Bernardo Lemos

Polymorphic Y Chromosomes Harbor Cryptic Variation with Manifold Functional Consequences

The paucity of polymorphisms in single-copy genes on the Y chromosome of Drosophila contrasts with data indicating that this chromosome has polymorphic phenotypic effects on sex ratio, temperature sensitivity, behavior, and fitness. We show that the Y chromosome of D. melanogaster harbors substantial genetic diversity in the form of polymorphisms for genetic elements that differentially affect the expression of hundreds of X-linked and autosomal genes. The affected genes are more highly expressed in males, more meagerly expressed in females, and more highly divergent between species. Functionally, they affect microtubule stability, lipid and mitochondrial metabolism, and the thermal sensitivity of spermatogenesis. Our findings provide a mechanism for adaptive phenotypic variation associated with the Y chromosome.

RATES OF DIVERGENCE IN GENE EXPRESSION PROFILES OF PRIMATES, MICE, AND FLIES: STABILIZING SELECTION AND VARIABILITY AMONG FUNCTIONAL CATEGORIES

Abstract The extent to which natural selection shapes phenotypic variation has long been a matter of debate among those studying organic evolution. We studied the patterns of gene expression polymorphism and divergence in several datasets that ranged from comparisons between two very closely related laboratory strains of mice to comparisons across a considerably longer time scale, such as between humans and chimpanzees, two species of mice, and two species of Drosophila. The results were analyzed and interpreted in view of neutral models of phenotypic evolution. Our analyses used a number of metrics to show that most mRNA levels are evolutionary stable, changing little across the range of taxonomic distances compared. This implies that, overall, widespread stabilizing selection on transcription levels has prevented greater evolutionary changes in mRNA levels. Nevertheless, the range of rates of divergence is large with highly significant differences in the rate and patterns of transcription divergence across functional classes defined on the basis of the gene ontology annotation (primates and mice datasets) or on the basis of the pattern of sex‐biased gene expression (Drosophila). Moreover, rates of divergence of sex‐biased genes in the contrast between Drosophila species show a distinct pattern from that observed in the contrast between populations of D. melanogaster. Hence, we discuss the time scale of the changes observed and its consequences for the relationship between variation in gene expression within and between species. Finally, we argue that differences in mRNA levels of the magnitudes observed herein could be explained by a remarkably small number of generations of directional selection.

Epigenetic effects of polymorphic Y chromosomes modulate chromatin components, immune response, and sexual conflict

Genetic conflicts between sexes and generations provide a foundation for understanding the functional evolution of sex chromosomes and sexually dimorphic phenotypes. Y chromosomes of Drosophila contain multi-megabase stretches of satellite DNA repeats and a handful of protein-coding genes that are monomorphic within species. Nevertheless, polymorphic variation in heterochromatic Y chromosomes of Drosophila result in genome-wide gene expression variation. Here we show that such naturally occurring Y-linked regulatory variation (YRV) can be detected in somatic tissues and contributes to the epigenetic balance of heterochromatin/euchromatin at three distinct loci showing position-effect variegation (PEV). Moreover, polymorphic Y chromosomes differentially affect the expression of thousands of genes in XXY female genotypes in which Y-linked protein-coding genes are not transcribed. The data show a disproportionate influence of YRV on the variable expression of genes whose protein products localize to the nucleus, have nucleic-acid binding activity, and are involved in transcription, chromosome organization, and chromatin assembly. These include key components such as HP1, Trithorax-like (GAGA factor), Su(var)3–9, Brahma, MCM2, ORC2, and inner centromere protein. Furthermore, mitochondria-related genes, immune response genes, and transposable elements are also disproportionally affected by Y chromosome polymorphism. These functional clusterings may arise as a consequence of the involvement of Y-linked heterochromatin in the origin and resolution of genetic conflicts between males and females. Taken together, our results indicate that Y chromosome heterochromatin serves as a major source of epigenetic variation in natural populations that interacts with chromatin components to modulate the expression of biologically relevant phenotypic variation.

Dominance and the evolutionary accumulation of cis- and trans-effects on gene expression

Gene expression levels appear to be under pervasive stabilizing selection. Yet the genetic architecture underlying abundant gene expression diversity within and between populations remains elusive. Here, we investigated the role of dominance in the segregation of cis- and trans-regulation within and between populations. We used chromosome substitution lines of Drosophila melanogaster to show that (i) >70% of the genes that are differentially expressed between two homozygous lines are masked in the heterozygous, suggesting that one of the substituted chromosomes contains a recessive allele; (ii) such large masking is already obtained with heterozygous chromosomes originating from the same population, with the time of divergence between chromosomes in heterozygous lines making only a small but significant contribution to the masking of variation observed in homozygous lines; (iii) variation in gene expression due to trans-regulation is biased toward greater deviations from additivity because of recessive and dominant alleles, whereas variation due to cis-regulation shows higher additivity; and (iv) genetic divergence between second chromosomes is associated with increased cis-regulation, whereas the level of trans-regulation shows little increase over the time scale studied. Our results indicate that cis-acting alleles may be preferentially fixed by positive natural selection because of their higher additivity, and that the disruption of gene expression by recessive variation with pervasive trans-effects may be important for understanding gene expression variation within populations. We suggest that widespread regulatory effects of recessive low-frequency homozygous variation may provide a general mechanism mediating disease phenotypes and the genetic load of natural populations.

Male sterility at extreme temperatures: a significant but neglected phenomenon for understanding Drosophila climatic adaptations

The thermal range for viability is quite variable among Drosophila species and it has long been known that these variations are correlated with geographic distribution: temperate species are on average more cold tolerant but more heat sensitive than tropical species. At both ends of their viability range, sterile males have been observed in all species investigated so far. This symmetrical phenomenon restricts the temperature limits within which permanent cultures can be kept in the laboratory. Thermal heat sterility thresholds are very variable across species from 23 °C in heat sensitive species up to 31 °C in heat tolerant species. In Drosophila melanogaster, genetic variations are observed among geographic populations. Tropical populations are more tolerant to heat induced sterility and recover more rapidly than temperate ones. A genetic analysis revealed that about 50% of the difference observed between natural populations was due to the Y chromosome. Natural populations have not reached a selection limit, however: thermal tolerance was still increased by keeping strains at a high temperature, close to the sterility threshold. On the low temperature side, a symmetrical reverse phenomenon seems to exist: temperate populations are more tolerant to cold than tropical ones. Compared to Mammals, drosophilids exhibit two major differences: first, male sterility occurs not only at high temperature, but also at a low temperature; second, sterility thresholds are not evolutionarily constrained, but highly variable. Altogether, significant and sometimes major genetic variations have been observed between species, between geographic races of the same species, and even between strains kept in the laboratory under different thermal regimes. In each case, it is easily argued that the observed variations correspond to adaptations to climatic conditions, and that male sterility is a significant component of fitness and a target of natural selection.

Ribosomal DNA Deletions Modulate Genome-Wide Gene Expression: ‘‘rDNA–Sensitive’’ Genes and Natural Variation

The ribosomal rDNA gene array is an epigenetically-regulated repeated gene locus. While rDNA copy number varies widely between and within species, the functional consequences of subtle copy number polymorphisms have been largely unknown. Deletions in the Drosophila Y-linked rDNA modifies heterochromatin-induced position effect variegation (PEV), but it has been unknown if the euchromatic component of the genome is affected by rDNA copy number. Polymorphisms of naturally occurring Y chromosomes affect both euchromatin and heterochromatin, although the elements responsible for these effects are unknown. Here we show that copy number of the Y-linked rDNA array is a source of genome-wide variation in gene expression. Induced deletions in the rDNA affect the expression of hundreds to thousands of euchromatic genes throughout the genome of males and females. Although the affected genes are not physically clustered, we observed functional enrichments for genes whose protein products are located in the mitochondria and are involved in electron transport. The affected genes significantly overlap with genes affected by natural polymorphisms on Y chromosomes, suggesting that polymorphic rDNA copy number is an important determinant of gene expression diversity in natural populations. Altogether, our results indicate that subtle changes to rDNA copy number between individuals may contribute to biologically relevant phenotypic variation.

Copy-number variation: the balance between gene dosage and expression in Drosophila melanogaster.

Copy-number variants (CNVs) reshape gene structure, modulate gene expression, and contribute to significant phenotypic variation. Previous studies have revealed CNV patterns in natural populations of Drosophila melanogaster and suggested that selection and mutational bias shape genomic patterns of CNV. Although previous CNV studies focused on heterogeneous strains, here, we established a number of second-chromosome substitution lines to uncover CNV characteristics when homozygous. The percentage of genes harboring CNVs is higher than found in previous studies. More CNVs are detected in homozygous than heterozygous substitution strains, suggesting the comparative genomic hybridization arrays underestimate CNV owing to heterozygous masking. We incorporated previous gene expression data collected from some of the same substitution lines to investigate relationships between CNV gene dosage and expression. Most genes present in CNVs show no evidence of increased or diminished transcription, and the fraction of such dosage-insensitive CNVs is greater in heterozygotes. More than 70% of the dosage-sensitive CNVs are recessive with undetectable effects on transcription in heterozygotes. A deficiency of singletons in recessive dosage-sensitive CNVs supports the hypothesis that most CNVs are subject to negative selection. On the other hand, relaxed purifying selection might account for the higher number of protein–protein interactions in dosage-insensitive CNVs than in dosage-sensitive CNVs. Dosage-sensitive CNVs that are upregulated and downregulated coincide with copy-number increases and decreases. Our results help clarify the relation between CNV dosage and gene expression in the D. melanogaster genome.

Regulatory evolution across the protein interaction network

Protein-protein interactions may impose constraints on both structural and regulatory evolution. Here we show that protein-protein interactions are negatively associated with evolutionary variation in gene expression. Moreover, interacting proteins have similar levels of variation in expression, and their expression levels are positively correlated across strains. Our results suggest that interacting proteins undergo similar evolutionary dynamics, and that their expression levels are evolutionarily coupled. These patterns hold for organisms as diverse as budding yeast and fruit flies.

Concerted copy number variation balances ribosomal DNA dosage in human and mouse genomes

Significance Ribosomes are essential intracellular machines composed of proteins and RNA molecules. The DNA sequences [i.e., ribosomal DNA (rDNA)] encoding rRNAs are tandemly repeated and give rise to the nucleolus. The rRNAs are transcribed from two array kinds (the 5S and the 45S arrays). Here we show that variation in the 5S and 45S rDNA arrays is tightly coupled, despite their location on different chromosomes. Our observations suggest that natural selection contributes to maintain balanced rDNA dosage across unlinked rDNA arrays. Finally, we show that bisphenol A can induce parallel loss of rDNA units in 5S and 45S arrays. These observations raise the prospect that human diseases might be traced to disrupted rDNA dosage balance in the genome. Tandemly repeated ribosomal DNA (rDNA) arrays are among the most evolutionary dynamic loci of eukaryotic genomes. The loci code for essential cellular components, yet exhibit extensive copy number (CN) variation within and between species. CN might be partly determined by the requirement of dosage balance between the 5S and 45S rDNA arrays. The arrays are nonhomologous, physically unlinked in mammals, and encode functionally interdependent RNA components of the ribosome. Here we show that the 5S and 45S rDNA arrays exhibit concerted CN variation (cCNV). Despite 5S and 45S rDNA elements residing on different chromosomes and lacking sequence similarity, cCNV between these loci is strong, evolutionarily conserved in humans and mice, and manifested across individual genotypes in natural populations and pedigrees. Finally, we observe that bisphenol A induces rapid and parallel modulation of 5S and 45S rDNA CN. Our observations reveal a novel mode of genome variation, indicate that natural selection contributed to the evolution and conservation of cCNV, and support the hypothesis that 5S CN is partly determined by the requirement of dosage balance with the 45S rDNA array. We suggest that human disease variation might be traced to disrupted rDNA dosage balance in the genome.

Y Not a Dead End: Epistatic Interactions between Y-Linked Regulatory Polymorphisms and Genetic Background Affect Global Gene Expression in Drosophila Melanogaster

The Y chromosome, inherited without meiotic recombination from father to son, carries relatively few genes in most species. This is consistent with predictions from evolutionary theory that nonrecombining chromosomes lack variation and degenerate rapidly. However, recent work has suggested a dynamic role for the Y chromosome in gene regulation, a finding with important implications for spermatogenesis and male fitness. We studied Y chromosomes from two populations of Drosophila melanogaster that had previously been shown to have major effects on the thermal tolerance of spermatogenesis. We show that these Y chromosomes differentially modify the expression of hundreds of autosomal and X-linked genes. Genes showing Y-linked regulatory variation (YRV) also show an association with immune response and pheromone detection. Indeed, genes located proximal to the euchromatin–heterochromatin boundary of the X chromosome appear particularly responsive to Y-linked variation, including a substantial number of odorant-binding genes. Furthermore, the data show significant regulatory interactions between the Y chromosome and the genetic background of autosomes and X chromosome. Altogether, our findings support the view that interpopulation, Y-linked regulatory polymorphisms can differentially modulate the expression of many genes important to male fitness, and they also point to complex interactions between the Y chromosome and genetic background affecting global gene expression.