perturbation results in large-scale phenotypic effects and loss of robustness (Levy and Siegal 2008; Fu et al. 2009; Lempe et al. 2012; Lachowiec et al. 2015b). BES1 and BZR1 ChIP results (Sun et al. 2010; Yu et al. 2011) suggest that all other BEH family members are potential transcriptional targets of BES1 and BZR1 (Table S1), consistent with direct or indirect regulation among family members. Further, expression of BEH2 is up-regulated in RNAi lines in which BES1 is targeted (Wang et al. 2013), and BZR1 expression is reduced in bes1-1 mutants (Jeong et al. 2015). To test our hypothesis that BEH4 is the most highly connected genes in this gene family, we determined the relative expression of each BEH family member in each single lof mutant background. If mean gene expression was altered more than 2-fold in a given mutant background, we assumed a direct or indirect genetic interaction between the assayed and the mutated gene. Disproving our hypothesis, we found that BEH3 was the most highly connected gene among the BEH family, not BEH4 (Figure 3). Seven connections among BEH3 and other family members were counted, with BEH3 directly or indirectly regulating three family members and BEH3 expression affected in four mutants. Two of these interactions were reciprocal, in which BEH3 and BEH4 regulate each other, as well as BEH3 and BES1. Similar to BEH3, BEH4 directly or indirectly affected gene expression of three family members, but only two mutants influenced BEH4 expression. Notably, the lof beh3-1 mutant showed no decrease in developmental robustness; hence, connectivity, another frequently cited cause of developmental robustness (Levy and Siegal 2008; Lachowiec et al. 2015b), is apparently not majorly involved in robustness of hypocotyl growth. This interpretation does not consider possible interactions at the protein level through heterodimers among family members or connections of BEH4 with genes outside its gene family.
Although connectivity was not associated with phenotypic effects, gene duplicate age appeared to be associated with the number of connections among family members. BES1 and BZR1 are the most recently duplicated members of the family, followed by BEH1 and BEH2, with BEH3 and BEH4 being the most ancestral (Blanc et al. 2003). With three connections, BZR1 and BES1 were the least connected genes; BEH1 and BEH2 each showed four direct or indirect regulatory connections. These results are consistent with closely related transcription factors gaining regulatory complexity over time as paralogs are added.
To further explore the regulatory network underlying hypocotyl elongation in the dark, we analyzed recent DNaseI-seq data of dark grown seedlings (Sullivan et al. 2014). We and others have suggested that robustness regulators may be characterized by numerous regulatory inputs and few outputs, an architecture well suited to buffer noise (Sangster et al. 2004; Lehner et al. 2006; Levy and Siegal 2008; Rinott et al. 2011). Therefore, we identified and counted transcription factor (TF) binding motifs in the accessible chromatin marking the putative promoters of all BEH gene family members (Table S2). The promoter-proximal accessible chromatin of BEH4 and BEH3 each contained 25 TF binding motifs; 26 TF motifs were found for BEH2 and 35 for BZR1. In contrast, no TF motifs were detected for BEH1, and only six TF motifs were found for BES1. We conclude that at least for the BEH gene family the number of regulatory inputs (measured as number of promoter TF binding sites) is not associated with the severity of phenotypic effects on developmental robustness or trait mean. We were unable to assess regulatory outputs because the binding motifs of individual BEH family members are unknown. BES1 and BZR1 both recognize the BRZ motif, which resided in accessible, promoter-proximal chromatin of 230 genes. Although BEH4 most strongly affects phenotype among the BEH family members, neither connectivity nor regulatory architecture is consistent with the hypothesized role of BEH4 as a hub gene.
HSP90 likely maintains developmental robustness of dark-grown hypocotyls via BEH4
HSP90 function is crucial for developmental robustness of dark-grown hypocotyls and other traits (Queitsch et al. 2002; Sangster et al. 2007, 2008a; b). As HSP90 chaperones the BEH family member BES1 (Shigeta et al. 2013; Lachowiec et al. 2013), we hypothesized that the dominant role of BEH4 in developmental robustness may involve HSP90. To test this hypothesis, we assessed the genetic interaction of HSP90 and BEH4, using the potent and highly specific inhibitor geldanamycin (GdA) to reduce HSP90 function. As previously observed, HSP90 inhibition in wild-type seedlings decreased robustness (Figure 4a). HSP90 inhibition in bes1-2 mutant seedlings also decreased robustness, closely resembling the phenotypic effect observed in wild-type (Figure 4a). In stark contrast, beh4-1 exhibited no change in developmental robustness upon HSP90 inhibition (p=0.296, Levene’s test, n=210). In fact, BEH4 appeared to be epistatic to HSP90 in mediating developmental robustness of dark-grown hypocotyls, suggesting that HSP90 acts via BEH4 in this pathway.
The most obvious mechanism by which HSP90 would act via BEH4 to mediate developmental robustness is by chaperoning BEH4. The BEH family member BES1, but not BZR1, is an HSP90 client (Shigeta et al. 2013; Lachowiec et al. 2013). Due to the high similarity among BEH family members, it is certainly likely that others are also HSP90 substrates, as client status is often shared among family members (Taipale et al. 2012; Lachowiec et al. 2015a). HSP90 inhibition typically compromises the function of its clients due to mis-folding and degradation (Taipale et al. 2010). The observed epistasis of BEH4 with HSP90 in developmental robustness (lack of response in beh4-1 upon HSP90 inhibition) is consistent with the hypothesis that BEH4 is an HSP90 client.
To further test this hypothesis, we analyzed trait means of all single mutants of the BEH family members with and without HSP90 inhibition. As expected from our previous studies (Lachowiec et al. 2013), the lof mutant of the HSP90 client BES1, bes1-2, was significantly less sensitive than wild-type to HSP90 inhibition (p = 0.03, linear mixed effects model, n = 20, Figure 4b). Moreover, both beh3-1, and beh4-1 were significantly less affected than wild type (p = 0.01, p < 0.0001, respectively, linear mixed effects model, n = 20, Figure 4b). In contrast, BRZ1, which is not chaperoned by HSP90 (Shigeta et al. 2013; Lachowiec et al. 2013), BEH1 and BEH2 behave like wild type. These results are consistent with our hypothesis that BEH4 and possibly BEH3 are HSP90 clients.
DISCUSSION
Developmental robustness is thought to emerge from the topology of gene networks, including the activity of redundant genes, gene connectivity, and regulatory architecture (Lachowiec et al. 2015b). Here, we trace robustness of the model trait hypocotyl length to a specific member of the BEH gene family, BEH4. Contrary to our expectations, BEH4’s role in developmental robustness of dark-grown hypocotyls does not appear to arise through functional redundancy with closely related family members. Loss of another family member did not further decrease developmental robustness; rather, we observed partial rescue. BEH4, the ancestral member of the BEH family, also showed the largest effect on the trait mean phenotype. Our observations challenge a prior theory that additional connections (here paralogs), added later, may stabilize traits (Wagner 1996). Instead, at least for this particular trait and gene family, the ancestral gene remained the largest player for both trait mean and variance (developmental robustness). Previous studies frequently found that loci that affect trait robustness also affect trait mean (Hall et al. 2007; Sangster et al. 2008a; Ordas et al. 2008; Jimenez-Gomez et al. 2011). This frequently observed overlap makes intuitive sense: a gene that significantly affects trait mean when disrupted will perturb the underlying stabilizing genetic network and may so decrease trait robustness (Félix and Barkoulas 2015). As stabilizing selection on genetic variants that affect both mean and variance will be far stronger than selection on variants that affect only trait variance, genes such as BEH4 will play critical roles in maintaining phenotypic robustness.
Gene network hubs are thought to be crucial for developmental robustness, presumably due to their high number of connections with other loci. This assumption is certainly supported by several prior studies in plants, yeast and worms (Queitsch et al. 2002; Lehner et al. 2006; Sangster et al. 2007; Levy and Siegal 2008; Rinott et al. 2011). At the small scale of the BEH gene family this assumption did not hold true. We did, however, observe that the older gene duplicates, BEH3 and BEH4, tended to engage in more regulatory connections than other family members, consistent with previous studies finding that number of protein interactions correlates with gene age (Eisenberg and Levanon 2003; Kunin et al. 2004; Saeed and Deane 2006). However, beh3-1 did not exhibit altered developmental robustness, indicating that connectivity alone does not suffice to explain effects on developmental robustness.
One may argue that our experiments did not thoroughly test BEH4 as a hub, as we primarily restricted our analysis to the BEH family. The known genetic network underlying hypocotyl dark growth is certainly complex (Oh et al. 2014), and thus far BEH4’s role within this network has been unknown. Our analysis of DNAseI-seq data for dark-grown seedlings revealed the putative number of TFs regulating different BEH family members (Table S1). The number of potential regulatory inputs for individual family members did not correlate with the severity of the phenotypic effects in their mutants; several family members showed equal or more inputs that BEH4.
Our data best support the alternative hypothesis that BEH4’s role in developmental robustness arises through the topology of its connections with other family members. For example, feedback loops are known to promote robustness (Hornstein and Shomron 2006; Ebert and Sharp 2012; Cassidy et al. 2013; Lachowiec et al. 2015b). We found that BEH4 positively regulates BEH3 and BEH1, which in turn, both negatively regulate BEH4. Hence, loss of robustness in beh4 mutants likely arises through the loss of finely tuned regulation among family members. This hypothesis is supported by our observation that in the bes1-2;beh4-1 double mutant developmental robustness is partially rescued, possibly because the fine-tuned balance among family members is partially restored in the double mutant.
The BEH family member BES1 is known to be a client of the developmental robustness regulator HSP90 (Shigeta et al. 2013; Lachowiec et al. 2013). HSP90 presumably governs developmental robustness by chaperoning its client proteins, which function in diverse developmental pathways (Taipale et al. 2010). HSP90 inhibition leads to destabilization and loss of function for its many clients (Xu 1993; Taipale et al. 2012). Notably, loss of BES1 function did not affect robustness, indicating that HSP90 does not regulate robustness through its client BES1. Instead, we observed that HSP90-dependent robustness of hypocotyl growth is likely due to BEH4 function—unlike wild type, the beh4-1 mutant showed no response to HSP90 inhibition with regard to developmental robustness. Together, this result and the significantly diminished mean response of beh4-1 mutant to HSP90 suggest that BEH4 is also an HSP90 client. In sum, we propose that HSP90 regulates developmental robustness of dark-grown hypocotyls through the activity of BEH4, which is central for fine-tuned cross-regulation among all BEH family members.
ACKNOWLEDGMENTS
We thank Alessandra Sullivan for sharing BEH family DNaseI-seq results. This work was supported by grants from the National Human Genome Research Institute Interdisciplinary Training in Genomic Sciences (T32 HG00035 to J.L. and G.A.M.), the National Science Foundation (DGE-0718124 to J.L. and DGE–1256082 to G.A.M.) and National Institutes of Health (new innovator award no. DP2OD008371 to C.Q.).