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)

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

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.The open access fee for this work was funded through the Texas A&M University Open Access to Knowledge (OAK) Fund

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 (Pol ), 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 localization to the nucleus. This function of Pol32 in facilitating the nuclear import of Pol 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. Author summary Cancer etiological studies suggest that the majority of pathological mutations occurred under near normal DNA replication conditions, emphasizing the importance of understanding replication regulation under non-lethal conditions. To gain such a better understanding, we investigated the function of Pol32, a conserved ancillary subunit of the essential DNA polymerase Delta complex, through the development of the fruit fly Drosophila. We uncovered a previously unappreciated function of Pol32 in regulating the nuclear import of the polymerase complex, and this function is developmentally regulated. By utilizing mutations in pol32 and other replication factors, we have started to define basic features of Chromosome Fragile Sites (CFS) in Drosophila somatic cells. CFS is a major source of genome instability associated with replication stresses, and has been an important topic of cancer biology. We discovered that CFS formation does not favor genomic regions with repetitive sequences except the highly transcribed locus encoding ribosomal RNA. Our work lays the groundwork for future studies using Drosophila as an alternative system to uncover the most fundamental features of CFS.National Key R&D Program of China [2018YFA0107000]; National Natural Science Foundation of China [31830046, 31601162]; National Cancer Institute of America; Science and Technology Program of Guangzhou, China [201707010022]; National Institutes of Health of America [R01GM123640]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

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

Epigenetic regulation of centromeric chromatin: old dogs, new tricks?

The assembly of just a single kinetochore at the centromere of each sister chromatid is essential for accurate chromosome segregation during cell division. Surprisingly, despite their vital function, centromeres show considerable plasticity with respect to their chromosomal locations and activity. The establishment and maintenance of centromeric chromatin, and therefore the location of kinetochores, is epigenetically regulated. The histone H3 variant CENP-A is the key determinant of centromere identity and kinetochore assembly. Recent studies have identified many factors that affect CENP-A localization, but their precise roles in this process are unknown. We build on these advances and on new information about the timing of CENP-A assembly during the cell cycle to propose new models for how centromeric chromatin is established and propagated

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