Inference and reconstruction of the heimdallarchaeial ancestry of eukaryotes.

In the ongoing debates about eukaryogenesis-the series of evolutionary events leading to the emergence of the eukaryotic cell from prokaryotic ancestors-members of the Asgard archaea play a key part as the closest archaeal relatives of eukaryotes1. However, the nature and phylogenetic identity of the last common ancestor of Asgard archaea and eukaryotes remain unresolved2-4. Here we analyse distinct phylogenetic marker datasets of an expanded genomic sampling of Asgard archaea and evaluate competing evolutionary scenarios using state-of-the-art phylogenomic approaches. We find that eukaryotes are placed, with high confidence, as a well-nested clade within Asgard archaea and as a sister lineage to Hodarchaeales, a newly proposed order within Heimdallarchaeia. Using sophisticated gene tree and species tree reconciliation approaches, we show that analogous to the evolution of eukaryotic genomes, genome evolution in Asgard archaea involved significantly more gene duplication and fewer gene loss events compared with other archaea. Finally, we infer that the last common ancestor of Asgard archaea was probably a thermophilic chemolithotroph and that the lineage from which eukaryotes evolved adapted to mesophilic conditions and acquired the genetic potential to support a heterotrophic lifestyle. Our work provides key insights into the prokaryote-to-eukaryote transition and a platform for better understanding the emergence of cellular complexity in eukaryotic cells.

A closed Candidatus Odinarchaeum chromosome exposes Asgard archaeal viruses.

Asgard archaea have recently been identified as the closest archaeal relatives of eukaryotes. Their ecology, and particularly their virome, remain enigmatic. We reassembled and closed the chromosome of Candidatus Odinarchaeum yellowstonii LCB_4, through long-range PCR, revealing CRISPR spacers targeting viral contigs. We found related viruses in the genomes of diverse prokaryotes from geothermal environments, including other Asgard archaea. These viruses open research avenues into the ecology and evolution of Asgard archaea.

Proposal of the reverse flow model for the origin of the eukaryotic cell based on comparative analyses of Asgard archaeal metabolism.

The origin of eukaryotes represents an unresolved puzzle in evolutionary biology. Current research suggests that eukaryotes evolved from a merger between a host of archaeal descent and an alphaproteobacterial endosymbiont. The discovery of the Asgard archaea, a proposed archaeal superphylum that includes Lokiarchaeota, Thorarchaeota, Odinarchaeota and Heimdallarchaeota suggested to comprise the closest archaeal relatives of eukaryotes, has helped to elucidate the identity of the putative archaeal host. Whereas Lokiarchaeota are assumed to employ a hydrogen-dependent metabolism, little is known about the metabolic potential of other members of the Asgard superphylum. We infer the central metabolic pathways of Asgard archaea using comparative genomics and phylogenetics to be able to refine current models for the origin of eukaryotes. Our analyses indicate that Thorarchaeota and Lokiarchaeota encode proteins necessary for carbon fixation via the Wood-Ljungdahl pathway and for obtaining reducing equivalents from organic substrates. By contrast, Heimdallarchaeum LC2 and LC3 genomes encode enzymes potentially enabling the oxidation of organic substrates using nitrate or oxygen as electron acceptors. The gene repertoire of Heimdallarchaeum AB125 and Odinarchaeum indicates that these organisms can ferment organic substrates and conserve energy by coupling ferredoxin reoxidation to respiratory proton reduction. Altogether, our genome analyses suggest that Asgard representatives are primarily organoheterotrophs with variable capacity for hydrogen consumption and production. On this basis, we propose the 'reverse flow model', an updated symbiogenetic model for the origin of eukaryotes that involves electron or hydrogen flow from an organoheterotrophic archaeal host to a bacterial symbiont.

Complex Evolutionary History of Translation Elongation Factor 2 and Diphthamide Biosynthesis in Archaea and Parabasalids.

Diphthamide is a modified histidine residue which is uniquely present in archaeal and eukaryotic elongation factor 2 (EF-2), an essential GTPase responsible for catalyzing the coordinated translocation of tRNA and mRNA through the ribosome. In part due to the role of diphthamide in maintaining translational fidelity, it was previously assumed that diphthamide biosynthesis genes (dph) are conserved across all eukaryotes and archaea. Here, comparative analysis of new and existing genomes reveals that some archaea (i.e., members of the Asgard superphylum, Geoarchaea, and Korarchaeota) and eukaryotes (i.e., parabasalids) lack dph. In addition, while EF-2 was thought to exist as a single copy in archaea, many of these dph-lacking archaeal genomes encode a second EF-2 paralog missing key residues required for diphthamide modification and for normal translocase function, perhaps suggesting functional divergence linked to loss of diphthamide biosynthesis. Interestingly, some Heimdallarchaeota previously suggested to be most closely related to the eukaryotic ancestor maintain dph genes and a single gene encoding canonical EF-2. Our findings reveal that the ability to produce diphthamide, once thought to be a universal feature in archaea and eukaryotes, has been lost multiple times during evolution, and suggest that anticipated compensatory mechanisms evolved independently.

Functional reconstruction of a eukaryotic-like E1/E2/(RING) E3 ubiquitylation cascade from an uncultured archaeon.

The covalent modification of protein substrates by ubiquitin regulates a diverse range of critical biological functions. Although it has been established that ubiquitin-like modifiers evolved from prokaryotic sulphur transfer proteins it is less clear how complex eukaryotic ubiquitylation system arose and diversified from these prokaryotic antecedents. The discovery of ubiquitin, E1-like, E2-like and small-RING finger (srfp) protein components in the Aigarchaeota and the Asgard archaea superphyla has provided a substantive step toward addressing this evolutionary question. Encoded in operons, these components are likely representative of the progenitor apparatus that founded the modern eukaryotic ubiquitin modification systems. Here we report that these proteins from the archaeon Candidatus 'Caldiarchaeum subterraneum' operate together as a bona fide ubiquitin modification system, mediating a sequential ubiquitylation cascade reminiscent of the eukaryotic process. Our observations support the hypothesis that complex eukaryotic ubiquitylation signalling pathways have developed from compact systems originally inherited from an archaeal ancestor.

Genomic exploration of the diversity, ecology, and evolution of the archaeal domain of life.

About 40 years ago, Archaea were recognized as a major prokaryotic domain of life besides Bacteria. Recently, cultivation-independent sequencing methods have produced a wealth of genomic data for previously unidentified archaeal lineages, several of which appear to represent newly revealed branches in the tree of life. Analyses of some recently obtained genomes have uncovered previously unknown metabolic traits and provided insights into the evolution of archaea and their relationship to eukaryotes. On the basis of our current understanding, much archaeal diversity still defies genomic exploration. Efforts to obtain and study genomes and enrichment cultures of uncultivated microbial lineages will likely further expand our knowledge about archaeal phylogenetic and metabolic diversity and their cell biology and ecological function.

CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart.

Cardiac progenitors are specified early in development and progressively differentiate and mature into fully functional cardiomyocytes. This process is controlled by an extensively studied transcriptional program. However, the regulatory events coordinating the progression of such program from development to maturation are largely unknown. Here, we show that the genome organizer CTCF is essential for cardiogenesis and that it mediates genomic interactions to coordinate cardiomyocyte differentiation and maturation in the developing heart. Inactivation of Ctcf in cardiac progenitor cells and their derivatives in vivo during development caused severe cardiac defects and death at embryonic day 12.5. Genome wide expression analysis in Ctcf mutant hearts revealed that genes controlling mitochondrial function and protein production, required for cardiomyocyte maturation, were upregulated. However, mitochondria from mutant cardiomyocytes do not mature properly. In contrast, multiple development regulatory genes near predicted heart enhancers, including genes in the IrxA cluster, were downregulated in Ctcf mutants, suggesting that CTCF promotes cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Accordingly, loss of CTCF disrupts gene expression and chromatin interactions as shown by chromatin conformation capture followed by deep sequencing. Furthermore, CRISPR-mediated deletion of an intergenic CTCF site within the IrxA cluster alters gene expression in the developing heart. Thus, CTCF mediates local regulatory interactions to coordinate transcriptional programs controlling transitions in morphology and function during heart development.

Asgard archaea illuminate the origin of eukaryotic cellular complexity.

The origin and cellular complexity of eukaryotes represent a major enigma in biology. Current data support scenarios in which an archaeal host cell and an alphaproteobacterial (mitochondrial) endosymbiont merged together, resulting in the first eukaryotic cell. The host cell is related to Lokiarchaeota, an archaeal phylum with many eukaryotic features. The emergence of the structural complexity that characterizes eukaryotic cells remains unclear. Here we describe the 'Asgard' superphylum, a group of uncultivated archaea that, as well as Lokiarchaeota, includes Thor-, Odin- and Heimdallarchaeota. Asgard archaea affiliate with eukaryotes in phylogenomic analyses, and their genomes are enriched for proteins formerly considered specific to eukaryotes. Notably, thorarchaeal genomes encode several homologues of eukaryotic membrane-trafficking machinery components, including Sec23/24 and TRAPP domains. Furthermore, we identify thorarchaeal proteins with similar features to eukaryotic coat proteins involved in vesicle biogenesis. Our results expand the known repertoire of 'eukaryote-specific' proteins in Archaea, indicating that the archaeal host cell already contained many key components that govern eukaryotic cellular complexity.