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

Lemos B (2011) Ribosomal DNA Deletions Modulate Genome-Wide Gene Expression: ''rDNA-Sensitive'' Genes and Natural Variation. PLoS Genet 7(4): e1001376. doi:10.1371/journal.pgen.1001376 Ribosomal DNA Deletions Modulate Genome-Wide Gene Expression: ''rDNA -Sensitive'' Genes and Natural Variation Silvana Paredes 0 Alan T. Branco 0 Daniel L. Hartl 0 Keith A. Maggert 0 Bernardo Lemos 0 Andrew G. Clark, Cornell University, United States of America 0 1 Department of Biology, Texas A&M University, College Station, Texas, United States of America, 2 Department of Organismic and Evolutionary Biology, Harvard University , Cambridge, Massachusetts , United States of America 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. - Funding: This work was supported by NIH R01 (GM076092) and by NIH R01 (GM084236). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The ribosomal rDNA gene array is an epigenetically-regulated repeated locus responsible for producing over 90% of cellular RNAs. The large (35S) rDNA array gives origin to the Nucleolus Organizer Region (NOR) and the 35S rRNA genes are present from fewer than 50 to more than 25,000 copies among eukaryotes [1]. However, even though copy number varies widely between and within species [2,3], the functional consequences of copy number polymorphisms have been largely unknown, and often assumed to have negligible functional consequence. This is in part because redundant rDNA arrays are found in eukaryotic genomes. In humans, for example, rDNA arrays containing the 35S rRNA genes are found on chromosomes 13, 14, 15, 21, and 22. Similarly, redundant rDNA genes within each array can compensate for copy number polymorphisms to maintain a sufficient supply of rRNAs [46]; indeed, only a fraction of the rDNA units in a particular array are transcribed at any given time [7], and flexibility in the transcriptional elongation rate allows for control of rRNA output in conditions of rapid cell division and growth. Hence, even though rDNA copy number and rRNA production is regulated by the cell, the relevance of supernumerary or inactive rDNA repeat units has remained elusive. Recent work has investigated whether inactive rDNA copies are necessary for genome stability [8]. In Drosophila melanogaster, rDNA arrays exist as a pair of functionally-redundant loci on the X and Y chromosomes [5,6], which have further suggested that variable copy number in the Ylinked rDNA array might have little, if any, functional significance. Indeed, even though X0 males are sterile due to loss of Y-linked fertility genes, they appear morphologically normal despite having no Y-linked rDNA [9]. Nevertheless, Paredes and Maggert [10] have recently shown that induced variation in the Y-linked rDNA copy number modifies heterochromatin-induced position effect variegation (PEV), and natural changes in rDNA copy number through development correlate with PEV. Taken together, these findings suggested that polymorphisms in rDNA copy number might be relevant to the maintenance of genome-wide chromatin structure. Polymorphic naturally occurring Y chromosomes induce Ylinked Regulatory Variation (YRV), which affects the expression of autosomal and X-linked genes [11]. In Drosophila melanogaster, YRV is observed in males differing only in the origin of their Y chromosomes and it is manifested as the differential expression of hundreds of non-Y-linked genes [11]. The source of YRV cannot be simply ascribed to polymorphisms in protein-coding genes [12,13], nor is it easily mapped to sub-regions of the Y chromosome because of the lack of recombination along the Ychromosome and the difficulty in manipulating large segments of The repeated rDNA array gives rise to the nucleolus, which is one of the first described intracellular structures and is known to be involved in key cellular processes such as stress response, cell cycle regulation, RNA modification, and production of more than 90% of all cellular RNAs (the ribosomal RNAs). The rDNA exists in excess; and, although many copies are inactivated through epigenetic mechanisms, the biological significance of inactive copies has been a matter of debate. We present a system that allows for the identification of global gene expression effects stemming from differences in rDNA copy number. We have discovered that deletions in the rDNA locus result in the differential expression of hundreds to thousands of genes. This raises the expectation that important phenotypic variation affecting health and disease might be traced to polymorphic variation in rDNA copy number. Furthermore, the manifold effects of rDNA copy number indicate that considering polymorphisms in the rDNA might bring new light to studies of epigenetic inheritance and its contribution to the heritability of complex traits. heterochromatin. The similar contributions of induced deletions in the Y-linked rDNA array and naturally occurring Y chromosome variation to PEV [1013], suggested that naturally occurring rDNA copy number polymorphism might be a significant source of regulatory variation. The hypothesized association between Ylinked regulatory variation and rDNA copy number might provide a molecular framework for the contribution of the Drosophila melanogaster Y chromosome to adaptive phenotypic variation and fitness. Here we tested the hypothesis that induced deletions in the Ylinked rDNA in an otherwise isogenic background might modulat (...truncated)