CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping.

Broad-scale protein-protein interaction mapping is a major challenge given the cost, time, and sensitivity constraints of existing technologies. Here, we present a massively multiplexed yeast two-hybrid method, CrY2H-seq, which uses a Cre recombinase interaction reporter to intracellularly fuse the coding sequences of two interacting proteins and next-generation DNA sequencing to identify these interactions en masse. We applied CrY2H-seq to investigate sparsely annotated Arabidopsis thaliana transcription factors interactions. By performing ten independent screens testing a total of 36 million binary interaction combinations, and uncovering a network of 8,577 interactions among 1,453 transcription factors, we demonstrate CrY2H-seq's improved screening capacity, efficiency, and sensitivity over those of existing technologies. The deep-coverage network resource we call AtTFIN-1 recapitulates one-third of previously reported interactions derived from diverse methods, expands the number of known plant transcription factor interactions by three-fold, and reveals previously unknown family-specific interaction module associations with plant reproductive development, root architecture, and circadian coordination

Protein interaction network of alternatively spliced isoforms from brain links genetic risk factors for autism

Increased risk for autism spectrum disorders (ASD) is attributed to hundreds of genetic loci. The convergence of ASD variants have been investigated using various approaches, including protein interactions extracted from the published literature. However, these datasets are frequently incomplete, carry biases and are limited to interactions of a single splicing isoform, which may not be expressed in the disease-relevant tissue. Here we introduce a new interactome mapping approach by experimentally identifying interactions between brain-expressed alternatively spliced variants of ASD risk factors. The Autism Spliceform Interaction Network reveals that almost half of the detected interactions and about 30% of the newly identified interacting partners represent contribution from splicing variants, emphasizing the importance of isoform networks. Isoform interactions greatly contribute to establishing direct physical connections between proteins from the de novo autism CNVs. Our findings demonstrate the critical role of spliceform networks for translating genetic knowledge into a better understanding of human diseases

High-resolution molecular networks from novel 'omics' approaches elucidate survival strategies in organisms from land to sea

Molecular networks drive nearly all cellular processes. With the advent of omics technologies involving next-generation sequencing and mass spectrometry, we have started to uncover highly complex gene, protein, and biochemical networks that underlie survival mechanisms like growth and stress tolerance.However, the study of cell wide of protein-protein interactions that importantly link genomic, transcriptomic, and proteomic data to cellular activity has been hindered by technical limitations, and the need to survey and track billions of possible combinatorial protein interactions. Moreover, many omics technologies have not yet been developed or applied to non-model organisms, particularly to ecologically and economically important species that may be negatively impacted by climate change. To address these shortcomings, I developed a modified yeast two-hybrid technology called CrY2H-seq that enables massively multiplexed protein interaction screening through a Cre-lox reporter and next generation sequencing, and demonstrated its applications for generating interactome resources by screening a comprehensive set of Arabidopsis transcription factors. Lastly, I applied metabolomics analysis to investigate how ocean acidification might impact the Dungeness crab. I found transcription factor families preferentially interact with others, and relationships among families supported by recent independent studies may drive mechanisms underlying reproductive development and hormone signaling. I also found that metabolomes of developing Dungeness crab show treatment specific responses to low oxygen and low pH seawater treatments. Taken all together, the new biological insight gained from these novel omics approaches can valuably be used to inform targeted future experiments aimed at optimizing crop cultivation and predicting how economically and economically important species might response to future environmental stress

Seawater carbonate chemistry and performance and oxidative status in a tolerant burrowing clam

Whereas low levels of thermal stress, irradiance, and dietary restriction can have beneficial effects for many taxa, stress acclimation remains understudied in marine invertebrates, despite being threatened by climate change stressors such as ocean acidification. To test for life-stage and stress-intensity dependence in eliciting enhanced tolerance under subsequent stress encounters, we initially conditioned pediveliger Pacific geoduck (Panopea generosa) larvae to (i) ambient and moderately elevated pCO2 (920 µatm and 2800 µatm, respectively) for 110 days, (ii) secondarily applied a 7-day exposure to ambient, moderate, and severely elevated pCO2 (750 µatm, 2800 µatm, and 4900 µatm, respectively), followed by 7 days in ambient conditions, and (iii) implemented a 7-day third exposure to ambient (970 µatm) and moderate pCO2 (3000 µatm). Initial conditioning to moderate pCO2 stress followed by second and third exposure to severe and moderate pCO2 stress increased respiration rate, organic biomass, and shell size suggesting a stress-intensity-dependent effect on energetics. Additionally, stress-acclimated clams had lower antioxidant capacity compared to clams under ambient conditions, supporting the hypothesis that stress over postlarval-to-juvenile development affects oxidative status later in life. Time series and stress intensity-specific approaches can reveal life-stages and magnitudes of exposure, respectively, that may elicit beneficial phenotypic variation

Repeat exposure to hypercapnic seawater modifies growth and oxidative status in a tolerant burrowing clam

Although low levels of thermal stress, irradiance and dietary restriction can have beneficial effects for many taxa, stress acclimation remains little studied in marine invertebrates, even though they are threatened by climate change stressors such as ocean acidification. To test the role of life-stage and stress-intensity dependence in eliciting enhanced tolerance under subsequent stress encounters, we initially conditioned pediveliger Pacific geoduck (Panopea generosa) larvae to ambient and moderately elevated PCO2 (920 μatm and 2800 μatm, respectively) for 110 days. Then, clams were exposed to ambient, moderate or severely elevated PCO2 (750, 2800 or 4900 μatm, respectively) for 7 days and, following 7 days in ambient conditions, a 7-day third exposure to ambient (970 μatm) or moderate PCO2 (3000 μatm). Initial conditioning to moderate PCO2 stress followed by second and third exposure to severe and moderate PCO2 stress increased respiration rate, organic biomass and shell size, suggesting a stress-intensity-dependent effect on energetics. Additionally, stressacclimated clams had lower antioxidant capacity compared with clams under ambient conditions, supporting the hypothesis that stress over postlarval-to-juvenile development affects oxidative status later in life. Time series and stress intensity-specific approaches can reveal life-stages and magnitudes of exposure, respectively, that may elicit beneficial phenotypic variation

Seawater carbonate chemistry of experiment on acclimatory gene expression of primed clams enhances robustness to elevated pCO2

Sublethal exposure to environmental challenges may enhance ability to cope with chronic or repeated change, a process known as priming. In a previous study, pre-exposure to seawater enriched with pCO2 improved growth and reduced antioxidant capacity of juvenile Pacific geoduck Panopea generosa clams, suggesting that transcriptional shifts may drive phenotypic modifications post-priming. To this end, juvenile clams were sampled and TagSeq gene expression data were analysed after (i) a 110-day acclimation under ambient (921 μatm, naïve) and moderately elevated pCO2 (2870 μatm, pre-exposed); then following (ii) a second 7-day exposure to three pCO2 treatments (ambient: 754 μatm; moderately elevated: 2750 μatm; severely elevated: 4940 μatm), a 7-day return to ambient pCO2 and a third 7-day exposure to two pCO2 treatments (ambient: 967 μatm; moderately elevated: 3030 μatm). Pre-exposed geoducks frontloaded genes for stress and apoptosis/innate immune response, homeostatic processes, protein degradation and transcriptional modifiers. Pre-exposed geoducks were also responsive to subsequent encounters, with gene sets enriched for mitochondrial recycling and immune defence under elevated pCO2 and energy metabolism and biosynthesis under ambient recovery. In contrast, gene sets with higher expression in naïve clams were enriched for fatty-acid degradation and glutathione components, suggesting naïve clams could be depleting endogenous fuels, with unsustainable energetic requirements if changes in carbonate chemistry persist. Collectively, our transcriptomic data indicate that pCO2 priming during post-larval periods could, via gene expression regulation, enhance robustness in bivalves to environmental change. Such priming approaches may be beneficial for aquaculture, as seafood demand intensifies concurrent with increasing climate change in marine systems

Invertebrate methylomes provide insight into mechanisms of environmental tolerance and reveal methodological biases

There is a growing focus on the role of DNA methylation in the ability of marine invertebrates to rapidly respond to changing environmental factors and anthropogenic impacts. However, genome-wide DNA methylation studies in nonmodel organisms are currently hampered by a limited understanding of methodological biases. Here, we compare three methods for quantifying DNA methylation at single base-pair resolution—whole genome bisulfite sequencing (WGBS), reduced representation bisulfite sequencing (RRBS), and methyl-CpG binding domain bisulfite sequencing (MBDBS)—using multiple individuals from two reef-building coral species with contrasting environmental sensitivity. All methods reveal substantially greater methylation in Montipora capitata (11.4%) than the more sensitive Pocillopora acuta (2.9%). The majority of CpG methylation in both species occurs in gene bodies and flanking regions. In both species, MBDBS has the greatest capacity for detecting CpGs in coding regions at our sequencing depth, but MBDBS may be influenced by intrasample methylation heterogeneity. RRBS yields robust information for specific loci albeit without enrichment of any particular genome feature and with significantly reduced genome coverage. Relative genome size strongly influences the number and location of CpGs detected by each method when sequencing depth is limited, illuminating nuances in cross-species comparisons. As genome-wide methylation differences, supported by data across bisulfite sequencing methods, may contribute to environmental sensitivity phenotypes in critical marine invertebrate taxa, these data provide a genomic resource for investigating the functional role of DNA methylation in environmental tolerance

CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping.

Broad-scale protein-protein interaction mapping is a major challenge given the cost, time, and sensitivity constraints of existing technologies. Here, we present a massively multiplexed yeast two-hybrid method, CrY2H-seq, which uses a Cre recombinase interaction reporter to intracellularly fuse the coding sequences of two interacting proteins and next-generation DNA sequencing to identify these interactions en masse. We applied CrY2H-seq to investigate sparsely annotated Arabidopsis thaliana transcription factors interactions. By performing ten independent screens testing a total of 36 million binary interaction combinations, and uncovering a network of 8,577 interactions among 1,453 transcription factors, we demonstrate CrY2H-seq's improved screening capacity, efficiency, and sensitivity over those of existing technologies. The deep-coverage network resource we call AtTFIN-1 recapitulates one-third of previously reported interactions derived from diverse methods, expands the number of known plant transcription factor interactions by three-fold, and reveals previously unknown family-specific interaction module associations with plant reproductive development, root architecture, and circadian coordination

Widespread Expansion of Protein Interaction Capabilities by Alternative Splicing.

While alternative splicing is known to diversify the functional characteristics of some genes, the extent to which protein isoforms globally contribute to functional complexity on a proteomic scale remains unknown. To address this systematically, we cloned full-length open reading frames of alternatively spliced transcripts for a large number of human genes and used protein-protein interaction profiling to functionally compare hundreds of protein isoform pairs. The majority of isoform pairs share less than 50% of their interactions. In the global context of interactome network maps, alternative isoforms tend to behave like distinct proteins rather than minor variants of each other. Interaction partners specific to alternative isoforms tend to be expressed in a highly tissue-specific manner and belong to distinct functional modules. Our strategy, applicable to other functional characteristics, reveals a widespread expansion of protein interaction capabilities through alternative splicing and suggests that many alternative "isoforms" are functionally divergent (i.e., "functional alloforms")