Sergey Igorievich Nikolaev

Phylogenomics Reshuffles the Eukaryotic Supergroups

Background Resolving the phylogenetic relationships between eukaryotes is an ongoing challenge of evolutionary biology. In recent years, the accumulation of molecular data led to a new evolutionary understanding, in which all eukaryotic diversity has been classified into five or six supergroups. Yet, the composition of these large assemblages and their relationships remain controversial. Methodology/Principle Findings Here, we report the sequencing of expressed sequence tags (ESTs) for two species belonging to the supergroup Rhizaria and present the analysis of a unique dataset combining 29908 amino acid positions and an extensive taxa sampling made of 49 mainly unicellular species representative of all supergroups. Our results show a very robust relationship between Rhizaria and two main clades of the supergroup chromalveolates: stramenopiles and alveolates. We confirm the existence of consistent affinities between assemblages that were thought to belong to different supergroups of eukaryotes, thus not sharing a close evolutionary history. Conclusions This well supported phylogeny has important consequences for our understanding of the evolutionary history of eukaryotes. In particular, it questions a single red algal origin of the chlorophyll-c containing plastids among the chromalveolates. We propose the abbreviated name ‘SAR’ (Stramenopiles+Alveolates+Rhizaria) to accommodate this new super assemblage of eukaryotes, which comprises the largest diversity of unicellular eukaryotes.

Exome sequencing identifies recurrent somatic MAP2K1 and MAP2K2 mutations in melanoma

We performed exome sequencing to detect somatic mutations in protein-coding regions in seven melanoma cell lines and donor-matched germline cells. All melanoma samples had high numbers of somatic mutations, which showed the hallmark of UV-induced DNA repair. Such a hallmark was absent in tumor sample–specific mutations in two metastases derived from the same individual. Two melanomas with non-canonical BRAF mutations harbored gain-of-function MAP2K1 and MAP2K2 (MEK1 and MEK2, respectively) mutations, resulting in constitutive ERK phosphorylation and higher resistance to MEK inhibitors. Screening a larger cohort of individuals with melanoma revealed the presence of recurring somatic MAP2K1 and MAP2K2 mutations, which occurred at an overall frequency of 8%. Furthermore, missense and nonsense somatic mutations were frequently found in three candidate melanoma genes, FAT4, LRP1B and DSC1.

Early history of mammals is elucidated with the ENCODE multiple species sequencing data.

Understanding the early evolution of placental mammals is one of the most challenging issues in mammalian phylogeny. Here, we addressed this question by using the sequence data of the ENCODE consortium, which include 1% of mammalian genomes in 18 species belonging to all main mammalian lineages. Phylogenetic reconstructions based on an unprecedented amount of coding sequences taken from 218 genes resulted in a highly supported tree placing the root of Placentalia between Afrotheria and Exafroplacentalia (Afrotheria hypothesis). This topology was validated by the phylogenetic analysis of a new class of genomic phylogenetic markers, the conserved noncoding sequences. Applying the tests of alternative topologies on the coding sequence dataset resulted in the rejection of the Atlantogenata hypothesis (Xenarthra grouping with Afrotheria), while this test rejected the second alternative scenario, the Epitheria hypothesis (Xenarthra at the base), when using the noncoding sequence dataset. Thus, the two datasets support the Afrotheria hypothesis; however, none can reject both of the remaining topological alternatives.

Genomic analysis identifies new drivers and progression pathways in skin basal cell carcinoma

Basal cell carcinoma (BCC) of the skin is the most common malignant neoplasm in humans. BCC is primarily driven by the Sonic Hedgehog (Hh) pathway. However, its phenotypic variation remains unexplained. Our genetic profiling of 293 BCCs found the highest mutation rate in cancer (65 mutations/Mb). Eighty-five percent of the BCCs harbored mutations in Hh pathway genes (PTCH1, 73% or SMO, 20% (P = 6.6 × 10−8) and SUFU, 8%) and in TP53 (61%). However, 85% of the BCCs also harbored additional driver mutations in other cancer-related genes. We observed recurrent mutations in MYCN (30%), PPP6C (15%), STK19 (10%), LATS1 (8%), ERBB2 (4%), PIK3CA (2%), and NRAS, KRAS or HRAS (2%), and loss-of-function and deleterious missense mutations were present in PTPN14 (23%), RB1 (8%) and FBXW7 (5%). Consistent with the mutational profiles, N-Myc and Hippo-YAP pathway target genes were upregulated. Functional analysis of the mutations in MYCN, PTPN14 and LATS1 suggested their potential relevance in BCC tumorigenesis.

Life-history traits drive the evolutionary rates of mammalian coding and noncoding genomic elements

A comprehensive phylogenetic framework is indispensable for investigating the evolution of genomic features in mammals as a whole, and particularly in humans. Using the ENCODE sequence data, we estimated mammalian neutral evolutionary rates and selective pressures acting on conserved coding and noncoding elements. We show that neutral evolutionary rates can be explained by the generation time (GT) hypothesis. Accordingly, primates (especially humans), having longer GTs than other mammals, display slower rates of neutral evolution. The evolution of constrained elements, particularly of nonsynonymous sites, is in agreement with the expectations of the nearly neutral theory of molecular evolution. We show that rates of nonsynonymous substitutions (dN) depend on the population size of a species. The results are robust to the exclusion of hypermutable CpG prone sites. The average rate of evolution in conserved noncoding sequences (CNCs) is 1.7 times higher than in nonsynonymous sites. Despite this, CNCs evolve at similar or even lower rates than nonsynonymous sites in the majority of basal branches of the eutherian tree. This observation could be the result of an overall gradual or, alternatively, lineage-specific relaxation of CNCs. The latter hypothesis was supported by the finding that 3 of the 20 longest CNCs displayed significant relaxation of individual branches. This observation may explain why the evolution of CNCs fits the expectations of the nearly neutral theory less well than the evolution of nonsynonymous sites.

A Single-Nucleotide Substitution Mutator Phenotype Revealed by Exome Sequencing of Human Colon Adenomas

Oncogene-induced DNA replication stress is thought to drive genomic instability in cancer. In particular, replication stress can explain the high prevalence of focal genomic deletions mapping within very large genes in human tumors. However, the origin of single-nucleotide substitutions (SNS) in nonfamilial cancers is strongly debated. Some argue that cancers have a mutator phenotype, whereas others argue that the normal DNA replication error rates are sufficient to explain the number of observed SNSs. Here, we sequenced the exomes of 24, mostly precancerous, colon polyps. Analysis of the sequences revealed mutations in the APC, CTNNB1, and BRAF genes as the presumptive cancer-initiating events and many passenger SNSs. We used the number of SNSs in the various lesions to calculate mutation rates for normal colon and adenomas and found that colon adenomas exhibit a mutator phenotype. Interestingly, the SNSs in the adenomas mapped more often than expected within very large genes, where focal deletions in response to DNA replication stress also map. We propose that single-stranded DNA generated in response to oncogene-induced replication stress compromises the repair of deaminated cytosines and other damaged bases, leading to the observed SNS mutator phenotype.

Purifying Selection in Mammalian Mitochondrial Protein-Coding Genes Is Highly Effective and Congruent with Evolution of Nuclear Genes

The mammalian mitochondrial genomes differ from the nuclear genomes by maternal inheritance, absence of recombination, and higher mutation rate. All these differences decrease the effective population size of mitochondrial genome and make it more susceptible to accumulation of slightly deleterious mutations. It was hypothesized that mitochondrial genes, especially in species with low effective population size, irreversibly degrade leading to decrease of organismal fitness and even to extinction of species through the mutational meltdown. To interrogate this hypothesis, we compared the purifying selections acting on the representative set of mitochondrial (potentially degrading) and nuclear (potentially not degrading) protein-coding genes in species with different effective population size. For 21 mammalian species, we calculated the ratios of accumulation of slightly deleterious mutations approximated by Kn/Ks separately for mitochondrial and nuclear genomes. The 75% of variation in Kn/Ks is explained by two independent variables: type of a genome (mitochondrial or nuclear) and effective population size of species approximated by generation time. First, we observed that purifying selection is more effective in mitochondria than in the nucleus that implies strong evolutionary constraints of mitochondrial genome. Mitochondrial de novo nonsynonymous mutations have at least 5-fold more harmful effect when compared with nuclear. Second, Kn/Ks of mitochondrial and nuclear genomes is positively correlated with generation time of species, indicating relaxation of purifying selection with decrease of species-specific effective population size. Most importantly, the linear regression lines of mitochondrial and nuclear Kn/Kss from generation times of species are parallel, indicating congruent relaxation of purifying selection in both genomes. Thus, our results reveal that the distribution of selection coefficients of de novo nonsynonymous mitochondrial mutations has a similar shape with the distribution of de novo nonsynonymous nuclear mutations, but its mean is five times smaller. The harmful effect of mitochondrial de novo nonsynonymous mutations triggers highly effective purifying selection, which maintains the fitness of the mammalian mitochondrial genome.

The Not3/5 subunit of the Ccr4-Not complex: A central regulator of gene expression that integrates signals between the cytoplasm and the nucleus in eukaryotic cells

The Ccr4-Not complex is a conserved multi-subunit complex in eukaryotes that carries 2 enzymatic activities: ubiquitination mediated by the Not4 RING E3 ligase and deadenylation mediated by the Ccr4 and Caf1 orthologs. This complex has been implicated in all aspects of the mRNA life cycle, from synthesis of mRNAs in the nucleus to their degradation in the cytoplasm. More recently the complex has also been implicated in many aspects of the life cycle of proteins, from quality control during synthesis of peptides, to assembly of protein complexes and protein degradation. Consistently, the Ccr4-Not complex is found both in the nucleus, where it is connected to transcribing ORFs, and in the cytoplasm, where it was revealed to be both associated with translating ribosomes and in RNA processing bodies. This functional and physical presence of the Ccr4-Not complex at all stages of gene expression raises the question of its fundamental role. This review will summarize recent evidence designing the Not3/5 module of the Ccr4-Not complex as a functional module involved in coordination of the regulation of gene expression between the nucleus and the cytoplasm.

Frequent cases of RAS-mutated Down syndrome acute lymphoblastic leukaemia lack JAK2 mutations

Children with Down syndrome (DS) and acute lymphoblastic leukaemia (ALL) have poorer survival and more relapses than non-DS children with ALL, highlighting an urgent need for deeper mechanistic understanding of DS-ALL. Here, using full-exome or cancer genes-targeted sequencing of 42 ALL samples from 39 DS patients, we uncover driver mutations in RAS, (KRAS and NRAS) recurring to a similar extent (15/42) as JAK2 (12/42) mutations or P2RY8-CRLF2 fusions (14/42). RAS mutations are almost completely mutually exclusive with JAK2 mutations (P=0.016), driving a combined total of two-thirds of analysed cases. Clonal architecture analysis reveals that both RAS and JAK2 drove sub-clonal expansions primarily initiated by CRLF2 rearrangements, and/or mutations in chromatin remodellers and lymphocyte differentiation factors. Remarkably, in 2/3 relapsed cases, there is a switch from a primary JAK2- or PTPN11-mutated sub-clone to a RAS-mutated sub-clone in relapse. These results provide important new insights informing the patient stratification strategies for targeted therapeutic approaches for DS-ALL.

Gene Age Predicts the Strength of Purifying Selection Acting on Gene Expression Variation in Humans

Gene expression levels can be subject to selection. We hypothesized that the age of gene origin is associated with expression constraints, given that it affects the level of gene integration into the functional cellular environment. By studying the genetic variation affecting gene expression levels (cis expression quantitative trait loci [cis-eQTLs]) and protein levels (cis protein QTLs [cis-pQTLs]), we determined that young, primate-specific genes are enriched in cis-eQTLs and cis-pQTLs. Compared to cis-eQTLs of old genes originating before the zebrafish divergence, cis-eQTLs of young genes have a higher effect size, are located closer to the transcription start site, are more significant, and tend to influence genes in multiple tissues and populations. These results suggest that the expression constraint of each gene increases throughout its lifespan. We also detected a positive correlation between expression constraints (approximated by cis-eQTL properties) and coding constraints (approximated by Ka/Ks) and observed that this correlation might be driven by gene age. To uncover factors associated with the increase in gene-age-related expression constraints, we demonstrated that gene connectivity, gene involvement in complex regulatory networks, gene haploinsufficiency, and the strength of posttranscriptional regulation increase with gene age. We also observed an increase in heritability of gene expression levels with age, implying a reduction of the environmental component. In summary, we show that gene age shapes key gene properties during evolution and is therefore an important component of genome function.