Robert G. Wisotzkey

Ontology-Based Meta-Analysis of Global Collections of High-Throughput Public Data

Background The investigation of the interconnections between the molecular and genetic events that govern biological systems is essential if we are to understand the development of disease and design effective novel treatments. Microarray and next-generation sequencing technologies have the potential to provide this information. However, taking full advantage of these approaches requires that biological connections be made across large quantities of highly heterogeneous genomic datasets. Leveraging the increasingly huge quantities of genomic data in the public domain is fast becoming one of the key challenges in the research community today. Methodology/Results We have developed a novel data mining framework that enables researchers to use this growing collection of public high-throughput data to investigate any set of genes or proteins. The connectivity between molecular states across thousands of heterogeneous datasets from microarrays and other genomic platforms is determined through a combination of rank-based enrichment statistics, meta-analyses, and biomedical ontologies. We address data quality concerns through dataset replication and meta-analysis and ensure that the majority of the findings are derived using multiple lines of evidence. As an example of our strategy and the utility of this framework, we apply our data mining approach to explore the biology of brown fat within the context of the thousands of publicly available gene expression datasets. Conclusions Our work presents a practical strategy for organizing, mining, and correlating global collections of large-scale genomic data to explore normal and disease biology. Using a hypothesis-free approach, we demonstrate how a data-driven analysis across very large collections of genomic data can reveal novel discoveries and evidence to support existing hypothesis.

dSno Facilitates Baboon Signaling in the Drosophila Brain by Switching the Affinity of Medea Away From Mad and Toward dSmad2

A screen for modifiers of Dpp adult phenotypes led to the identification of the Drosophila homolog of the Sno oncogene (dSno). The dSno locus is large, transcriptionally complex and contains a recent retrotransposon insertion that may be essential for dSno function, an intriguing possibility from the perspective of developmental evolution. dSno is highly transcribed in the embryonic central nervous system and transcripts are most abundant in third instar larvae. dSno mutant larvae have proliferation defects in the optic lobe of the brain very similar to those seen in baboon (Activin type I receptor) and dSmad2 mutants. This suggests that dSno is a mediator of Baboon signaling. dSno binds to Medea and Medea/dSno complexes have enhanced affinity for dSmad2. Alternatively, Medea/dSno complexes have reduced affinity for Mad such that, in the presence of dSno, Dpp signaling is antagonized. We propose that dSno functions as a switch in optic lobe development, shunting Medea from the Dpp pathway to the Activin pathway to ensure proper proliferation. Pathway switching in target cells is a previously unreported mechanism for regulating TGFβ signaling and a novel function for Sno/Ski family proteins.

Lysine Conservation and Context in TGFβ and Wnt Signaling Suggest New Targets and General Themes for Posttranslational Modification

TGFβ and Wnt pathways play important roles in the development of animals from sponges to humans. In both pathways posttranslational modification as a means of regulating their function, such as lysine modification by ubiquitination and sumoylation, has been observed. However, a gap exists between the immunological observation of posttranslational modification and the identification of the target lysine. To fill this gap, we conducted a phylogenetic analysis of lysine conservation and context in TGFβ and Wnt pathway receptors and signal transducers and suggest numerous high-probability candidates for posttranslational modification. Further comparison of results from both pathways suggests two general features for biochemical regulation of intercellular signaling: receptors are less frequent targets for modification than signal transduction agonists, and a lysine adjacent to an upstream hydrophobic residue may be a preferred context for modification. Overall the results suggest numerous applications for an evolutionary approach to the biochemical regulation of developmental pathways, including (1) streamlining of the identification of the target lysine, (2) determination of when members of a multigene family acquire distinct activities, (3) application to any conserved protein family, and (4) application to any modification of a specific amino acid.

Molecular Evolution of Smad Proteins

To date, Smad family members have been found only in eumetazoan animals. To understand the evolutionary relationship between family members we conducted a phylogenetic analysis. To simplify the analysis but retain its explanatory power, we focused on Smad proteins from organisms in three distinct phyla: human, fly, and nematode. Overall, we found that human and fly proteins always cluster together in four subfamilies while three subfamilies contain only nematode proteins. Sequence alignments of distinct regions of were also analyzed. Data from the alignments confirmed that the MH1 (DNA-binding) and MH2 (protein-protein interaction) domains are highly conserved family-wide. The linker region between these domains is also highly conserved but only within subfamilies. Conservation in the C-terminal receptor phosphorylation region provides new insight into a unique subfamily containing three interacting nematode proteins that signal for DAF-7. From a larger perspective, our analysis strongly supports the traditional view that flies are more closely related to humans than to nematodes

Distinct molecular evolutionary mechanisms underlie the functional diversification of the Wnt and TGFβ signaling pathways.

The canonical Wnt pathway is one of the oldest and most functionally diverse of animal intercellular signaling pathways. Though much is known about loss-of-function phenotypes for Wnt pathway components in several model organisms, the question of how this pathway achieved its current repertoire of functions has not been addressed. Our phylogenetic analyses of 11 multigene families from five species belonging to distinct phyla, as well as additional analyses employing the 12 Drosophila genomes, suggest frequent gene duplications affecting ligands and receptors as well as co-evolution of new ligand–receptor pairs likely facilitated the expansion of this pathway’s capabilities. Further, several examples of recent gene loss are visible in Drosophila when compared to family members in other phyla. By comparison the TGFβ signaling pathway is characterized by ancient gene duplications of ligands, receptors, and signal transducers with recent duplication events restricted to the vertebrate lineage. Overall, the data suggest that two distinct molecular evolutionary mechanisms can create a functionally diverse developmental signaling pathway. These are the recent dynamic generation of new genes and ligand–receptor interactions as seen in the Wnt pathway and the conservative adaptation of ancient pre-existing genes to new roles as seen in the TGFβ pathway. From a practical perspective, the former mechanism limits the investigator’s ability to transfer knowledge of specific pathway functions across species while the latter facilitates knowledge transfer.

α/β Hydrolase2, a predicated gene adjacent to mad in Drosophila melanogaster, belongs to a new global multigene family and is associated with obesity

The experimental validation of genes predicted from genomic sequence and the identification of functions for these genes is an increasingly important task. We report a multidisciplinary analysis of CG3488, a predicted gene adjacent to Mothers against dpp in Drosophila melanogaster. We cloned and sequenced a cDNA corresponding to CG3488 and we show that it is expressed in embryos. A computational analysis shows that CG3488 contains a number of conserved domains present in enzymes capable of lipid hydrolysis. A phylogenetic analysis shows that CG3488 is the homolog of human α/β hydrolase2 and that these genes belong to a novel multigene family with members in animals, plants, fungi, and bacteria. A genetic analysis shows that heterozygosity for a chromosomal deletion that removes CG3488 dominantly enhances the excess lipid phenotype associated with a mutation in adipose, an uncloned obesity gene. Further, overexpression of a CG3488 transgene rescues this obesity phenotype. Overall, the data suggests that CG3488 functions as a lipase and that analyses of its homologs will provide unique insights into lipid metabolism in many species.

New Gene Evolution in the Bonus-TIF1-γ/TRIM33 Family Impacted the Architecture of the Vertebrate Dorsal–Ventral Patterning Network

Uncovering how a new gene acquires its function and understanding how the function of a new gene influences existing genetic networks are important topics in evolutionary biology. Here, we demonstrate nonconservation for the embryonic functions of Drosophila Bonus and its newest vertebrate relative TIF1-γ/TRIM33. We showed previously that TIF1-γ/TRIM33 functions as an ubiquitin ligase for the Smad4 signal transducer and antagonizes the Bone Morphogenetic Protein (BMP) signaling network underlying vertebrate dorsal-ventral axis formation. Here, we show that Bonus functions as an agonist of the Decapentaplegic (Dpp) signaling network underlying dorsal-ventral axis formation in flies. The absence of conservation for the roles of Bonus and TIF1-γ/TRIM33 reveals a shift in the dorsal-ventral patterning networks of flies and mice, systems that were previously considered wholly conserved. The shift occurred when the new gene TIF1-γ/TRIM33 replaced the function of the ubiquitin ligase Nedd4L in the lineage leading to vertebrates. Evidence of this replacement is our demonstration that Nedd4 performs the function of TIF1-γ/TRIM33 in flies during dorsal-ventral axis formation. The replacement allowed vertebrate Nedd4L to acquire novel functions as a ubiquitin ligase of vertebrate-specific Smad proteins. Overall our data reveal that the architecture of the Dpp/BMP dorsal-ventral patterning network continued to evolve in the vertebrate lineage, after separation from flies, via the incorporation of new genes.

Hippo Pathway Phylogenetics Predicts Monoubiquitylation of Salvador and Merlin/Nf2

Recently we employed phylogenetics to predict that the cellular interpretation of TGF-β signals is modulated by monoubiquitylation cycles affecting the Smad4 signal transducer/tumor suppressor. This prediction was subsequently validated by experiments in flies, frogs and mammalian cells. Here we apply a phylogenetic approach to the Hippo pathway and predict that two of its signal transducers, Salvador and Merlin/Nf2 (also a tumor suppressor) are regulated by monoubiquitylation. This regulatory mechanism does not lead to protein degradation but instead serves as a highly efficient “off/on” switch when the protein is subsequently deubiquitylated. Overall, our study shows that the creative application of phylogenetics can predict new roles for pathway components and new mechanisms for regulating intercellular signaling pathways.

lolal Is an Evolutionarily New Epigenetic Regulator of dpp Transcription during Dorsal–Ventral Axis Formation

Secreted ligands in the Dpp/BMP family drive dorsal–ventral (D/V) axis formation in all Bilaterian species. However, maternal factors regulating Dpp/BMP transcription in this process are largely unknown. We identified the BTB domain protein longitudinals lacking-like (lolal) as a modifier of decapentaplegic (dpp) mutations. We show that Lolal is evolutionarily related to the Trithorax group of chromatin regulators and that lolal interacts genetically with the epigenetic factor Trithorax-like during Dpp D/V signaling. Maternally driven LolalHA is found in oocytes and translocates to zygotic nuclei prior to the point at which dpp transcription begins. lolal maternal and zygotic mutant embryos display significant reductions in dpp, pMad, and zerknullt expression, but they are never absent. The data suggest that lolal is required to maintain dpp transcription during D/V patterning. Phylogenetic data revealed that lolal is an evolutionarily new gene present only in insects and crustaceans. We conclude that Lolal is the first maternal protein identified with a role in dpp D/V transcriptional maintenance, that Lolal and the epigenetic protein Trithorax-like are essential for Dpp D/V signaling and that the architecture of the Dpp D/V pathway evolved in the arthropod lineage after the separation from vertebrates via the incorporation of new genes such as lolal.

Molecular Screening of Familial Hypercholesterolemia in the Icelandic Population

Familial hypercholesterolemia (FH) is a monogenic disease characterized by a lifelong exposure to high LDL-C levels that can lead to early onset coronary heart disease (CHD). The main causes of FH identified to date include loss-of-function mutations in LDLR or APOB, or gain-of-function mutations in PCSK9. Early diagnosis and genetic testing of FH suspects is critical for improved prognosis of affected individuals as lipid lowering treatments are effective in preventing CHD related morbidity and mortality. In the present manuscript, we developed a comprehensive next generation sequencing (NGS) panel which we applied on two different resources of FH in the Icelandic population: 62 subjects from 23 FH families with known or unknown culprit mutations, and a population-based sampling of 315 subjects selected for total cholesterol levels above the 95th percentile cut-point. The application of the NGS panel revealed significant diagnostic yields in identifying pathogenic LDLR mutations in both family and population-based genetic testing.