Polyploidy Promotes Hypertranscription, Apoptosis Resistance, and Ciliogenesis in Cancer Cells and Mesenchymal Stem Cells of Various Origins: Comparative Transcriptome In Silico Study.

Mesenchymal stem cells (MSC) attract an increasing amount of attention due to their unique therapeutic properties. Yet, MSC can undergo undesirable genetic and epigenetic changes during their propagation in vitro. In this study, we investigated whether polyploidy can compromise MSC oncological safety and therapeutic properties. For this purpose, we compared the impact of polyploidy on the transcriptome of cancer cells and MSC of various origins (bone marrow, placenta, and heart). First, we identified genes that are consistently ploidy-induced or ploidy-repressed through all comparisons. Then, we selected the master regulators using the protein interaction enrichment analysis (PIEA). The obtained ploidy-related gene signatures were verified using the data gained from polyploid and diploid populations of early cardiomyocytes (CARD) originating from iPSC. The multistep bioinformatic analysis applied to the cancer cells, MSC, and CARD indicated that polyploidy plays a pivotal role in driving the cell into hypertranscription. It was evident from the upregulation of gene modules implicated in housekeeping functions, stemness, unicellularity, DNA repair, and chromatin opening by means of histone acetylation operating via DNA damage associated with the NUA4/TIP60 complex. These features were complemented by the activation of the pathways implicated in centrosome maintenance and ciliogenesis and by the impairment of the pathways related to apoptosis, the circadian clock, and immunity. Overall, our findings suggest that, although polyploidy does not induce oncologic transformation of MSC, it might compromise their therapeutic properties because of global epigenetic changes and alterations in fundamental biological processes. The obtained results can contribute to the development and implementation of approaches enhancing the therapeutic properties of MSC by removing polyploid cells from the cell population.

Long-Term Transcriptomic Changes and Cardiomyocyte Hyperpolyploidy after Lactose Intolerance in Neonatal Rats.

Many cardiovascular diseases originate from growth retardation, inflammation, and malnutrition during early postnatal development. The nature of this phenomenon is not completely understood. Here we aimed to verify the hypothesis that systemic inflammation triggered by neonatal lactose intolerance (NLI) may exert long-term pathologic effects on cardiac developmental programs and cardiomyocyte transcriptome regulation. Using the rat model of NLI triggered by lactase overloading with lactose and the methods of cytophotometry, image analysis, and mRNA-seq, we evaluated cardiomyocyte ploidy, signs of DNA damage, and NLI-associated long-term transcriptomic changes of genes and gene modules that differed qualitatively (i.e., were switched on or switched off) in the experiment vs. the control. Our data indicated that NLI triggers the long-term animal growth retardation, cardiomyocyte hyperpolyploidy, and extensive transcriptomic rearrangements. Many of these rearrangements are known as manifestations of heart pathologies, including DNA and telomere instability, inflammation, fibrosis, and reactivation of fetal gene program. Moreover, bioinformatic analysis identified possible causes of these pathologic traits, including the impaired signaling via thyroid hormone, calcium, and glutathione. We also found transcriptomic manifestations of increased cardiomyocyte polyploidy, such as the induction of gene modules related to open chromatin, e.g., "negative regulation of chromosome organization", "transcription" and "ribosome biogenesis". These findings suggest that ploidy-related epigenetic alterations acquired in the neonatal period permanently rewire gene regulatory networks and alter cardiomyocyte transcriptome. Here we provided first evidence indicating that NLI can be an important trigger of developmental programming of adult cardiovascular disease. The obtained results can help to develop preventive strategies for reducing the NLI-associated adverse effects of inflammation on the developing cardiovascular system.

Systemic Alterations of Cancer Cells and Their Boost by Polyploidization: Unicellular Attractor (UCA) Model.

Using meta-analyses, we introduce a unicellular attractor (UCA) model integrating essential features of the 'atavistic reversal', 'cancer attractor', 'somatic mutation', 'genome chaos', and 'tissue organization field' theories. The 'atavistic reversal' theory is taken as a keystone. We propose a possible mechanism of this reversal, its refinement called 'gradual atavism', and evidence for the 'serial atavism' model. We showed the gradual core-to-periphery evolutionary growth of the human interactome resulting in the higher protein interaction density and global interactome centrality in the UC center. In addition, we revealed that UC genes are more actively expressed even in normal cells. The modeling of random walk along protein interaction trajectories demonstrated that random alterations in cellular networks, caused by genetic and epigenetic changes, can result in a further gradual activation of the UC center. These changes can be induced and accelerated by cellular stress that additionally activates UC genes (especially during cell proliferation), because the genes involved in cellular stress response and cell cycle are mostly of UC origin. The functional enrichment analysis showed that cancer cells demonstrate the hyperactivation of energetics and the suppression of multicellular genes involved in communication with the extracellular environment (especially immune surveillance). Collectively, these events can unleash selfish cell behavior aimed at survival at all means. All these changes are boosted by polyploidization. The UCA model may facilitate an understanding of oncogenesis and promote the development of therapeutic strategies.

Gradistics: An underappreciated dimension in evolutionary space.

The growth of complexity is an unsolved and underappreciated problem. We consider possible causes of this growth, hypotheses testing, molecular mechanisms, complexity measures, cases of simplification, and significance for biomedicine. We focus on a general ability of regulation, which is based on the growing information storage and processing capacities, as the main proxy of complexity. Natural selection is indifferent to complexity. However, complexification can be inferred from the same first principle, on which natural selection is founded. Natural selection depends on potentially unlimited reproduction under limited environmental conditions. Because of the demographic pressure, the simple ecological niches become fulfilled and diversified (due to species splitting and divergence). Diversification increases complexity of biocenoses. After the filling and diversification of simple niches, the more complex niches can arise. This is the 'atomic orbitals' (AO) model. Complexity has many shortcomings but it has an advantage. This advantage is ability to regulatory adaptation, including behavioral, formed in the evolution by means of genetic adaptation. Regulatory adaptation is much faster than genetic one because it is based on the information previously accumulated via genetic adaptation and learning. Regulatory adaptation further increases complexity of biocenoses. This is the 'regulatory advantage' (RA) model. The comparison of both models allows testable predictions. We focus on the animal evolution because of the appearance of higher regulatory level (nervous system), which is absent in other lineages, and relevance to humans (including biomedical aspects).

Cellular Biogenetic Law and Its Distortion by Protein Interactions: A Possible Unified Framework for Cancer Biology and Regenerative Medicine.

The biogenetic law (recapitulation law) states that ontogenesis recapitulates phylogenesis. However, this law can be distorted by the modification of development. We showed the recapitulation of phylogenesis during the differentiation of various cell types, using a meta-analysis of human single-cell transcriptomes, with the control for cell cycle activity and the improved phylostratigraphy (gene dating). The multipotent progenitors, differentiated from pluripotent embryonic stem cells (ESC), showed the downregulation of unicellular (UC) genes and the upregulation of multicellular (MC) genes, but only in the case of those originating up to the Euteleostomi (bony vertebrates). This picture strikingly resembles the evolutionary profile of regulatory gene expansion due to gene duplication in the human genome. The recapitulation of phylogenesis in the induced pluripotent stem cells (iPSC) during their differentiation resembles the ESC pattern. The unipotent erythroblasts differentiating into erythrocytes showed the downregulation of UC genes and the upregulation of MC genes originating after the Euteleostomi. The MC interactome neighborhood of a protein encoded by a UC gene reverses the gene expression pattern. The functional analysis showed that the evolved environment of the UC proteins is typical for protein modifiers and signaling-related proteins. Besides a fundamental aspect, this approach can provide a unified framework for cancer biology and regenerative/rejuvenation medicine because oncogenesis can be defined as an atavistic reversal to a UC state, while regeneration and rejuvenation require an ontogenetic reversal.

Polyploidy and Myc Proto-Oncogenes Promote Stress Adaptation via Epigenetic Plasticity and Gene Regulatory Network Rewiring.

Polyploid cells demonstrate biological plasticity and stress adaptation in evolution; development; and pathologies, including cardiovascular diseases, neurodegeneration, and cancer. The nature of ploidy-related advantages is still not completely understood. Here, we summarize the literature on molecular mechanisms underlying ploidy-related adaptive features. Polyploidy can regulate gene expression via chromatin opening, reawakening ancient evolutionary programs of embryonality. Chromatin opening switches on genes with bivalent chromatin domains that promote adaptation via rapid induction in response to signals of stress or morphogenesis. Therefore, stress-associated polyploidy can activate Myc proto-oncogenes, which further promote chromatin opening. Moreover, Myc proto-oncogenes can trigger polyploidization de novo and accelerate genome accumulation in already polyploid cells. As a result of these cooperative effects, polyploidy can increase the ability of cells to search for adaptive states of cellular programs through gene regulatory network rewiring. This ability is manifested in epigenetic plasticity associated with traits of stemness, unicellularity, flexible energy metabolism, and a complex system of DNA damage protection, combining primitive error-prone unicellular repair pathways, advanced error-free multicellular repair pathways, and DNA damage-buffering ability. These three features can be considered important components of the increased adaptability of polyploid cells. The evidence presented here contribute to the understanding of the nature of stress resistance associated with ploidy and may be useful in the development of new methods for the prevention and treatment of cardiovascular and oncological diseases.

Polyploidy as a Fundamental Phenomenon in Evolution, Development, Adaptation and Diseases.

DNA replication during cell proliferation is 'vertical' copying, which reproduces an initial amount of genetic information. Polyploidy, which results from whole-genome duplication, is a fundamental complement to vertical copying. Both organismal and cell polyploidy can emerge via premature cell cycle exit or via cell-cell fusion, the latter giving rise to polyploid hybrid organisms and epigenetic hybrids of somatic cells. Polyploidy-related increase in biological plasticity, adaptation, and stress resistance manifests in evolution, development, regeneration, aging, oncogenesis, and cardiovascular diseases. Despite the prevalence in nature and importance for medicine, agri- and aquaculture, biological processes and epigenetic mechanisms underlying these fundamental features largely remain unknown. The evolutionarily conserved features of polyploidy include activation of transcription, response to stress, DNA damage and hypoxia, and induction of programs of morphogenesis, unicellularity, and longevity, suggesting that these common features confer adaptive plasticity, viability, and stress resistance to polyploid cells and organisms. By increasing cell viability, polyploidization can provide survival under stressful conditions where diploid cells cannot survive. However, in somatic cells it occurs at the expense of specific function, thus promoting developmental programming of adult cardiovascular diseases and increasing the risk of cancer. Notably, genes arising via evolutionary polyploidization are heavily involved in cancer and other diseases. Ploidy-related changes of gene expression presumably originate from chromatin modifications and the derepression of bivalent genes. The provided evidence elucidates the role of polyploidy in evolution, development, aging, and carcinogenesis, and may contribute to the development of new strategies for promoting regeneration and preventing cardiovascular diseases and cancer.

Growth of Biological Complexity from Prokaryotes to Hominids Reflected in the Human Genome.

The growth of complexity in evolution is a most intriguing phenomenon. Using gene phylostratigraphy, we showed this growth (as reflected in regulatory mechanisms) in the human genome, tracing the path from prokaryotes to hominids. Generally, the different regulatory gene families expanded at different times, yet only up to the Euteleostomi (bony vertebrates). The only exception was the expansion of transcription factors (TF) in placentals; however, we argue that this was not related to increase in general complexity. Surprisingly, although TF originated in the Prokaryota while chromatin appeared only in the Eukaryota, the expansion of epigenetic factors predated the expansion of TF. Signaling receptors, tumor suppressors, oncogenes, and aging- and disease-associated genes (indicating vulnerabilities in terms of complex organization and strongly enrichment in regulatory genes) also expanded only up to the Euteleostomi. The complexity-related gene properties (protein size, number of alternative splicing mRNA, length of untranslated mRNA, number of biological processes per gene, number of disordered regions in a protein, and density of TF-TF interactions) rose in multicellular organisms and declined after the Euteleostomi, and possibly earlier. At the same time, the speed of protein sequence evolution sharply increased in the genes that originated after the Euteleostomi. Thus, several lines of evidence indicate that molecular mechanisms of complexity growth were changing with time, and in the phyletic lineage leading to humans, the most salient shift occurred after the basic vertebrate body plan was fixed with bony skeleton. The obtained results can be useful for evolutionary medicine.

Isolation and Characterization of Human Colon Adenocarcinoma Stem-Like Cells Based on the Endogenous Expression of the Stem Markers.

BACKGROUND: Cancer stem cells' (CSCs) self-maintenance is regulated via the pluripotency pathways promoting the most aggressive tumor phenotype. This study aimed to use the activity of these pathways for the CSCs' subpopulation enrichment and separating cells characterized by the OCT4 and SOX2 expression. METHODS: To select and analyze CSCs, we used the SORE6x lentiviral reporter plasmid for viral transduction of colon adenocarcinoma cells. Additionally, we assessed cell chemoresistance, clonogenic, invasive and migratory activity and the data of mRNA-seq and intrinsic disorder predisposition protein analysis (IDPPA). RESULTS: We obtained the line of CSC-like cells selected on the basis of the expression of the OCT4 and SOX2 stem cell factors. The enriched CSC-like subpopulation had increased chemoresistance as well as clonogenic and migration activities. The bioinformatic analysis of mRNA seq data identified the up-regulation of pluripotency, development, drug resistance and phototransduction pathways, and the downregulation of pathways related to proliferation, cell cycle, aging, and differentiation. IDPPA indicated that CSC-like cells are predisposed to increased intrinsic protein disorder. CONCLUSION: The use of the SORE6x reporter construct for CSCs enrichment allows us to obtain CSC-like population that can be used as a model to search for the new prognostic factors and potential therapeutic targets for colon cancer treatment.

Phylostratic Shift of Whole-Genome Duplications in Normal Mammalian Tissues towards Unicellularity Is Driven by Developmental Bivalent Genes and Reveals a Link to Cancer.

Tumours were recently revealed to undergo a phylostratic and phenotypic shift to unicellularity. As well, aggressive tumours are characterized by an increased proportion of polyploid cells. In order to investigate a possible shared causation of these two features, we performed a comparative phylostratigraphic analysis of ploidy-related genes, obtained from transcriptomic data for polyploid and diploid human and mouse tissues using pairwise cross-species transcriptome comparison and principal component analysis. Our results indicate that polyploidy shifts the evolutionary age balance of the expressed genes from the late metazoan phylostrata towards the upregulation of unicellular and early metazoan phylostrata. The up-regulation of unicellular metabolic and drug-resistance pathways and the downregulation of pathways related to circadian clock were identified. This evolutionary shift was associated with the enrichment of ploidy with bivalent genes (p < 10-16). The protein interactome of activated bivalent genes revealed the increase of the connectivity of unicellulars and (early) multicellulars, while circadian regulators were depressed. The mutual polyploidy-c-MYC-bivalent genes-associated protein network was organized by gene-hubs engaged in both embryonic development and metastatic cancer including driver (proto)-oncogenes of viral origin. Our data suggest that, in cancer, the atavistic shift goes hand-in-hand with polyploidy and is driven by epigenetic mechanisms impinging on development-related bivalent genes.

Systemic evolutionary changes in mammalian gene expression.

Changes in gene expression play an important role in evolution and can be relevant to evolutionary medicine. In this work, a strong relationship was found between the statistical significance of evolutionary changes in the expression of orthologous genes in the five or six homologous mammalian tissues and the across-tissues unidirectionality of changes (i.e., they occur in the same direction in different tissues -- all upward or all downward). In the area of highly significant changes, the fraction of unidirectionally changed genes (UCG) was above 0.9 (random expectation is 0.03). This observation indicates that the most pronounced evolutionary changes in mammalian gene expression are systemic (i.e., they operate at the whole-organism level). The UCG are strongly enriched in the housekeeping genes. More specifically, in the human-chimpanzee comparison, the UCG are enriched in the pathways belonging to gene expression (translation is prominent), cell cycle control, ubiquitin-dependent protein degradation (mostly related to cell cycle control), apoptosis, and Parkinson's disease. In the human-macaque comparison, the two other neurodegenerative diseases (Alzheimer's and Huntington's) are added to the enriched pathways. The consolidation of gene expression changes at the level of pathways indicates that they are not neutral but functional. The systemic expression changes probably maintain the across-tissues balance of basic physiological processes in the course of evolution (e.g., during the movement along the fast-slow life axis). These results can be useful for understanding the variation in longevity and susceptibility to cancer and widespread neurodegenerative diseases. This approach can also guide the choice of prospective genes for studies aiming to decipher cis-regulatory code (the gene list is provided).

Cell-cycle dependence of transcriptome gene modules: comparison of regression lines.

The transcriptome consists of various gene modules that can be mutually dependent, and ignoring these dependencies may lead to misinterpretation. The most important problem is module dependence on cell-cycle activity. Using meta-analysis of over 30 000 single-cell transcriptomes, we show gene module dependencies on cell-cycle signature, which can be consistently observed in various normal and cancer cells. Transcript levels of receptors, plasma membrane, and differentiation-related genes are negatively regressed on cell-cycle signature. Pluripotency, stress response, DNA repair, chromatin remodeling, proteasomal protein degradation, protein network connectivity, and unicellular evolutionary origin are regressed positively. These effects cannot be explained by partial overlap of corresponding gene sets because they remain if the overlapped genes were removed. We propose a visual analysis of gene module-specific regression lines as complement to an uncurated enrichment analysis. The different lines for a same gene module indicate different cell conditions. The approach is tested on several problems (polyploidy, pluripotency, cancer, phylostratigraphy). Intriguingly, we found variation in cell-cycle activity, which is independent of cell progression through the cycle. The upregulation of G2/M checkpoint genes with downregulation of G2/M transition and cytokinesis is revealed in polyploid cells. A temporal increase in cell-cycle activity at transition from pluripotent to more differentiated state is found in human embryonic stem cells. The upregulation of unicellular interactome cluster in human cancers is shown in single cells with control for cell-cycle activity. The greater scatter around regression line in cancer cells suggests greater heterogeneity caused by deviation from a line of normal cells.

Cytogenetic and Transcriptomic Analysis of Human Endometrial MSC Retaining Proliferative Activity after Sublethal Heat Shock.

Temperature is an important exogenous factor capable of leading to irreversible processes in the vital activity of cells. However, the long-term effects of heat shock (HS) on mesenchymal stromal cells (MSC) remain unstudied. We investigated the karyotype and DNA repair drivers and pathways in the human endometrium MSC (eMSC) survived progeny at passage 6 after sublethal heat stress (sublethal heat stress survived progeny (SHS-SP)). G-banding revealed an outbreak of random karyotype instability caused by chromosome breakages and aneuploidy. Molecular karyotyping confirmed the random nature of this instability. Transcriptome analysis found homologous recombination (HR) deficiency that most likely originated from the low thermostability of the AT-rich HR driving genes. SHS-SP protection from transformation is provided presumably by low oncogene expression maintained by tight co-regulation between thermosensitive HR drivers BRCA, ATM, ATR, and RAD51 (decreasing expression after SHS), and oncogenes mTOR, MDM2, KRAS, and EGFR. The cancer-related transcriptomic features previously identified in hTERT transformed MSC in culture were not found in SHS-SP, suggesting no traits of malignancy in them. The entrance of SHS-SP into replicative senescence after 25 passages confirms their mortality and absence of transformation features. Overall, our data indicate that SHS may trigger non-tumorigenic karyotypic instability due to HR deficiency and decrease of oncogene expression in progeny of SHS-survived MSC. These data can be helpful for the development of new therapeutic approaches in personalized medicine.

Somatic polyploidy is associated with the upregulation of c-MYC interacting genes and EMT-like signature.

The dependence of cancer on overexpressed c-MYC and its predisposition for polyploidy represents a double puzzle. We address this conundrum by cross-species transcription analysis of c-MYC interacting genes in polyploid vs. diploid tissues and cells, including human vs. mouse heart, mouse vs. human liver and purified 4n vs. 2n mouse decidua cells. Gene-by-gene transcriptome comparison and principal component analysis indicated that c-MYC interactants are significantly overrepresented among ploidy-associated genes. Protein interaction networks and gene module analysis revealed that the most upregulated genes relate to growth, stress response, proliferation, stemness and unicellularity, as well as to the pathways of cancer supported by MAPK and RAS coordinated pathways. A surprising feature was the up-regulation of epithelial-mesenchymal transition (EMT) modules embodied by the N-cadherin pathway and EMT regulators from SNAIL and TWIST families. Metabolic pathway analysis also revealed the EMT-linked features, such as global proteome remodeling, oxidative stress, DNA repair and Warburg-like energy metabolism. Genes associated with apoptosis, immunity, energy demand and tumour suppression were mostly down-regulated. Noteworthy, despite the association between polyploidy and ample features of cancer, polyploidy does not trigger it. Possibly it occurs because normal polyploidy does not go that far in embryonalisation and linked genome destabilisation. In general, the analysis of polyploid transcriptome explained the evolutionary relation of c-MYC and polyploidy to cancer.

Accelerated pathway evolution in mouse-like rodents involves cell cycle control.

Rodents include both the cancer-susceptible short-lived mouse and the two unrelated cancer-resistant long-lived mole-rats. In this work, their genomes were analyzed with the goal to reveal pathways enriched in genes, which are more similar between the mole-rats than between the mouse and the naked mole-rat. The pathways related to cell cycle control were prominent. They include external signal transduction and all cell cycle stages. There are several stem cell pathways among them. The other enriched pathways involve ubiquitin-dependent protein degradation, immunity, mRNA splicing, and apoptosis. The ubiquitin-dependent protein degradation is a core of network of enriched pathways. However, this phenomenon is not specific for the mouse and the mole-rats. The other muroid species show features similar to the mouse, whereas the non-muroid rodents and the human show features similar to the mole-rats. The higher ratio of non-synonymous to synonymous nucleotide substitutions (dN/dS) indicates the accelerated evolution of revealed pathways in the muroid rodents (except the blind mole-rat). Paradoxically, the dN/dS averaged over the whole genome is lower in the muroids, i.e., the purifying selection is generally stronger in them. In practical sense, these data suggest caveat for using muroid rodents (mouse, rat, and hamsters) as biomedical models of human conditions involving cell cycle and show the network of pathways where muroid genes are most different (compared with non-muroid) from human genes. The guinea pig is emphasized as a more suitable rodent model for biomedical research involving cell cycle.

Consolidation of slow or fast but not moderately evolving genes at the level of pathways and processes.

Conservatism versus innovation is probably the most important dichotomy of all evolving systems. In molecular evolution the distinction between conservative (negative) selection, innovative (positive) selection and unconstrained evolution (drift) is usually ambiguous at the gene level. Only rare cases with the ratio of nonsynonymous to synonymous nucleotide substitutions above unity (dN/dS>1) are thought to be due to positive selection, whereas the lower dN/dS ratio may indicate negative selection in combination with drift. The density of the dN/dS ratio for orthologous genes forms a unimodal distribution where no particular regions can be discerned. Here it is shown that at the level of overrepresented pathways and processes the picture is strikingly different. The distribution is strongly polarized with a wide completely depressed middle part. This three-phase distribution is very robust. It is observed with various substitution models and remains at very low significance of overrepresentation (up to p<0.99). This fact suggests consolidation of either negative or positive selection but not of unconstrained evolution at the level of pathways/processes. The effect is demonstrated for different phylogenetic distances: from human to other primates, mammals and vertebrates. This approach suggests estimating the boundaries for conservative and innovative selection using the pathway/process level. Emphasizing the role of a critical mass of negatively or positively selected genes in a pathway/process, it can elucidate how the bridge between 'tinkering' at the gene level and 'design' at the higher levels is forming.

Density peaks of paralog pairs in human and mouse genomes.

Paralog gene trees, which reflect the increase of genomic complexity in the evolution, can be complicated and ambiguous. A simpler complementary approach is analysis of density distribution of paralog pairs. It can reveal general features of genome evolution, which may be hidden in the forest of gene trees. It is known that distribution of human paralog pairs along the axis of protein divergence between pair members forms two main peaks. Here I show that there are three main peaks in the mouse genome. Thus, the multimodality of paralog pair distribution seems to be a fundamental feature of mammalian genomes. Despite the great diversity of domains presented in small amounts or in multidomain architectures with a few predominant domains, both in human and mouse the first peak consists mostly of gene pairs with zinc finger domains or olfactory receptor domain. In the mouse the olfactory receptor predominates, which stipulates the three-peak distribution (since in the olfactory receptors the second peak is closer to the first peak than in other genes). The mammalian-wide zinc finger orthologs are biased towards the second peak. Thus, the marsupial orthologs are nearly absent in the first peak of human and mouse. The gene pairs in the first peak show a lower ratio of nonsynonymous to synonymous substitutions, which suggests that their evolution is more constrained. The plausible explanation is that they are in subfunctionalization state (partition of initial function of ancestral gene), whereas the second peak contains gene pairs that are already in neofunctionalization state (acquiring of novel functions). These data suggest that the adaptive radiation of mammals was accompanied by a burst of duplication of zinc finger genes, which are located in the first (most recent) peak of paralog pairs.

Large scale of human duplicate genes divergence.

Proteome complexity increases in the evolution mostly by means of gene duplication followed by divergence. In this genome-scale study of human genome I show that density distribution of duplicate gene pairs along the axis of protein divergence between pair members forms two main peaks with a small peak and plateau before the first main peak. This picture indicates the existence of three evolutionary stages of duplicate gene evolution. The analysis of various functional parameters (gene expression level and breadth, transcription factor targets, protein interaction networks) suggests that subfunctionalization (partition of function) is a predominant mode of divergence in the first main peak, whereas neofunctionalization (acquiring of novel functions) prevails in the second main peak. The young duplicate pairs show a much higher expression level compared with singleton genes and more diverged duplicates, which indicates that requirement for high gene dosage is important for retention of duplicates just after the duplication event. Thus, a prevailing route of duplicate evolution seems to be the high gene dosage-subfunctionalization-neofunctionalization. This adaptationist model suggests that an organism is evolving in the direction of its most intensively used functions.

Minority of mammalian orthologs can be regarded as physiologically closest genes.

Orthologs are genes from different genomes that originate from a common ancestor gene by speciation event. They are most similar by the structure of encoded proteins and therefore should have a similar function. Here I apply the principle used for detection of structural orthology for a genome-wide analysis of gene expression. For this purpose, I determine the mutual similarity rank in all-by-all comparison of among-tissues expression patterns. The expression of most part of human-mouse orthologs in homologous tissues is poorly correlated (average mutual coexpression rank is only 4835 out of 18,092). Genes from evolutionarily labile gene families, which experience rapid turnover of family composition, are among those with the strongest expression change. However, the revealed phenomenon is not limited to them. There is no or very weak relationship between protein sequence divergence and mutual coexpression rank. Also, generally there is no relationship between the ratio of nonsynonymous to synonymous nucleotide substitutions and coexpression rank. This relationship is tangible only within evolutionarily labile gene families. These results indicate that despite of a similar biochemical function of orthologs reflected in the conserved protein sequence, the physiological (systemic) context of this function can be changed. Also, these results suggest that gene biochemical function and its physiological role in the organism can evolve independently.

Human more complex than mouse at cellular level.

The family of transcription factors with the C2H2 zinc finger domain is expanding in the evolution of vertebrates, reaching its highest numbers in the mammals. The question arises: whether an increased amount of these transcription factors is related to embryogenesis, nervous system, pathology or more of them are expressed in individual cells? Among mammals, the primates have a more complex anatomical structure than the rodents (e.g., brain). In this work, I show that a greater number of C2H2-ZF genes are expressed in the human cells than in the mouse cells. The effect is especially pronounced for C2H2-ZF genes accompanied with the KRAB domain. The relative difference between the numbers of C2H2-ZF(-KRAB) genes in the human and mouse cellular transcriptomes even exceeds their difference in the genomes (i.e. a greater subset of existing in the genome genes is expressed in the human cellular transcriptomes compared to the mouse transcriptomes). The evolutionary turnover of C2H2-ZF(-KRAB) genes acts in the direction of the revealed phenomenon, i.e. gene duplication and loss enhances the difference in the relative number of C2H2-ZF(-KRAB) genes between human and mouse cellular transcriptomes. A higher amount of these genes is expressed in the brain and embryonic cells (compared with other tissues), whereas a lower amount--in the cancer cells. It is specifically the C2H2-ZF transcription factors whose repertoire is poorer in the cancer and richer in the brain (other transcription factors taken together do not show this trend). These facts suggest that increase of anatomical complexity is accompanied by a more complex intracellular regulation involving these transcription factors. Malignization is associated with simplification of this regulation. These results agree with the known fact that human cells are more resistant to oncogenic transformation than mouse cells. The list of C2H2-ZF genes whose suppression might be involved in malignization is provided.