Rel/NF-κB Transcription Factors Emerged at the Onset of Opisthokonts

Rel/NF-jB Transcription Factors Emerged at the Onset of Opisthokonts Michelle M. Leger 1, * ,† ,N  uria Ros-Rocher 1,† , Sebasti an R. Najle 1,4 ,andI ~ naki Ruiz-Trillo 1,2,3 1 Institute of Evolutionary Biology (Consejo Superior de Investigaciones Cient ıficas-Universitat Pompeu Fabra), Barcelona, Catalonia, Spain 2 Department of Genetics, Microbiology and Statistics, Institute for Research on Biodiversity, University of Barcelona, Catalonia, Spain 3 Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Catalonia, Spain 4 Present address: Center for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Barcelona, Spain † These authors contributed equally to this work. *Corresponding author: E-mail: michelle.lege[email protected]. Accepted: 27 December 2021 Abstract The Rel/NF-jB transcription factor family has myriad roles in immunity, development, and differentiation in animals, and was considered a key innovation for animal multicellularity. Rel homology domain-containing proteins were previously hypothesized to have originated in a last common ancestor of animals and some of their closest unicellular relatives. However, key taxa were missing from previous analyses, necessitating a systematic investigation into the distribution and evolution of these proteins. Here, we address this knowledge gap by surveying taxonomically broad data from eukaryotes, with a special emphasis on lineages closely related to animals. We report an earlier origin for Rel/NF-jB proteins than previously described, in the last common ancestor of animals and fungi, and show that even in the sister group to fungi, these proteins contain elements that in animals are necessary for the subcellular regulation of Rel/NF-jB. Key words: NF-jB, Rel homology domain, transcription factors, opisthokonts, NF-kappa B. The Rel homology region (RHR) is an evolutionarily conserved N-terminal DNA-binding region present in two major paralogous families of animal transcription factors with crucial roles in immunity and development: the Rel/Nuclear Factor-jB(NFjB) and the Nuclear Factor of Activated T-cells (NFAT) families. Members of the Rel/NF-jB family were originally described in the late 1980s as oncogenes (Gilmore and Temin 1986)and immunoglobulin jlight chain enhancer-binding proteins in vertebrates (Sen and Baltimore 1986).Membersofthisfamily have since been implicated in a wide range of cellular processes in animals (Ghosh and Hayden 2012), including innate and adaptive immunity (Hayden and Ghosh 2011), cell cycle regulation (Ledoux and Perkins 2014), apoptosis (Kucharczak et al. 2003), autophagy (Salminen et al. 2012), and regulation of oxidative stress responses (Lingappan 2018). Despite these crucial roles in animals, their functions and domain architectures in other taxa remain to be fully explored. Pinpointing the origins and taxonomic distribution of Rel/NF-jB proteins is a fundamental first step to tackle these questions and understand the evolutionary history of Significance The Rel/NF-jB transcription factor family plays a fundamental role in animal innate immunity, programmed cell death, intercellular signaling, and transcriptional regulation. Here, we show that proteins sharing the characteristic features of animal Rel/NF-jB transcription factors originate much earlier than previously described: not in a relatively recent common ancestor of animals and some of their unicellular relatives, but prior to the divergence of animals and fungi. Intriguingly, we show that even the earliest-diverging nonmetazoan Rel/NF-jB-like protein has sequence features consistent with an animal-like mode of regulation. ßThe Author(s) 2022. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Genome Biol. Evol. 14(1) doi:10.1093/gbe/evab289 Advance Access publication 6 January 2022 1 GBE Downloaded from https://academic.oup.com/gbe/article/14/1/evab289/6499270 by CSIC - Instituto De Ganaderia De Montana user on 09 June 2022 Rel/NF-jB proteins. Rel/NF-jB proteins are characterized by the presence of an RHR, consisting of an N-terminal DNAbinding and dimerization domain that facilitates homoor heterodimerization, and a short stretch of arginine and/or lysine residues forming a nuclear localization signal (NLS) that mediates the translocation of these proteins into the nucleus (fig. 1A) (reviewed in Napetschnig and Wu [2013] and Williams and Gilmore [2020]). The Rel/NF-jB family can be further subdivided into two classes according to the transactivation potential of its members. The first, collectively termed Rel proteins, include the vertebrate RelA (p65), RelB, and c-Rel and their orthologs (fig. 1A). Besides the RHR, Rel proteins also contain a poorly conserved C-terminal transactivation domain (TAD) that is acidic, and may be rich in proline, serine, glutamine, and/or hydrophobic residues (Bull et al. 1990;Blair et al. 1994;Gross et al. 1999), that allows them to activate target gene expression. RelB proteins additionally possess an N-terminal leucine zipper domain critical for their activity (Dobrzanski et al. 1993). Rel proteins can homodimerize, or heterodimerize with other Rel/NF-jB family members lacking a classical TAD (reviewed in Napetschnig and Wu [2013]). The second class includes the vertebrate p50 and p52 and their orthologs (fig. 1A). p50 and p52 are synthetized as larger precursors termed NF-jB1/p105 and NF-jB2/p100, respectively. These precursors include an RHR followed by a glycine-rich region (GRR) and a variable number of C-terminal ankyrin repeats. In their inactive state, ankyrin repeats inhibit nuclear localization and transcriptional activity and keep the NF-jB dimers sequestered in the cytosol. They additionally possess a death domain that mediates interaction with other death domain-containing signaling proteins (Hayden and Ghosh 2008). The p105 and p100 precursors are classified as inhibitors of NF-jB(IjBs). This category also includes a family of separate C-terminal ankyrin repeat-containing proteins that carry out the same inhibitory function for Rel proteins, by sequestering them in the cytosol (Kanarek et al. 2010)(fig. 1A). Upon upstream activation, C-terminal serine residues in ankyrin-repeat-rich regions of IjBs are phosphorylated by an IjB kinase complex (IKK) (Karin 1999). The IKK complex is also involved in the processing of the p105 and p100 precursors, leading to the ubiquitination and proteasomal degradation of their C-terminal regions (fig. 1A). In this case, the proteasome falls off at the GRR located between the RHR and the C-terminal ankyrin repeats (Lin and Ghosh 1996; Williams and Gilmore 2020). This process releases an intact Nterminal part of the NF-jB protein, including the GRR (Moorthy et al. 2006), and leads to its nuclear translocation for gene expression activation (reviewed in Napetschnig and Wu [2013]). A key scaffolding component of the IKK complex, the IKKc/NF-jB essential modulator (NEMO), is also required for IKK recruitment and NF-jB activation (fig. 1A) (reviewed in Napetschnig and Wu [2013]). The NFAT family constitutes the paralogous group of RHRcontaining proteins. NFAT proteins were first described almost three decades ago as calcium-dependent transcription factors implicated in T-cell activation (Shaw et al. 1988), cell proliferation, migration, and angiogenesis (reviewed in Mu¨ ller and Rao [2010]). NFAT proteins contain a more centrally located RHR flanked by longer Nand C-terminal regions, and lack ankyrin repeats (fig. 1A). The NFAT NLS is contained not within the RHR, but within the N-terminal regulatory region known as the NFAT homology region (NHR) (fig. 1A). This region also contains calcineurin-binding sites required for nuclear translocation (Park et al. 2000). NFAT1-4 also possess N-terminal TADs (Serfling et al. 2004). NFAT5, the only noncalcium regulatedNFATproteininhumans,lacksanNHR(fig. 1A) but is generally located in the nucleus, and plays a role in osmotic stress response and immune cell development (Leeetal.2019). Animals and their closest unicellular relatives together comprise the eukaryotic group Holozoa; the larger eukaryotic group comprising Holozoa, fungi, and their closest unicellular relatives, is known as Opisthokonta (fig. 1B). Rel homology domain-containing proteins were initially believed to be specific to animals (Metazoa), in which these proteins have been extensively studied. However, they were later reported in two lineages of unicellular holozoans: the filastereans (Mikhailov et al. 2009;Seb e-Pedr os et al. 2011) and choanoflagellates (Richter et al. 2018;Williams and Gilmore 2020). These sequences branched sister to all metazoan NF-jB and NFAT sequences, but contained RHRs, GRR, NLS, and/or ankyrin repeats more characteristic of the Rel/NF-jBfamily(Seb ePedr os et al. 2011;Williams and Gilmore 2020). Rel homology DNA-binding domains had also been reported as being present in more distantly related taxa, including a nucleariid and possibly one or more ichthyosporeans and/or pluriformeans ([de Mendoza et al. 2013;de Mendoza and Seb e-Pedr os 2019]; fig. 1Band supplementary table S1,Supplementary Material online). However, these studies focused solely on the Rel homology DNA-binding domain, and obtained conflicting results that cast doubt on the domain’s true taxonomic distribution (de Mendoza et al. 2013;Richter et al. 2018;de Mendoza and Seb e-Pedr os 2019). As a result, the precise origin, early evolution, and molecular context of Rel homology domain-containing transcription factors remained unknown. To resolve these questions, we performed a taxonomically broad survey of genomic and transcriptomic sequence data representing all major eukaryotic supergroups, including newly sequenced protistan lineages within Holozoa (GrauBov e et al. 2017;Hehenberger et al. 2017;Tikhonenkov et al. 2020;Urrutia et al. 2021). We surveyed data from 180 species for key Rel homology DNA-binding and dimerization domain-containing proteins, examined their domain architecture, and constructed phylogenies of the proteins identified. We additionally extended the search to homologs of IKK components. Leger et al. GBE 2Genome Biol. Evol. 14(1) doi:10.1093/gbe/evab289 Advance Access publication 6 January 2022 Downloaded from https://academic.oup.com/gbe/article/14/1/evab289/6499270 by CSIC - Instituto De Ganaderia De Montana user on 09 June 2022 Syssomonas multiformis Corallochytrium limacisporum Sphaerothecum destruens Creolimax fragrantissima Amoebidium parasiticum Abeoforma whisleri Ichthyophonus hoferi Sphaeroforma arctica Pirum gemmata Chromosphaera perkinsii Ministeria vibrans Pigoraptor chileana Capsaspora owczarzaki Pigoraptor vietnamica Tunicaraptor unikontum Txikispora philomaios Nematostella vectensis Homo sapiens Sycon ciliatum Mnemiopsis leidyi Trichoplax adhaerens Caenorhabditis elegans Crassostrea gigas Daphnia pulex Choanoeca perplexa Choanoeca flexa Diaphanoeca grandis Acanthoeca spectabilis Didymoeca costata Salpingoeca dolichothecata Helgoeca nana Stephanoeca diplocostata (FR) Savillea parva Codosiga hollandica Salpingoeca kvevrii Salpingoeca helianthica Salpingoeca macrocollata Salpingoeca urceolata Mylnosiga fluctuans Salpingoeca punica Stephanoeca diplocostata (AU) Salpingoeca rosetta Microstomoeca roanoka Hartaetosiga balthica Hartaetosiga gracilis Salpingoeca infusionum Monosiga brevicollis Amphimedon queenslandica Oscarella carmela Ephydatia muelleri Hydra magnipapillata Saccoglossus kowalevskii Saccharomyces cerevisiae Coprinopsis cinerea Fonticula alba Parvularia atlantis B RHR RHD DB Dim Ct TAD RelB LZ RelB TAD GRR NF-κB proteins Rel proteins Ct TAD A PEST Death IκB-like regions Kinase NBD IκB Family IκBα, IκBβ IκBε, IκBζ, Bcl-3 NF-κB1/p105 NF-κB2/p100 IKK Complex IKKα p50 p52 Rel/NF-κB RelB RelA (p65) c-Rel IKKγ/NEMO Ct TAD NHR RHR NFAT NFAT1-4 NFAT5 Ct TADNt TAD Kinase ULD IKKβ NEMO LZ ZFUBD Rel homology DNA-binding domain (PF00554) Rel homology dimerisation domain (PF16179) RelB Transactivation domain (PF16181) RHD DB GRR RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim GRR GRR Death RHD DB Dim RHD DB Dim NBD Domains and regions key RelB Leucine Zipper domain (PF16180) RelB LZ RelB TAD Dim Ankyrin repeats (PF00023, PF12796, PF13606, PF13637, PF13857) Proline-, glutamic acid-, serine-, threonine-rich regions PEST Taxonomic Group Metazoa Choanoflagellatea Filasterea Ichthyosporea Pluriformea/Corallochytrea Holomycota Present Absent from genome Absent from transcriptome Previously reported domains outside Metazoa Domain presence/absence LxVP motif PxIxIT motif Death domain (PF00531) Nuclear localization signal Glycine-rich region GRR Death Nt or Ct Transactivation domains Ct/Nt TAD Zinc finger domain Ubiquitin-binding domain Leucine Zipper domain Ubiquitin-like domain ULD UBD LZ ZF Rel/NF-κB RHD DB Dim Ankyrin repeats NBD NEMO Death Holomycota Holozoa Opisthokonta SDD IKKβ NEMO-binding domain (PF12179) NF-κB Essential Modulator domain (PF11577) Kinase domain (PF00069) Kinase NEMO NBD IQBAL Scaffold dimerization domain (PF18397) SDD SDD ULD FIG.1.—Rel/NF-jB proteins emerged at the onset of Opisthokonta (A) Domain architecture representation of members of the Rel/NF-jB, and NFAT protein families, IjB, and the IKK complex. Details of the represented features are shown in the domains and regions key. The Rel Homology Region, characteristic of Rel/NF-jB and NFAT proteins, contains conserved Rel homology DNA-binding (RHD DB) and dimerization domains (Dim) and, in the case of Rel/NF-jB proteins, an NLS (orange bar). Rel proteins also contain a C-terminal, serine-rich Transactivation Domain (Ct TAD) or a RelB TAD; and RelB proteins additionally possess an N-terminal leucine zipper domain (RelB LZ). The NF-jB1 precursor (p105) and NF-jB2 precursor (p100) contain a more centrally located GRR and C-terminal Death domain (Death). These precursors share with IjB proteins C-terminal ankyrin repeats (light green bars). Other domains present in IjB families include proline-, glutamic acid-, serine-, and threonine-rich regions (PEST). Key domains specific to NFAT proteins, include an Nterminal TAD (Nt TAD) inside an NHR, an NLS (orange bar) and C-terminal TAD (Ct TAD). PxIxIT and LxVP Calcineurin-binding motifs in the NHR are depicted with a magenta and a green bar, respectively. Domains for IKKalpha and beta include kinase domains (Kinase); ubiquitin-like domains (ULD); ubiquitinbinding domains (UBD); IQBAL scaffold dimerization domain (SDD); NEMO-binding domains (NBD). Domains for IKKc/NF-jB essential modulator (NEMO) include NEMO domain (NEMO); UBD; leucine-zipper domains (LZ); and zinc finger domains (ZF). (B) Presence or absence of key Pfam domains analyzed in this study are represented in columns and color-coded according to genome or transcriptome data source (indicated in the Domain presence/absence key). The phylogenetic relationships of selected taxa are based on several recent phylogenomic studies (Torruella et al. 2015;Grau-Bov e et al. 2017;Hehenberger et al. 2017;Tikhonenkov et al. 2020;Urrutia et al. 2021). Taxa are color-coded according to the Taxonomic Group key. Evolution of Rel/NF-jB proteins GBE Genome Biol. Evol. 14(1) doi:10.1093/gbe/evab289 Advance Access publication 6 January 2022 3 Downloaded from https://academic.oup.com/gbe/article/14/1/evab289/6499270 by CSIC - Instituto De Ganaderia De Montana user on 09 June 2022 We identified RHRs in all but one of the holozoan groups examined, including the filastereans Pigoraptor spp., the recently sequenced Txikispora philomaios and Tunicaraptor unikontum, and several ichthyosporean species (fig. 1B-2; supplementary fig. S1, tables S1 and S2,Supplementary Material online). A candidate protein was also present in Parvularia atlantis (formerly referred to as Nuclearia sp. ATCC 50694 [L opez-Escard o et al. 2018]), a member of the sister group to Fungi (Nucleariida), suggesting that Rel homology proteins were present in the last common ancestor of Opisthokonta, and were secondarily lost in Fungi (de Mendoza et al. 2013;de Mendoza and Seb e-Pedr os 2019). Strikingly, at least one sequence from each of these lineages included both DNA-binding and dimerization domains (fig. 1B-3; supplementary fig. S1 and table S2, Supplementary Material online), and C-terminal ankyrin repeat-rich regions preceded by a GRR were found in at least one sequence from Filasterea, from Ichthyosporea, and from P. atlantis (fig. 2;supplementary table S2,Supplementary Material online). The finding of these traits in the nucleariid P. atlantis, in particular, not only confirms the origin of Rel/ NF-jB proteins in the opisthokont stem lineage, but also is consistent with an ancient conserved mechanism of cytosolic sequestration for these proteins. Similarly, nonmetazoan Rel/NF-jB-like sequences from all of these groups share with their animal homologs key domains for DNAbinding specificity, including a highly conserved specific recognition loop (RL) within the RHR, as well as a dimerization domain and a highly conserved monopartite NLS (figs. 3 and 4;supplementary table S2,Supplementary Material online). In contrast, the linker region between the DNA-binding and dimerization domains (Ghosh et al. 1995;Mu¨ller 1995) appears to be animal-specific (fig. 3). Metazoan Rel/NF-jB proteins and metazoan NFAT proteins each formed a well-supported clade, within a larger wellsupported clade of metazoan sequences. Sequences from choanoflagellates, filastereans, and ichthyosporeans formed a clade sister to all metazoan sequences (fig. 2). The most parsimonious explanation suggested by the phylogeny is a duplication of ancestral Rel/NF-jB proteins in the metazoan stem lineage followed by loss of the GRR and ankyrin repeats from NFAT proteins (fig. 4;Seb e-Pedr os et al. [2011] and Gilmore and Wolenski [2012]), and additional duplications in individual metazoan and choanoflagellate lineages (Williams and Gilmore 2020). None of the nonmetazoan Rel/NF-jB sequences contained death domains. The animal NFAT sequences recovered in our survey contain the calcineurin-binding motifs LxVP, and, in the case of chordates, PxIxIT (Wigington et al. 2020) near the N-terminus. Seven of the nonmetazoan Rel/NF-jB-like proteins contained a LxVP motif. In the choanoflagellates Codosiga hollandica and Savillea parva (two out of three proteins), and in the filasterean Pigoraptor vietnamica, this motif was found near the Nterminus, before the RHR (fig. 2;supplementary table S2, Supplementary Material online). This raises the possibility that calcineurin may be an additional regulator of some nonmetazoan Rel/NF-jB-like proteins, as it is in NFAT proteins. Despite the presence of C-terminal ankyrin-rich repeats preceded by a GRR in some sequences from nonmetazoan opisthokonts, and consistent with earlier reports (Williams and Gilmore 2020), we were unable to retrieve any apparent orthologs of NEMO (fig. 1B;supplementary fig. S1, Supplementary Material online) or the other IKK subunits (IKKa,IKKb,andIKKe) outside Metazoa (data not shown). If the C-terminal region of nonmetazoan Rel/NF-jB-like proteins is processed, it may be phosphorylated by another kinase, or targeted for degradation by a different mechanism. The diversity of nonmetazoan Rel/NF-jB-like proteins likely reflect the variety of lifestyles of the organisms in which they are found. Choanoflagellates are free-living, mostly marine or freshwater bacterivores, some of which form clonal multicellular structures in response to specific bacterial molecules (Alegado et al. 2012;Leadbeater 2015); filastereans include both free-living freshwater bacterivores and endobiotic species (Stibbs et al. 1979;Tong 1997;Hehenberger et al. 2017; Tikhonenkov et al. 2020;Urrutia et al. 2021), at least some of which can form multicellular aggregates (Seb e-Pedr os et al. 2013;Hehenberger et al. 2017;Mylnikov et al. 2019); ichthyosporeans include free-living species and parasites of invertebrates or fish, with diverse life cycles and cell states including multinucleate coenocytic stages (reviewed in Mendoza et al. [2002]); and Parvularia is a free-living freshwater bacterivorous amoeboid (L opez-Escard o et al. 2018). Rel/NF-jB-like proteins may play similar or very different roles in how these organisms interact with a variety of prey or host organisms, and/or environmental factors. Interactions with newly evolved partners and gene duplications may have been key to increasing their combinatorial regulatory capabilities in different lineages, including along the animal stem. Overall, we provide an updated evolutionary reconstruction of Rel/NF-jB and NFAT transcription factor families, based on a broad taxon sampling including representatives of all major eukaryotic lineages. We show that Rel/NF-jB-like proteins emerged earlier than previously known, prior to the split between animals and fungi. We further highlight conserved, animal-like architecture in these proteins from diverse opisthokonts. Together, our results suggest that localization and regulatory mechanisms found in animal Rel/NF-jBproteins were likewise present in the last common ancestor of animals and fungi. Materials and Methods Raw Hidden Markov Models (HMMs) of Rel homology DNAbinding domain (RHD_DNA_bind v.21, PF00554), Rel homology Dimerization domain (RHD_dimer v.4, PF16179), Death domain (Death v.21, PF00531), and the IKK component domains Inhibitor of Kappa B Kinase Beta NEMO-binding Leger et al. GBE 4Genome Biol. Evol. 14(1) doi:10.1093/gbe/evab289 Advance Access publication 6 January 2022 Downloaded from https://academic.oup.com/gbe/article/14/1/evab289/6499270 by CSIC - Instituto De Ganaderia De Montana user on 09 June 2022 domain (IKKbetaNEMObind v.7, PF12179) and NF-Kappa B Essential Modulator (NEMO v.7, PF11577) were retrieved from Pfam v.34.0 (Mistry et al. 2021),andusedasqueries in hmmscan (hmmer 3.1b2-2; Eddy 1998;So¨ ding 2005) searches against a paneukaryotic predicted proteome database enriched in holozoan representatives (supplementary table S1,Supplementary Material online). BLAST searches for IKK complex components were carried out using Homo sapiens (GenBank accession numbers O15111.2 [Inhibitor of nuclear factor kappa-B kinase subunit alpha], O14920.1 [Inhibitor of nuclear factor kappa-B kinase subunit beta], Q9Y6K9.2 [NF-kappa-B essential modulator], Q14164.1 Amoebidium parasiticum Apar_comp14023_c0_seq1_fr6 Aplysia californica Acal_524868441 Helgoeca nana Hnan_m.33607 Savillea parva Spar_m.68229 Daphnia pulex Dpul_52849 Daphnia pulex Dpul_237874 Savillea parva Spar_m.44316 Acanthoeca spectabilis Aspe_m.49642 Homo sapiens Hsap_ENSP00000295025 Acanthoeca spectabilis Aspe_m.114880 Strongylocentrotus purpuratus Spur_XP780741 Homo sapiens Hsap_ENSP00000189444 Leucosolenia complicata Lcom_115644 Saccoglossus kowalevskii Skow_NP001158473 Daphnia pulex Dpul_329057 Leucosolenia complicata Lcom_38056 Sphaeroforma arctica Sarc4_g42T Oscarella carmela Ocar_g6369t1 Salpingoeca helianthica Shel_m.70197 Acropora digitifera Adig_6054v104467 Leucosolenia complicata Lcom_85983 Salpingoeca punica Spun_m.10159 Capsaspora owczarzaki Cowc_CAOG_01632 Stephanoeca diplocostata Sdip_AU_m.415255 Xenopus tropicalis Xtro_ENSXETP00000063270 Creolimax fragrantissima Cfra_2801T1 Xenopus tropicalis Xtro_ENSXETP00000004697 Xenopus tropicalis Xtro_ENSXETP00000055326 Stephanoeca diplocostata Sdip_FR_m.262780 Crassostrea gigas Cgig_10028750 Leucosolenia complicata Lcom_55111 Capitella teleta Ctel_220294 Ichthyophonus hoferi Ihof_3724 Helobdella robusta Hrob_69490 Lottia gigantea Lgig_107626 Crassostrea gigas Cgig_10021567 Abeoforma whisleri Awhi_c11394_fr6 Sycon ciliatum Scil_23899 Amphimedon queenslandica Aque_XP003382641 Sycon ciliatum Scil_73441 Chromosphaera perkinsii Cper_evm57s1073 Helobdella robusta Hrob_125959 Tribolium castaneum Tcas_TC011191 Strigamia maritima Smar_011690 Mylnosiga fluctuans Mflu_m.24231 Diaphanoeca grandis Dgra_m.53837 Savillea parva Spar_m.25201 Ixodes scapularis Isca_XP002434504 Salpingoeca helianthica Shel_m.70211 Didymoeca costata Dcos_m.57667 Leucosolenia complicata Lcom_32738 Strongylocentrotus purpuratus Spur_NP999819 Tunicaraptor unikontum Tuni_GIQG01089954.1.p1 Homo sapiens Hsap_ENSP00000384273 Ministeria vibrans Mvib_comp15962_c0_seq1_fr6 Mylnosiga fluctuans Mflu_m.258041 Stephanoeca diplocostata Sdip_FR_m.557985 Sycon ciliatum Scil_42065 Salpingoeca helianthica Shel_m.70222 Pigoraptor vietnamica Pvie_g7289 Drosophila melanogaster Dmel_FBpp0088375 Txikispora philomaios Tphi_evm10s388 Capitella teleta Ctel_181359 Drosophila melanogaster Dmel_FBpp0290792 Diaphanoeca grandis Dgra_m.68592 Daphnia pulex Dpul_300285 Saccoglossus kowalevskii Skow_NP001158458 Parvularia atlantis Patl_3322_fr6 Pirum gemmata Pgem_c67824_fr1 Sycon ciliatum Scil_74006 Helgoeca nana Hnan_m.58794 Sycon ciliatum Scil_86512 Ephydatia muelleri Emue_m.65151 Pigoraptor chileana Pchi_g11706 Xenopus tropicalis Xtro_ENSXETP00000061578 Salpingoeca macrocollata Smac_m.102663 Leucosolenia complicata Lcom_51781 Lottia gigantea Lgig_106471 Homo sapiens Hsap_ENSP00000221452 Nematostella vectensis Nvec_XP001625023 Xenopus tropicalis Xtro_ENSXETP00000046127 Drosophila melanogaster Dmel_FBpp0080560 Leucosolenia complicata Lcom_45816 Ixodes scapularis Isca_XP002399379 Homo sapiens Hsap_ENSP00000226574 Helobdella robusta Hrob_77620 Codosiga hollandica Chol_m.661230 Acanthoeca spectabilis Aspe_m.431975 Stephanoeca diplocostata Sdip_AU_m.283612 Diaphanoeca grandis Dgra_m.73238 96.4/- 98.3/97 85.7/68 99/98 95.5/76 99.6/100 99.9/100 89.3/65 81.2/- 93.3/77 93.6/77 99.9/100 90.8/78 99.8/100 94.9/93 99.8/100 98.1/100 96.6/86 95.8/100 97.3/83 98.5/99 94/81 97.6/99 99.8/100 89.9/81 99.3/95 99.3/100 97.1/76 96.8/97 93.6/70 90/78 93.6/70 99.6/100 87.8/66 97/100 92.8/86 94.5/76 98.1/100 95.4/88 99.4/100 97.1/91 91.7/86 99.4/100 97/100 99.3/99 89.3/96 98.8/100 NFAT 93.2/70 RelB c-Rel RelA/p65 NF-κB1/p105 NF-κB2/p100 DimRHD DB DimRHD DB DimRHD DB DimRHD DB RHD DB Dim GRR RHD DB Dim GRR RHD DB Dim GRR RHD DB Dim GRR Dim GRR RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim DimRHD DB RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim GRR RHD DB Dim RHD DB Dim RHD DB Dim Dim RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim DimRHD DB RHD DB DimRelB LZ RelB TAD RHD DB Dim GRR Death RHD DB RHD DB RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim GRR Death RHD DB Dim RHD DB Dim GRR RHD DB Dim GRR RHD DB DimRHD DB RHD DB RHD DB Dim GRR RHD DB Dim GRR RHD DB Dim RHD DB Dim GRR RHD DB Dim GRR RHD DB Dim RHD DB Dim RHD DB Dim GRR RHD DB Dim GRR RHD DB Dim GRR RHD DB Dim RHD DB Dim GRR RHD DB Dim GRR Death RHD DB Dim GRR RHD DB Dim GRR RHD DB Dim GRR Death RHD DB Dim RHD DB Dim GRR RHD DB Dim RHD DB Dim Death RHD DB Dim RHD DB RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim GRR RHD DB Dim GRR Death RHD DB Dim RHD DB Dim GRR RHD DB Dim RHD DB Dim RHD DB Dim RHD DB Dim GRR RHD DB Dim GRR RHD DB NHR RHD DB Death RHR 1 SH-aLRT/BS 92.8/- 80/- 93.9/- 85/69 -/66 -/66 88/- 89.3/- -/71 -/67 93.1/- 86.4/- 91.2/77 RHD DB DimNt TAD Ct TAD Ct TAD Ct TAD Ct TAD FIG.2.—Rel homology domain phylogeny and domain architecture of Rel homology domain-containing proteins in NF-jB and NFAT families Only SHaLRT and nonparametric bootstrap support values above 80 and 60, respectively, are shown. Fully supported bipartitions are indicated with filled circles. Sequences are color-coded according to taxonomic group: red, Metazoa; yellow, Choanoflagellatea; orange, Filasterea; green, Ichthyosporea; and blue, Holomycota (Fungi and their closest relatives). The two side lengths of the collapsed NFAT clade are proportional to the distances between the node andits closest and furthest leaves. Schematic representation of Pfam domains and conserved regions related to Rel/NF-jB and NFAT proteins in the RHR are depicted to the right of each gene identifier; colors and abbreviations as in figure 1A. Domains or regions depicted in gray match domains or regions that were identified in previous Pfam versions or that share high sequence similarity in the protein alignment (at least four Lysine or Arginine residues in the case of NLS). Evolution of Rel/NF-jB proteins GBE Genome Biol. Evol. 14(1) doi:10.1093/gbe/evab289 Advance Access publication 6 January 2022 5 Downloaded from https://academic.oup.com/gbe/article/14/1/evab289/6499270 by CSIC - Instituto De Ganaderia De Montana user on 09 June 2022 [Inhibitor of nuclear factor kappa-B kinase subunit epsilon]) and Nematostella vectensis (ADQ57374.1 [single IKK-like protein]) IKK complex components as queries. Using custom Perl scripts, the resulting output files were parsed and reanalyzed using PfamScan v.1.5 (Gish and States 1993), and all sequences containing a Rel homology DNAbinding domain were retrieved and examined using reciprocal best BlastP searches against the nonredundant protein database (nr) of the National Center for Biotechnology Information (NCBI). The domain architecture of all retrieved sequences was inferred with PfamScan using the gathering threshold as cutoff value. The number of ankyrin repeats was verified using InterProScan 5.26-65.0 (Jones et al. 2014). Sequences were aligned using MAFFT v7.299b E-INS-i (Katoh et al. 2002,2005;Katoh and Standley 2013)with the gap extension parameter set to 0, trimmed using BMGE v.1.0 (Criscuolo and Gribaldo 2010)usingtheBLOSUM45 matrix, and alignments and trimming were verified by eye. Partial sequences with fewer than 50% of positions represented in the final trimmed alignment were excluded. Preliminary phylogenies were constructed using FastTree v. 2.1.9 (Price et al. 2009,2010); the number of metazoan representatives was reduced. Final alignments were constructed using MAFFT E-INS-i with the gap extension parameter set to 0, and trimmed using trimAl v1.4.rev22 build[2015-05-21] (Capella-Guti errez et al. 2009), and final phylogenies were constructed using IQ-TREE multicore version 2.0-rc1 (Nguyen et al. 2015;Minh et al. 2020) with 1,000 ultrafast bootstrap resamplings (Minh et al. 2013;Hoang et al. 2018), using LG þFþR7 for Rel homology domain proteins (chosen by ModelFinder [Kalyaanamoorthy et al. 2017) as the best fitting model according to the Bayesian H. sapiens (Hsap_ENSP00000396538) D. melanogaster (Dmel_FBpp0111893) D. melanogaster (Dmel_FBpp0290792) D. melanogaster (Dmel_FBpp0088375) H. sapiens (Hsap_ENSP00000221452) H. sapiens (Hsap_ENSP00000384273) N. vectensis (Nvec_XP_001632550) N. vectensis (Nvec_XP_001625023) O. carmela (Ocar_g116_t1) O. carmela (Ocar_g6369_t1) S. ciliatum (Scil_60447) S. ciliatum (Scil_23899) E. muelleri (Emue_m.48144) E. muelleri (Emue_m.65151) A. queenslandica (Aque_XP_003382641) M. vibrans (Mvib_comp15962_c0_seq1_fr6) P. chileana (Pchi_g11706) P. vietnamica (Pvie_g7289) T. philomaios (Tphi_evm10s388) T. unikontum (Tuni_GIQG01089954.1.p1) C. fragrantissima (Cfra_2801T1) S. arctica (Sarc4_g42T) A. parasiticum (Apar_comp14023_c0_seq1_fr6) I. hoferi (Ihof_3724) A. whisleri (Awhi_c11394_fr6) P. gemmata (Pgem_c67824_fr1) C. perkinsii (Cper_evm57s1073) P. atlantis (Patl_3322_fr6) C. owczarzaki (Cowc_CAOG_01632) S. macrocollata (Smac_m.102663) D. grandis (Dgra_m.68592) D. grandis (Dgra_m.73238) D. grandis (Dgra_m.53837) D. costata (Dcos_m.57667) S. diplocostata (Sdip_FR_m.262780) S. diplocostata (Sdip_FR_m.557985) S. diplocostata (Sdip_AU_m.415255) S. diplocostata (Sdip_AU_m.283612) S. punica (Spun_m.10159) S. parva (Spar_m.25201) S. parva (Spar_m.44316) S. parva (Spar_m.68229) S. helianthica (Shel_m.70211) S. helianthica (Shel_m.70197) S. helianthica (Shel_m.70222) M. fluctuans (Mflu_m.24231) M. fluctuans (Mflu_m.258041) C. hollandica (Chol_m.661230) H. nana (Hnan_m.33607) H. nana (Hnan_m.58794) A. spectabilis (Aspe_m.114880) A. spectabilis (Aspe_m.431975) A. spectabilis (Aspe_m.49642) Conservation 207 165 122 22 287 31 321 333 186 161 153 222 163 247 154 43 251 221 205 56 89 205 130 156 116 122 159 85 190 208 293 385 41 409 254 379 148 46 152 80 21 127 67 128 148 517 502 298 398 398 50 61 47 0% 100% Rel/NF-κB NFAT Taxonomic Group Metazoa Choanoflagellatea Filasterea Ichthyosporea Holomycota 473 446 414 315 573 316 606 618 495 465 419 489 436 524 428 315 526 499 475 335 368 475 404 422 386 388 425 355 456 483 563 647 358 667 510 643 412 369 453 371 300 409 333 391 407 792 794 580 657 660 389 384 372 340 307 272 173 428 173 461 473 356 327 291 360 303 392 298 191 399 367 346 200 233 346 276 299 263 269 302 232 333 356 432 519 230 542 388 518 287 241 332 248 183 292 217 279 308 676 661 454 532 532 264 263 249 Rel-homology Dimerisation Domain NLS Nt Ct Rel-homology DNA-binding Domain 0% 100% 0% 100% recognition loop linker region NLS FIG.3.—Partial multiple protein sequence alignment of key Rel/NF-jB domains in Opisthokonta Partial protein sequence alignment (generated by MAFFT v7.299b E-INS-i multiple sequence alignment with the gap extension parameter set to 0) depicting the Nand C-terminal regions of the Rel homology DNA-binding domain, the N-terminal region of the Rel homology dimerization domain and the complete NLS. Background shading of individual amino acids reflects the degree of conservation at a given position; this degree of conservation is also depicted in the histogram shown below the alignment. Dashes indicate gaps in the alignment. Key DNA-binding amino acid residues in the recognition loop, residues comprising the complete linker region between the Rel homology DNA-binding domain and dimerization domain, and key residues in the NLS are highlighted in orange. The amino acid positions in the original sequences are shown to the left of each alignment series. Leger et al. GBE 6Genome Biol. Evol. 14(1) doi:10.1093/gbe/evab289 Advance Access publication 6 January 2022 Downloaded from https://academic.oup.com/gbe/article/14/1/evab289/6499270 by CSIC - Instituto De Ganaderia De Montana user on 09 June 2022 Information Criterion). Subsequently, 100 nonparametric bootstrap replicates were also performed under the same model. Supplementary Material Supplementary data areavailableatGenome Biology and Evolution online. Acknowledgments This work was supported by a European Research Council Consolidator Grant (ERC-2012-CO-616960) grant and a grant (BFU2017-90114-P) from Ministerio de Econom ıa y Competitividad (MINECO), Agencia Estatal de Investigaci on (AEI), and Fondo Europeo de Desarrollo Regional (FEDER) to I.R.-T. M.M.L. was supported by a Marie Skłodowska-Curie Individual Fellowship under the EU Framework Programme for Research and Innovation Horizon 2020 (Project ID 747789) and an Ayuda Juan de la Cierva-Incorporaci on postdoctoral fellowship (IJC2018-036657-I) from the Spanish Ministry of Sciece and Innovation. N.R.-R. was supported by a“Formaci  on del Profesorado Universitario (FPU13/01840)” PhD scholarship from the Spanish Ministerio de Educaci on, Cultura y Deporte (MECD). We thank the anonymous reviewers for their helpful and insightful feedback. Data Availability The data underlying this article are available in the supplementary materials (supplementary table S2 and dataset S1, Supplementary Material online); the data sources are listed in supplementary table S1,Supplementary Material online. Literature Cited Alegado RA, et al. 2012. A bacterial sulfonolipid triggers multicellular development in the closest living relatives of animals. Elife 1:e00013. Filasterea Ichthyosporea Pluriformea/Corallochytrea Fungi Nucleariida Other eukaryotes Archaea & Bacteria RHD DB Dim GRR RHD DB Dim RHD DB Dim GRR RHD DB Dim GRR Opisthokonta ancestor Origin of Rel/NF-κB proteins RHD DB Dim GRR secondary losses domain loss Rel homology DNA-binding domain (PF00554) Rel homology dimerisation domain (PF16179) Domains and regions key Ankyrin repeats (PF00023, PF12796, PF13606, PF13637, PF13857) Death domain (PF00531) RHD DB Dim Death RelB Transactivation domain (PF16181) RelB Leucine Zipper domain (PF16180) RelB LZ RelB TAD Nt or Ct Transactivation domains Ct/Nt TAD Nuclear localization signal Glycine-rich region GRR LxVP motif PxIxIT motif Choanoflagellatea Metazoa ancestor Gene duplication of Rel/NF-κB proteins and expansion of Rel/NF-κB pathway components RHD DB Dim RHD DB Dim Incorporation of Death domain and diversification of NF-κB proteins Loss of GRR and Ankyrin repeats, addition of TADs and diversification of Rel proteins Emergence of IκB proteins Loss of GRR and Ankyrin repeats, addition of TADs and diversification of NFAT proteins RHR RelB LZ RelB TAD Ct TAD NHR RHD DB Dim RHD DB Dim GRR RHD DB Dim Ct TAD Ct TAD Nt TAD RHD DB Dim RHD DB Dim RHD DB Dim Death GRR Metazoa NFAT Rel/NF-κB Loss of GRR and Ankyrin repeats; duplications in some lineages gene duplication Loss of GRR and Ankyrin repeats and duplications in some lineages FIG.4.—Evolution and diversification of Rel/NF-jB proteins in Opisthokonta The Rel/NF-jB proteins originated along the Opisthokonta stem and diversified through gene duplication events and secondary losses and gains of key domains. 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Williams LM, Gilmore TD. 2020. Looking down on NF-jB.MolCellBiol. 40(15):e00104-20. Associate editor: Sandra Baldauf Evolution of Rel/NF-jB proteins GBE Genome Biol. Evol. 14(1) doi:10.1093/gbe/evab289 Advance Access publication 6 January 2022 9 Downloaded from https://academic.oup.com/gbe/article/14/1/evab289/6499270 by CSIC - Instituto De Ganaderia De Montana user on 09 June 2022