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Whole genome duplications (WGDs) are major events that drastically reshape genome architecture and are causally associated with organismal innovations and radiations1. The 2R Hypothesis suggests that two WGD events (1R and 2R) occurred during early vertebrate evolution2,3. However, the veracity and timing of the 2R event relative to the divergence of gnathostomes (jawed vertebrates) and cyclostomes (jawless hagfishes and lampreys) is unresolved4-6 and whether these WGD events underlie vertebrate phenotypic diversification remains elusive7. Here we present the genome of the inshore hagfish, Eptatretus burgeri. Through comparative analysis with lamprey and gnathostome genomes, we reconstruct the early events in cyclostome genome evolution, leveraging insights into the ancestral vertebrate genome. Genome-wide synteny and phylogenetic analyses support a scenario in which 1R occurred in the vertebrate stem-lineage during the early Cambrian, and the 2R event occurred in the gnathostome stem-lineage in the late Cambrian after its divergence from cyclostomes. We find that the genome of stem-cyclostomes experienced two additional, independent genome duplications (herein CR1 and CR2). Functional genomic and morphospace analyses demonstrate that WGD events generally contribute to developmental evolution with similar changes in the regulatory genome of both vertebrate groups. However, appreciable morphological diversification occurred only after the 2R event, questioning the general expectation that WGDs lead to leaps of morphological complexity7.

Specific DNA methylation at CpG and non-CpG sites is essential for chromatin regulation. The DNA methyltransferase DNMT3A interacts with target sites surrounded by variable DNA sequences with its TRD and RD loops, but the functional necessity of these interactions is unclear. We investigated CpG and non-CpG methylation in a randomized sequence context using WT DNMT3A and several DNMT3A variants containing mutations at DNA-interacting residues. Our data revealed that the flanking sequence of target sites between the -2 and up to the +8 position modulates methylation rates100-fold. Non-CpG methylation flanking preferences were even stronger and favor C(+1). R836 and N838 in concert mediate recognition of the CpG guanine. R836 changes its conformation in a flanking sequence-dependent manner and either contacts the CpG guanine or the +1/+2 flank, thereby coupling the interaction with both sequence elements. R836 suppresses activity at CNT sites but supports methylation of CAC substrates, the preferred target for non-CpG methylation of DNMT3A in cells. N838 helps to balance this effect and prevent the preference for C(+1) from becoming too strong. Surprisingly, we found L883 reduces DNMT3A activity despite being highly conserved in evolution. However, mutations at L883 disrupt the DNMT3A-specific DNA interactions of the RD loop, leading to altered flanking sequence preferences. Similar effects occur after the R882H mutation in cancer cells. Our data reveal that DNMT3A forms flexible and interdependent interaction networks with the CpG guanine and flanking residues that ensure recognition of the CpG and efficient methylation of the cytosine in contexts of variable flanking sequences.

In vertebrates, DNA methylation predominantly occurs at CG dinucleotides however, widespread non-CG methylation (mCH) has been reported in mammalian embryonic stem cells and in the brain. In mammals, mCH is found at CAC trinucleotides in the nervous system, where it is associated with transcriptional repression, and at CAG trinucleotides in embryonic stem cells, where it positively correlates with transcription. Moreover, CAC methylation appears to be a conserved feature of adult vertebrate brains. Unlike any of those methylation signatures, here we describe a novel form of mCH that occurs in the TGCT context within zebrafish mosaic satellite repeats. TGCT methylation is inherited from both male and female gametes, remodelled during mid-blastula transition, and re-established during gastrulation in all embryonic layers. Moreover, we identify DNA methyltransferase 3ba (Dnmt3ba) as the primary enzyme responsible for the deposition of this mCH mark. Finally, we observe that TGCT-methylated repeats are specifically associated with H3K9me3-marked heterochromatin suggestive of a functional interplay between these two gene-regulatory marks. Altogether, this work provides insight into a novel form of vertebrate mCH and highlights the substrate diversity of vertebrate DNA methyltransferases.

Reproduction induces increased food intake across females of many animal species1–4, providing a physiologically relevant paradigm for the exploration of appetite regulation. Here, by examining the diversity of enteric neurons in Drosophila melanogaster, we identify a key role for gut-innervating neurons with sex- and reproductive state-specific activity in sustaining the increased food intake of mothers during reproduction. Steroid and enteroendocrine hormones functionally remodel these neurons, which leads to the release of their neuropeptide onto the muscles of the crop—a stomach-like organ—after mating. Neuropeptide release changes the dynamics of crop enlargement, resulting in increased food intake, and preventing the post-mating remodelling of enteric neurons reduces both reproductive hyperphagia and reproductive fitness. The plasticity of enteric neurons is therefore key to reproductive success. Our findings provide a mechanism to attain the positive energy balance that sustains gestation, dysregulation of which could contribute to infertility or weight gain. A multi-organ circuit is activated in female flies after mating, leading to changes in enteric neurons that increase food intake.

Background Cytosine DNA methylation is widely described as a transcriptional repressive mark with the capacity to silence promoters. Epigenome engineering techniques enable direct testing of the effect of induced DNA methylation on endogenous promoters; however, the downstream effects have not yet been comprehensively assessed. Results Here, we simultaneously induce methylation at thousands of promoters in human cells using an engineered zinc finger-DNMT3A fusion protein, enabling us to test the effect of forced DNA methylation upon transcription, chromatin accessibility, histone modifications, and DNA methylation persistence after the removal of the fusion protein. We find that transcriptional responses to DNA methylation are highly context-specific, including lack of repression, as well as cases of increased gene expression, which appears to be driven by the eviction of methyl-sensitive transcriptional repressors. Furthermore, we find that some regulatory networks can override DNA methylation and that promoter methylation can cause alternative promoter usage. DNA methylation deposited at promoter and distal regulatory regions is rapidly erased after removal of the zinc finger-DNMT3A fusion protein, in a process combining passive and TET-mediated demethylation. Finally, we demonstrate that induced DNA methylation can exist simultaneously on promoter nucleosomes that possess the active histone modification H3K4me3, or DNA bound by the initiated form of RNA polymerase II. Conclusions These findings have important implications for epigenome engineering and demonstrate that the response of promoters to DNA methylation is more complex than previously appreciated.

Mammalian brains feature exceptionally high levels of non-CpG DNA methylation alongside the canonical form of CpG methylation. Non-CpG methylation plays a critical regulatory role in cognitive function, which is mediated by the binding of MeCP2, the transcriptional regulator that when mutated causes Rett syndrome. However, it is unclear whether the non-CpG neural methylation system is restricted to mammalian species with complex cognitive abilities or has deeper evolutionary origins. To test this, we investigated brain DNA methylation across 12 distantly related animal lineages, revealing that non-CpG methylation is restricted to vertebrates. We discovered that in vertebrates, non-CpG methylation is enriched within a highly conserved set of developmental genes transcriptionally repressed in adult brains, indicating that it demarcates a deeply conserved regulatory program. We also found that the writer of non-CpG methylation, DNMT3A, and the reader, MeCP2, originated at the onset of vertebrates as a result of the ancestral vertebrate whole-genome duplication. Together, we demonstrate how this novel layer of epigenetic information assembled at the root of vertebrates and gained new regulatory roles independent of the ancestral form of the canonical CpG methylation. This suggests that the emergence of non-CpG methylation may have fostered the evolution of sophisticated cognitive abilities found in the vertebrate lineage., This work was supported by the Australian Research Council (ARC) Centre of Excellence programme in Plant Energy Biology (grant no. CE140100008). R.L. was supported by a Sylvia and Charles Viertel Senior Medical Research Fellowship, ARC Future Fellowship (no. FT120100862) and Howard Hughes Medical Institute International Research Scholarship. A.d.M. was funded by an EMBO long-term fellowship (no. ALTF 144-2014). J.L.G.-S. was supported by the Spanish government (grant no. BFU2016- 74961-P) and the institutional grant Unidad de Excelencia María de Maeztu (no. MDM-2016-0687). B.V. was supported by the Biomedical Research Council of the Agency for Science, Technology and Research of Singapore. F.G. was supported by an ARC Future Fellowship (no. FT160100267). C.W.R. was supported by an NSF grant (no. IOS-1354898). J.R.E. is an investigator of the Howard Hughes Medical Institute. Genomic data was generated at the Australian Cancer Research Foundation Centre for Advanced Cancer Genomics.

Almost all animals undergo embryonic development, going from a single-celled zygote to a complex multicellular adult. We know that the patterning and morphogenetic processes involved in development are deeply conserved within the animal kingdom. However, the origins of these developmental processes are just beginning to be unveiled. Here, we focus on how the protist lineages sister to animals are reshaping our view of animal development. Most intriguingly, many of these protistan lineages display transient multicellular structures, which are governed by similar morphogenetic and gene regulatory processes as animal development. We discuss here two potential alternative scenarios to explain the origin of animal embryonic development: either it originated concomitantly at the onset of animals or it evolved from morphogenetic processes already present in their unicellular ancestors. We propose that an integrative study of several unicellular taxa closely related to animals will allow a more refined picture of how the last common ancestor of animals underwent embryonic development., Work in the I.R.-T. lab has been funded by a European Research Council Consolidator Grant (ERC-2012-Co-616960), a Gordon and Betty Moore Foundation grant (MMI Experimental Model Systems grant, 4973.01) and a grant (BFU2017- 90114-P) from Ministerio de Economıa y Competitividad (MINECO), Agencia Estatal ́ de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER; European Regional Development Fund).

In vertebrates, DNA methylation predominantly occurs at CG dinucleotides even though widespread non-CG methylation (mCH) has been reported in mammalian embryonic and neural cells. Unlike in mammals, where mCH is found enriched at CAC/G trinucleotides and is tissue-restricted, we find that zebrafish embryos as well as adult somatic and germline tissues display robust methylation enrichment at TGCT positions associated with mosaic satellite repeats. These repeats reside in H3K9me3-marked heterochromatin and display mCH reprogramming coincident with zygotic genome activation. Altogether, this work provides insight into a novel form of vertebrate mCH and highlights the substrate diversity of vertebrate DNA methyltransferases.

The evolution of winged insects revolutionized terrestrial ecosystems and led to the largest animal radiation on Earth. However, we still have an incomplete picture of the genomic changes that underlay this diversification. Mayflies, as one of the sister groups of all other winged insects, are key to understanding this radiation. Here, we describe the genome of the mayfly Cloeon dipterum and its gene expression throughout its aquatic and aerial life cycle and specific organs. We discover an expansion of odorant-binding-protein genes, some expressed specifically in breathing gills of aquatic nymphs, suggesting a novel sensory role for this organ. In contrast, flying adults use an enlarged opsin set in a sexually dimorphic manner, with some expressed only in males. Finally, we identify a set of wing-associated genes deeply conserved in the pterygote insects and find transcriptomic similarities between gills and wings, suggesting a common genetic program. Globally, this comprehensive genomic and transcriptomic study uncovers the genetic basis of key evolutionary adaptations in mayflies and winged insects., This project was mainly funded by the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie Grant Agreement 657732 to I.A., Grant BFU2015-66040-P to F.Ca., institutional Grant MDM-2016-0687 (MINECO, Spain). Additional funding was provided by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (ERC-StG-LS2-637591 to M.I.), the Spanish Ministerio de Ciencia (BFU2017-89201-P to M.I., RYC-2016-20089 and PGC2018-099392-A-I00 to I.M.).

The genomes of non-bilaterian metazoans are key to understanding the molecular basis of early animal evolution. However, a full comprehension of how animal-specific traits, such as nervous systems, arose is hindered by the scarcity and fragmented nature of genomes from key taxa, such as Porifera. Ephydatia muelleri is a freshwater sponge found across the northern hemisphere. Here, we present its 326 Mb genome, assembled to high contiguity (N50: 9.88 Mb) with 23 chromosomes on 24 scaffolds. Our analyses reveal a metazoan-typical genome architecture, with highly shared synteny across Metazoa, and suggest that adaptation to the extreme temperatures and conditions found in freshwater often involves gene duplication. The pancontinental distribution and ready laboratory culture of E. muelleri make this a highly practical model system which, with RNAseq, DNA methylation and bacterial amplicon data spanning its development and range, allows exploration of genomic changes both within sponges and in early animal evolution., Reconstructing the early molecular evolution of animals requires genomic resources for non-bilaterian animals. Here, the authors present the chromosome-level genome of a freshwater sponge together with analyses of its genome architecture, methylation, developmental gene expression, and microbiome.

The first winged insects underwent profound morphological and functional transformations leading to the most successful animal radiations in the history of earth. Despite this, we still have a very incomplete picture of the changes in their genomes that underlay this radiation. Mayflies (Ephemeroptera) are one of the extant sister groups of all other winged insects and therefore are at a key phylogenetic position to understand this radiation. Here, we describe the genome of the cosmopolitan mayfly Cloeon dipterum and study its expression along development and in specific organs. We discover an expansion of odorant-binding proteins, some expressed specifically in the breathing gills of aquatic nymphs, suggesting a novel sensory role for gills. In contrast, as flying adults, mayflies make use of an enlarged set of opsins and utilise these visual genes in a sexually dimorphic manner, with some opsins expressed only in males. Finally, to illuminate the origin of wings, we identify a core set of deeply conserved wing-specific genes at the root of the pterygote insects. Globally, this is the first comprehensive study of the structure and expression of the genome of a paleopteran insect and shows how its genome has kept a record of its functional adaptations.

Cytosine DNA methylation (5mC) is a widespread base modification in eukaryotic genomes with critical roles in transcriptional regulation. In recent years, our understanding of 5mC has changed because of advances in 5mC detection techniques that allow mapping of this mark on the whole genome scale. Profiling DNA methylomes from organisms across the eukaryotic tree of life has reshaped our views on the evolution of 5mC. In this review, we explore the macroevolution of 5mC in major eukaryotic groups, and then focus on recent advances made in animals. Genomic 5mC patterns as well as the mechanisms of 5mC deposition tend to be evolutionary labile across large phylogenetic distances; however, some common patterns are starting to emerge. Within the animal kingdom, 5mC diversity has proven to be much greater than anticipated. For example, a previously held common view that genome hypermethylation is a trait exclusive to vertebrates has recently been challenged. Also, data from genome-wide studies are starting to yield insights into the potential roles of 5mC in invertebrate cis regulation. Here we provide an evolutionary perspective of both the well-known and enigmatic roles of 5mC across the eukaryotic tree of life.

Transcription factors (TFs) have a central role in genome regulation directing gene transcription through binding specific DNA sequences. Eukaryotic genomes encode a large diversity of TF classes, each defined by unique DNA-interaction domains. Recent advances in genome sequencing and phylogenetic placement of diverse eukaryotic and archaeal species are re-defining the evolutionary history of eukaryotic TFs. The emerging view from a comparative genomics perspective is that the Last Eukaryotic Common Ancestor (LECA) had an extensive repertoire of TFs, most of which represent eukaryotic evolutionary novelties. This burst of TF innovation coincides with the emergence of genomic nuclear segregation and complex chromatin organization.

Metazoans evolved from a single-celled eukaryotic ancestor more than 650 Ma ago. While the investigations on the origin of animal multicellularity remained purely theoretical for over a century, the recent discovery of the closest extant unicellular relatives of metazoans opened the way to the comparative study of this major evolutionary transition. In this chapter, we will describe how the sequencing of the genomes of multiple protists phylogenetically related to animals and the study of the biology of these species are illuminating the emergence of metazoans. We will show that recent data support a scenario with a complex unicellular ancestor of all animals. From comparisons of animal and protist genomes, we can infer that this unicellular ancestor had many of the genes involved in animal multicellularity (adhesion, signalling, transcriptional regulation). Moreover, the regulatory biology and diversity of cell behaviours in extant unicellular relatives suggest that the unicellular ancestor of animals had multiple temporally separated cell types. These cell types might have become spatially integrated in the first multicellular animals through the advent of genomic regulatory novelties.

Vertebrates have highly methylated genomes at CpG positions, whereas invertebrates have sparsely methylated genomes. This increase in methylation content is considered a major regulatory innovation of vertebrate genomes. However, here we report that a sponge, proposed as the potential sister group to the rest of animals, has a highly methylated genome. Despite major differences in genome size and architecture, we find similarities between the independent acquisitions of the hypermethylated state. Both lineages show genome-wide CpG depletion, conserved strong transcription factor methyl-sensitivity and developmental methylation dynamics at 5-hydroxymethylcytosine enriched regions. Together, our findings trace back patterns associated with DNA methylation in vertebrates to the early steps of animal evolution. Thus, the sponge methylome challenges previous hypotheses concerning the uniqueness of vertebrate genome hypermethylation and its implications for regulatory complexity.

The original version of this Article contained an error in the spelling of the author Hongfei Li, which was incorrectly given as Fei Hong. This has now been corrected in both the PDF and HTML versions of the Article.

Transposable elements are in a constant arms race with the silencing mechanisms of their host genomes. One silencing mechanism commonly used by many eukaryotes is dependent on cytosine methylation, a covalent modification of DNA deposited by C5 cytosine methyltransferases (DNMTs). Here, we report how two distantly related eukaryotic lineages, dinoflagellates and charophytes, have independently incorporated DNMTs into the coding regions of distinct retrotransposon classes. Concomitantly, we show that dinoflagellates of the genus Symbiodinium have evolved cytosine methylation patterns unlike any other eukaryote, with most of the genome methylated at CG dinucleotides. Finally, we demonstrate the ability of retrotransposon DNMTs to methylate CGs de novo, suggesting that retrotransposons could self-methylate retrotranscribed DNA. Together, this is an example of how retrotransposons incorporate host-derived genes involved in DNA methylation. In some cases, this event could have implications for the composition and regulation of the host epigenomic environment., Cytosine methyltransferases (DNMTs) often silence transposons in eukaryotic genomes. Here the authors describe the recurrent acquisition of DNMTs by transposons from two distantly-related eukaryotes and suggest that methylation of CG dinucleotides by transposon DNMTs could modify the host epigenome in dinoflagellates.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, Hemerythrins and hemocyanins are respiratory proteins present in some of the most ecologically diverse animal lineages; however, the precise evolutionary history of their enzymatic domains (hemerythrin, hemocyanin M, and tyrosinase) is still not well understood. We survey a wide dataset of prokaryote and eukaryote genomes and RNAseq data to reconstruct the phylogenetic origins of these proteins. We identify new species with hemerythrin, hemocyanin M, and tyrosinase domains in their genomes, particularly within animals, and demonstrate that the current distribution of respiratory proteins is due to several events of lateral gene transfer and/or massive gene loss. We conclude that the last common metazoan ancestor had at least two hemerythrin domains, one hemocyanin M domain, and six tyrosinase domains. The patchy distribution of these proteins among animal lineages can be partially explained by physiological adaptations, making these genes good targets for investigations into the interplay between genomic evolution and physiological constraints. © The Author(s) 2013., This work was funded by the Sars International Centre for Marine Molecular Biology to J.M.M.-D. and A.H., and an ICREA contract, an European Research Council Starting Grant (ERC-2007-StG-206883), and a grant (BFU2011-23434) from Ministerio de Economía y Competitividad (MINECO) to I.R.-T. A.S.-P.’s salary was supported by a pregraduate FPU grant and A.d.M.’s salary from a FPI grant, both from MICINN.

Molecular fossils (or biomarkers) are key to unraveling the deep history of eukaryotes, especially in the absence of traditional fossils. In this regard, the sterane 24-isopropylcholestane has been proposed as a molecular fossil for sponges, and could represent the oldest evidence for animal life. The sterane is found in rocks ∼650–540 million y old, and its sterol precursor (24-isopropylcholesterol, or 24-ipc) is synthesized today by certain sea sponges. However, 24-ipc is also produced in trace amounts by distantly related pelagophyte algae, whereas only a few close relatives of sponges have been assayed for sterols. In this study, we analyzed the sterol and gene repertoires of four taxa (Salpingoeca rosetta, Capsaspora owczarzaki, Sphaeroforma arctica, and Creolimax fragrantissima), which collectively represent the major living animal outgroups. We discovered that all four taxa lack C30 sterols, including 24-ipc. By building phylogenetic trees for key enzymes in 24-ipc biosynthesis, we identified a candidate gene (carbon-24/28 sterol methyltransferase, or SMT) responsible for 24-ipc production. Our results suggest that pelagophytes and sponges independently evolved C30 sterol biosynthesis through clade-specific SMT duplications. Using a molecular clock approach, we demonstrate that the relevant sponge SMT duplication event overlapped with the appearance of 24-isopropylcholestanes in the Neoproterozoic, but that the algal SMT duplication event occurred later in the Phanerozoic. Subsequently, pelagophyte algae and their relatives are an unlikely alternative to sponges as a source of Neoproterozoic 24-isopropylcholestanes, consistent with growing evidence that sponges evolved long before the Cambrian explosion ∼542 million y ago., We gratefully acknowledge funding from the Agouron Institute Geobiology Fellowship and the NASA Astrobiology Institute (NNA13AA90A) Foundations of Complex Life, Evolution, Preservation, and Detection on Earth and Beyond.

Molecular signatures that characterize a clade are an important tool to create or corroborate phylogenetic groupings (reviewed in Telford and Copley 2011). Examples of such molecular synapomorphies include the presence of Hox/ParaHox genes in the ParaHoxozoa (Placozoa, Cnidaria and Bilateria) (Ryan et al. 2010), an indel in the gene elongation-factor 1-alpha as a character of opisthokonts (Steenkamp, Wright and Baldauf 2006) or the the NAD5 mithocondrial gene that is exclusive to protostomes (Papillon et al 2004). The monophyly of deuterostomes (the clade that comprises chordates, echinoderms, and hemichordates) has been consistently recovered on phylogenetic and phylogenomic studies (Hejnol et al. 2009; Paps, Baguna and Riutort 2009). However, it remains contentious whether Xenoturbella and/or the acoelomorphs (acoels and nemertodermatids) are members of the deuterostomes or basal bilaterians (Ruiz-Trillo et al. 1999; Ruiz-Trillo et al. 2002; Bourlat et al. 2003; Bourlat et al. 2006; Philippe et al. 2007; Dunn et al. 2008; Hejnol et al. 2009; Paps, Baguna and Riutort 2009; Mwinyi et al. 2010; Philippe et al. 2011). Thus the identification of diagnostic molecular synapomorphies for deuterostomes is important to both corroborate previous molecular analyses and independently test the putative deuterostome affiliation of acoels and Xenoturbella. We here show that the bi-functional enzyme UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase (GNE), is exclusive to deuterostomes, acoels and Xenoturbella, being absent from all sequenced protostomes and non-bilaterian taxa. Our data show that GNE is encoded in the genomes of all sequenced deuterostomes except for the urochordates Ciona savignyi, C. intestinalis and Oikopleura dioica, most likely an effect of secondary gene loss (D’Aniello et al. 2008; Churcher and Taylor 2009). Moreover, a small fragment of the gene is present in the expressed sequence tags (EST) of Xenoturbella bocki and we have amplified the gene GNE from the acoel Symsagittifera roscoffensis (GenBank {"type":"entrez-nucleotide","attrs":{"text":"JF826132","term_id":"334878555","term_text":"JF826132"}}JF826132). We searched publicly available EST data as well as unpublished transcriptome data from nemertodermatids and did not get any hit. However, since a complete genome of a nemertodermatid is not available, we can not discard the presence of GNE in this group. Interestingly, the GNE encoded by chordates, echinoderms and hemichordates all share the same 9 introns (both in position and phase), while the GNE of the acoel S. roscoffensis does not share any intron with deuterostomes (Figure S1). Unfortunately, we could not elucidate the intronexon structure of the GNE encoded by X. bocki since only a small cDNA fragment is available. GNE is known to play an important role in the biosynthesis of sialic acids, which are monosaccharides that act in a wide range of biological and pathological events, such as cellular adhesion, recognition determinants, tumorigenesis and stem cells (Effertz, Hinderlich and Reutter 1999; Tanner 2005; Weidemann et al. 2010). In mammals, the metabolic precursor of sialic acids is the N-acetylneuraminic (Neu5Ac) acid, which derives from UDP-N-acetylglucosamine (UDP-GlcNAc). The first two steps of this reaction are catalyzed by the bi-functional enzyme UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase (GNE) (Figure 1). The bi-functional activity of GNE comes from two different protein domains: the UDP-N-acetylglucosamine 2-epimerase domain (PF02350) (from herein the “epimerase-2 domain”) and a kinase domain known as ROK (Repressor, ORF, Kinase PF00480) (Tanner 2005). The epimerase-2 domain converts UDP-GlcNAc to N-acetylmannosamine (ManNAc), which is consecutively phosphorylated to ManNAc-6-P by the ROK domain (Figure 1). Interestingly, while in prokaryotes the epimerase and kinase functions are carried out by two separate enzymes, in vertebrates those two domains have been fused, allowing an allosteric site to appear, and thus conferring the potential for new functions to arise. Figure 1 A) Schematic representation of the biosynthesis pathway of CMP-NeuNAc both in bacteria and mammals (adapted from (Tanner 2005). Steps 1 and 2 are performed by GNE in deuterostomes. Step 3 is exclusive of Bacteria; while the additional step 4 has so-far ... Thus, to gain further insights into the evolutionary origin of GNE, we performed both exhaustive searches across the public databases and phylogenetic analyses of the two domains independently. Our data show that both domains have a patchy distribution across eukaryotes. The Epimerase-2 domain is encoded in a few eukaryotic genomes, being, in contrast, ubiquitous among Archaea and Eubacteria (Figure 2). The phylogenetic analyses show two major clades, one comprising the bilaterian-specific GNE genes within a mostly prokaryotic clade (clade A in Figure 2), the other comprising most other (non-metazoan) eukaryotes branching also within a prokaryotic clade (clade B in Figure 2). Both clades are divided with high nodal support (Bootstrap Value (BV) = 100%, and Bayesian Posterior Probability (PP) = 1.00), and both have specific indel characters (see Figure S2). The general topology of the epimerase-2 domain is probably due to either i) an extreme case of hidden paralogy (i.e, this protein domain was present at the origin of eukaryotes and lost in all lineages except in deuterostomes, Xenoturbella, Acoela and a few other eukaryotes), ii) domain convergence, or iii) the consequence of several independent Lateral Gene Transfer (LGT) events to the different eukaryotic lineages, one being a LGT event to the last common bilaterian ancestor (and then subsequently lost in protostomes and in urochordates), or to the last common ancestor of deuterostomes (and lost in urochordates), if xenacoelomorphs (Xenoturbella + acoelomorphs) are indeed deuterostomes as recently suggested (Philippe et al. 2011). Interestingly, the sequence from Micromonas sp. falls as sister group to bilaterians. However, the Micromonas gene, in contrast to the deuterostome sequences, has no introns. Moreover, neither M. pusilla (the other congeneric species with its complete genome sequenced), or any other sequenced clorophyte (except Ostreococcus lucimarinus, whose epimerase gene is far related; see Figure 2) encode this domain. Thus, Micromonas epimerase-2 sequence, as well as Ricinus homolog which branches within another independent bacterial clade (Figure 2), most probably come from independent LGT events (see Supplementary Material). Figure 2 Maximum likelihood (ML) phylogenetic tree of the Epimerase-2 domain as obtained by RAxML (WAG + Γ + I). The tree is rooted using the midpoint-rooted tree option. Nodal support was obtained by RAxML 100-bootstrap replicates (BV) and Bayesian Posterior ... The ROK domain also bears a complex evolutionary history (Figure 3). The phylogenetic analysis shows most eukaryotic sequences in a single monophyletic group that also includes Eubacteria. The bilaterian GNE genes are monophyletic and unrelated to the other eukaryotic sequences (also supported by indel characters, see Figure S2B). Again, this topology can either be explained by LGT or by hidden paralogy. Figure 3 Maximum likelihood (ML) phylogenetic tree of the ROK domain as obtained by RAxML (WAG + Γ + I). The tree is rooted using the eukaryotes as out-group. RAxML 100-bootstrap replicates (BV) and Bayesian Posterior Probabilities (PP) values are shown ... Our data show that GNE (the derived gene fusion of epimerase-2 and ROK domains) is exclusive to most deuterostomes, acoelomorphs and Xenoturbella. How this presence/absence scheme is interpreted remains unclear. One could easily propose the presence of the gene GNE as a molecular synapomorphy of deuterostomes (Figure 1B). This would corroborate the recent, although lowly supported, proposal that xenacoelomorphs are deuterostomes (Philippe, H. et al 2011). However if Xenacoelomorpha or just the Acoelomorpha are not deuterostomes but basal bilaterians, as most phylogenetic analyses suggest (Ruiz-Trillo et al. 1999; Ruiz-Trillo et al. 2002; Hejnol et al. 2009; Paps, Baguna and Riutort 2009; Mwinyi et al. 2010), then GNE was secondarily lost in the last common protostome ancestor. This is indeed not a difficult scenario, specially considering that gene loss must already be hypothesized for urochordates (Figure 1B). The analysis of intron composition shows the GNE encoded by acoels and deuterostomes independently evolved their own introns (Figure S1), somehow supporting acoels are not deuterostomes. However, independent intron evolution within acoels could easily be argued as well; as has been described, for example, in the urochordate Oikopleura dioica (Edvardsen et al. 2004). To sum up, although rare genomic changes can be important phylogenetic markers, they should be used with caution, since gene loss has been shown to play an important role in eukaryotic evolution (see for example Sebe-Pedros et al. 2011 and Zmasek and Godzik 2011). Additional data, especially from phylogenomics analyses, should be taken into account. Finally, the two domains that make up the GNE gene have complex evolutionary histories, most likely involving LGT events or extreme hidden paralogy.

Somatic cell reprogramming into induced pluripotent stem cells (iPSCs) induces changes in genome architecture reflective of the embryonic stem cell (ESC) state. However, only a small minority of cells typically transition to pluripotency, which has limited our understanding of the process. Here, we characterize the DNA regulatory landscape during reprogramming by time-course profiling of isolated sub-populations of intermediates poised to become iPSCs. Widespread reconfiguration of chromatin states and transcription factor (TF) occupancy occurs early during reprogramming, and cells that fail to reprogram partially retain their original chromatin states. A second wave of reconfiguration occurs just prior to pluripotency acquisition, where a majority of early changes revert to the somatic cell state and many of the changes that define the pluripotent state become established. Our comprehensive characterization of reprogramming-associated molecular changes broadens our understanding of this process and sheds light on how TFs access and change the chromatin during cell-fate transitions.

The transition from a unicellular life-style, with temporal cell differentiation to a multicellular life-style, with both temporal and spatial cell differentiation, required the expansion of the regulatory capabilities of ancient animals. In this chapter, we describe howthis change occurred from the perspective of transcription factor (TF) evolution. First, we revise TF diversification throughout eukaryotes. We trace the evolutionary origins of major TF classes and describe general patterns of TF content and how they correlate with multicellular life-styles in eukaryotes. We then focus on the animal TF developmental toolkit at the gene family level. Many of the metazoan developmental TFs originated in a unicellular context; yet there are also many TFs that evolved at the onset of Metazoa. Finally, we describe the changes that led to the establishment of gene regulatory networks that control animal multicellularity and review different case-examples that have provided illuminating insights into this question.

This article appears in:Special Issue: Plant Cell Cycle and Chromatin and Epigenetics., The retinoblastoma (Rb) protein was identified as a human tumour suppressor protein that controls various stages of cell proliferation through the interaction with members of the E2F family of transcription factors. It was originally thought to be specific to animals but plants contain homologues of Rb, called RETINOBLASTOMA-RELATED (RBR). In fact, the Rb-E2F module seems to be a very early acquisition of eukaryotes. The activity of RBR depends on phosphorylation of certain amino acid residues, which in most cases are well conserved between plant and animal proteins. In addition to its role in cell-cycle progression, RBR has been shown to participate in various cellular processes such as endoreplication, transcriptional regulation, chromatin remodelling, cell growth, stem cell biology, and differentiation. Here, we discuss the most recent advances to define the role of RBR in cell proliferation and asymmetric cell division. These and other reports clearly support the idea that RBR is used as a landing platform of a plethora of cellular proteins and complexes to control various aspects of cell physiology and plant development. © The Author 2013., This work is supported by MINECO (grant BFU2012-34821 to CG and grant BFU2011-23434 to IRT) and by an institutional grant of Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa.

The G-protein-coupled receptor (GPCR) signaling system is one of themain signaling pathways in eukaryotes. Here, we analyze the evolutionary history of all its components, from receptors to regulators, to gain a broad picture of its system-level evolution. Using eukaryotic genomes covering most lineages sampled to date, we find that the various components of the GPCR signaling pathway evolved independently, highlighting the modular nature of this system. Our data show that some GPCR families, G proteins, and regulators of G proteins diversified through lineage-specific diversifications and recurrent domain shuffling. Moreover, most of the gene families involved in the GPCR signaling system were already present in the last common ancestor of eukaryotes. Furthermore, we show that the unicellular ancestor of Metazoa already had most of the cytoplasmic components of the GPCR signaling system, including, remarkably, all the G protein alpha subunits, which are typical of metazoans. Thus, we show how the transition to multicellularity involved conservation of the signaling transduction machinery, as well as a burst of receptor diversification to cope with the new multicellular necessities. © The Author(s) 2014., This work was supported by a contract from the Institució Catalana de Recerca i Estudis Avançats, a European Research Council Starting Grant (ERC-2007-StG-206883), a grant (BFU2011-23434) from Ministerio de Economía y Competitividad (MINECO) to I.R.-T, and a pregraduate Formación del Personal Investigador and a pregraduate Formación Profesorado Universitario grant from MINECO to A.d.M. and A.S.-P.

Suga, Hiroshi et al.-- This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License., To reconstruct the evolutionary origin of multicellular animals from their unicellular ancestors, the genome sequences of diverse unicellular relatives are essential. However, only the genome of the choanoflagellate Monosiga brevicollis has been reported to date. Here we completely sequence the genome of the filasterean Capsaspora owczarzaki, the closest known unicellular relative of metazoans besides choanoflagellates. Analyses of this genome alter our understanding of the molecular complexity of metazoans' unicellular ancestors showing that they had a richer repertoire of proteins involved in cell adhesion and transcriptional regulation than previously inferred only with the choanoflagellate genome. Some of these proteins were secondarily lost in choanoflagellates. In contrast, most intercellular signalling systems controlling development evolved later concomitant with the emergence of the first metazoans. We propose that the acquisition of these metazoan-specific developmental systems and the co-option of pre-existing genes drove the evolutionary transition from unicellular protists to metazoans. © 2013 Macmillan Publishers Limited. All rights reserved., H.S. was supported by the Marie Curie Intra-European Fellowship within the 7th European Community Framework Programme. Genome sequencing, assembly and some supporting analysis was supported by grants from the National Human Genome Research Institute (HG003067-05 through HG003067-10), as were C.N., C.R., B.H. and Z.C. B.F.L. and A.J.R. acknowledge financial support through the Canadian Research Chair program. This study was supported by an ICREA contract, a European Research Council Starting Grant (ERC-2007-StG-206883) and a grant (BFU2011-23434) from the Spanish Ministry of the Economy and Competitiveness (MINECO) awarded to I.R.-T. M.V. was supported by CNRS, the Agence Nationale de la Recherche (ANR grant BLAN-0294) and the Institut Universitaire de France.

Trabajo presentado en la XIII Jornada de Biologia Evolutiva, celebrada en Barcelona el 2 de julio de 2013., Control over gene transcription is one of the main regulatory systems in eukaryotes. Transcription Factors (TFs) have a central role in this system, as they bind to DNA and enhance or inhibit gene expression. Although previous studies analysed the evolutionary history of TFs, it remains unclear what role TFs played in the origin of all the different eukaryotic multicellular lineages. In this paper, we explore how the origin of TF repertoires shaped eukaryotic evolution and, in particular, their role into the emergence of multicellular lineages. We traced the origin and expansion of all known TFs through the eukaryotic tree of life, using the broadest possible taxon sampling and an updated phylogenetic background. Our results show that the most complex multicellular lineages (i.e., those with with embryonic development, Metazoa and Embryophyta) have the most complex TF repertoires, and that these repertoires were assembled in a step-wise manner. We also show that a significant part of the metazoan and embryophyte TF toolkits evolved much earlier, in their respective unicellular ancestors. To gain insights into the role of TFs in the development of both embryophytes and metazoans, we analysed TF expression patterns throughout their ontogeny. The expression patterns observed in both groups recapitulate those of the whole transcriptome, but reveal important differences. In particular, gastrulation in animals is defined by metazoan-specific TFs, while later development in embryophytes is characterised by an enrichment in archaeplastid-specific TFs. We suggest that these differences are due to the difference between the closed and open development of extant metazoans and embryophytes, respectively. Our comparative genomics and expression data re-shapes our view on how TFs contributed to eukaryotic evolution and reveals the importance of TFs to the origins of multicellularity and embryonic development.

The evolution of multicellular metazoans from a unicellular ancestor is one of the most important advances in the history of life. Protein tyrosine kinases play important roles in cell-to-cell communication, cell adhesion, and differentiation in metazoans; thus, elucidating their origins and early evolution is crucial for understanding the origin of metazoans. Although tyrosine kinases exist in choanoflagellates, few data are available about their existence in other premetazoan lineages. To unravel the origin of tyrosine kinases, we performed a genomic and polymerase chain reaction (PCR)-based survey of the genes that encode tyrosine kinases in the two described filasterean species, Capsaspora owczarzaki and Ministeria vibrans, the closest relatives to the Metazoa and Choanoflagellata clades.We present 103 tyrosine kinase-encoding genes identified in the whole genome sequence of C. owczarzaki and 15 tyrosine kinase-encoding genes cloned by PCR from M. vibrans. Through detailed phylogenetic analysis, comparison of the organizations of the protein domains, and resequencing and revision of tyrosine kinase sequences previously found in some whole genome sequences, we demonstrate that the basic repertoire ofmetazoan cytoplasmic tyrosine kinases was established before the divergence of filastereans from the Metazoa and Choanoflagellata clades. In contrast, the receptor tyrosine kinases diversified extensively in each of the filasterean, choanoflagellate, and metazoan clades. This difference in the divergence patterns between cytoplasmic tyrosine kinases and receptor tyrosine kinases suggests that receptor tyrosine kinases that had been used for receiving environmental cues were subsequently recruited as a communication tool between cells at the onset of metazoan multicellularity.

A report on the meeting Comparative Genomics of Eukaryotic Microorganisms: understanding the complexity of diversity. Sant Feliu de Guíxols, Spain. October 15-20, 2011., Research in IR-T’s lab is supported by a European Research Council Starting Grant (ERC-2007-StG-206883) and a grant (BFU2008-02839/BMC) from Ministerio de Ciencia e Innovación (MICINN) to IR-T. AdM is supported by a pre-graduate FPI grant from MICINN.

The Opisthokonta are a eukaryotic supergroup divided in two main lineages: animals and related protistan taxa, and fungi and their allies [1, 2]. There is a great diversity of lifestyles and morphologies among unicellular opisthokonts, from free-living phagotrophic flagellated bacterivores and filopodiated amoebas to cell-walled osmotrophic parasites and saprotrophs. However, these characteristics do not group into monophyletic assemblages, suggesting rampant convergent evolution within Opisthokonta. To test this hypothesis, we assembled a new phylogenomic dataset via sequencing 12 new strains of protists. Phylogenetic relationships among opisthokonts revealed independent origins of filopodiated amoebas in two lineages, one related to fungi and the other to animals. Moreover, we observed that specialized osmotrophic lifestyles evolved independently in fungi and protistan relatives of animals, indicating convergent evolution. We therefore analyzed the evolution of two key fungal characters in Opisthokonta, the flagellum and chitin synthases. Comparative analyses of the flagellar toolkit showed a previously unnoticed flagellar apparatus in two close relatives of animals, the filasterean Ministeria vibrans and Corallochytrium limacisporum. This implies that at least four different opisthokont lineages secondarily underwent flagellar simplification. Analysis of the evolutionary history of chitin synthases revealed significant expansions in both animals and fungi, and also in the Ichthyosporea and C. limacisporum, a group of cell-walled animal relatives. This indicates that the last opisthokont common ancestor had a complex toolkit of chitin synthases that was differentially retained in extant lineages. Thus, our data provide evidence for convergent evolution of specialized lifestyles in close relatives of animals and fungi from a generalist ancestor. © 2015 Elsevier Ltd., This work was supported by two grants (BFU2011-23434 and BFU2014-57779-P) from Ministerio de Economía y Competitividad (MINECO) and an ERC Starting Grant (ERC-2007-StG-206883) to I.R.-T. We also acknowledge support from Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya (project 2014 SGR 619). G.T. was supported by a pre-graduate FI grant from the Catalan Government. A.d.M. and X.G.-B. were supported by a pre-graduate FPI grant from MINECO. L.E. was supported by a CGEB postdoctoral fellowship from the Tula Foundation, and A.J.R. was supported by a CIHR-NSHRF RPP grant (FRN# 62809). Assembly of the I. hoferi genome (USA) was supported by NSF grant number ACI-1053575. G.P.-C. was supported by a “Juan de la Cierva” grant, and A.S.-B. was supported by Generalitat Valenciana (PROMETEO FASE II-2014/085).

Background Cell-to-cell communication is a key process in multicellular organisms. In multicellular animals, scaffolding proteins belonging to the family of membrane-associated guanylate kinases (MAGUK) are involved in the regulation and formation of cell junctions. These MAGUK proteins were believed to be exclusive to Metazoa. However, a MAGUK gene was recently identified in an EST survey of Capsaspora owczarzaki, an unicellular organism that branches off near the metazoan clade. To further investigate the evolutionary history of MAGUK, we have undertook a broader search for this gene family using available genomic sequences of different opisthokont taxa. Results Our survey and phylogenetic analyses show that MAGUK proteins are present not only in Metazoa, but also in the choanoflagellate Monosiga brevicollis and in the protist Capsaspora owczarzaki. However, MAGUKs are absent from fungi, amoebozoans or any other eukaryote. The repertoire of MAGUKs in Placozoa and eumetazoan taxa (Cnidaria + Bilateria) is quite similar, except for one class that is missing in Trichoplax, while Porifera have a simpler MAGUK repertoire. However, Vertebrata have undergone several independent duplications and exhibit two exclusive MAGUK classes. Three different MAGUK types are found in both M. brevicollis and C. owczarzaki: DLG, MPP and MAGI. Furthermore, M. brevicollis has suffered a lineage-specific diversification. Conclusions The diversification of the MAGUK protein gene family occurred, most probably, prior to the divergence between Metazoa+choanoflagellates and the Capsaspora+Ministeria clade. A MAGI-like, a DLG-like, and a MPP-like ancestral genes were already present in the unicellular ancestor of Metazoa, and new gene members have been incorporated through metazoan evolution within two major periods, one before the sponge-eumetazoan split and another within the vertebrate lineage. Moreover, choanoflagellates have suffered an independent MAGUK diversification. This study highlights the importance of generating enough genome data from the broadest possible taxonomic sampling, in order to fully understand the evolutionary history of major protein gene families.