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A massively parallel pipeline to clone DNA variants and examine molecular phenotypes of human disease mutations.

Wei X, Das J, Fragoza R, Liang J, Bastos de Oliveira FM, Lee HR, Wang X, Mort M, Stenson PD, Cooper DN, Lipkin SM, Smolka MB, Yu H - PLoS Genet. (2014)

Bottom Line: We describe a massively-parallel site-directed mutagenesis approach, "Clone-seq", leveraging next-generation sequencing to rapidly and cost-effectively generate a large number of mutant alleles.We use this pipeline to show that disease mutations on protein-protein interaction interfaces are significantly more likely than those away from interfaces to disrupt corresponding interactions.The general scheme of our experimental pipeline can be readily expanded to other types of interactome-mapping methods to comprehensively evaluate the functional relevance of all DNA variants, including those in non-coding regions.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Weill Cornell College of Medicine, New York, New York, United States of America; Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Understanding the functional relevance of DNA variants is essential for all exome and genome sequencing projects. However, current mutagenesis cloning protocols require Sanger sequencing, and thus are prohibitively costly and labor-intensive. We describe a massively-parallel site-directed mutagenesis approach, "Clone-seq", leveraging next-generation sequencing to rapidly and cost-effectively generate a large number of mutant alleles. Using Clone-seq, we further develop a comparative interactome-scanning pipeline integrating high-throughput GFP, yeast two-hybrid (Y2H), and mass spectrometry assays to systematically evaluate the functional impact of mutations on protein stability and interactions. We use this pipeline to show that disease mutations on protein-protein interaction interfaces are significantly more likely than those away from interfaces to disrupt corresponding interactions. We also find that mutation pairs with similar molecular phenotypes in terms of both protein stability and interactions are significantly more likely to cause the same disease than those with different molecular phenotypes, validating the in vivo biological relevance of our high-throughput GFP and Y2H assays, and indicating that both assays can be used to determine candidate disease mutations in the future. The general scheme of our experimental pipeline can be readily expanded to other types of interactome-mapping methods to comprehensively evaluate the functional relevance of all DNA variants, including those in non-coding regions.

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Identifying interactions of Mlh1 that are affected by the I107R mutation using SILAC-based mass spectrometry.(a) Schematic illustrating criteria used to identify interactions that are lost/weakened, unchanged, and gained/enhanced due to the I107R mutation on Mlh1. Blue denotes samples cultured in light media and black denotes samples cultured in heavy media. (b) Scatter plot illustrating fold change (FC; log scale) in the amount of protein pulled down by wild-type Mlh1 and mutant Mlh1 (I107R). Values are computed based on the wild-type (heavy) vs. mutant (light) (X-axis) and mutant (heavy) vs. wild-type (light) (Y-axis) experiments. Green denotes enhancement of interaction, red denotes weakening of interaction, and gold denotes no change. Mlh1 is shown in grey. (c) Fold changes and read counts (r) for interactors of Mlh1 that can be reliably identified as weakened, unchanged, and enhanced due to the I107R mutation. (d) Anti-HA immunoprecipitation followed by Western blotting with anti-V5 antibody confirming that the Mlh1-Brip1 interaction remains unchanged and that the Mlh1-Hspa8 interaction is dramatically enhanced due to the I107R mutation.
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pgen-1004819-g006: Identifying interactions of Mlh1 that are affected by the I107R mutation using SILAC-based mass spectrometry.(a) Schematic illustrating criteria used to identify interactions that are lost/weakened, unchanged, and gained/enhanced due to the I107R mutation on Mlh1. Blue denotes samples cultured in light media and black denotes samples cultured in heavy media. (b) Scatter plot illustrating fold change (FC; log scale) in the amount of protein pulled down by wild-type Mlh1 and mutant Mlh1 (I107R). Values are computed based on the wild-type (heavy) vs. mutant (light) (X-axis) and mutant (heavy) vs. wild-type (light) (Y-axis) experiments. Green denotes enhancement of interaction, red denotes weakening of interaction, and gold denotes no change. Mlh1 is shown in grey. (c) Fold changes and read counts (r) for interactors of Mlh1 that can be reliably identified as weakened, unchanged, and enhanced due to the I107R mutation. (d) Anti-HA immunoprecipitation followed by Western blotting with anti-V5 antibody confirming that the Mlh1-Brip1 interaction remains unchanged and that the Mlh1-Hspa8 interaction is dramatically enhanced due to the I107R mutation.

Mentions: While we use only those interactions that are supported by co-crystal structures to estimate the fraction of interactions that are disrupted by mutations at different structural loci, the described procedures can also be applied to interactions with predicted interfaces and structural models [30], [31], [32], [33]. This is of particular importance because over 90% of known interactions do not currently have corresponding co-crystal structures [33], [34]. For example, Mlh1 is known to interact with Pms2, both of which are well-studied DNA mismatch repair genes frequently mutated in hereditary nonpolyposis colorectal cancer [35]. Although the structural basis of the Mlh1-Pms2 interaction still remains unknown, both our previous 3D reconstruction of the human interactome network [7], [32] and the newly-established Interactome3D [33] database suggest that the HATPase_c domain is part of the interface for Mlh1's interaction with Pms2. Previous work has shown that a point mutation (I107R) on the HATPase_c domain of Mlh1 is associated with colorectal cancer and disrupts the Mlh1-Pms2 interaction [7], [35], [36]. First, using Y2H, we were able to confirm the disruption (Figure S4). Next, we developed a high-throughput-amenable mass spectrometry pipeline using Stable Isotope Labeling by Amino acids in Cell culture (SILAC) [37], [38], which was designed to reveal both lost/weakened and gained/enhanced interactions of the target proteins (Fig. 1d) [39]. We added an HA-tag to the N-terminus of both wild-type and mutant Mlh1, as well as to GFP as a control, and performed four SILAC experiments: wild-type Mlh1 (heavy) vs. GFP control (light), mutant Mlh1 (heavy) vs. GFP control (light), wild-type (heavy) vs. mutant (light) Mlh1, and mutant (heavy) vs. wild-type (light) Mlh1 (Fig. 6a; Materials and Methods). Interactors of wild-type/mutant Mlh1 are defined as those that bind wild-type/mutant Mlh1 more than 2× stronger than GFP control (Materials and Methods). For a lost/weakened interaction, we required that the interaction be more than 2× stronger with wild-type Mlh1 than with mutant Mlh1 as confirmed both in wild-type (heavy) vs. mutant (light) and in mutant (heavy) vs. wild-type (light) experiments; we further required that the interaction be detected in the wild-type vs. control experiment (Fig. 6a; Materials and Methods). For a gained/enhanced interaction, we required that the interaction be more than 2× stronger with mutant Mlh1 than with wild-type Mlh1 as confirmed both in wild-type (heavy) vs. mutant (light) and in mutant (heavy) vs. wild-type (light) experiments; we further required that the interaction be detected in the mutant vs. control experiment (Fig. 6a; Materials and Methods). We were able to detect Pms2 as the only specifically weakened interactor caused by the mutation (Figs. 6b,c; E = −1.77; P = 3×10−4), in agreement with our Y2H results and previous studies [7], [36]. Additionally, we were able to detect Hspa8 as the only specifically enhanced interactor of the mutant protein (Figs. 6b,c; E = 2.71; P = 7×10−8). Two other known interactors of Mlh1, Pms1 (Figs. 6b,c; E = −0.32; P = 0.21) [40] and Brip1 (Fig. 6b,c; E = 0.18; P = 0.32) [41], were also detected, although their interactions with Mlh1 are not affected by this particular mutation (Materials and Methods).


A massively parallel pipeline to clone DNA variants and examine molecular phenotypes of human disease mutations.

Wei X, Das J, Fragoza R, Liang J, Bastos de Oliveira FM, Lee HR, Wang X, Mort M, Stenson PD, Cooper DN, Lipkin SM, Smolka MB, Yu H - PLoS Genet. (2014)

Identifying interactions of Mlh1 that are affected by the I107R mutation using SILAC-based mass spectrometry.(a) Schematic illustrating criteria used to identify interactions that are lost/weakened, unchanged, and gained/enhanced due to the I107R mutation on Mlh1. Blue denotes samples cultured in light media and black denotes samples cultured in heavy media. (b) Scatter plot illustrating fold change (FC; log scale) in the amount of protein pulled down by wild-type Mlh1 and mutant Mlh1 (I107R). Values are computed based on the wild-type (heavy) vs. mutant (light) (X-axis) and mutant (heavy) vs. wild-type (light) (Y-axis) experiments. Green denotes enhancement of interaction, red denotes weakening of interaction, and gold denotes no change. Mlh1 is shown in grey. (c) Fold changes and read counts (r) for interactors of Mlh1 that can be reliably identified as weakened, unchanged, and enhanced due to the I107R mutation. (d) Anti-HA immunoprecipitation followed by Western blotting with anti-V5 antibody confirming that the Mlh1-Brip1 interaction remains unchanged and that the Mlh1-Hspa8 interaction is dramatically enhanced due to the I107R mutation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263371&req=5

pgen-1004819-g006: Identifying interactions of Mlh1 that are affected by the I107R mutation using SILAC-based mass spectrometry.(a) Schematic illustrating criteria used to identify interactions that are lost/weakened, unchanged, and gained/enhanced due to the I107R mutation on Mlh1. Blue denotes samples cultured in light media and black denotes samples cultured in heavy media. (b) Scatter plot illustrating fold change (FC; log scale) in the amount of protein pulled down by wild-type Mlh1 and mutant Mlh1 (I107R). Values are computed based on the wild-type (heavy) vs. mutant (light) (X-axis) and mutant (heavy) vs. wild-type (light) (Y-axis) experiments. Green denotes enhancement of interaction, red denotes weakening of interaction, and gold denotes no change. Mlh1 is shown in grey. (c) Fold changes and read counts (r) for interactors of Mlh1 that can be reliably identified as weakened, unchanged, and enhanced due to the I107R mutation. (d) Anti-HA immunoprecipitation followed by Western blotting with anti-V5 antibody confirming that the Mlh1-Brip1 interaction remains unchanged and that the Mlh1-Hspa8 interaction is dramatically enhanced due to the I107R mutation.
Mentions: While we use only those interactions that are supported by co-crystal structures to estimate the fraction of interactions that are disrupted by mutations at different structural loci, the described procedures can also be applied to interactions with predicted interfaces and structural models [30], [31], [32], [33]. This is of particular importance because over 90% of known interactions do not currently have corresponding co-crystal structures [33], [34]. For example, Mlh1 is known to interact with Pms2, both of which are well-studied DNA mismatch repair genes frequently mutated in hereditary nonpolyposis colorectal cancer [35]. Although the structural basis of the Mlh1-Pms2 interaction still remains unknown, both our previous 3D reconstruction of the human interactome network [7], [32] and the newly-established Interactome3D [33] database suggest that the HATPase_c domain is part of the interface for Mlh1's interaction with Pms2. Previous work has shown that a point mutation (I107R) on the HATPase_c domain of Mlh1 is associated with colorectal cancer and disrupts the Mlh1-Pms2 interaction [7], [35], [36]. First, using Y2H, we were able to confirm the disruption (Figure S4). Next, we developed a high-throughput-amenable mass spectrometry pipeline using Stable Isotope Labeling by Amino acids in Cell culture (SILAC) [37], [38], which was designed to reveal both lost/weakened and gained/enhanced interactions of the target proteins (Fig. 1d) [39]. We added an HA-tag to the N-terminus of both wild-type and mutant Mlh1, as well as to GFP as a control, and performed four SILAC experiments: wild-type Mlh1 (heavy) vs. GFP control (light), mutant Mlh1 (heavy) vs. GFP control (light), wild-type (heavy) vs. mutant (light) Mlh1, and mutant (heavy) vs. wild-type (light) Mlh1 (Fig. 6a; Materials and Methods). Interactors of wild-type/mutant Mlh1 are defined as those that bind wild-type/mutant Mlh1 more than 2× stronger than GFP control (Materials and Methods). For a lost/weakened interaction, we required that the interaction be more than 2× stronger with wild-type Mlh1 than with mutant Mlh1 as confirmed both in wild-type (heavy) vs. mutant (light) and in mutant (heavy) vs. wild-type (light) experiments; we further required that the interaction be detected in the wild-type vs. control experiment (Fig. 6a; Materials and Methods). For a gained/enhanced interaction, we required that the interaction be more than 2× stronger with mutant Mlh1 than with wild-type Mlh1 as confirmed both in wild-type (heavy) vs. mutant (light) and in mutant (heavy) vs. wild-type (light) experiments; we further required that the interaction be detected in the mutant vs. control experiment (Fig. 6a; Materials and Methods). We were able to detect Pms2 as the only specifically weakened interactor caused by the mutation (Figs. 6b,c; E = −1.77; P = 3×10−4), in agreement with our Y2H results and previous studies [7], [36]. Additionally, we were able to detect Hspa8 as the only specifically enhanced interactor of the mutant protein (Figs. 6b,c; E = 2.71; P = 7×10−8). Two other known interactors of Mlh1, Pms1 (Figs. 6b,c; E = −0.32; P = 0.21) [40] and Brip1 (Fig. 6b,c; E = 0.18; P = 0.32) [41], were also detected, although their interactions with Mlh1 are not affected by this particular mutation (Materials and Methods).

Bottom Line: We describe a massively-parallel site-directed mutagenesis approach, "Clone-seq", leveraging next-generation sequencing to rapidly and cost-effectively generate a large number of mutant alleles.We use this pipeline to show that disease mutations on protein-protein interaction interfaces are significantly more likely than those away from interfaces to disrupt corresponding interactions.The general scheme of our experimental pipeline can be readily expanded to other types of interactome-mapping methods to comprehensively evaluate the functional relevance of all DNA variants, including those in non-coding regions.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Weill Cornell College of Medicine, New York, New York, United States of America; Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Understanding the functional relevance of DNA variants is essential for all exome and genome sequencing projects. However, current mutagenesis cloning protocols require Sanger sequencing, and thus are prohibitively costly and labor-intensive. We describe a massively-parallel site-directed mutagenesis approach, "Clone-seq", leveraging next-generation sequencing to rapidly and cost-effectively generate a large number of mutant alleles. Using Clone-seq, we further develop a comparative interactome-scanning pipeline integrating high-throughput GFP, yeast two-hybrid (Y2H), and mass spectrometry assays to systematically evaluate the functional impact of mutations on protein stability and interactions. We use this pipeline to show that disease mutations on protein-protein interaction interfaces are significantly more likely than those away from interfaces to disrupt corresponding interactions. We also find that mutation pairs with similar molecular phenotypes in terms of both protein stability and interactions are significantly more likely to cause the same disease than those with different molecular phenotypes, validating the in vivo biological relevance of our high-throughput GFP and Y2H assays, and indicating that both assays can be used to determine candidate disease mutations in the future. The general scheme of our experimental pipeline can be readily expanded to other types of interactome-mapping methods to comprehensively evaluate the functional relevance of all DNA variants, including those in non-coding regions.

Show MeSH