The structure of a virus-encoded nucleosome.

Certain large DNA viruses, including those in the Marseilleviridae family, encode histones. Here we show that fused histone pairs Hß-Hα and Hδ-Hγ from Marseillevirus are structurally analogous to the eukaryotic histone pairs H2B-H2A and H4-H3. These viral histones form 'forced' heterodimers, and a heterotetramer of four such heterodimers assembles DNA to form structures virtually identical to canonical eukaryotic nucleosomes.

Analysis of zebrafish periderm enhancers facilitates identification of a regulatory variant near human KRT8/18.

Genome-wide association studies for non-syndromic orofacial clefting (OFC) have identified single nucleotide polymorphisms (SNPs) at loci where the presumed risk-relevant gene is expressed in oral periderm. The functional subsets of such SNPs are difficult to predict because the sequence underpinnings of periderm enhancers are unknown. We applied ATAC-seq to models of human palate periderm, including zebrafish periderm, mouse embryonic palate epithelia, and a human oral epithelium cell line, and to complementary mesenchymal cell types. We identified sets of enhancers specific to the epithelial cells and trained gapped-kmer support-vector-machine classifiers on these sets. We used the classifiers to predict the effects of 14 OFC-associated SNPs at 12q13 near KRT18. All the classifiers picked the same SNP as having the strongest effect, but the significance was highest with the classifier trained on zebrafish periderm. Reporter and deletion analyses support this SNP as lying within a periderm enhancer regulating KRT18/KRT8 expression.

A Screen for Gene Paralogies Delineating Evolutionary Branching Order of Early Metazoa.

The evolutionary diversification of animals is one of Earth's greatest marvels, yet its earliest steps are shrouded in mystery. Animals, the monophyletic clade known as Metazoa, evolved wildly divergent multicellular life strategies featuring ciliated sensory epithelia. In many lineages epithelial sensoria became coupled to increasingly complex nervous systems. Currently, different phylogenetic analyses of single-copy genes support mutually-exclusive possibilities that either Porifera or Ctenophora is sister to all other animals. Resolving this dilemma would advance the ecological and evolutionary understanding of the first animals and the evolution of nervous systems. Here we describe a comparative phylogenetic approach based on gene duplications. We computationally identify and analyze gene families with early metazoan duplications using an approach that mitigates apparent gene loss resulting from the miscalling of paralogs. In the transmembrane channel-like (TMC) family of mechano-transducing channels, we find ancient duplications that define separate clades for Eumetazoa (Placozoa + Cnidaria + Bilateria) vs. Ctenophora, and one duplication that is shared only by Eumetazoa and Porifera. In the Max-like protein X (MLX and MLXIP) family of bHLH-ZIP regulators of metabolism, we find that all major lineages from Eumetazoa and Porifera (sponges) share a duplicated gene pair that is sister to the single-copy gene maintained in Ctenophora. These results suggest a new avenue for deducing deep phylogeny by choosing rather than avoiding ancient gene paralogies.

Episodic evolution of a eukaryotic NADK repertoire of ancient provenance.

NAD kinase (NADK) is the sole enzyme that phosphorylates nicotinamide adenine dinucleotide (NAD+/NADH) into NADP+/NADPH, which provides the chemical reducing power in anabolic (biosynthetic) pathways. While prokaryotes typically encode a single NADK, eukaryotes encode multiple NADKs. How these different NADK genes are all related to each other and those of prokaryotes is not known. Here we conduct phylogenetic analysis of NADK genes and identify major clade-defining patterns of NADK evolution. First, almost all eukaryotic NADK genes belong to one of two ancient eukaryotic sister clades corresponding to cytosolic ("cyto") and mitochondrial ("mito") clades. Secondly, we find that the cyto-clade NADK gene is duplicated in connection with loss of the mito-clade NADK gene in several eukaryotic clades or with acquisition of plastids in Archaeplastida. Thirdly, we find that horizontal gene transfers from proteobacteria have replaced mitochondrial NADK genes in only a few rare cases. Last, we find that the eukaryotic cyto and mito paralogs are unrelated to independent duplications that occurred in sporulating bacteria, once in mycelial Actinobacteria and once in aerobic endospore-forming Firmicutes. Altogether these findings show that the eukaryotic NADK gene repertoire is ancient and evolves episodically with major evolutionary transitions.

Wilhelm His' lasting insights into hindbrain and cranial ganglia development and evolution.

Wilhelm His (1831-1904) provided lasting insights into the development of the central and peripheral nervous system using innovative technologies such as the microtome, which he invented. 150 years after his resurrection of the classical germ layer theory of Wolff, von Baer and Remak, his description of the developmental origin of cranial and spinal ganglia from a distinct cell population, now known as the neural crest, has stood the test of time and more recently sparked tremendous advances regarding the molecular development of these important cells. In addition to his 1868 treatise on 'Zwischenstrang' (now neural crest), his work on the development of the human hindbrain published in 1890 provided novel ideas that more than 100 years later form the basis for penetrating molecular investigations of the regionalization of the hindbrain neural tube and of the migration and differentiation of its constituent neuron populations. In the first part of this review we briefly summarize the major discoveries of Wilhelm His and his impact on the field of embryology. In the second part we relate His' observations to current knowledge about the molecular underpinnings of hindbrain development and evolution. We conclude with the proposition, present already in rudimentary form in the writings of His, that a primordial spinal cord-like organization has been molecularly supplemented to generate hindbrain 'neomorphs' such as the cerebellum and the auditory and vestibular nuclei and their associated afferents and sensory organs.

Phylogenetic analysis of the core histone doublet and DNA topo II genes of Marseilleviridae: evidence of proto-eukaryotic provenance.

BACKGROUND: While the genomes of eukaryotes and Archaea both encode the histone-fold domain, only eukaryotes encode the core histone paralogs H2A, H2B, H3, and H4. With DNA, these core histones assemble into the nucleosomal octamer underlying eukaryotic chromatin. Importantly, core histones for H2A and H3 are maintained as neofunctionalized paralogs adapted for general bulk chromatin (canonical H2 and H3) or specialized chromatin (H2A.Z enriched at gene promoters and cenH3s enriched at centromeres). In this context, the identification of core histone-like "doublets" in the cytoplasmic replication factories of the Marseilleviridae (MV) is a novel finding with possible relevance to understanding the origin of eukaryotic chromatin. Here, we analyze and compare the core histone doublet genes from all known MV genomes as well as other MV genes relevant to the origin of the eukaryotic replisome. RESULTS: Using different phylogenetic approaches, we show that MV histone domains encode obligate H2B-H2A and H4-H3 dimers of possible proto-eukaryotic origin. MV core histone moieties form sister clades to each of the four eukaryotic clades of canonical and variant core histones. This suggests that MV core histone moieties diverged prior to eukaryotic neofunctionalizations associated with paired linear chromosomes and variant histone octamer assembly. We also show that MV genomes encode a proto-eukaryotic DNA topoisomerase II enzyme that forms a sister clade to eukaryotes. This is a relevant finding given that DNA topo II influences histone deposition and chromatin compaction and is the second most abundant nuclear protein after histones. CONCLUSIONS: The combined domain architecture and phylogenomic analyses presented here suggest that a primitive origin for MV histone genes is a more parsimonious explanation than horizontal gene transfers + gene fusions + sufficient divergence to eliminate relatedness to eukaryotic neofunctionalizations within the H2A and H3 clades without loss of relatedness to each of the four core histone clades. We thus suggest MV histone doublet genes and their DNA topo II gene possibly were acquired from an organism with a chromatinized replisome that diverged prior to the origin of eukaryotic core histone variants for H2/H2A.Z and H3/cenH3. These results also imply that core histones were utilized ancestrally in viral DNA compaction and/or protection from host endonucleases.

Correction: Recessive coding and regulatory mutations in FBLIM1 underlie the pathogenesis of chronic recurrent multifocal osteomyelitis (CRMO).

[This corrects the article DOI: 10.1371/journal.pone.0169687.].

Recessive coding and regulatory mutations in FBLIM1 underlie the pathogenesis of chronic recurrent multifocal osteomyelitis (CRMO).

Chronic recurrent multifocal osteomyelitis (CRMO) is a rare, pediatric, autoinflammatory disease characterized by bone pain due to sterile osteomyelitis, and is often accompanied by psoriasis or inflammatory bowel disease. There are two syndromic forms of CRMO, Majeed syndrome and DIRA, for which the genetic cause is known. However, for the majority of cases of CRMO, the genetic basis is unknown. Via whole-exome sequencing, we detected a homozygous mutation in the filamin-binding domain of FBLIM1 in an affected child with consanguineous parents. Microarray analysis of bone marrow macrophages from the CRMO murine model (cmo) determined that the Fblim1 ortholog is the most differentially expressed gene, downregulated over 20-fold in the cmo mouse. We sequenced FBLIM1 in 96 CRMO subjects and found a second proband with a novel frameshift mutation in exon 6 and a rare regulatory variant. In SaOS2 cells, overexpressing the regulatory mutation showed the flanking region acts as an enhancer, and the mutation ablates enhancer activity. Our data implicate FBLIM1 in the pathogenesis of sterile bone inflammation and our findings suggest CRMO is a disorder of chronic inflammation and imbalanced bone remodeling.

Evolving Notch polyQ tracts reveal possible solenoid interference elements.

Polyglutamine (polyQ) tracts in regulatory proteins are extremely polymorphic. As functional elements under selection for length, triplet repeats are prone to DNA replication slippage and indel mutations. Many polyQ tracts are also embedded within intrinsically disordered domains, which are less constrained, fast evolving, and difficult to characterize. To identify structural principles underlying polyQ tracts in disordered regulatory domains, here I analyze deep evolution of metazoan Notch polyQ tracts, which can generate alleles causing developmental and neurogenic defects. I show that Notch features polyQ tract turnover that is restricted to a discrete number of conserved "polyQ insertion slots". Notch polyQ insertion slots are: (i) identifiable by an amphipathic "slot leader" motif; (ii) conserved as an intact C-terminal array in a 1-to-1 relationship with the N-terminal solenoid-forming ankyrin repeats (ARs); and (iii) enriched in carboxamide residues (Q/N), whose sidechains feature dual hydrogen bond donor and acceptor atoms. Correspondingly, the terminal loop and ß-strand of each AR feature conserved carboxamide residues, which would be susceptible to folding interference by hydrogen bonding with residues outside the ARs. I thus suggest that Notch polyQ insertion slots constitute an array of AR interference elements (ARIEs). Notch ARIEs would dynamically compete with the delicate serial folding induced by adjacent ARs. Huntingtin, which harbors solenoid-forming HEAT repeats, also possesses a similar number of polyQ insertion slots. These results suggest that intrinsically disordered interference arrays featuring carboxamide and polyQ enrichment may constitute coupled proteodynamic modulators of solenoids.

Integration of Orthogonal Signaling by the Notch and Dpp Pathways in Drosophila.

The transcription factor Suppressor of Hairless and its coactivator, the Notch intracellular domain, are polyglutamine (pQ)-rich factors that target enhancer elements and interact with other locally bound pQ-rich factors. To understand the functional repertoire of such enhancers, we identify conserved regulatory belts with binding sites for the pQ-rich effectors of both Notch and BMP/Dpp signaling, and the pQ-deficient tissue selectors Apterous (Ap), Scalloped (Sd), and Vestigial (Vg). We find that the densest such binding site cluster in the genome is located in the BMP-inducible nab locus, a homolog of the vertebrate transcriptional cofactors NAB1/NAB2 We report three major findings. First, we find that this nab regulatory belt is a novel enhancer driving dorsal wing margin expression in regions of peak phosphorylated Mad in wing imaginal discs. Second, we show that Ap is developmentally required to license the nab dorsal wing margin enhancer (DWME) to read out Notch and Dpp signaling in the dorsal compartment. Third, we find that the nab DWME is embedded in a complex of intronic enhancers, including a wing quadrant enhancer, a proximal wing disc enhancer, and a larval brain enhancer. This enhancer complex coordinates global nab expression via both tissue-specific activation and interenhancer silencing. We suggest that DWME integration of BMP signaling maintains nab expression in proliferating margin descendants that have divided away from Notch-Delta boundary signaling. As such, uniform expression of genes like nab and vestigial in proliferating compartments would typically require both boundary and nonboundary lineage-specific enhancers.

The Nature, Extent, and Consequences of Genetic Variation in the opa Repeats of Notch in Drosophila.

Polyglutamine (pQ) tracts are abundant in proteins co-interacting on DNA. The lengths of these pQ tracts can modulate their interaction strengths. However, pQ tracts >40 residues are pathologically prone to amyloidogenic self-assembly. Here, we assess the extent and consequences of variation in the pQ-encoding opa repeats of Notch in Drosophila melanogaster. We use Sanger sequencing to genotype opa sequences ([Formula: see text]-CAX repeats), which have resisted assembly using short sequence reads. While most sampled lines carry the major allele opa31 encoding Q13HQ17 or the opa32 allele encoding Q13HQ18, many lines carry rare alleles encoding pQ tracts >32 residues: opa33a (Q14HQ18), opa33b (Q15HQ17), opa34 (Q16HQ17), opa35a1/opa35a2 (Q13HQ21), opa36 (Q13HQ22), and opa37 (Q13HQ23). Only one rare allele encodes a tract <31 residues: opa23 (Q13-Q10). This opa23 allele shortens the pQ tract while simultaneously eliminating the interrupting histidine. We introgressed these opa variant alleles into common backgrounds and measured the frequency of Notch-type phenotypes. Homozygotes for the short and long opa alleles have defects in embryonic survival and sensory bristle organ patterning, and sometimes show wing notching. Consistent with functional differences between Notch opa variants, we find that a scute inversion carrying the rare opa33b allele suppresses the bristle patterning defect caused by achaete/scute insufficiency, while an equivalent scute inversion carrying opa31 manifests the patterning defect. Our results demonstrate the existence of potent pQ variants of Notch and the need for long read genotyping of key repeat variables underlying gene regulatory networks.

Genes conserved in bilaterians but jointly lost with Myc during nematode evolution are enriched in cell proliferation and cell migration functions.

Animals use a stereotypical set of developmental genes to build body architectures of varying sizes and organizational complexity. Some genes are critical to developmental patterning, while other genes are important to physiological control of growth. However, growth regulator genes may not be as important in small-bodied "micro-metazoans" such as nematodes. Nematodes use a simplified developmental strategy of lineage-based cell fate specifications to produce an adult bilaterian body composed of a few hundreds of cells. Nematodes also lost the MYC proto-oncogenic regulator of cell proliferation. To identify additional regulators of cell proliferation that were lost with MYC, we computationally screened and determined 839 high-confidence genes that are conserved in bilaterians/lost in nematodes (CIBLIN genes). We find that 30 % of all CIBLIN genes encode transcriptional regulators of cell proliferation, epithelial-to-mesenchyme transitions, and other processes. Over 50 % of CIBLIN genes are unnamed genes in Drosophila, suggesting that there are many understudied genes. Interestingly, CIBLIN genes include many Myc synthetic lethal (MycSL) hits from recent screens. CIBLIN genes include key regulators of heparan sulfate proteoglycan (HSPG) sulfation patterns, and lysyl oxidases involved in cross-linking and modification of the extracellular matrix (ECM). These genes and others suggest the CIBLIN repertoire services critical functions in ECM remodeling and cell migration in large-bodied bilaterians. Correspondingly, CIBLIN genes are co-expressed with Myc in cancer transcriptomes, and include a preponderance of known determinants of cancer progression and tumor aggression. We propose that CIBLIN gene research can improve our understanding of regulatory control of cellular growth in metazoans.

Metabolic and chaperone gene loss marks the origin of animals: evidence for Hsp104 and Hsp78 chaperones sharing mitochondrial enzymes as clients.

The evolution of animals involved acquisition of an emergent gene repertoire for gastrulation. Whether loss of genes also co-evolved with this developmental reprogramming has not yet been addressed. Here, we identify twenty-four genetic functions that are retained in fungi and choanoflagellates but undetectable in animals. These lost genes encode: (i) sixteen distinct biosynthetic functions; (ii) the two ancestral eukaryotic ClpB disaggregases, Hsp78 and Hsp104, which function in the mitochondria and cytosol, respectively; and (iii) six other assorted functions. We present computational and experimental data that are consistent with a joint function for the differentially localized ClpB disaggregases, and with the possibility of a shared client/chaperone relationship between the mitochondrial Fe/S homoaconitase encoded by the lost LYS4 gene and the two ClpBs. Our analyses lead to the hypothesis that the evolution of gastrulation-based multicellularity in animals led to efficient extraction of nutrients from dietary sources, loss of natural selection for maintenance of energetically expensive biosynthetic pathways, and subsequent loss of their attendant ClpB chaperones.

Microsatellite repeat instability fuels evolution of embryonic enhancers in Hawaiian Drosophila.

For ∼30 million years, the eggs of Hawaiian Drosophila were laid in ever-changing environments caused by high rates of island formation. The associated diversification of the size and developmental rate of the syncytial fly embryo would have altered morphogenic gradients, thus necessitating frequent evolutionary compensation of transcriptional responses. We investigate the consequences these radiations had on transcriptional enhancers patterning the embryo to see whether their pattern of molecular evolution is different from non-Hawaiian species. We identify and functionally assay in transgenic D. melanogaster the Neurogenic Ectoderm Enhancers from two different Hawaiian Drosophila groups: (i) the picture wing group, and (ii) the modified mouthparts group. We find that the binding sites in this set of well-characterized enhancers are footprinted by diverse microsatellite repeat (MSR) sequences. We further show that Hawaiian embryonic enhancers in general are enriched in MSR relative to both Hawaiian non-embryonic enhancers and non-Hawaiian embryonic enhancers. We propose embryonic enhancers are sensitive to Activator spacing because they often serve as assembly scaffolds for the aggregation of transcription factor activator complexes. Furthermore, as most indels are produced by microsatellite repeat slippage, enhancers from Hawaiian Drosophila lineages, which experience dynamic evolutionary pressures, would become grossly enriched in MSR content.

New insights into the roles of Xin repeat-containing proteins in cardiac development, function, and disease.

Since the discovery of Xin repeat-containing proteins in 1996, the importance of Xin proteins in muscle development, function, regeneration, and disease has been continuously implicated. Most Xin proteins are localized to myotendinous junctions of the skeletal muscle and also to intercalated discs (ICDs) of the heart. The Xin gene is only found in vertebrates, which are characterized by a true chambered heart. This suggests that the evolutionary origin of the Xin gene may have played a key role in vertebrate origins. Diverse vertebrates including mammals possess two paralogous genes, Xinα (or Xirp1) and Xinß (or Xirp2), and this review focuses on the role of their encoded proteins in cardiac muscles. Complete loss of mouse Xinß (mXinß) results in the failure of forming ICD, severe growth retardation, and early postnatal lethality. Deletion of mouse Xinα (mXinα) leads to late-onset cardiomyopathy with conduction defects. Molecular studies have identified three classes of mXinα-interacting proteins: catenins, actin regulators/modulators, and ion-channel subunits. Thus, mXinα acts as a scaffolding protein modulating the N-cadherin-mediated adhesion and ion-channel surface expression. Xin expression is significantly upregulated in early stages of stressed hearts, whereas Xin expression is downregulated in failing hearts from various human cardiomyopathies. Thus, mutations in these Xin loci may lead to diverse cardiomyopathies and heart failure.

A Schnurri/Mad/Medea complex attenuates the dorsal-twist gradient readout at vnd.

Morphogen gradients are used in developing embryos, where they subdivide a field of cells into territories characterized by distinct cell fate potentials. Such systems require both a spatially-graded distribution of the morphogen, and an ability to encode different responses at different target genes. However, the potential for different temporal responses is also present because morphogen gradients typically provide temporal cues, which may be a potential source of conflict. Thus, a low threshold response adapted for an early temporal onset may be inappropriate when the desired spatial response is a spatially-limited, high-threshold expression pattern. Here, we identify such a case with the Drosophila vnd locus, which is a target of the dorsal (dl) nuclear concentration gradient that patterns the dorsal/ventral (D/V) axis of the embryo. The vnd gene plays a critical role in the "ventral dominance" hierarchy of vnd, ind, and msh, which individually specify distinct D/V neural columnar fates in increasingly dorsal ectodermal compartments. The role of vnd in this regulatory hierarchy requires early temporal expression, which is characteristic of low-threshold responses, but its specification of ventral neurogenic ectoderm demands a relatively high-threshold response to dl. We show that the Neurogenic Ectoderm Enhancer (NEE) at vnd takes additional input from the complementary Dpp gradient via a conserved Schnurri/Mad/Medea silencer element (SSE) unlike NEEs at brk, sog, rho, and vn. These results show how requirements for conflicting temporal and spatial responses to the same gradient can be solved by additional inputs from complementary gradients.

A model of proto-anti-codon RNA enzymes requiring L-amino acid homochirality.

All living organisms encode the 20 natural amino acid units of polypeptides using a universal scheme of triplet nucleotide "codons". Disparate features of this codon scheme are potentially informative of early molecular evolution: (i) the absence of any codons for D-amino acids; (ii) the odd combination of alternate codon patterns for some amino acids; (iii) the confinement of synonymous positions to a codon's third nucleotide; (iv) the use of 20 specific amino acids rather than a number closer to the full coding potential of 64; and (v) the evolutionary relationship of patterns in stop codons to amino acid codons. Here I propose a model for an ancestral proto-anti-codon RNA (pacRNA) auto-aminoacylation system and show that pacRNAs would naturally manifest features of the codon table. I show that pacRNAs could implement all the steps for auto-aminoacylation: amino acid coordination, intermediate activation of the amino acid by the 5'-end of the pacRNA, and 3'-aminoacylation of the pacRNA. The anti-codon cradles of pacRNAs would have been able to recognize and coordinate only a small number of L-amino acids via hydrogen bonding. A need for proper spatial coordination would have limited the number of chargeable amino acids for all anti-codon sequences, in addition to making some anti-codon sequences unsuitable. Thus, the pacRNA model implies that the idiosyncrasies of the anti-codon table and L-amino acid homochirality co-evolved during a single evolutionary period. These results further imply that early life consisted of an aminoacylated RNA world with a richer enzymatic potential than ribonucleotides alone.

Dynamic evolution of precise regulatory encodings creates the clustered site signature of enhancers.

Concentration gradients of morphogenic proteins pattern the embryonic axes of Drosophila by activating different genes at different concentrations. The neurogenic ectoderm enhancers (NEEs) activate different genes at different threshold levels of the Dorsal (Dl) morphogen, which patterns the dorsal/ventral axis. NEEs share a unique arrangement of highly constrained DNA-binding sites for Dl, Twist (Twi), Snail (Sna) and Suppressor of Hairless (Su(H)), and encode the threshold variable in the precise length of DNA that separates one well-defined Dl element from a Twi element. However, NEEs also possess dense clusters of variant Dl sites. Here, we show that these increasingly variant sites are eclipsed relic elements, which were superseded by more recently evolved threshold encodings. Given the divergence in egg size during Drosophila lineage evolution, the observed characteristic clusters of divergent sites indicate a history of frequent selection for changes in threshold responses to the Dl morphogen gradient and confirm the NEE structure/function model.

Temporal regulation of the muscle gene cascade by Macho1 and Tbx6 transcription factors in Ciona intestinalis.

For over a century, muscle formation in the ascidian embryo has been representative of 'mosaic' development. The molecular basis of muscle-fate predetermination has been partly elucidated with the discovery of Macho1, a maternal zinc-finger transcription factor necessary and sufficient for primary muscle development, and of its transcriptional intermediaries Tbx6b and Tbx6c. However, the molecular mechanisms by which the maternal information is decoded by cis-regulatory modules (CRMs) associated with muscle transcription factor and structural genes, and the ways by which a seamless transition from maternal to zygotic transcription is ensured, are still mostly unclear. By combining misexpression assays with CRM analyses, we have identified the mechanisms through which Ciona Macho1 (Ci-Macho1) initiates expression of Ci-Tbx6b and Ci-Tbx6c, and we have unveiled the cross-regulatory interactions between the latter transcription factors. Knowledge acquired from the analysis of the Ci-Tbx6b CRM facilitated both the identification of a related CRM in the Ci-Tbx6c locus and the characterization of two CRMs associated with the structural muscle gene fibrillar collagen 1 (CiFCol1). We use these representative examples to reconstruct how compact CRMs orchestrate the muscle developmental program from pre-localized ooplasmic determinants to differentiated larval muscle in ascidian embryos.