Peter Heger

The chromatin insulator CTCF and the emergence of metazoan diversity

The great majority of metazoans belong to bilaterian phyla. They diversified during a short interval in Earth’s history known as the Cambrian explosion, ∼540 million years ago. However, the genetic basis of these events is poorly understood. Here we argue that the vertebrate genome organizer CTCF (CCCTC-binding factor) played an important role for the evolution of bilaterian animals. We provide evidence that the CTCF protein and a genome-wide abundance of CTCF-specific binding motifs are unique to bilaterian phyla, but absent in other eukaryotes. We demonstrate that CTCF-binding sites within vertebrate and Drosophila Hox gene clusters have been maintained for several hundred million years, suggesting an ancient origin of the previously known interaction between Hox gene regulation and CTCF. In addition, a close correlation between the presence of CTCF and Hox gene clusters throughout the animal kingdom suggests conservation of the Hox-CTCF link across the Bilateria. On the basis of these findings, we propose the existence of a Hox-CTCF kernel as principal organizer of bilaterian body plans. Such a kernel could explain (i) the formation of Hox clusters in Bilateria, (ii) the diversity of bilaterian body plans, and (iii) the uniqueness and time of onset of the Cambrian explosion.

Loss of the insulator protein CTCF during nematode evolution

BackgroundThe zinc finger (ZF) protein CTCF (CCCTC-binding factor) is highly conserved in Drosophila and vertebrates where it has been shown to mediate chromatin insulation at a genomewide level. A mode of genetic regulation that involves insulators and insulator binding proteins to establish independent transcriptional units is currently not known in nematodes including Caenorhabditis elegans. We therefore searched in nematodes for orthologs of proteins that are involved in chromatin insulation.ResultsWhile orthologs for other insulator proteins were absent in all 35 analysed nematode species, we find orthologs of CTCF in a subset of nematodes. As an example for these we cloned the Trichinella spiralis CTCF-like gene and revealed a genomic structure very similar to the Drosophila counterpart. To investigate the pattern of CTCF occurrence in nematodes, we performed phylogenetic analysis with the ZF protein sets of completely sequenced nematodes. We show that three ZF proteins from three basal nematodes cluster together with known CTCF proteins whereas no zinc finger protein of C. elegans and other derived nematodes does so.ConclusionOur findings show that CTCF and possibly chromatin insulation are present in basal nematodes. We suggest that the insulator protein CTCF has been secondarily lost in derived nematodes like C. elegans. We propose a switch in the regulation of gene expression during nematode evolution, from the common vertebrate and insect type involving distantly acting regulatory elements and chromatin insulation to a so far poorly characterised mode present in more derived nematodes. Here, all or some of these components are missing. Instead operons, polycistronic transcriptional units common in derived nematodes, seemingly adopted their function.

The genome of Romanomermis culicivorax: revealing fundamental changes in the core developmental genetic toolkit in Nematoda.

BackgroundThe genetics of development in the nematode Caenorhabditis elegans has been described in exquisite detail. The phylum Nematoda has two classes: Chromadorea (which includes C. elegans) and the Enoplea. While the development of many chromadorean species resembles closely that of C. elegans, enoplean nematodes show markedly different patterns of early cell division and cell fate assignment. Embryogenesis of the enoplean Romanomermis culicivorax has been studied in detail, but the genetic circuitry underpinning development in this species has not been explored.ResultsWe generated a draft genome for R. culicivorax and compared its gene content with that of C. elegans, a second enoplean, the vertebrate parasite Trichinella spiralis, and a representative arthropod, Tribolium castaneum. This comparison revealed that R. culicivorax has retained components of the conserved ecdysozoan developmental gene toolkit lost in C. elegans. T. spiralis has independently lost even more of this toolkit than has C. elegans. However, the C. elegans toolkit is not simply depauperate, as many novel genes essential for embryogenesis in C. elegans are not found in, or have only extremely divergent homologues in R. culicivorax and T. spiralis. Our data imply fundamental differences in the genetic programmes not only for early cell specification but also others such as vulva formation and sex determination.ConclusionsDespite the apparent morphological conservatism, major differences in the molecular logic of development have evolved within the phylum Nematoda. R. culicivorax serves as a tractable system to contrast C. elegans and understand how divergent genomic and thus regulatory backgrounds nevertheless generate a conserved phenotype. The R. culicivorax draft genome will promote use of this species as a research model.

SUCCESSIVE GAIN OF INSULATOR PROTEINS IN ARTHROPOD EVOLUTION

Alteration of regulatory DNA elements or their binding proteins may have drastic consequences for morphological evolution. Chromatin insulators are one example of such proteins and play a fundamental role in organizing gene expression. While a single insulator protein, CTCF (CCCTC‐binding factor), is known in vertebrates, Drosophila melanogaster utilizes six additional factors. We studied the evolution of these proteins and show here that—in contrast to the bilaterian‐wide distribution of CTCF—all other D. melanogaster insulators are restricted to arthropods. The full set is present exclusively in the genus Drosophila whereas only two insulators, Su(Hw) and CTCF, existed at the base of the arthropod clade and all additional factors have been acquired successively at later stages. Secondary loss of factors in some lineages further led to the presence of different insulator subsets in arthropods. Thus, the evolution of insulator proteins within arthropods is an ongoing and dynamic process that reshapes and supplements the ancient CTCF‐based system common to bilaterians. Expansion of insulator systems may therefore be a general strategy to increase an organisms gene regulatory repertoire and its potential for morphological plasticity.

Conservation of MAP kinase activity and MSP genes in parthenogenetic nematodes

BackgroundMAP (mitogen-activated protein) kinase activation is a prerequisite for oocyte maturation, ovulation and fertilisation in many animals. In the hermaphroditic nematode Caenorhabditis elegans, an MSP (major sperm protein) dependent pathway is utilised for MAP kinase activation and successive oocyte maturation with extracellular MSP released from sperm acting as activator. How oocyte-to-embryo transition is triggered in parthenogenetic nematode species that lack sperm, is not known.ResultsWe investigated two key elements of oocyte-to-embryo transition, MSP expression and MAP kinase signaling, in two parthenogenetic nematodes and their close hermaphroditic relatives. While activated MAP kinase is present in all analysed nematodes irrespective of the reproductive mode, MSP expression differs. In contrast to hermaphroditic or bisexual species, we do not find MSP expression at the protein level in parthenogenetic nematodes. However, genomic sequence analysis indicates that functional MSP genes are present in several parthenogenetic species.ConclusionsWe present three alternative interpretations to explain our findings. (1) MSP has lost its function as a trigger of MAP kinase activation and is not expressed in parthenogenetic nematodes. Activation of the MAP kinase pathway is achieved by another, unknown mechanism. Functional MSP genes are required for occasionally emerging males found in some parthenogenetic species. (2) Because of long-term disadvantages, parthenogenesis is of recent origin. MSP genes remained intact during this short intervall although they are useless. As in the first scenario, an unknown mechanism is responsible for MAP kinase activation. (3) The molecular machinery regulating oocyte-to-embryo transition in parthenogenetic nematodes is conserved with respect to C. elegans, thus requiring intact MSP genes. However, MSP expression has been shifted to non-sperm cells and is reduced below the detection limits, but is still sufficient to trigger MAP kinase activation and embryogenesis.

New tools in the box: An evolutionary synopsis of chromatin insulators

Despite progress in understanding genome organization and gene expression during the last decade, the evolutionary pathways that led to the intricate patterns of gene expression in different cells of an organism are still poorly understood. Important steps in this regulation take place at the level of chromatin, where the (epi)genomic environment of a gene determines its expression in time and space. Although the basic mechanisms of gene expression apply to all eukaryotes, multicellular organisms face the additional challenge of coordinating gene expression during development. In this review we summarize and put into evolutionary context current knowledge about chromatin insulators, an important class of regulatory factors mediating these tasks. Our interpretation of historical and recent findings points to a dynamic and ongoing evolution of insulator proteins characterized by multiple instances of convergent evolution, gene loss, and binding site changes in different organisms. The idea of two autonomously evolving insulator functions (as a barrier element and an enhancer blocker) further suggests that the evolution of metazoans and their enhancer-rich gene regulatory repertoire might be connected to the radiation of enhancer blocking insulators. Although speculative at the moment, such coevolution might create tools for complex gene regulation and therefore influence the evolutionary roadmaps of metazoans.

Molecular evolutionary trends and feeding ecology diversification in the Hemiptera, anchored by the milkweed bug genome

Background The Hemiptera (aphids, cicadas, and true bugs) are a key insect order whose members offer a close outgroup to the Holometabola, with high diversity within the order for feeding ecology and excellent experimental tractability for molecular genetics. Sequenced genomes have recently become available for hemipteran pest species such as phloem-feeding aphids and blood-feeding bed bugs. To complement and build upon these resources, we present the genome sequence and comparative analyses centered on the large milkweed bug, Oncopeltus fasciatus, a seed feeder of the family Lygaeidae. Results The 926-Mb genome of Oncopeltus is relatively well represented by the current assembly and official gene set, which supports Oncopeltus as a fairly conservative hemipteran species for anchoring molecular comparisons. We use our genomic and RNA-seq data not only to characterize features of the protein-coding gene repertoire and perform isoform-specific RNAi, but also to elucidate patterns of molecular evolution and physiology. We find ongoing, lineage-specific expansion and diversification of repressive C2H2 zinc finger proteins and of intron gain and turnover in the Hemiptera. These analyses also weigh the relative importance of lineage and genome size as predictors of gene structure evolution in insects. Furthermore, we identify enzymatic gains and losses that correlate with hemipteran feeding biology, particularly for reductions in chemoreceptor family size and loss of metabolic reactions within species with derived, fluid-nutrition feeding modes. Conclusions With the milkweed bug genome, for the first time we have a critical mass of sequenced species representing a hemimetabolous insect order, substantially improving the diversity of insect genomics beyond holometabolans such as flies and ants. We use this addition to define commonalities among the Hemiptera and then delve into how hemipteran species’ genomes reflect their feeding ecology types. Our novel and detailed analyses integrate global and rigorous manual approaches, generating hypotheses and identifying specific sets of genes for future investigation. Given Oncopeltus’s strength as an experimental research model, we take particular care to evaluate the sequence resources presented here, augmenting its foundation for molecular research and highlighting potentially general considerations exemplified in the assembly and annotation of this medium-sized genome.

Transcriptomic data from panarthropods shed new light on the evolution of insulator binding proteins in insects

BackgroundBody plan development in multi-cellular organisms is largely determined by homeotic genes. Expression of homeotic genes, in turn, is partially regulated by insulator binding proteins (IBPs). While only a few enhancer blocking IBPs have been identified in vertebrates, the common fruit fly Drosophila melanogaster harbors at least twelve different enhancer blocking IBPs. We screened recently compiled insect transcriptomes from the 1KITE project and genomic and transcriptomic data from public databases, aiming to trace the origin of IBPs in insects and other arthropods.ResultsOur study shows that the last common ancestor of insects (Hexapoda) already possessed a substantial number of IBPs. Specifically, of the known twelve insect IBPs, at least three (i.e., CP190, Su(Hw), and CTCF) already existed prior to the evolution of insects. Furthermore we found GAF orthologs in early branching insect orders, including Zygentoma (silverfish and firebrats) and Diplura (two-pronged bristletails). Mod(mdg4) is most likely a derived feature of Neoptera, while Pita is likely an evolutionary novelty of holometabolous insects. Zw5 appears to be restricted to schizophoran flies, whereas BEAF-32, ZIPIC and the Elba complex, are probably unique to the genus Drosophila. Selection models indicate that insect IBPs evolved under neutral or purifying selection.ConclusionsOur results suggest that a substantial number of IBPs either pre-date the evolution of insects or evolved early during insect evolution. This suggests an evolutionary history of insulator binding proteins in insects different to that previously thought. Moreover, our study demonstrates the versatility of the 1KITE transcriptomic data for comparative analyses in insects and other arthropods.

The Diverging Routes of BORIS and CTCF: An Interactomic and Phylogenomic Analysis

The CCCTC-binding factor (CTCF) is multi-functional, ubiquitously expressed, and highly conserved from Drosophila to human. It has important roles in transcriptional insulation and the formation of a high-dimensional chromatin structure. CTCF has a paralog called “Brother of Regulator of Imprinted Sites” (BORIS) or “CTCF-like” (CTCFL). It binds DNA at sites similar to those of CTCF. However, the expression profiles of the two proteins are quite different. We investigated the evolutionary trajectories of the two proteins after the duplication event using a phylogenomic and interactomic approach. We find that CTCF has 52 direct interaction partners while CTCFL only has 19. Almost all interactors already existed before the emergence of CTCF and CTCFL. The unique secondary loss of CTCF from several nematodes is paralleled by a loss of two of its interactors, the polycomb repressive complex subunit SuZ12 and the multifunctional transcription factor TYY1. In contrast to earlier studies reporting the absence of BORIS from birds, we present evidence for a multigene synteny block containing CTCFL that is conserved in mammals, reptiles, and several species of birds, indicating that not the entire lineage of birds experienced a loss of CTCFL. Within this synteny block, BORIS and its genomic neighbors seem to be partitioned into two nested chromatin loops. The high expression of SPO11, RAE1, RBM38, and PMEPA1 in male tissues suggests a possible link between CTCFL, meiotic recombination, and fertility-associated phenotypes. Using the 65,700 exomes and the 1000 genomes data, we observed a higher number of intergenic, non-synonymous, and loss-of-function mutations in CTCFL than in CTCF, suggesting a reduced strength of purifying selection, perhaps due to less functional constraint.

A catalogue of Bilaterian-specific genes { their function and expression profiles in early development

Bilateria constitute a monophyletic group of organisms comprising about 99% of all living animals. Since their initial radiation about 540Mya they have evolved a plethora of traits and body forms allowing them to conquer almost any habitat on earth. There are only few truly uniting and shared morphological features retained across the phylum. Unsurprisingly, also the genetic toolkit of bilateria is highly diverged. In the light of this divergence we investigated if a set of bilaterian-specific genes exists and, beyond this, if such genes are related with respect to function and expression patterns among organisms as distant as Drosophila, Caenorhabditis and Danio. Using a conservative pyramidal approach of orthology inference we collected a set of protein-coding genes which have orthologs in all major branches of Bilateria, but no homologs in non-bilaterian species. To characterize the proteins with respect to function, we employ a novel method for multi-species GO analysis and augmented it by a human-curated annotation based on an extensive literature search. Finally, we extracted characteristic developmental expression profiles for Bilateria from the extensive data available for three model organisms and we explored the relation between expression and function. Among an initial set of several hundred candidates we identified 85 clusters of orthologous proteins which passed our filter criteria for bilaterian specificity. Although some of these proteins belong to common developmental processes, they cover a wide range of biological components, from transcription factors to metabolic enzymes. For instance, the clusters include myoD, an important regulator of mesodermal cell fate and muscle development, and prospero and several other factors involved in nervous system development. Our results reveal a so far unknown connection between morphological key innovations of bilateria, such as the mesoderm and a complex nervous system, and their genetic basis. Furthermore, we find typical expression profiles for these bilaterian specific genes, with the majority of them being highly expressed when the adult body plan is constructed. These observations are compatible with the idea that bilaterians are characterized by the unfolding of a new developmental phase, namely the transition of the larva to morphologically distinct adults. Author Summary Bilateria represent by far the largest and morphologically most diverse clade of all extant animals. The bilaterian radiation dates back to the so-called Cambrian explosion of species. Although bilateria show a large variety of very distinct body plans, they are also characterized by several common developmental and morphological traits, on which their monophyly is based. Here, we wanted to know whether these common phenotypic features may also have a shared and conserved genetic basis. To address this question we compared the proteomes of bilaterian and non-bilaterian species and extracted an initial set of a few hundred candidate proteins. Their underlying genes were further post-processed by means of orthology clustering, multi-species GO enrichment, expression analysis and extensive literature mining. This resulted in a thorough set of genes with roles in body morphology-, neuronal system‐ and muscle development, as well as in cell-cell signalling processes. This gene catalogue can be regarded as blue-print of a common bilaterian pheno‐ or morphotype and should contain highly interesting targets for further functional studies in model and non-model organisms.Bilateria constitute a monophyletic group of organisms comprising about 99% of all living animals. Since their initial radiation about 540Mya they have evolved a plethora of traits and body forms allowing them to conquer almost any habitat on earth. There are only few truly uniting and shared morphological features retained across the phylum. Unsurprisingly, also the genetic toolkit of bilateria is highly diverged. In the light of this divergence we investigated if a set of Bilaterian-specific genes exists and, beyond this, if such genes are related with respect to function and expression among organisms as distant as fly, worm and fish. Using a conservative pyramidal approach of orthology inference we first employed BLAST to extract a set of protein-coding genes which has orthologs in all major branches of Bilateria, but no homologs in non-Bilaterian species. To characterize the proteins with respect to function, we developed a new method for multi-species GO analysis and augmented it by a human-curated annotation based on an extensive literature search. Finally, we extracted characteristic developmental expression profiles for Bilateria from the detailed data available for three model organisms and we explored the relation between expression and function. We find 85 clusters of pan-bilaterian proteins. Although some of these proteins belong to common developmental processes, they cover a wide range of biological components, from transcription factors to metabolic enzymes. The 85 groups include myoD, an important regulator of mesodermal cell fate and muscle development, and prospero and several other factors involved in nervous system development. They reveal a so far unknown connection between bilaterian key innovations (mesoderm, complex nervous system) and the emergence of new genes in the ancestor of bilaterians. In addition, the majority of these genes tends to be highly expressed when the adult body plan is constructed. These observations are compatible with the idea that bilaterians are characterized by the unfolding of an additional developmental phase, namely the transition of the larva to morphologically distinct adults.