A Family of Vertebrate-Specific Polycombs Encoded by the LCOR/LCORL Genes Balance PRC2 Subtype Activities.

The polycomb repressive complex 2 (PRC2) consists of core subunits SUZ12, EED, RBBP4/7, and EZH1/2 and is responsible for mono-, di-, and tri-methylation of lysine 27 on histone H3. Whereas two distinct forms exist, PRC2.1 (containing one polycomb-like protein) and PRC2.2 (containing AEBP2 and JARID2), little is known about their differential functions. Here, we report the discovery of a family of vertebrate-specific PRC2.1 proteins, "PRC2 associated LCOR isoform 1" (PALI1) and PALI2, encoded by the LCOR and LCORL gene loci, respectively. PALI1 promotes PRC2 methyltransferase activity in vitro and in vivo and is essential for mouse development. Pali1 and Aebp2 define mutually exclusive, antagonistic PRC2 subtypes that exhibit divergent H3K27-tri-methylation activities. The balance of these PRC2.1/PRC2.2 activities is required for the appropriate regulation of polycomb target genes during differentiation. PALI1/2 potentially link polycombs with transcriptional co-repressors in the regulation of cellular identity during development and in cancer.

The deceptive simplicity of mendelian genetics.

Mendel, a genius experimentalist, meticulously uncovered the genetic basis of heredity in work that transformed the science of biology. But does the alluring simplicity of Mendel's laws sometimes obscure the true complexity of genetics?

A chromatin-independent role of Polycomb-like 1 to stabilize p53 and promote cellular quiescence.

Polycomb-like proteins 1-3 (PCL1-3) are substoichiometric components of the Polycomb-repressive complex 2 (PRC2) that are essential for association of the complex with chromatin. However, it remains unclear why three proteins with such apparent functional redundancy exist in mammals. Here we characterize their divergent roles in both positively and negatively regulating cellular proliferation. We show that while PCL2 and PCL3 are E2F-regulated genes expressed in proliferating cells, PCL1 is a p53 target gene predominantly expressed in quiescent cells. Ectopic expression of any PCL protein recruits PRC2 to repress the INK4A gene; however, only PCL2 and PCL3 confer an INK4A-dependent proliferative advantage. Remarkably, PCL1 has evolved a PRC2- and chromatin-independent function to negatively regulate proliferation. We show that PCL1 binds to and stabilizes p53 to induce cellular quiescence. Moreover, depletion of PCL1 phenocopies the defects in maintaining cellular quiescence associated with p53 loss. This newly evolved function is achieved by the binding of the PCL1 N-terminal PHD domain to the C-terminal domain of p53 through two unique serine residues, which were acquired during recent vertebrate evolution. This study illustrates the functional bifurcation of PCL proteins, which act in both a chromatin-dependent and a chromatin-independent manner to regulate the INK4A and p53 pathways.

Open questions in the study of de novo genes: what, how and why.

The study of de novo protein-coding genes is maturing from the ad hoc reporting of individual cases to the systematic analysis of extensive genomic data from several species. We identify three key challenges for this emerging field: understanding how best to identify de novo genes, how they arise and why they spread. We highlight the intellectual challenges of understanding how a de novo gene becomes integrated into pre-existing functions and becomes essential. We suggest that, as with protein sequence evolution, antagonistic co-evolution may be key to de novo gene evolution, particularly for new essential genes and new cancer-associated genes.

Pseudogenes Provide Evolutionary Evidence for the Competitive Endogenous RNA Hypothesis.

The competitive endogenous RNA (ceRNA) hypothesis is an attractively simple model to explain the biological role of many putatively functionless noncoding RNAs. Under this model, there exist transcripts in the cell whose role is to titrate out microRNAs such that the expression level of another target sequence is altered. That it is logistically possible for expression of one microRNA recognition element (MRE)-containing transcript to affect another is seen in the multiple examples of pathogenic effects of inappropriate expression of MRE-containing RNAs. However, the role, if any, of ceRNAs in normal biological processes and at physiological levels is disputed. By comparison of parent genes and pseudogenes we show, both for a specific example and genome-wide, that the pseudo-3' untranslated regions (3'UTRs) of expressed pseudogenes are frequently retained and are under selective constraint in mammalian genomes. We found that the pseudo-3'UTR of BRAFP1, a previously described oncogenic ceRNA, has reduced substitutions relative to its pseudo-coding sequence, and we show sequence constraint on MREs shared between the parent gene, BRAF, and the pseudogene. Investigation of RNA-seq data reveals expression of BRAFP1 in normal somatic tissues in human and in other primates, consistent with biological ceRNA functionality of this pseudogene in nonpathogenic cellular contexts. Furthermore, we find that on a genome-wide scale pseudo-3'UTRs of mammalian pseudogenes (n = 1,629) are under stronger selective constraint than their pseudo-coding sequence counterparts, and are more often retained and expressed. Our results suggest that many human pseudogenes, often considered nonfunctional, may have an evolutionarily constrained role, consistent with the ceRNA hypothesis.

Faster Evolving Primate Genes Are More Likely to Duplicate.

An attractive and long-standing hypothesis regarding the evolution of genes after duplication posits that the duplication event creates new evolutionary possibilities by releasing a copy of the gene from constraint. Apparent support was found in numerous analyses, particularly, the observation of higher rates of evolution in duplicated as compared with singleton genes. Could it, instead, be that more duplicable genes (owing to mutation, fixation, or retention biases) are intrinsically faster evolving? To uncouple the measurement of rates of evolution from the determination of duplicate or singleton status, we measure the rates of evolution in singleton genes in outgroup primate lineages but classify these genes as to whether they have duplicated or not in a crown group of great apes. We find that rates of evolution are higher in duplicable genes prior to the duplication event. In part this is owing to a negative correlation between coding sequence length and rate of evolution, coupled with a bias toward smaller genes being more duplicable. The effect is masked by difference in expression rate between duplicable genes and singletons. Additionally, in contradiction to the classical assumption, we find no convincing evidence for an increase in dN/dS after duplication, nor for rate asymmetry between duplicates. We conclude that high rates of evolution of duplicated genes are not solely a consequence of the duplication event, but are rather a predictor of duplicability. These results are consistent with a model in which successful gene duplication events in mammals are skewed toward events of minimal phenotypic impact.

Evolutionary journey and characterisation of a novel pan-gene associated with beer strains of Saccharomyces cerevisiae.

The sequencing of over a thousand Saccharomyces cerevisiae genomes revealed a complex pangenome. Over one third of the discovered genes are not present in the S. cerevisiae core genome but instead are often restricted to a subset of yeast isolates and thus may be important for adaptation to specific environmental niches. We refer to these genes as "pan-genes," being part of the pangenome but not the core genome. Here, we describe the evolutionary journey and characterisation of a novel pan-gene, originally named hypothetical (HYPO) open-reading frame. Phylogenetic analysis reveals that HYPO has been predominantly retained in S. cerevisiae strains associated with brewing but has been repeatedly lost in most other fungal species during evolution. There is also evidence that HYPO was horizontally transferred at least once, from S. cerevisiae to Saccharomyces paradoxus. The phylogenetic analysis of HYPO exemplifies the complexity and intricacy of evolutionary trajectories of genes within the S. cerevisiae pangenome. To examine possible functions for Hypo, we overexpressed a HYPO-GFP fusion protein in both S. cerevisiae and Saccharomyces pastorianus. The protein localised to the plasma membrane where it accumulated initially in distinct foci. Time-lapse fluorescent imaging revealed that when cells are grown in wort, Hypo-gfp fluorescence spreads throughout the membrane during cell growth. The overexpression of Hypo-gfp in S. cerevisiae or S. pastorianus strains did not significantly alter cell growth in medium-containing glucose, maltose, maltotriose, or wort at different concentrations.

Genomes as documents of evolutionary history: a probabilistic macrosynteny model for the reconstruction of ancestral genomes.

MOTIVATION: It has been argued that whole-genome duplication (WGD) exerted a profound influence on the course of evolution. For the purpose of fully understanding the impact of WGD, several formal algorithms have been developed for reconstructing pre-WGD gene order in yeast and plant. However, to the best of our knowledge, those algorithms have never been successfully applied to WGD events in teleost and vertebrate, impeded by extensive gene shuffling and gene losses. RESULTS: Here, we present a probabilistic model of macrosynteny (i.e. conserved linkage or chromosome-scale distribution of orthologs), develop a variational Bayes algorithm for inferring the structure of pre-WGD genomes, and study estimation accuracy by simulation. Then, by applying the method to the teleost WGD, we demonstrate effectiveness of the algorithm in a situation where gene-order reconstruction algorithms perform relatively poorly due to a high rate of rearrangement and extensive gene losses. Our high-resolution reconstruction reveals previously overlooked small-scale rearrangements, necessitating a revision to previous views on genome structure evolution in teleost and vertebrate. CONCLUSIONS: We have reconstructed the structure of a pre-WGD genome by employing a variational Bayes approach that was originally developed for inferring topics from millions of text documents. Interestingly, comparison of the macrosynteny and topic model algorithms suggests that macrosynteny can be regarded as documents on ancestral genome structure. From this perspective, the present study would seem to provide a textbook example of the prevalent metaphor that genomes are documents of evolutionary history. AVAILABILITY AND IMPLEMENTATION: The analysis data are available for download at http://www.gen.tcd.ie/molevol/supp_data/MacrosyntenyTGD.zip , and the software written in Java is available upon request. CONTACT: yoichiro.nakatani@tcd.ie or aoife.mclysaght@tcd.ie. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Dosage-sensitive genes in evolution and disease.

For a subset of genes in our genome a change in gene dosage, by duplication or deletion, causes a phenotypic effect. These dosage-sensitive genes may confer an advantage upon copy number change, but more typically they are associated with disease, including heart disease, cancers and neuropsychiatric disorders. This gene copy number sensitivity creates characteristic evolutionary constraints that can serve as a diagnostic to identify dosage-sensitive genes. Though the link between copy number change and disease is well-established, the mechanism of pathogenicity is usually opaque. We propose that gene expression level may provide a common basis for the pathogenic effects of many copy number variants.

Spatial Colocalization of Human Ohnolog Pairs Acts to Maintain Dosage-Balance.

Ohnologs -paralogous gene pairs generated by whole genome duplication- are enriched for dosage sensitive genes, that is, genes that have a phenotype due to copy number changes. Dosage sensitive genes frequently occur in the same metabolic pathway and in physically interacting proteins. Accumulating evidence reveals that functionally related genes tend to co-localize in the three-dimensional (3D) arrangement of chromosomes. We query whether the spatial distribution of ohnologs has implications for their dosage balance. We analyzed the colocalization frequency of ohnologs based on chromatin interaction datasets of seven human cell lines and found that ohnolog pairs exhibit higher spatial proximity in 3D nuclear organization than other paralog pairs and than randomly chosen ohnologs in the genome. We also found that colocalized ohnologs are more resistant to copy number variations and more likely to be disease-associated genes, which indicates a stronger dosage balance in ohnologs with high spatial proximity. This phenomenon is further supported by the stronger similarity of gene co-expression and of gene ontology terms of colocalized ohnologs. In addition, for a large fraction of ohnologs, the spatial colocalization is conserved in mouse cells, suggestive of functional constraint on their 3D positioning in the nucleus.

Ohnologs are overrepresented in pathogenic copy number mutations.

A number of rare copy number variants (CNVs), including both deletions and duplications, have been associated with developmental disorders, including schizophrenia, autism, intellectual disability, and epilepsy. Pathogenicity may derive from dosage sensitivity of one or more genes contained within the CNV locus. To understand pathophysiology, the specific disease-causing gene(s) within each CNV need to be identified. In the present study, we test the hypothesis that ohnologs (genes retained after ancestral whole-genome duplication events, which are frequently dosage sensitive) are overrepresented in pathogenic CNVs. We selected three sets of genes implicated in copy number pathogenicity: (i) genes mapping within rare disease-associated CNVs, (ii) genes within de novo CNVs under negative genetic selection, and (iii) genes identified by clinical array comparative genome hybridization studies as potentially pathogenic. We compared the proportion of ohnologs between these gene sets and control genes, mapping to CNVs not known to be disease associated. We found that ohnologs are significantly overrepresented in genes mapping to pathogenic CNVs, irrespective of how CNVs were identified, with over 90% containing an ohnolog, compared with control CNVs >100 kb, where only about 30% contained an ohnolog. In some CNVs, such as del15p11.2 (CYFIP1) and dup/del16p13.11 (NDE1), the most plausible prior candidate gene was also an ohnolog, as were the genes VIPR2 and NRXN1, each found in short CNVs containing no other genes. Our results support the hypothesis that ohnologs represent critical dosage-sensitive elements of the genome, possibly responsible for some of the deleterious phenotypes observed for pathogenic CNVs and as such are readily identifiable candidate genes for further study.

Positionally biased gene loss after whole genome duplication: evidence from human, yeast, and plant.

Whole genome duplication (WGD) has made a significant contribution to many eukaryotic genomes including yeast, plants, and vertebrates. Following WGD, some ohnologs (WGD paralogs) remain in the genome arranged in blocks of conserved gene order and content (paralogons). However, the most common outcome is loss of one of the ohnolog pair. It is unclear what factors, if any, govern gene loss from paralogons. Recent studies have reported physical clustering (genetic linkage) of functionally linked (interacting) genes in the human genome and propose a biological significance for the clustering of interacting genes such as coexpression or preservation of epistatic interactions. Here we conduct a novel test of a hypothesis that functionally linked genes in the same paralogon are preferentially retained in cis after WGD. We compare the number of protein-protein interactions (PPIs) between linked singletons within a paralogon (defined as cis-PPIs) with that of PPIs between singletons across paralogon pairs (defined as trans-PPIs). We find that paralogons in which the number of cis-PPIs is greater than that of trans-PPIs are significantly enriched in human and yeast. The trend is similar in plants, but it is difficult to assess statistical significance due to multiple, overlapping WGD events. Interestingly, human singletons participating in cis-PPIs tend to be classified into "response to stimulus." We uncover strong evidence of biased gene loss after WGD, which further supports the hypothesis of biologically significant gene clusters in eukaryotic genomes. These observations give us new insight for understanding the evolution of genome structure and of protein interaction networks.

Mammalian X chromosome inactivation evolved as a dosage-compensation mechanism for dosage-sensitive genes on the X chromosome.

How and why female somatic X-chromosome inactivation (XCI) evolved in mammals remains poorly understood. It has been proposed that XCI is a dosage-compensation mechanism that evolved to equalize expression levels of X-linked genes in females (2X) and males (1X), with a prior twofold increase in expression of X-linked genes in both sexes ("Ohno's hypothesis"). Whereas the parity of X chromosome expression between the sexes has been clearly demonstrated, tests for the doubling of expression levels globally along the X chromosome have returned contradictory results. However, changes in gene dosage during sex-chromosome evolution are not expected to impact on all genes equally, and should have greater consequences for dosage-sensitive genes. We show that, for genes encoding components of large protein complexes (≥ 7 members)--a class of genes that is expected to be dosage-sensitive--expression of X-linked genes is similar to that of autosomal genes within the complex. These data support Ohno's hypothesis that XCI acts as a dosage-compensation mechanism, and allow us to refine Ohno's model of XCI evolution. We also explore the contribution of dosage-sensitive genes to X aneuploidy phenotypes in humans, such as Turner (X0) and Klinefelter (XXY) syndromes. X aneuploidy in humans is common and is known to have mild effects because most of the supernumerary X genes are inactivated and not affected by aneuploidy. Only genes escaping XCI experience dosage changes in X-aneuploidy patients. We combined data on dosage sensitivity and XCI to compute a list of candidate genes for X-aneuploidy syndromes.

Ohnologs in the human genome are dosage balanced and frequently associated with disease.

About 30% of protein-coding genes in the human genome are related through two whole genome duplication (WGD) events. Although WGD is often credited with great evolutionary importance, the processes governing the retention of these genes and their biological significance remain unclear. One increasingly popular hypothesis is that dosage balance constraints are a major determinant of duplicate gene retention. We test this hypothesis and show that WGD-duplicated genes (ohnologs) have rarely experienced subsequent small-scale duplication (SSD) and are also refractory to copy number variation (CNV) in human populations and are thus likely to be sensitive to relative quantities (i.e., they are dosage-balanced). By contrast, genes that have experienced SSD in the vertebrate lineage are more likely to also display CNV. This supports the hypothesis of biased retention of dosage-balanced genes after WGD. We also show that ohnologs have a strong association with human disease. In particular, Down Syndrome (DS) caused by trisomy 21 is widely assumed to be caused by dosage effects, and 75% of previously reported candidate genes for this syndrome are ohnologs that experienced no other copy number changes. We propose the remaining dosage-balanced ohnologs on chromosome 21 as candidate DS genes. These observations clearly show a persistent resistance to dose changes in genes duplicated by WGD. Dosage balance constraints simultaneously explain duplicate gene retention and essentiality after WGD.

Extensive genomic duplication during early chordate evolution.

Opinions on the hypothesis that ancient genome duplications contributed to the vertebrate genome range from strong skepticism to strong credence. Previous studies concentrated on small numbers of gene families or chromosomal regions that might not have been representative of the whole genome, or used subjective methods to identify paralogous genes and regions. Here we report a systematic and objective analysis of the draft human genome sequence to identify paralogous chromosomal regions (paralogons) formed during chordate evolution and to estimate the ages of duplicate genes. We found that the human genome contains many more paralogons than would be expected by chance. Molecular clock analysis of all protein families in humans that have orthologs in the fly and nematode indicated that a burst of gene duplication activity took place in the period 350 650 Myr ago and that many of the duplicate genes formed at this time are located within paralogons. Our results support the contention that many of the gene families in vertebrates were formed or expanded by large-scale DNA duplications in an early chordate. Considering the incompleteness of the sequence data and the antiquity of the event, the results are compatible with at least one round of polyploidy.

Ohnologs and SSD Paralogs Differ in Genomic and Expression Features Related to Dosage Constraints.

Gene duplication is recognized as a critical process in genome evolution; however, many questions about this process remain unanswered. Although gene duplicability has been observed to differ by duplication mechanism and evolutionary rate, there is so far no broad characterization of its determinants. Many features correlate with this difference in duplicability; however, our ability to exploit these observations to advance our understanding of the role of duplication in evolution is hampered by limitations within existing work. In particular, the existence of methodological differences across studies impedes meaningful comparison. Here, we use consistent definitions of duplicability in the human lineage to explore these associations, allow resolution of the impact of confounding factors, and define the overall relevance of individual features. Using a classifier approach and controlling for the confounding effect of duplicate longevity, we find a subset of gene features important in differentiating genes duplicable by small-scale duplication from those duplicable by whole-genome duplication, revealing critical roles for gene dosage and expression costs in duplicability. We further delve into patterns of functional enrichment and find a lack of constraint on duplicate retention in any context for genes duplicable by small-scale duplication.

Independent rediploidization masks shared whole genome duplication in the sturgeon-paddlefish ancestor.

Whole genome duplication (WGD) is a dramatic evolutionary event generating many new genes and which may play a role in survival through mass extinctions. Paddlefish and sturgeon are sister lineages that both show genomic evidence for ancient WGD. Until now this has been interpreted as two independent WGD events due to a preponderance of duplicate genes with independent histories. Here we show that although there is indeed a plurality of apparently independent gene duplications, these derive from a shared genome duplication event occurring well over 200 million years ago, likely close to the Permian-Triassic mass extinction period. This was followed by a prolonged process of reversion to stable diploid inheritance (rediploidization), that may have promoted survival during the Triassic-Jurassic mass extinction. We show that the sharing of this WGD is masked by the fact that paddlefish and sturgeon lineage divergence occurred before rediploidization had proceeded even half-way. Thus, for most genes the resolution to diploidy was lineage-specific. Because genes are only truly duplicated once diploid inheritance is established, the paddlefish and sturgeon genomes are thus a mosaic of shared and non-shared gene duplications resulting from a shared genome duplication event.