Keith A. Maggert

Centromere proteins and chromosome inheritance: a complex affair

Centromeres and the associated kinetochores are involved in essential aspects of chromosome transmission. Recent advances have included the identification and understanding of proteins that have a pivotal role in centromere structure, kinetochore formation, and the coordination of chromosome inheritance with the cell cycle in several organisms. A picture is beginning to emerge of the centromere-kinetechore as a complex and dynamic structure with conservation of function at the protein level across diverse species.

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.

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.

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.

Uncoupling gastrulation and mesoderm differentiation in the Drosophila embryo.

In Drosophila, ventral furrow formation and mesoderm differentiation are initiated by two regulatory genes, twist (twi) and snail (sna). Both genes are evolutionarily conserved and have also been implicated in vertebrate gastrulation. Evidence is presented that sna is sufficient to initiate the invagination of the ventral‐most embryonic cells in the absence of twi+ gene activity. The invaginated cells fail to express mesoderm regulatory genes, suggesting that ventral furrow formation can be uncoupled from mesoderm differentiation. Despite the previous demonstration that sna functions as a sequence‐specific transcriptional repressor, low levels of sna that fail to repress neuroectoderm determinants in the presumptive mesoderm are nonetheless able to promote invagination. Cells that possess an ambiguous developmental identity can initiate the invagination process, providing further evidence that ventral furrow formation need not be linked to mesoderm differentiation.

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.

Functional analysis of conserved cysteine residues in the catalytic subunit of the yeast vacuolar H+-ATPase

The A subunit of the yeast vacuolar ATPase contains three highly conserved cysteines: Cys-261, Cys-284, and Cys-538. Cys-261 is located within the nucleotide-binding P-loop. Each of the conserved cysteines, and one nonconserved cysteine, Cys-254, were altered to serine by site-directed mutagenesis, and the effects on growth at pH 7.5 were determined. The Cys-254-->Ser, Cys-261-->Ser and the double mutants all grew at pH 7.5 and contained nitrate- and bafilomycin-sensitive ATPase activity. However, the ATPase activities of the Cys-261-->Ser and the double mutants were insensitive to the sulfhydryl group inhibitor, N-ethylmaleimide, demonstrating that Cys-261 is the site of inhibition by N-ethylmaleimide. Changing either Cys-284 or Cys-538 to serine prevented growth at pH 7.5. Cys-284 and Cys-538 thus appear to be essential cysteine residues which are required either for assembly or catalysis.

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 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.

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.

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.