Under the magnifying glass: The ups and downs of rDNA copy number.

The ribosomal DNA (rDNA) in Drosophila is found as two additive clusters of individual 35 S cistrons. The multiplicity of rDNA is essential to assure proper translational demands, but the nature of the tandem arrays expose them to copy number variation within and between populations. Here, we discuss means by which a cell responds to insufficient rDNA copy number, including a historical view of rDNA magnification whose mechanism was inferred some 35 years ago. Recent work has revealed that multiple conditions may also result in rDNA loss, in response to which rDNA magnification may have evolved. We discuss potential models for the mechanism of magnification, and evaluate possible consequences of rDNA copy number variation.

Live analysis of position-effect variegation in Drosophila reveals different modes of action for HP1a and Su(var)3-9.

Position-effect variegation (PEV) results from the juxtaposition of euchromatic and heterochromatic components of eukaryotic genomes, silencing genes near the new euchromatin/heterochromatin junctions. Silencing is itself heritable through S phase, giving rise to distinctive random patterns of cell clones expressing the genes intermixed with clones in which the genes are silenced. Much of what we know about epigenetic inheritance in the soma stems from work on PEV aimed at identifying the components of the silencing machinery and its mechanism of inheritance. The roles of two central gene activities-the Su(var)3-9-encoded histone H3-lysine-9 methyltransferase and the Su(var)205-encoded methyl-H3-lysine-9 binding protein heterochromatin protein 1 (HP1a)-have been inferred from terminal phenotypes, leaving considerable gaps in understanding of how PEV behaves through development. Here, we investigate the PEV phenotypes of Su(var)3-9 and Su(var)205 mutations in live developing tissues. We discovered that mutation in Su(var)205 compromises the initial establishment of PEV in early embryogenesis. Later gains of heterochromatin-induced gene silencing are possible but are unstable and lost rapidly. In contrast, a strain with mutation in Su(var)3-9 exhibits robust silencing early in development but fails to maintain it through subsequent cell divisions. Our analyses show that, while the terminal phenotypes of these mutations may appear identical, they have arrived at them through different developmental trajectories. We discuss how our findings expand and clarify existing models for epigenetic inheritance of heterochromatin-induced gene silencing.

Human rDNA copy number is unstable in metastatic breast cancers.

Chromatin-mediated silencing, including the formation of heterochromatin, silent chromosome territories, and repressed gene promoters, acts to stabilize patterns of gene regulation and the physical structure of the genome. Reduction of chromatin-mediated silencing can result in genome rearrangements, particularly at intrinsically unstable regions of the genome such as transposons, satellite repeats, and repetitive gene clusters including the rRNA gene clusters (rDNA). It is thus expected that mutational or environmental conditions that compromise heterochromatin function might cause genome instability, and diseases associated with decreased epigenetic stability might exhibit genome changes as part of their aetiology. We find the support of this hypothesis in invasive ductal breast carcinoma, in which reduced epigenetic silencing has been previously described, by using a facile method to quantify rDNA copy number in biopsied breast tumours and pair-matched healthy tissue. We found that rDNA and satellite DNA sequences had significant copy number variation - both losses and gains of copies - compared to healthy tissue, arguing that these genome rearrangements are common in developing breast cancer. Thus, any proposed aetiology onset or progression of breast cancer should consider alterations to the epigenome, but must also accommodate concomitant changes to genome sequence at heterochromatic loci.

Monitoring of switches in heterochromatin-induced silencing shows incomplete establishment and developmental instabilities.

Position effect variegation (PEV) in Drosophila results from new juxtapositions of euchromatic and heterochromatic chromosomal regions, and manifests as striking bimodal patterns of gene expression. The semirandom patterns of PEV, reflecting clonal relationships between cells, have been interpreted as gene-expression states that are set in development and thereafter maintained without change through subsequent cell divisions. The rate of instability of PEV is almost entirely unexplored beyond the final expression of the modified gene; thus the origin of the expressivity and patterns of PEV remain unexplained. Many properties of PEV are not predicted from currently accepted biochemical and theoretical models. In this work we investigate the time at which expressivity of silencing is set, and find that it is determined before heterochromatin exists. We employ a mathematical simulation and a corroborating experimental approach to monitor switching (i.e., gains and losses of silencing) through development. In contrast to current views, we find that gene silencing is incompletely set early in embryogenesis, but nevertheless is repeatedly lost and gained in individual cells throughout development. Our data support an alternative to locus-specific "epigenetic" silencing at variegating gene promoters that more fully accounts for the final patterns of PEV.

The processivity factor Pol32 mediates nuclear localization of DNA polymerase delta and prevents chromosomal fragile site formation in Drosophila development.

The Pol32 protein is one of the universal subunits of DNA polymerase δ (Pol δ), which is responsible for genome replication in eukaryotic cells. Although the role of Pol32 in DNA repair has been well-characterized, its exact function in genome replication remains obscure as studies in single cell systems have not established an essential role for Pol32 in the process. Here we characterize Pol32 in the context of Drosophila melanogaster development. In the rapidly dividing embryonic cells, loss of Pol32 halts genome replication as it specifically disrupts Pol δ localization to the nucleus. This function of Pol32 in facilitating the nuclear import of Pol δ would be similar to that of accessory subunits of DNA polymerases from mammalian Herpes viruses. In post-embryonic cells, loss of Pol32 reveals mitotic fragile sites in the Drosophila genome, a defect more consistent with Pol32's role as a polymerase processivity factor. Interestingly, these fragile sites do not favor repetitive sequences in heterochromatin, with the rDNA locus being a striking exception. Our study uncovers a possibly universal function for DNA polymerase ancillary factors and establishes a powerful system for the study of chromosomal fragile sites in a non-mammalian organism.

The peculiar genetics of the ribosomal DNA blurs the boundaries of transgenerational epigenetic inheritance.

Our goal is to draw a line-hypothetical in its totality but experimentally supported at each individual step-connecting the ribosomal DNA and the phenomenon of transgenerational epigenetic inheritance of induced phenotypes. The reasonableness of this hypothesis is offset by its implication, that many (or most) (or all) of the cases of induced-and-inherited phenotypes that are seen to persist for generations are instead unmapped induced polymorphisms in the ribosomal DNA, and thus are the consequence of the peculiar and enduringly fascinating genetics of the highly transcribed repeat DNA structure at that locus.

Transgenerational inheritance of diet-induced genome rearrangements in Drosophila.

Ribosomal RNA gene (rDNA) copy number variation modulates heterochromatin formation and influences the expression of a large fraction of the Drosophila genome. 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 transgenerationally heritable. This finding explains one source of natural rDNA copy number variation revealing a clear long-term consequence of diet.

What do you mean, "epigenetic"?

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.

Simple quantitative PCR approach to reveal naturally occurring and mutation-induced repetitive sequence variation on the Drosophila Y chromosome.

Heterochromatin is a significant component of the human genome and the genomes of most model organisms. Although heterochromatin is thought to be largely non-coding, it is clear that it plays an important role in chromosome structure and gene regulation. Despite a growing awareness of its functional significance, the repetitive sequences underlying some heterochromatin remain relatively uncharacterized. We have developed a real-time quantitative PCR-based method for quantifying simple repetitive satellite sequences and have used this technique to characterize the heterochromatic Y chromosome of Drosophila melanogaster. In this report, we validate the approach, identify previously unknown satellite sequence copy number polymorphisms in Y chromosomes from different geographic sources, and show that a defect in heterochromatin formation can induce similar copy number polymorphisms in a laboratory strain. These findings provide a simple method to investigate the dynamic nature of repetitive sequences and characterize conditions which might give rise to long-lasting alterations in DNA sequence.

Reduced rDNA copy number does not affect "competitive" chromosome pairing in XYY males of Drosophila melanogaster.

The ribosomal DNA (rDNA) arrays are causal agents in X-Y chromosome pairing in meiosis I of Drosophila males. Despite broad variation in X-linked and Y-linked rDNA copy number, polymorphisms in regulatory/spacer sequences between rRNA genes, and variance in copy number of interrupting R1 and R2 retrotransposable elements, there is little evidence that different rDNA arrays affect pairing efficacy. I investigated whether induced rDNA copy number polymorphisms affect chromosome pairing in a "competitive" situation in which complex pairing configurations were possible using males with XYY constitution. Using a common normal X chromosome, one of two different full-length Y chromosomes, and a third chromosome from a series of otherwise-isogenic rDNA deletions, I detected no differences in X-Y or Y-Y pairing or chromosome segregation frequencies that could not be attributed to random variation alone. This work was performed in the context of an undergraduate teaching program at Texas A&M University, and I discuss the pedagogical utility of this and other such experiments.

Genetics: polymorphisms, epigenetics, and something in between.

At its broadest sense, to say that a phenotype is epigenetic suggests that it occurs without changes in DNA sequence, yet is heritable through cell division and occasionally from one organismal generation to the next. Since gene regulatory changes are oftentimes in response to environmental stimuli and may be retained in descendent cells, there is a growing expectation that one's experiences may have consequence for subsequent generations and thus impact evolution by decoupling a selectable phenotype from its underlying heritable genotype. But the risk of this overbroad use of "epigenetic" is a conflation of genuine cases of heritable non-sequence genetic information with trivial modes of gene regulation. A look at the term "epigenetic" and some problems with its increasing prevalence argues for a more reserved and precise set of defining characteristics. Additionally, questions arising about how we define the "sequence independence" aspect of epigenetic inheritance suggest a form of genome evolution resulting from induced polymorphisms at repeated loci (e.g., the rDNA or heterochromatin).

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.

The CCCTC-binding factor (CTCF) of Drosophila contributes to the regulation of the ribosomal DNA and nucleolar stability.

In the repeat array of ribosomal DNA (rDNA), only about half of the genes are actively transcribed while the others are silenced. In arthropods, transposable elements interrupt a subset of genes, often inactivating transcription of those genes. Little is known about the establishment or separation of juxtaposed active and inactive chromatin domains, or preferential inactivation of transposable element interrupted genes, despite identity in promoter sequences. CTCF is a sequence-specific DNA binding protein which is thought to act as a transcriptional repressor, block enhancer-promoter communication, and delimit juxtaposed domains of active and inactive chromatin; one or more of these activities might contribute to the regulation of this repeated gene cluster. In support of this hypothesis, we show that the Drosophila nucleolus contains CTCF, which is bound to transposable element sequences within the rDNA. Reduction in CTCF gene activity results in nucleolar fragmentation and reduced rDNA silencing, as does disruption of poly-ADP-ribosylation thought to be necessary for CTCF nucleolar localization. Our data establish a role for CTCF as a component necessary for proper control of transposable element-laden rDNA transcription and nucleolar stability.

Drosophila SAF-B links the nuclear matrix, chromosomes, and transcriptional activity.

Induction of gene expression is correlated with alterations in nuclear organization, including proximity to other active genes, to the nuclear cortex, and to cytologically distinct domains of the nucleus. Chromosomes are tethered to the insoluble nuclear scaffold/matrix through interaction with Scaffold/Matrix Attachment Region (SAR/MAR) binding proteins. Identification and characterization of proteins involved in establishing or maintaining chromosome-scaffold interactions is necessary to understand how the nucleus is organized and how dynamic changes in attachment are correlated with alterations in gene expression. We identified and characterized one such scaffold attachment factor, a Drosophila homolog of mammalian SAF-B. The large nuclei and chromosomes of Drosophila have allowed us to show that SAF-B inhabits distinct subnuclear compartments, forms weblike continua in nuclei of salivary glands, and interacts with discrete chromosomal loci in interphase nuclei. These interactions appear mediated either by DNA-protein interactions, or through RNA-protein interactions that can be altered during changes in gene expression programs. Extraction of soluble nuclear proteins and DNA leaves SAF-B intact, showing that this scaffold/matrix-attachment protein is a durable component of the nuclear matrix. Together, we have shown that SAF-B links the nuclear scaffold, chromosomes, and transcriptional activity.

Ribosomal DNA contributes to global chromatin regulation.

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.

Expression of I-CreI endonuclease generates deletions within the rDNA of Drosophila.

The rDNA arrays in Drosophila contain the cis-acting nucleolus organizer regions responsible for forming the nucleolus and the genes for the 28S, 18S, and 5.8S/2S RNA components of the ribosomes and so serve a central role in protein synthesis. Mutations or alterations that affect the nucleolus organizer region have pleiotropic effects on genome regulation and development and may play a role in genomewide phenomena such as aging and cancer. We demonstrate a method to create an allelic series of graded deletions in the Drosophila Y-linked rDNA of otherwise isogenic chromosomes, quantify the size of the deletions using real-time PCR, and monitor magnification of the rDNA arrays as their functions are restored. We use this series to define the thresholds of Y-linked rDNA required for sufficient protein translation, as well as establish the rate of Y-linked rDNA magnification in Drosophila. Finally, we show that I-CreI expression can revert rDNA deletion phenotypes, suggesting that double-strand breaks are sufficient to induce rDNA magnification.

Methods for homologous recombination in Drosophila.

We present detailed protocols for two methods of gene targeting in Drosophila. The first, ends-out targeting, is identical in concept to gene replacement techniques used routinely in mammalian and yeast cells. In Drosophila, the targeted gene is replaced by the marker gene white + (although options exist to generate unmarked targeted alleles). This approach is simple in both the molecular cloning and the genetic manipulations. Ends-out will likely serve most investigators' purposes to generate simple gene deletions or reporter gene "knock-ins." The second method, ends-in targeting, targets a wild-type gene with an engineered mutated copy and generates a duplication structure at the target locus. This duplication can subsequently be reduced to one copy, removing the wild-type gene and leaving only the introduced mutation. Although more complicated in the cloning and genetic manipulations (see Note 1), this approach has the benefit that the mutations may be introduced with no other remnant of the targeting procedure. This "surgical" approach will appeal to investigators who desire minimal perturbation to the genome, such as single nucleotide mutation. Although both approaches appear to be approximately equally efficient (see Note 2), each method has separate strengths and drawbacks. The choice of which approach is best depends on the researcher's goal.

Methylation of tRNAAsp by the DNA methyltransferase homolog Dnmt2.

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.

Highly efficient sex chromosome interchanges produced by I-CreI expression in Drosophila.

The homing endonuclease I-CreI recognizes a site in the gene encoding the 23S rRNA of Chlamydomonas reinhardtii. A very similar sequence is present in the 28S rRNA genes that are located on the X and Y chromosomes of Drosophila melanogaster. In this work we show that I-CreI expression in Drosophila is capable of causing induced DNA damage and eliciting cell cycle arrest. Expression also caused recombination between the X and Y chromosomes in the heterochromatic regions where the rDNA is located, presumably as a result of a high frequency of double-strand breaks in these regions. Approximately 20% of the offspring of males expressing I-CreI showed exceptional inheritance of X- and Y-linked markers, consistent with chromosome exchange at rDNA loci. Cytogenetic analysis confirmed the structures of many of these products. Exchange between the X and Y chromosomes can be induced in males and females to produce derivative-altered Y chromosomes, attached-XY, and attached-X chromosomes. This method has advantages over the traditional use of X rays for generating X-Y interchanges because it is very frequent and it generates predictable products.