ABSTRACT
Animals capable of whole-body regeneration can replace any missing cell type and regenerate fully functional new organs, including new brains, de novo. The regeneration of a new brain requires the formation of diverse neural cell types and their assembly into an organized structure with correctly wired circuits. Recent work in various regenerative animals has revealed transcriptional programs required for the differentiation of distinct neural subpopulations, however, how these transcriptional programs are initiated in response to injury remains unknown. Here, we focused on the highly regenerative acoel worm, Hofstenia miamia, to study wound-induced transcriptional regulatory events that lead to the production of neurons and subsequently a functional brain. Footprinting analysis using chromatin accessibility data on a chromosome-scale genome assembly revealed that binding sites for the Nuclear Factor Y (NFY) transcription factor complex were significantly bound during regeneration, showing a dynamic increase in binding within one hour upon amputation specifically in tail fragments, which will regenerate a new brain. Strikingly, NFY targets were highly enriched for genes with neuronal function. Single-cell transcriptome analysis combined with functional studies identified soxC+ stem cells as a putative progenitor population for multiple neural subtypes. Further, we found that wound-induced soxC expression is likely under direct transcriptional control by NFY, uncovering a mechanism for the initiation of a neural differentiation pathway by early wound-induced binding of a transcriptional regulator.
Subject(s)
Cell Differentiation , Neurons , Animals , Neurons/metabolism , Neurons/cytology , Regeneration/physiology , Regeneration/genetics , Brain/metabolism , Brain/cytologyABSTRACT
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.
Subject(s)
DNA/chemistry , Homeodomain Proteins/chemistry , Animals , Base Sequence , Computational Biology , Conserved Sequence , DNA/metabolism , Evolution, Molecular , Homeodomain Proteins/metabolism , Mice , Models, Molecular , Protein Binding , Transcription Factors/chemistry , Transcription Factors/metabolismABSTRACT
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.
Subject(s)
Animal Fins/embryology , Biological Evolution , Extremities/embryology , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Zebrafish/embryology , Zebrafish/genetics , Animal Fins/metabolism , Animals , Cell Lineage , Enhancer Elements, Genetic/genetics , Gene Deletion , Gene Knockout Techniques , Mice , Multigene Family/genetics , PhenotypeABSTRACT
Differential gene expression is the core of development, mediating the genetic changes necessary for determining cell identity. The regulation of gene activity by cis-acting elements (e.g., enhancers) is a crucial mechanism for determining differential gene activity by precise control of gene expression in embryonic space and time. Modifications to regulatory regions can have profound impacts on phenotype, and therefore developmental and evolutionary biologists have increasingly focused on elucidating the transcriptional control of genes that build and pattern body plans. Here, we trace the evolutionary history of transcriptional control of three loci key to vertebrate appendage development (Fgf8, Shh, and HoxD/A). Within and across these regulatory modules, we find both complex and flexible regulation in contrast with more fixed enhancers that appear unchanged over vast timescales of vertebrate evolution. The transcriptional control of vertebrate appendage development was likely already incredibly complex in the common ancestor of fish, implying that subtle changes to regulatory networks were more likely responsible for alterations in phenotype rather than the de novo addition of whole regulatory domains. Finally, we discuss the dangers of relying on inter-species transgenesis when testing enhancer function, and call for more controlled regulatory swap experiments when inferring the evolutionary history of enhancer elements.
Subject(s)
Biological Evolution , Extremities/embryology , Regulatory Sequences, Nucleic Acid/genetics , Vertebrates/genetics , Animals , Fibroblast Growth Factor 8/genetics , Gene Expression Regulation, DevelopmentalABSTRACT
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.
Subject(s)
Embryonic Development , Genomics , Organogenesis/genetics , Paleontology , Animal Fins/embryology , Animals , Biological Evolution , Epigenesis, Genetic , Extremities/embryology , Phenotype , Time Factors , Vertebrates/embryologyABSTRACT
Extreme novelties in the shape and size of paired fins are exemplified by extinct and extant cartilaginous and bony fishes. Pectoral fins of skates and rays, such as the little skate (Batoid, Leucoraja erinacea), show a strikingly unique morphology where the pectoral fin extends anteriorly to ultimately fuse with the head. This results in a morphology that essentially surrounds the body and is associated with the evolution of novel swimming mechanisms in the group. In an approach that extends from RNA sequencing to in situ hybridization to functional assays, we show that anterior and posterior portions of the pectoral fin have different genetic underpinnings: canonical genes of appendage development control posterior fin development via an apical ectodermal ridge (AER), whereas an alternative Homeobox (Hox)-Fibroblast growth factor (Fgf)-Wingless type MMTV integration site family (Wnt) genetic module in the anterior region creates an AER-like structure that drives anterior fin expansion. Finally, we show that GLI family zinc finger 3 (Gli3), which is an anterior repressor of tetrapod digits, is expressed in the posterior half of the pectoral fin of skate, shark, and zebrafish but in the anterior side of the pelvic fin. Taken together, these data point to both highly derived and deeply ancestral patterns of gene expression in skate pectoral fins, shedding light on the molecular mechanisms behind the evolution of novel fin morphologies.
Subject(s)
Adaptation, Physiological/genetics , Animal Fins/metabolism , Fish Proteins/genetics , Skates, Fish/genetics , Animal Fins/anatomy & histology , Animal Fins/embryology , Animals , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/metabolism , Fibroblast Growth Factors/classification , Fibroblast Growth Factors/genetics , Fish Proteins/classification , Gene Expression Regulation, Developmental , Homeodomain Proteins/classification , Homeodomain Proteins/genetics , In Situ Hybridization , Phylogeny , Skates, Fish/embryologyABSTRACT
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.
Subject(s)
Fishes/anatomy & histology , Animals , Enhancer Elements, Genetic , Fishes/genetics , Gene Expression Regulation, Developmental , Genes, Homeobox , MiceABSTRACT
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.
Subject(s)
Body Patterning/genetics , Caenorhabditis elegans/growth & development , Caenorhabditis elegans/genetics , Amino Acid Sequence , Animals , Base Sequence , Body Patterning/physiology , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Consensus Sequence , Cyclic GMP/metabolism , DNA, Helminth/genetics , DNA, Helminth/metabolism , Gene Expression Regulation, Developmental , Genes, Helminth , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Larva/growth & development , Larva/metabolism , Molecular Sequence Data , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Olfactory Receptor Neurons/growth & development , Olfactory Receptor Neurons/metabolism , Sequence Homology, Amino Acid , Signal TransductionABSTRACT
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.
Subject(s)
DNA/metabolism , Genome-Wide Association Study , Proto-Oncogene Proteins c-ets/metabolism , Animals , Base Sequence , Binding Sites , Cell Line , DNA/chemistry , Humans , Mice , Models, Molecular , Protein Binding , Proto-Oncogene Proteins c-ets/chemistry , Sequence Analysis, DNAABSTRACT
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.
Subject(s)
Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Regulatory Sequences, Nucleic Acid/genetics , Vertebrates/genetics , Animal Fins/anatomy & histology , Animal Fins/embryology , Animal Fins/metabolism , Animals , Animals, Genetically Modified , Biological Evolution , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/metabolism , Extremities/anatomy & histology , Extremities/embryology , Gene Expression Profiling , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Homeodomain Proteins/classification , Homeodomain Proteins/metabolism , Humans , Mice , Mice, Transgenic , Molecular Sequence Data , Phylogeny , Poly-ADP-Ribose Binding Proteins , Skates, Fish/embryology , Skates, Fish/genetics , Transcription Factors/classification , Transcription Factors/genetics , Transcription Factors/metabolism , Vertebrates/classification , Vertebrates/embryology , Zebrafish/embryology , Zebrafish/geneticsABSTRACT
Animals capable of whole-body regeneration can replace any missing cell type and regenerate fully-functional new organs, de novo . The regeneration of a new brain requires the formation of diverse neuronal cell types and their assembly into an organized structure and correctly-wired circuits. Recent work in various regenerative animals has revealed transcriptional programs required for the differentiation of distinct neuronal subpopulations, however how these transcriptional programs are initiated upon amputation remains unknown. Here, we focused on the highly regenerative acoel worm, Hofstenia miamia , to study wound-induced transcriptional regulatory events that lead to the production of neurons. Footprinting analysis using chromatin accessibility data on an improved genome assembly revealed that binding sites for the NFY transcription factor complex were significantly bound during regeneration, showing a dynamic increase in binding within one hour upon amputation specifically in tail fragments, which will regenerate a new brain. Strikingly, NFY targets were highly enriched for genes with neuronal functional. Single-cell transcriptome analysis combined with functional studies identified sox4 + stem cells as the likely progenitor population for multiple neuronal subtypes. Further, we found that wound-induced sox4 expression is likely under direct transcriptional control by NFY, uncovering a mechanism for how early wound-induced binding of a transcriptional regulator results in the initiation of a neuronal differentiation pathway. Highlights: A new chromosome-scale assembly for Hofstenia enables comprehensive analysis of transcription factor binding during regeneration NFY motifs become dynamically bound by 1hpa in regenerating tail fragments, particularly in the loci of neural genes A sox4 + neural-specialized stem cell is identified using scRNA-seq sox4 is wound-induced and required for differentiation of multiple neural cell types NFY regulates wound-induced expression of sox4 during regeneration.
ABSTRACT
Classifying proteins into subgroups with similar molecular function on the basis of sequence is an important step in deriving reliable functional annotations computationally. So far, however, available classification procedures have been evaluated against protein subgroups that are defined by experts using mainly qualitative descriptions of molecular function. Recently, in vitro DNA-binding preferences to all possible 8-nt DNA sequences have been measured for 178 mouse homeodomains using protein-binding microarrays, offering the unprecedented opportunity of evaluating the classification methods against quantitative measures of molecular function. To this end, we automatically derive homeodomain subtypes from the DNA-binding data and independently group the same domains using sequence information alone. We test five sequence-based methods, which use different sequence-similarity measures and algorithms to group sequences. Results show that methods that optimize the classification robustness reflect well the detailed functional specificity revealed by the experimental data. In some of these classifications, 73-83% of the subfamilies exactly correspond to, or are completely contained in, the function-based subtypes. Our findings demonstrate that certain sequence-based classifications are capable of yielding very specific molecular function annotations. The availability of quantitative descriptions of molecular function, such as DNA-binding data, will be a key factor in exploiting this potential in the future.
Subject(s)
Homeodomain Proteins/classification , Animals , DNA/metabolism , Homeodomain Proteins/chemical synthesis , Homeodomain Proteins/metabolism , Mice , Sequence Analysis, ProteinABSTRACT
Dynamic gene expression seen during whole-body regeneration is likely controlled by genomic regulatory elements that dictate the spatiotemporal activity of the regeneration transcriptome. Identifying and characterizing these non-coding regulatory sequences are key to understanding how genes are connected into networks to deploy the process of whole-body regeneration. Here, we describe the application of the Assay for Transposase Accessible Chromatin (ATAC-seq) in the acoel Hofstenia miamia to identify regions of open chromatin that represent putative regulatory elements. Notably, when paired with gene knockdown techniques such as RNAi, ATAC-seq can be implemented in a functional genomics approach to validate putative regulatory elements. ATAC-seq requires no species-specific reagents, is amenable to small input cell numbers, and can be completed in a single day, making it an ideal assay to identify dynamic chromatin at high resolution during whole-body regeneration in virtually any species with a quality genome assembly.
Subject(s)
Chromatin Immunoprecipitation Sequencing , Chromatin , Chromatin/genetics , Regulatory Sequences, Nucleic Acid , Sequence Analysis, DNA/methods , Transposases/genetics , Transposases/metabolismABSTRACT
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.
Subject(s)
DNA, Protozoan/metabolism , Gene Regulatory Networks , Genes, Protozoan , Plasmodium falciparum/genetics , Protozoan Proteins/metabolism , Transcription Factor AP-2/metabolism , Amino Acid Sequence , Animals , Base Sequence , Electrophoretic Mobility Shift Assay , Gene Expression Regulation , Molecular Sequence Data , Protein Structure, Tertiary , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Regulatory Elements, Transcriptional , Regulon , Transcription Factor AP-2/chemistry , Transcription Factor AP-2/geneticsABSTRACT
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.
Subject(s)
Computational Biology/methods , DNA/chemistry , Sequence Analysis, DNA/methods , Transcription Factors/metabolism , Binding Sites , DNA/metabolism , Transcription Factors/chemistryABSTRACT
Whole-body regeneration is accompanied by complex transcriptomic changes, yet the chromatin regulatory landscapes that mediate this dynamic response remain unexplored. To decipher the regulatory logic that orchestrates regeneration, we sequenced the genome of the acoel worm Hofstenia miamia, a highly regenerative member of the sister lineage of other bilaterians. Epigenomic profiling revealed thousands of regeneration-responsive chromatin regions and identified dynamically bound transcription factor motifs, with the early growth response (EGR) binding site as the most variably accessible during Hofstenia regeneration. Combining egr inhibition with chromatin profiling suggests that Egr functions as a pioneer factor to directly regulate early wound-induced genes. The genetic connections inferred by this approach allowed the construction of a gene regulatory network for whole-body regeneration, enabling genomics-based comparisons of regeneration across species.
Subject(s)
Early Growth Response Transcription Factors/metabolism , Gene Expression Regulation , Gene Regulatory Networks , Invertebrates/genetics , Invertebrates/physiology , Regeneration/genetics , Animals , Binding Sites , Chromatin/metabolism , Genome , Transcriptome , Wound Healing/geneticsABSTRACT
The molecular mechanisms underlying whole-body regeneration are best understood in the planarian flatworm Schmidtea mediterranea, where a heterogeneous population of somatic stem cells called neoblasts provides new tissue for regeneration of essentially any missing body part. Studies on Schmidtea have provided a detailed description of neoblasts and their role in regeneration, but comparatively little is known about the evolutionary history of these cells and their underlying developmental programs. Acoels, an understudied group of aquatic worms that are also capable of extensive whole-body regeneration, have arisen as an attractive group to study the evolution of regenerative processes due to their phylogenetically distant position relative to flatworms. Here, we review the phylogenetic distribution of neoblast cells and compare their anatomical locations, transcriptional profiles, and roles during regeneration in flatworms and acoels to understand the evolution of whole-body regeneration. While the general role of neoblasts appears conserved in species separated by 550 million years of evolution, the extrinsic inputs they receive during regeneration can vary, making the distinction between homology and convergence of mechanism unclear. A more detailed understanding of the precise mechanisms behind whole-body regeneration in diverse phyla is necessary to understand the evolutionary history of this powerful process.
Subject(s)
Biological Evolution , Planarians/genetics , Regeneration/genetics , Stem Cells , Animals , Cell Differentiation/genetics , Phylogeny , Planarians/growth & developmentABSTRACT
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.
Subject(s)
Fishes/genetics , Animals , Evolution, Molecular , Female , Fishes/metabolism , Genome , Humans , Karyotype , Models, Genetic , Organ Specificity , Sequence Analysis, DNA , TranscriptomeABSTRACT
Circadian clocks provide an adaptive advantage by allowing organisms to anticipate daily and seasonal environmental changes [1, 2]. Eukaryotic oscillators rely on complex hierarchical networks composed of transcriptional and posttranslational regulatory circuits [3]. In Arabidopsis, current representations of the circadian clock consist of three or four interlocked transcriptional feedback loops [3, 4]. Although molecular components contributing to different domains of these circuits have been described, how the loops are connected at the molecular level is not fully understood. Genetic screens previously identified LUX ARRHYTHMO (LUX) [5], also known as PHYTOCLOCK1 (PCL1) [6], an evening-expressed putative transcription factor essential for circadian rhythmicity. We determined the in vitro DNA-binding specificity for LUX by using universal protein binding microarrays; we then demonstrated that LUX directly regulates the expression of PSEUDO RESPONSE REGULATOR9 (PRR9), a major component of the morning transcriptional feedback circuit, through association with the newly discovered DNA binding site. We also show that LUX binds to its own promoter, defining a new negative autoregulatory feedback loop within the core clock. These novel connections between the archetypal loops of the Arabidopsis clock represent a significant advance toward defining the molecular dynamics underlying the circadian network in plants and provide the first mechanistic insight into the molecular function of the previously orphan clock factor LUX.