'Edgetic' perturbation of a C. elegans BCL2 ortholog

Abstract

Genes and gene products do not function in isolation but within highly interconnected 'interactome' networks, modeled as graphs of nodes and edges representing macromolecules and interactions between them, respectively. We propose to investigate genotype-phenotype associations by methodical use of alleles that lack single interactions, while retaining all others, in contrast to genetic approaches designed to eliminate gene products completely. We describe an integrated strategy based on the reverse yeast two-hybrid system to isolate and characterize such edge-specific, or 'edgetic', alleles. We established a proof of concept with CED-9, a Caenorhabditis elegans BCL2 ortholog. Using ced-9 edgetic alleles, we uncovered a new potential functional link between apoptosis and a centrosomal protein. This approach is amenable to higher throughput and is particularly applicable to interactome network analysis in organisms for which transgenesis is straightforward.

Figure 1

Schematic representations of genotype-phenotype associations. (a) Possible phenotypes resulting from distinct network perturbations caused by different experimental strategies or mutation types. Lightning bolts, nonsense mutations; stars, missense mutations. (b) Edgetic strategy applied to a protein of interest (grey node). Colors represent specific edges, their specific perturbation, and the specific corresponding phenotypes.

Figure 2

Isolation of ced-9 alleles insensitive to EGL-1. (a) Schematic of modified Y2H assay used to identify edgetic alleles that maintain CED-9 interaction with CED-4 in presence of EGL-1. DB: Gal4-DNA binding domain. AD: Gal4-activation domain. (b) Y2H phenotypes of the interaction between CED-4 and CED-9 (wild-type or G169E) in the absence or presence of EGL-1. Each combination is shown in quadruplicate. Panels show a filter β-galactosidase assay (left), growth assay on media without uracil (middle) and a quantitative β-galactosidase assay (right). Error bars represent standard error of the mean (n = 4). (c) CED-9ΔTM mutant library generation. ORFs mutagenized by PCR are cloned by Gateway reaction into pDONR-Express, a bacterial expression vector containing a kanamycin (Kan) resistance-encoding gene (Km^r) placed in frame with the ORF cloning site. The selection of E. coli transformants on Kan-containing plates is designed to eliminate nonsense mutations and out-of-frame changes, enriching the library with full-length ORFs that can then be transferred into the pDEST-DB Y2H vector by Gateway reaction for R-Y2H selections. White boxes surrounding ced-9 ORF represent Gateway recombination sites. (d) Crystal structure of a CED-9 (grey)/EGL-1 (light green) complex (PDB ID code 1TY4). Blue, residues mutated in ced-9 edgetic alleles insensitive to EGL-1. Substitutions are indicated. EGL-1 residues less than 4 A away from CED-9 mutated residues and CED-9 residues less than 4 A away from A183 are shown as sticks. For clarity hydrogen atoms have been omitted.

Figure 3

Schema of the edgetic strategy. The interaction network of CED-9 is mapped by Y2H and confirmed by Co-AP in human HEK293T cells (Supplementary Fig. 2). In parallel, a CED-9ΔTM mutant library enriched for full-length ORFs is generated (Fig. 2c). Interaction defective alleles are isolated by R-Y2H from the CED-9ΔTM mutant library (Supplementary Data 2). PCR amplicons of ced-9 alleles obtained directly from yeast colonies are sequenced to identify potential mutations and reintroduced by gap repair into fresh yeast cells to confirm loss-of-interaction phenotypes by Y2H (Supplementary Figs. 3–5, Supplementary Tables 2–4, and Supplementary Data 2). Interaction-defective alleles are subsequently tested by Y2H against other CED-9 partners to distinguish between edgetic and non-edgetic alleles (Supplementary Figs. 3–5, Supplementary Tables 2–4, and Supplementary Data 2). The interaction profiles of a subset of interaction-specific edgetic alleles are validated by co-AP in human HEK293T cells (Supplementary Fig. 6 and Supplementary Data 2). Validated alleles are expressed in vivo and the phenotypic consequences of their expression examined (Fig. 6).

Figure 4

Edgetic and non-edgetic residues in CED-9 and CED-9/CED-4 structures. (a) Ribbon diagram of CED-9 (PDB ID code 1OHU) (left); residues mutated in R-Y2H alleles are shown as sticks (hydrogen atoms omitted). Space filling representation of the same structure in the identical (middle) and opposite (right) orientations. Residues mutated in alleles defective for only one interaction are labeled. G82 (dashed line) is buried. (b) Fraction of residues (all residues versus the residues mutated in the indicated sets of ced-9 alleles) accessible in at least one of the three CED-9 structures,, using a 10% solvent-accessible surface area cutoff. Error bars represent standard error for a binomial distribution. (c) Ribbon diagram of CED-9 complexed with one CED-4 monomer (PDB ID code 2A5Y) (left); residues mutated in CED-4-interaction defective alleles are shown as sticks. In the zoom-in of the same view (right), CED-4 residues that interact with CED-9 residues mutated in CED-4-specific edgetic alleles are also shown as sticks. Red dashed lines, interactions. Oxygen atoms are red and nitrogen atoms blue. Hydrogen atoms are omitted. (d) Distribution of the average distance to CED-4 in the CED-9/CED-4 co-crystal obtained for 1,000,000 random sets of 6, 14 or 24 residues as compared to the average distance (Obs. <dist.>) of the residues mutated in the indicated sets of CED-4-interaction defective alleles.

Figure 5

Positioning edgetic residues in CED-9 structures. (a) Positions of edgetic residues in the CED-9 sequence. The portion of CED-9 present in the crystal (PDB ID code 1OHU) and the α-helices observed in the corresponding structure are indicated above the sequence; BCL2 Homology (BH) domains are indicated under the sequence. Edgetic and non-edgetic residues are in bold font, edgetic residues are colored as indicated. (b) Ribbon diagram of the CED-9 structure (PDB ID code 2A5Y) (left); residues mutated in edgetic alleles defective for CED-4 and/or SPD-5 interaction are shown as sticks. Helix α4 (the region undergoing EGL-1-induced conformational changes) is indicated. Van der Waals surface of the same structure in identical orientation (right). The CED-4 binding site and the hypothetical SPD-5 binding site are shown. (c) Ribbon diagram of the CED-9 structure (PDB ID code 2A5Y) (left) at opposite orientation with respect to (b). Residues mutated in edgetic alleles defective for SPD-5 and/or F25F8.1 interactions are shown as sticks. Van der Waals surface of the same structure in identical orientation (right). The EGL-1 binding site is shown.

Figure 6

Node removal and edgetic perturbation in vivo. (a) Corpse count per germ line arm in wild-type (N2) or apoptosis-defective ced-3(n717) worms treated with the indicated RNAi. Vector(RNAi) is the negative control. (b) Average number of embryos laid in the indicated strains (n~10 worms for each strain). Error bars represent standard error of the mean (c) Fraction of worm broods reaching adulthood in the indicated strains. Error bars represent standard error for a binomial distribution. (d) Corpse count per germ line arm in the indicated strains treated with RNAi targeting gfp (negative control), ced-4, or cpb-3. Error bars represent standard error of the mean. (e) Schematic of phenotypic consequences of selected network perturbations. Node removal is induced either by ced-9 or spd-5(RNAi), while edgetic perturbation is caused by the CED-9(G169E),, CED-9(W214R), or CED-9(K207E) mutations. WT: wild-type [unc-69(e587)] worm strain. ced-9^−/−: worm strain carrying a ced-9 null allele [ced-9(n1950n2161)]. CED-9 wild-type, CED-9(K207E) and CED-9(W214R) are expressed from constructs integrated into the ced-9 null allele worm strain genetic background.