Enhanced evolution by stochastically variable modification of epigenetic marks in the early embryo.

Evolution by gene duplication is generally accepted as one of the crucial driving forces for the gain of new complexity and functions, but the formation of pseudogenes remains a problem for this mechanism. Here we expand on earlier ideas that epigenetic modifications can drive neo- and subfunctionalization in evolution by gene duplication. We explore the effects of stochastic epigenetic modifications on the evolution (and thus development) of complex organisms in a constant environment. Modeling is done both using a modified genetic drift analytical treatment and computer simulations, which were found to agree. A transposon silencing model is also explored. Some key assumptions made include (i) stochastic, incomplete removal (or addition) of repressive epigenetic marks takes place during a window(s) of opportunity in the zygote and early embryo; (ii) there is no statistical variation of the marks after the window closes; and (iii) the genes affected are sensitive to dosage. Our genetic drift treatment takes into account that after gene duplication the prevailing case upon which selection operates is a duplicate/singlet heterozygote; to the best of our knowledge, this has not been considered in previous treatments. We conclude from our modeling that stochastic epigenetic modifications, with rates consistent with experimental observation, can both increase the rate of gene fixation and decrease pseudogenization, thus dramatically improving the efficacy of evolution by gene duplication. We also find that a transposon silencing model is advantageous for fixation of recessive genes in diploid organisms, especially with large effective population sizes.

CpG island clusters and pro-epigenetic selection for CpGs in protein-coding exons of HOX and other transcription factors.

CpG dinucleotides contribute to epigenetic mechanisms by being the only site for DNA methylation in mammalian somatic cells. They are also mutation hotspots and approximately 5-fold depleted genome-wide. We report here a study focused on CpG sites in the coding regions of Hox and other transcription factor genes, comparing methylated genomes of Homo sapiens, Mus musculus, and Danio rerio with nonmethylated genomes of Drosophila melanogaster and Caenorhabditis elegans. We analyzed 4-fold degenerate, synonymous codons with the potential for CpG. That is, we studied "silent" changes that do not affect protein products but could damage epigenetic marking. We find that DNA-binding transcription factors and other developmentally relevant genes show, only in methylated genomes, a bimodal distribution of CpG usage. Several genetic code-based tests indicate, again for methylated genomes only, that the frequency of silent CpGs in Hox genes is much greater than expectation. Also informative are NCG-GNN and NCC-GNN codon doublets, for which an unusually high rate of G to C and C to G transversions was observed at the third (silent) position of the first codon. Together these results are interpreted as evidence for strong "pro-epigenetic" selection acting to preserve CpG sites in coding regions of many genes controlling development. We also report that DNA-binding transcription factors and developmentally important genes are dramatically overrepresented in or near clusters of three or more CpG islands, suggesting a possible relationship between evolutionary preservation of CpG dinucleotides in both coding regions and CpG islands.

On primordial sense-antisense coding.

The genetic code is implemented by aminoacyl-tRNA synthetases (aaRS). These 20 enzymes are divided into two classes that, despite performing same functions, have nothing common in structure. The mystery of this striking partition of aaRSs might have been concealed in their sterically complementary modes of tRNA recognition that, as we have found recently, protect the tRNAs with complementary anticodons from confusion in translation. This finding implies that, in the beginning, life increased its coding repertoire by the pairs of complementary codons (rather than one-by-one) and used both complementary strands of genes as templates for translation. The class I and class II aaRSs may represent one of the most important examples of such primordial sense-antisense (SAS) coding (Rodin and Ohno, Orig Life Evol Biosph 25:565-589, 1995). In this report, we address the issue of SAS coding in a wider scope. We suggest a variety of advantages that such coding would have had in exploring a wider sequence space before translation became highly specific. In particular, we confirm that in Achlya klebsiana a single gene might have originally coded for an HSP70 chaperonin (class II aaRS homolog) and an NAD-specific GDH-like enzyme (class I aaRS homolog) via its sense and antisense strands. Thus, in contrast to the conclusions in Williams et al. (Mol Biol Evol 26:445-450, 2009), this could indeed be a "Rosetta stone" gene (Carter and Duax, Mol Cell 10:705-708, 2002) (eroded somewhat, though) for the SAS origin of the two aaRS classes.

Origin of the genetic code: first aminoacyl-tRNA synthetases could replace isofunctional ribozymes when only the second base of codons was established.

Analysis of the updated compilation of more than 8,000 tRNA gene sequences confirmed our previously reported finding that in pairs of consensus tRNAs with complementary anticodons, their second bases in the acceptor stems are also complementary. This dual complementarity points to the following: (1) the operational code embodied in the acceptor stem, and the classic genetic code embodied in the anticodon could have had the same common ancestor; (2) new tRNAs most likely entered primitive translation in pairs with complementary anticodons; and (3) this process of code expansion was directed by the primordial double-strand coding. However, we did not find the dual complementarity when testing all tRNA pairs in which anticodons were complementary only at the central position, but not complementary at least at one of the flanking two positions. This observation, together with certain additional evidence, suggests that both codes were still being shaped (with only the second base established at the time) when the first protein aminoacyl-tRNA synthetases could have already started replacing their ribozymic precursors.

Partitioning of aminoacyl-tRNA synthetases in two classes could have been encoded in a strand-symmetric RNA world.

The "chicken-or-egg" dilemma dictates that archaic tRNAs be aminoacylated by ribozymic aminoacyl-tRNA synthetases, rAARSs, with protein synthetases (pAARSs) emerging later and, strikingly in two versions. However, the distribution of these two versions among the codons also suggests their involvement in development of the genetic code. Here we propose a solution to this controversy, which relies on a primordial complementarity hypothesis that in a strand-symmetric RNA world both complementary replicas of many genes could encode the first proteins. Accordingly, if one rearranges the code table in a manner that puts complementary codons directly against each other, an almost perfect mirror symmetry in tRNA aminoacylation by the two groups of synthetases is revealed. Specifically, the pairs of complementary anticodons from the same pAARS class tend to contain RR and YY dinucleotides at first and second versus third and second positions, whereas in pairs of pAARSs from the different classes these positions are occupied by YR and RY, including CG, GC, UA, and AU palindromes. The latter are indistinguishable in complementary anticodons, thus leading to erroneous aminoacylation (note that there is no such problem for RR- and YY-containing complementary anticodons). This can be averted by "spreading out" tRNA recognition by two rAARSs away from the anticodons in the opposite directions, giving two complementary rAARSs. The principle of evolutionary continuity suggests that their protein successors also arose on complementary strands. Our analyses support this hypothesis.

Repositioning-dependent fate of duplicate genes.

Gene duplication is the main source of evolutionary novelties. However, the problem with duplicates is that the purifying selection overlooks deleterious mutations in the redundant sequence, which therefore, instead of gaining a new function, often degrades into a functionless pseudogene. This risk of functional loss instead of gain is much higher for small populations of higher organisms with a slow and complex development. We propose that it is the epigenetic tissue/stage-complementary silencing of duplicates that makes them exposable to the purifying selection, thus saving them from pseudogenization and opening the way towards new function(s). Our genome-wide analyses of gene duplicates in several eukaryotic species combined with the phylogenetic comparison of vertebrate alpha- and beta-globin gene clusters strongly support this epigenetic complementation (EC) model. The distinctive condition for a new duplicate to survive by the EC mechanism seems to be its repositioning to an ectopic site, which is accompanied by changes in the rate and direction of mutagenesis. The most distinguished in this respect is the human genome. In this review, we extend and discuss the data on the EC- and repositioning-dependent fate of gene duplicates with the special emphasis on the problem of detecting brief postduplication period of adaptive evolution driven by positive selection. Accordingly, we propose a new CpG-focused measure of selection that is insensitive to translocation-caused biases in mutagenesis.

On the excess of G --> T transversions in the p53 gene in lung cancer cell lines. Reply to Pfeifer and Hainaut.

Our recent retrospective analysis of the lung cancer-associated p53 mutation data [Mutat. Res. 508 (2002) 1] showed the possibility of (i) inhibiting action of tobacco smoke on repair of G --> T primary lesions in the non-transcribed strand of the p53 gene and (ii) the origin of new p53 mutations, predominantly G --> T transversions, in lung cancer cell lines apparently unexposed to tobacco smoke. In summary, our arguments suggest that (i) in addition to polycyclic aromatic hydrocarbons (PAH)-DNA adducts there exist other lung cancer-specific, rather than smoke-specific sources of G --> T transversions and (ii) a direct mutagenic action is not the only smoke-associated cause of the prevalence of this class of p53 mutations in lung cancer. In the subsequent critical commentary [Mutat. Res. 526 (2003) 39], Pfeifer and Hainaut suggested our arguments to be "incompatible with available evidence". We would like to address their critique, and appreciate the editors of Mutation Research giving us an opportunity to do so.

On the origin of p53 G:C --> T:A transversions in lung cancers.

The high frequency of G-->T transversions in the p53 gene is a distinctive feature of lung cancer patients with a smoking history and is commonly believed to reflect the direct mutagenic signature of polycyclic aromatic hydrocarbon (PAH) adducts along the gene. Using the April 2000 update of the p53 mutation database of the International Agency for Research on Cancer together with the primary literature, we confirm that the frequency of p53 G-->T transversions in lung cancer of smokers is about three times higher than their frequency in lung cancer of nonsmokers and in most other smoke-unrelated cancers. In contrast, the frequency of C-->A transversions, the DNA-strand mirror counterpart of G-->T transversions, appears to be similar in virtually all human cancers. Along with other data, this strand bias leads us to suggest that smoking may inhibit repair of G-->T primary lesions on the non-transcribed strand. As to the origin of G-->T primary lesions in the p53 gene, we unexpectedly found that cell lines derived from lung cancers, but not from other cancers, demonstrate significant additional excess of G-->T transversions when compared to p53 mutations in parent primary tumors. A detailed codon-by-codon comparison provides evidence in favor of the in vitro origin of this culture-associated G-->T augmentation. Since in culture lung cancer cell lines are not exposed to the carcinogens from smoke, one would rather ascribe these new G-->T transversions to some other mutagens such as, for example, reactive oxygen and nitrogen species. These results are consistent with our previous report [Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 12244], and suggest that other factors, in addition to the direct mutagenic action of PAH-like carcinogens, contribute to p53 mutation-associated lung malignancy.

On origin of genetic code and tRNA before translation.

BACKGROUND: Synthesis of proteins is based on the genetic code - a nearly universal assignment of codons to amino acids (aas). A major challenge to the understanding of the origins of this assignment is the archetypal "key-lock vs. frozen accident" dilemma. Here we re-examine this dilemma in light of 1) the fundamental veto on "foresight evolution", 2) modular structures of tRNAs and aminoacyl-tRNA synthetases, and 3) the updated library of aa-binding sites in RNA aptamers successfully selected in vitro for eight amino acids. RESULTS: The aa-binding sites of arginine, isoleucine and tyrosine contain both their cognate triplets, anticodons and codons. We have noticed that these cases might be associated with palindrome-dinucleotides. For example, one-base shift to the left brings arginine codons CGN, with CG at 1-2 positions, to the respective anticodons NCG, with CG at 2-3 positions. Formally, the concomitant presence of codons and anticodons is also expected in the reverse situation, with codons containing palindrome-dinucleotides at their 2-3 positions, and anticodons exhibiting them at 1-2 positions. A closer analysis reveals that, surprisingly, RNA binding sites for Arg, Ile and Tyr "prefer" (exactly as in the actual genetic code) the anticodon(2-3)/codon(1-2) tetramers to their anticodon(1-2)/codon(2-3) counterparts, despite the seemingly perfect symmetry of the latter. However, since in vitro selection of aa-specific RNA aptamers apparently had nothing to do with translation, this striking preference provides a new strong support to the notion of the genetic code emerging before translation, in response to catalytic (and possibly other) needs of ancient RNA life. Consistently with the pre-translation origin of the code, we propose here a new model of tRNA origin by the gradual, Fibonacci process-like, elongation of a tRNA molecule from a primordial coding triplet and 5'DCCA3' quadruplet (D is a base-determinator) to the eventual 76 base-long cloverleaf-shaped molecule. CONCLUSION: Taken together, our findings necessarily imply that primordial tRNAs, tRNA aminoacylating ribozymes, and (later) the translation machinery in general have been co-evolving to ''fit'' the (likely already defined) genetic code, rather than the opposite way around. Coding triplets in this primal pre-translational code were likely similar to the anticodons, with second and third nucleotides being more important than the less specific first one. Later, when the code was expanding in co-evolution with the translation apparatus, the importance of 2-3 nucleotides of coding triplets "transferred" to the 1-2 nucleotides of their complements, thus distinguishing anticodons from codons. This evolutionary primacy of anticodons in genetic coding makes the hypothesis of primal stereo-chemical affinity between amino acids and cognate triplets, the hypothesis of coding coenzyme handles for amino acids, the hypothesis of tRNA-like genomic 3' tags suggesting that tRNAs originated in replication, and the hypothesis of ancient ribozymes-mediated operational code of tRNA aminoacylation not mutually contradicting but rather co-existing in harmony.

One ancestor for two codes viewed from the perspective of two complementary modes of tRNA aminoacylation.

BACKGROUND: The genetic code is brought into action by 20 aminoacyl-tRNA synthetases. These enzymes are evenly divided into two classes (I and II) that recognize tRNAs from the minor and major groove sides of the acceptor stem, respectively. We have reported recently that: (1) ribozymic precursors of the synthetases seem to have used the same two sterically mirror modes of tRNA recognition, (2) having these two modes might have helped in preventing erroneous aminoacylation of ancestral tRNAs with complementary anticodons, yet (3) the risk of confusion for the presumably earliest pairs of complementarily encoded amino acids had little to do with anticodons. Accordingly, in this communication we focus on the acceptor stem. RESULTS: Our main result is the emergence of a palindrome structure for the acceptor stem's common ancestor, reconstructed from the phylogenetic trees of Bacteria, Archaea and Eukarya. In parallel, for pairs of ancestral tRNAs with complementary anticodons, we present updated evidence of concerted complementarity of the second bases in the acceptor stems. These two results suggest that the first pairs of "complementary" amino acids that were engaged in primordial coding, such as Gly and Ala, could have avoided erroneous aminoacylation if and only if the acceptor stems of their adaptors were recognized from the same, major groove, side. The class II protein synthetases then inherited this "primary preference" from isofunctional ribozymes. CONCLUSION: Taken together, our results support the hypothesis that the genetic code per se (the one associated with the anticodons) and the operational code of aminoacylation (associated with the acceptor) diverged from a common ancestor that probably began developing before translation. The primordial advantage of linking some amino acids (most likely glycine and alanine) to the ancestral acceptor stem may have been selective retention in a protocell surrounded by a leaky membrane for use in nucleotide and coenzyme synthesis. Such acceptor stems (as cofactors) thus transferred amino acids as groups for biosynthesis. Later, with the advent of an anticodon loop, some amino acids (such as aspartic acid, histidine, arginine) assumed a catalytic role while bound to such extended adaptors, in line with the original coding coenzyme handle (CCH) hypothesis.

Translation of both complementary strands might govern early evolution of the genetic code.

The updated structural and phylogenetic analyses of tRNA pairs with complementary anticodons provide independent support for our earlier finding, namely that these tRNA pairs concertedly show complementary second bases in the acceptor stem. Two implications immediately follow: first, that a tRNA molecule gained its present, complete, cloverleaf shape via duplication(s) of a shorter precursor. Second, that common ancestry is shared by two major components of the genetic code within the tRNA molecule--the classic code per se embodied in anticodon triplets, and the operational code of aminoacylation embodied primarily in the first three base pairs of the acceptor stems. In this communication we show that it might have been a double, sense-antisense, in-frame translation of the very first protein-encoding genes that directed the code's earliest expansion, thus preserving this fundamental dual-complementary link between acceptors and anticodons. Furthermore, the dual complementarity appears to be consistent with two mirror-symmetrical modes by which class I and II aminoacyl-tRNA synthetases recognize the cognate tRNAs--from the minor and major groove side of the acceptor stem, respectively.

Origins and selection of p53 mutations in lung carcinogenesis.

Molecular epidemiologists usually consider the spectrum of p53 mutations found in human tumors to be a signature of the corresponding environmental carcinogen(s). In lung cancer, this signature is the spectrum of G --> T transversions, presumably induced by polycyclic aromatic hydrocarbons (PAH) from cigarette smoke. What complicates the situation, however, is that in the p53 gene the same codons are preferential targets for not only mutagenesis but also tumorigenic selection. In this review, we compare the G --> T spectra induced by PAH o-quinones and diol epoxides with those in lung cancer and show that the main "shaper" of the latter is selection, not mutagenesis. In addition, we propose the approach that allows to distinguish selection and mutagenesis components of the p53 spectra and, therefore, to test the suspect carcinogens for their "in vivo" mutagenic involvement. Collectively, the reviewed basic premises, concepts and data are consistent with the increasing recognition of environmental cancer risk conditions as selecting rather than inducing tumorigenic mutations.

Position-associated GC asymmetry of gene duplicates.

It is well known that repositioning of a gene often exerts a strong impact on its own expression and whole development. Here we report the results of genome-wide analyses suggesting that repositioning may also radically change the evolutionary fate of gene duplicates. As an indicator of these changes, we used the GC content of gene pairs which originated by duplication. This indicator turned out to be duplicate-asymmetric, which means that genes in a pair differ significantly in GC content despite their apparent origin from a common ancestor. Such an asymmetry necessarily implies that after duplication two originally identical genes mutated in opposite directions-toward GC-rich and GC-poor content, respectively. In mammalian genomes, this trend is definitely associated with presumably methylated hypermutable CpG sites, and in a typical GC-asymmetric gene pair, its two member genes are embedded in GC-contrasting isochores. However, we unexpectedly found similar significant GC asymmetry in fish, fly, worm, and yeast. This means that neither methylation alone nor methylation in combination with isochores can be counted as a primary cause of the GC asymmetry; rather they represent specific realizations of some universal principle of genome evolution. Remarkably, genes from pairs with the greatest GC asymmetry tend to be on different chromosomes, suggesting that the mutational difference between gene duplicates is associated with translocation of a new gene to a different place in the genome, whereas GC symmetric pairs demonstrate the opposite tendency. A recently emerged extra gene copy is usually on the same chromosome as is its parent but quickly, by 0.05 substitution per synonymous site, either has perished or occupies a different chromosome. During this earliest posttranslocation period, the ratio of nonsynonymous/synonymous base substitutions is unusually high, suggesting a rapid adaptive evolution of novel functions. In a general context of evolution by gene duplication, our interpretation of this position-dependent GC asymmetry between duplicated genes is that evolution of redundant genes toward a new function has often been associated with their very early, postduplication repositioning in the genome, with a concomitant abrupt change in epigenetic control of tissue/stage-specific expression and an increase in the mutation rate. Of eight eukaryotic genomes studied, the most distinguished in this respect is the human genome.

Epigenetic silencing may aid evolution by gene duplication.

Gene duplication is commonly regarded as the main evolutionary path toward the gain of a new function. However, even with gene duplication, there is a loss-versus-gain dilemma: most newly born duplicates degrade to pseudogenes, since degenerative mutations are much more frequent than advantageous ones. Thus, something additional seems to be needed to shift the loss versus gain equilibrium toward functional divergence. We suggest that epigenetic silencing of duplicates might play this role in evolution. This study began when we noticed in a previous publication (Lynch M, Conery JS [2000] Science 291:1151-1155) that the frequency of functional young gene duplicates is higher in organisms that have cytosine methylation (H. sapiens, M. musculus, and A. thaliana) than in organisms that do not have methylated genomes (S. cerevisiae, D. melanogaster, and C. elegans). We find that genome data analysis confirms the likelihood of much more efficient functional divergence of gene duplicates in mammals and plants than in yeast, nematode, and fly. We have also extended the classic model of gene duplication, in which newly duplicated genes have exactly the same expression pattern, to the case when they are epigenetically silenced in a tissue- and/or developmental stage-complementary manner. This exposes each of the duplicates to negative selection, thus protecting from "pseudogenization." Our analysis indicates that this kind of silencing (i) enhances evolution of duplicated genes to new functions, particularly in small populations, (ii) is quite consistent with the subfunctionalization model when degenerative but complementary mutations affect different subfunctions of the gene, and (iii) furthermore, may actually cooperate with the DDC (duplication-degeneration-complementation) process.