Transcription factors (TFs) are the main players in transcriptional regulation in eukaryotes. However, it remains unclear what role TFs played in the origin of all of 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 embryonic development, Metazoa and Embryophyta) have the most complex TF repertoires, and that these repertoires were assembled in a stepwise manner. We also show that a significant part of the metazoan and embryophyte TF toolkits evolved earlier, in their respective unicellular ancestors. To gain insights into the role of TFs in the development of both embryophytes and metazoans, we analyzed TF expression patterns throughout their ontogeny. The expression patterns observed in both groups recapitulate those of the whole transcriptome, but reveal some important differences. Our comparative genomics and expression data reshape our view on how TFs contributed to eukaryotic evolution and reveal the importance of TFs to the origins of multicellularity and embryonic development.phylotypic stage | Holozoa | LECA T ranscription factors (TFs) are proteins that bind to DNA in a sequence-specific manner (1) and enhance or repress gene expression (2-4). In response to a broad range of stimuli, TFs coordinate many important biological processes, from cell cycle progression and physiological responses, to cell differentiation and development (5, 6). Thus, TFs have a central role in the transcriptional regulation of all cellular organisms, being present in all branches of the tree of life (bacteria, archaea, and eukaryotes). There appears to be a correlation between elaborate regulation of gene expression and the complexity of organisms (7), such that the amount (as a proportion of an organism's total gene content) and diversity of TF proteins is expected to be directly correlated with this complexity (8). Indeed, TFs play a crucial role in multicellular eukaryotes. For example, TFs are the master regulators of embryonic development in embryophytes and metazoans (9), and analyses of their embryonic transcriptional profiles support the presence of a phylotypic stage in both lineages (10-14). These studies have also shown that evolutionarily younger genes tend to be expressed at earlier and later stages of development, whereas the transcriptomes of the middle stages (the phylotypic stage) are dominated by ancient genes (10, 13). It remains to be investigated how the evolutionary age and the expression patterns of the different TFs shift throughout the ontogeny of these lineages and whether TF expression profiles correlate with the general transcriptome profiles.Previous studies have analyzed the evolutionary ...

Phylostratigraphy is a computational framework for dating the emergence of DNA and protein sequences in a phylogeny. It has been extensively applied to make inferences on patterns of genome evolution, including patterns of disease gene evolution, ontogeny and de novo gene origination. Phylostratigraphy typically relies on BLAST searches along a species tree, but new simulation studies have raised concerns about the ability of BLAST to detect remote homologues and its impact on phylostratigraphic inferences. Here, we re-assessed these simulations. We found that, even with a possible overall BLAST false negative rate between 11–15%, the large majority of sequences assigned to a recent evolutionary origin by phylostratigraphy is unaffected by technical concerns about BLAST. Where the results of the simulations did cast doubt on previously reported findings, we repeated the original analyses but now excluded all questionable sequences. The originally described patterns remained essentially unchanged. These new analyses strongly support phylostratigraphic inferences, including: genes that emerged after the origin of eukaryotes are more likely to be expressed in the ectoderm than in the endoderm or mesoderm in Drosophila, and the de novo emergence of protein-coding genes from non-genic sequences occurs through proto-gene intermediates in yeast. We conclude that BLAST is an appropriate and sufficiently sensitive tool in phylostratigraphic analysis that does not appear to introduce significant biases into evolutionary pattern inferences.

The main family of serine/threonine/tyrosine protein kinases present in eukarya was defined and described by Hanks et al. in 1988 (Science, 241, 42-52). It was initially believed that these kinases do not exist in bacteria, but extensive genome sequencing revealed their existence in many bacteria. For historical reasons, the term "eukaryotic-type kinases" propagated in the literature to describe bacterial members of this protein family. Here, we argue that this term should be abandoned as a misnomer, and we provide several lines of evidence to support this claim. Our comprehensive phylostratigraphic analysis suggests that Hanks-type kinases present in eukarya, bacteria and archaea all share a common evolutionary origin in the lineage leading to the last universal common ancestor (LUCA). We found no evidence to suggest substantial horizontal transfer of genes encoding Hanks-type kinases from eukarya to bacteria. Moreover, our systematic structural comparison suggests that bacterial Hanks-type kinases resemble their eukaryal counterparts very closely, while their structures appear to be dissimilar from other kinase families of bacterial origin. This indicates that a convergent evolution scenario, by which bacterial kinases could have evolved a kinase domain similar to that of eukaryal Hanks-type kinases, is not very likely. Overall, our results strongly support a monophyletic origin of all Hanks-type kinases, and we therefore propose that this term should be adopted as a universal name for this protein family.

An elaborated tripartite brain is considered one of the important innovations of vertebrates. Other extant chordate groups have a more basic brain organization. For instance, cephalochordates possess a relatively simple brain possibly homologous to the vertebrate forebrain and hindbrain, whereas tunicates display the tripartite organization, but without the specialized brain centers. The difference in anatomical complexity is even more pronounced if one compares chordates with other deuterostomes that have only a diffuse nerve net or alternatively a rather simple central nervous system. To gain a new perspective on the evolutionary roots of the complex vertebrate brain, we made here a phylostratigraphic analysis of gene expression patterns in the developing zebrafish (Danio rerio). The recovered adaptive landscape revealed three important periods in the evolutionary history of the zebrafish brain. The oldest period corresponds to preadaptive events in the first metazoans and the emergence of the nervous system at the metazoan–eumetazoan transition. The origin of chordates marks the next phase, where we found the overall strongest adaptive imprint in almost all analyzed brain regions. This finding supports the idea that the vertebrate brain evolved independently of the brains within the protostome lineage. Finally, at the origin of vertebrates we detected a pronounced signal coming from the dorsal telencephalon, in agreement with classical theories that consider this part of the cerebrum a genuine vertebrate innovation. Taken together, these results reveal a stepwise adaptive history of the vertebrate brain where most of its extant organization was already present in the chordate ancestor.

Bacillus subtilis is a sporulating Gram-positive bacterium widely used in basic research and biotechnology. Despite being one of the best-characterized bacterial model organism, recent proteomics studies identified only about 50% of its theoretical protein count. Here we combined several hundred MS measurements to obtain a comprehensive map of the proteome, phosphoproteome and acetylome of B. subtilis grown at 37 °C in minimal medium. We covered 75% of the theoretical proteome (3,159 proteins), detected 1,085 phosphorylation and 4,893 lysine acetylation sites and performed a systematic bioinformatic characterization of the obtained data. A subset of analyzed MS files allowed us to reconstruct a network of Hanks-type protein kinases, Ser/Thr/Tyr phosphatases and their substrates. We applied genomic phylostratigraphy to gauge the evolutionary age of B. subtilis protein classes and revealed that protein modifications were present on the oldest bacterial proteins. Finally, we performed a proteogenomic analysis by mapping all MS spectra onto a six-frame translation of B. subtilis genome and found evidence for 19 novel ORFs. We provide the most extensive overview of the proteome and post-translational modifications for B. subtilis to date, with insights into functional annotation and evolutionary aspects of the B. subtilis genome.