The sequence and the structure of DNA methyltransferase-2 (Dnmt2) bear close affinities to authentic DNA cytosine methyltransferases. A combined genetic and biochemical approach revealed that human DNMT2 did not methylate DNA but instead methylated a small RNA; mass spectrometry showed that this RNA is aspartic acid transfer RNA (tRNA ^Asp ) and that DNMT2 specifically methylated cytosine 38 in the anticodon loop. The function of DNMT2 is highly conserved, and human DNMT2 protein restored methylation in vitro to tRNA ^Asp from Dnmt2-deficient strains of mouse, Arabidopsis thaliana, and Drosophila melanogaster in a manner that was dependent on preexisting patterns of modified nucleosides. Indirect sequence recognition is also a feature of eukaryotic DNA methyltransferases, which may have arisen from a Dnmt2-like RNA methyltransferase.

Interest in the field of epigenetics has increased rapidly over the last decade, with the term becoming more identifiable in biomedical research, scientific fields outside of the molecular sciences, such as ecology and physiology, and even mainstream culture. It has become increasingly clear, however, that different investigators ascribe different definitions to the term. Some employ epigenetics to explain changes in gene expression, others use it to refer to transgenerational effects and/or inherited expression states. This disagreement on a clear definition has made communication difficult, synthesis of epigenetic research across fields nearly impossible, and has in many ways biased methodologies and interpretations. This article discusses the history behind the multitude of definitions that have been employed since the conception of epigenetics, analyzes the components of these definitions, and offers solutions for clarifying the field and mitigating the problems that have arisen due to these definitional ambiguities.

The 35S ribosomal RNA genes ( rDNA ) are organized as repeated arrays in many organisms. Epigenetic regulation of transcription of the rRNA results in only a subset of copies being transcribed, making rDNA an important model for understanding epigenetic chromatin modification. We have created an allelic series of deletions within the rDNA array of the Drosophila Y chromosome that affect nucleolus size and morphology, but do not limit steady-state rRNA concentrations. These rDNA deletions result in reduced heterochromatin-induced gene silencing elsewhere in the genome, and the extent of the rDNA deletion correlates with the loss of silencing. Consistent with this, chromosomes isolated from strains mutated in genes required for proper heterochromatin formation have very small rDNA arrays, reinforcing the connection between heterochromatin and the rDNA . In wild-type cells, which undergo spontaneous natural rDNA loss, we observed the same correlation between loss of rDNA and loss of heterochromatin-induced silencing, showing that the volatility of rDNA arrays may epigenetically influence gene expression through normal development and differentiation. We propose that the rDNA contributes to a balance between heterochromatin and euchromatin in the nucleus, and alterations in rDNA —induced or natural—affect this balance.

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. 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.

Ribosomal RNA gene (rDNA) copy number variation modulates heterochromatin formation and influences the expression of a large fraction of the Drosophila ge-nome. This discovery, along with the link between rDNA, aging, and disease, high-lights the importance of understanding how natural rDNA copy number variation arises. Pursuing the relationship between rDNA expression and stability, we have discovered that increased dietary yeast concentration, emulating periods of dietary excess during life, results in somatic rDNA instability and copy number reduction. Modulation of Insulin/TOR signaling produces similar results, indicating a role for known nutrient sensing signaling pathways in this process. Furthermore, adults fed elevated dietary yeast concentrations produce offspring with fewer rDNA copies demonstrating that these effects also occur in the germline, and are transgenera-tionally heritable. This finding explains one source of natural rDNA copy number variation revealing a clear long-term consequence of diet. We show that altering the nutritional medium of Drosophila cultures, emulating dietary largess in the wild, increases expression of the high copy-number ribosomal RNA genes and results in rDNA instability and loss. The reduction in gene copy number occurs both in somatic and germ cells, such that altered copy numbers are transmitted to the next generation. Our findings have clear ecological and disease relevance. The reduction of supernumerary ribosomal RNA gene copy number has been previously shown to compromise both epigenetic and genomic stability and alter the expression of hundreds of genes, while rDNA instability itself is a key progression hallmark of some cancers. This study links diet and Insulin/Insulin-like Signaling modulation to changes in the genome and provides an accounting for natural copy number variation of rRNA genes.