Andrew R. Gehrke

Variation in homeodomain DNA-binding revealed by high-resolution analysis of sequence preferences

Most homeodomains are unique within a genome, yet many are highly conserved across vast evolutionary distances, implying strong selection on their precise DNA-binding specificities. We determined the binding preferences of the majority (168) of mouse homeodomains to all possible 8-base sequences, revealing rich and complex patterns of sequence specificity and showing that there are at least 65 distinct homeodomain DNA-binding activities. We developed a computational system that successfully predicts binding sites for homeodomain proteins as distant from mouse as Drosophila and C. elegans, and we infer full 8-mer binding profiles for the majority of known animal homeodomains. Our results provide an unprecedented level of resolution in the analysis of this simple domain structure and suggest that variation in sequence recognition may be a factor in its functional diversity and evolutionary success.

Genome-wide analysis of ETS-family DNA-binding in vitro and in vivo

Members of the large ETS family of transcription factors (TFs) have highly similar DNA‐binding domains (DBDs)—yet they have diverse functions and activities in physiology and oncogenesis. Some differences in DNA‐binding preferences within this family have been described, but they have not been analysed systematically, and their contributions to targeting remain largely uncharacterized. We report here the DNA‐binding profiles for all human and mouse ETS factors, which we generated using two different methods: a high‐throughput microwell‐based TF DNA‐binding specificity assay, and protein‐binding microarrays (PBMs). Both approaches reveal that the ETS‐binding profiles cluster into four distinct classes, and that all ETS factors linked to cancer, ERG, ETV1, ETV4 and FLI1, fall into just one of these classes. We identify amino‐acid residues that are critical for the differences in specificity between all the classes, and confirm the specificities in vivo using chromatin immunoprecipitation followed by sequencing (ChIP‐seq) for a member of each class. The results indicate that even relatively small differences in in vitro binding specificity of a TF contribute to site selectivity in vivo.

Specific DNA-binding by Apicomplexan AP2 transcription factors

Malaria remains one of the most prevalent infectious diseases worldwide, affecting more than half a billion people annually. Despite many years of research, the mechanisms underlying transcriptional regulation in the malaria-causing Plasmodium spp., and in Apicomplexan parasites generally, remain poorly understood. In Plasmodium, few regulatory elements sufficient to drive gene expression have been characterized, and their cognate DNA-binding proteins remain unknown. This study characterizes the DNA-binding specificities of two members of the recently identified Apicomplexan AP2 (ApiAP2) family of putative transcriptional regulators from Plasmodium falciparum. The ApiAP2 proteins contain AP2 domains homologous to the well characterized plant AP2 family of transcriptional regulators, which play key roles in development and environmental stress response pathways. We assayed ApiAP2 protein–DNA interactions using protein-binding microarrays and combined these results with computational predictions of coexpressed target genes to couple these putative trans factors to corresponding cis-regulatory motifs in Plasmodium. Furthermore, we show that protein–DNA sequence specificity is conserved in orthologous proteins between phylogenetically distant Apicomplexan species. The identification of the DNA-binding specificities for ApiAP2 proteins lays the foundation for the exploration of their role as transcriptional regulators during all stages of parasite development. Because of their origin in the plant lineage, ApiAP2 proteins have no homologues in the human host and may prove to be ideal antimalarial targets.

The spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences.

Deep conservation of wrist and digit enhancers in fish

Significance The fossil record shows that the wrist and digits have an aquatic origin, becoming recognizable in a group of (mostly extinct) fish that contained robust fins. Do the fins of living fishes have the equivalent of these structures? Because comparisons of fin and limb morphology have been inconclusive, we sought to investigate this question using developmental and molecular data. By utilizing a nonmodel fish (the spotted gar), we find that the regulatory networks that control “wrist and digit”-building genes (Hox) are deeply conserved between fish and tetrapods. The genomic architecture described here defines Hox gene activity in fins and limbs as equivalent, in turn suggesting equivalence between the distal bones of fish fins and the wrist and/or digits of tetrapods. There is no obvious morphological counterpart of the autopod (wrist/ankle and digits) in living fishes. Comparative molecular data may provide insight into understanding both the homology of elements and the evolutionary developmental mechanisms behind the fin to limb transition. In mouse limbs the autopod is built by a “late” phase of Hoxd and Hoxa gene expression, orchestrated by a set of enhancers located at the 5′ end of each cluster. Despite a detailed mechanistic understanding of mouse limb development, interpretation of Hox expression patterns and their regulation in fish has spawned multiple hypotheses as to the origin and function of “autopod” enhancers throughout evolution. Using phylogenetic footprinting, epigenetic profiling, and transgenic reporters, we have identified and functionally characterized hoxD and hoxA enhancers in the genomes of zebrafish and the spotted gar, Lepisosteus oculatus, a fish lacking the whole genome duplication of teleosts. Gar and zebrafish “autopod” enhancers drive expression in the distal portion of developing zebrafish pectoral fins, and respond to the same functional cues as their murine orthologs. Moreover, gar enhancers drive reporter gene expression in both the wrist and digits of mouse embryos in patterns that are nearly indistinguishable from their murine counterparts. These functional genomic data support the hypothesis that the distal radials of bony fish are homologous to the wrist and/or digits of tetrapods.

Appendage expression driven by the Hoxd Global Control Region is an ancient gnathostome feature.

The evolutionary transition of the fins of fish into tetrapod limbs involved genetic changes to developmental systems that resulted in novel skeletal patterns and functions. Approaches to understanding this issue have entailed the search for antecedents of limb structure in fossils, genes, and embryos. Comparative genetic analyses have produced ambiguous results: although studies of posterior Hox genes from homology group 13 (Hoxa-13 and Hoxd-13) reveal similarities in gene expression between the distal segments of fins and limbs, this functional homology has not been supported by genomic comparisons of the activity of their cis-regulatory elements, namely the Hoxd Global Control Region. Here, we show that cis-regulatory elements driving Hoxd gene expression in distal limbs are present in fish. Using an interspecies transgenesis approach, we find functional conservation between gnathostome Hoxd enhancers, demonstrating that orthologous sequences from tetrapods, zebrafish and skate can drive reporter gene expression in mouse limbs and zebrafish fins. Our results support the notion that some of the novelties associated with tetrapod limbs arose by modification of deeply conserved cis- and trans-acting mechanisms of Hox regulation in gnathostomes.

Digits and fin rays share common developmental histories

Understanding the evolutionary transformation of fish fins into tetrapod limbs is a fundamental problem in biology. The search for antecedents of tetrapod digits in fish has remained controversial because the distal skeletons of limbs and fins differ structurally, developmentally, and histologically. Moreover, comparisons of fins with limbs have been limited by a relative paucity of data on the cellular and molecular processes underlying the development of the fin skeleton. Here, we provide a functional analysis, using CRISPR/Cas9 and fate mapping, of 5′ hox genes and enhancers in zebrafish that are indispensable for the development of the wrists and digits of tetrapods. We show that cells marked by the activity of an autopodial hoxa13 enhancer exclusively form elements of the fin fold, including the osteoblasts of the dermal rays. In hox13 knockout fish, we find that a marked reduction and loss of fin rays is associated with an increased number of endochondral distal radials. These discoveries reveal a cellular and genetic connection between the fin rays of fish and the digits of tetrapods and suggest that digits originated via the transition of distal cellular fates.

Transcriptional regulation and stabilization of left-right neuronal identity in C. elegans.

At discrete points in development, transient signals are transformed into long-lasting cell fates. For example, the asymmetric identities of two Caenorhabditis elegans olfactory neurons called AWC(ON) and AWC(OFF) are specified by an embryonic signaling pathway, but maintained throughout the life of an animal. Here we show that the DNA-binding protein NSY-7 acts to convert a transient, partially differentiated state into a stable AWC(ON) identity. Expression of an AWC(ON) marker is initiated in nsy-7 loss-of-function mutants, but subsequently lost, so that most adult animals have two AWC(OFF) neurons and no AWC(ON) neurons. nsy-7 encodes a protein with distant similarity to a homeodomain. It is expressed in AWC(ON), and is an early transcriptional target of the embryonic signaling pathway that specifies AWC(ON) and AWC(OFF); its expression anticipates future AWC asymmetry. The NSY-7 protein binds a specific optimal DNA sequence that was identified through a complete biochemical survey of 8-mer DNA sequences. This sequence is present in the promoter of an AWC(OFF) marker and essential for its asymmetric expression. An 11-base-pair (bp) sequence required for AWC(OFF) expression has two activities: One region activates expression in both AWCs, and the overlapping NSY-7-binding site inhibits expression in AWC(ON). Our results suggest that NSY-7 responds to transient embryonic signaling by repressing AWC(OFF) genes in AWC(ON), thus acting as a transcriptional selector for a randomly specified neuronal identity.

Predicting the binding preference of transcription factors to individual DNA k-mers

Motivation: Recognition of specific DNA sequences is a central mechanism by which transcription factors (TFs) control gene expression. Many TF-binding preferences, however, are unknown or poorly characterized, in part due to the difficulty associated with determining their specificity experimentally, and an incomplete understanding of the mechanisms governing sequence specificity. New techniques that estimate the affinity of TFs to all possible k-mers provide a new opportunity to study DNA–protein interaction mechanisms, and may facilitate inference of binding preferences for members of a given TF family when such information is available for other family members. Results: We employed a new dataset consisting of the relative preferences of mouse homeodomains for all eight-base DNA sequences in order to ask how well we can predict the binding profiles of homeodomains when only their protein sequences are given. We evaluated a panel of standard statistical inference techniques, as well as variations of the protein features considered. Nearest neighbour among functionally important residues emerged among the most effective methods. Our results underscore the complexity of TF–DNA recognition, and suggest a rational approach for future analyses of TF families. Contact: t.hughes@utorotno.ca Supplementary information: Supplementary data are available at Bioinformatics online.

Organogenesis in deep time: A problem in genomics, development, and paleontology

Significance Phylogenetic data inclusive of rich paleontological records can be used to inform hypotheses on evolutionary transformations. These data, when combined with developmental studies and functional genomic assays in model and nonmodel organisms, expand our understanding of the evolutionary processes that build and pattern the vertebrate body plan. Here, we highlight a direction of the fossil record, one of “experimental paleontology,” where morphological transformations inferred from the fossil record can be experimentally assayed in the lab. With the addition of genomic techniques to test hypotheses, researchers can now begin to explore genomic states that have influenced both past and present morphological diversity. The fossil record is a unique repository of information on major morphological transitions. Increasingly, developmental, embryological, and functional genomic approaches have also conspired to reveal evolutionary trajectory of phenotypic shifts. Here, we use the vertebrate appendage to demonstrate how these disciplines can mutually reinforce each other to facilitate the generation and testing of hypotheses of morphological evolution. We discuss classical theories on the origins of paired fins, recent data on regulatory modulations of fish fins and tetrapod limbs, and case studies exploring the mechanisms of digit loss in tetrapods. We envision an era of research in which the deep history of morphological evolution can be revealed by integrating fossils of transitional forms with direct experimentation in the laboratory via genome manipulation, thereby shedding light on the relationship between genes, developmental processes, and the evolving phenotype.