Divergence of Grainy head affects chromatin accessibility, gene expression, and embryonic viability in Drosophila melanogaster.

Pioneer factors are critical for gene regulation and development because they bind chromatin and make DNA more accessible for binding by other transcription factors. The pioneer factor Grainy head (Grh) is present across metazoans and has been shown to retain a role in epithelium development in fruit flies, nematodes, and mice despite extensive divergence in both amino acid sequence and length. Here, we investigate the evolution of Grh function by comparing the effects of the fly (Drosophila melanogaster) and worm (Caenorhabditis elegans) Grh orthologs on chromatin accessibility, gene expression, embryonic development, and viability in transgenic D. melanogaster. We found that the Caenorhabditis elegans ortholog rescued cuticle development but not full embryonic viability in Drosophila melanogaster grh null mutants. At the molecular level, the C. elegans ortholog only partially rescued chromatin accessibility and gene expression. Divergence in the disordered N-terminus of the Grh protein contributes to these differences in embryonic viability and molecular phenotypes. These data show how pioneer factors can diverge in sequence and function at the molecular level while retaining conserved developmental functions at the organismal level.

Differential Grainy head binding correlates with variation in chromatin structure and gene expression in Drosophila melanogaster.

Phenotypic evolution is often caused by variation in gene expression resulting from altered gene regulatory mechanisms. Genetic variation affecting chromatin remodeling has been identified as a potential source of variable gene expression; however, the roles of specific chromatin remodeling factors remain unclear. Here, we address this knowledge gap by examining the relationship between variation in gene expression, variation in chromatin structure, and variation in binding of the pioneer factor Grainy head between imaginal wing discs of two divergent strains of Drosophila melanogaster and their F1 hybrid. We find that (1) variation in Grainy head binding is mostly due to sequence changes that act in cis but are located outside of the canonical Grainy head binding motif, (2) variation in Grainy head binding correlates with changes in chromatin accessibility, and (3) this variation in chromatin accessibility, coupled with variation in Grainy head binding, correlates with variation in gene expression in some cases but not others. Interactions among these three molecular layers is complex, but these results suggest that genetic variation affecting the binding of pioneer factors contributes to variation in chromatin remodeling and the evolution of gene expression.

The Role of Blm Helicase in Homologous Recombination, Gene Conversion Tract Length, and Recombination Between Diverged Sequences in Drosophilamelanogaster.

DNA double-strand breaks (DSBs) are a particularly deleterious class of DNA damage that threatens genome integrity. DSBs are repaired by three pathways: nonhomologous-end joining (NHEJ), homologous recombination (HR), and single-strand annealing (SSA). Drosophila melanogaster Blm (DmBlm) is the ortholog of Saccharomyces cerevisiae SGS1 and human BLM, and has been shown to suppress crossovers in mitotic cells and repair mitotic DNA gaps via HR. To further elucidate the role of DmBlm in repair of a simple DSB, and in particular recombination mechanisms, we utilized the Direct Repeat of white (DR-white) and Direct Repeat of whitewith mutations (DR-white.mu) repair assays in multiple mutant allele backgrounds. DmBlm null and helicase-dead mutants both demonstrated a decrease in repair by noncrossover HR, and a concurrent increase in non-HR events, possibly including SSA, crossovers, deletions, and NHEJ, although detectable processing of the ends was not significantly impacted. Interestingly, gene conversion tract lengths of HR repair events were substantially shorter in DmBlm null but not helicase-dead mutants, compared to heterozygote controls. Using DR-white.mu, we found that, in contrast to Sgs1, DmBlm is not required for suppression of recombination between diverged sequences. Taken together, our data suggest that DmBlm helicase function plays a role in HR, and the steps that contribute to determining gene conversion tract length are helicase-independent.

Aging impairs double-strand break repair by homologous recombination in Drosophila germ cells.

Aging is characterized by genome instability, which contributes to cancer formation and cell lethality leading to organismal decline. The high levels of DNA double-strand breaks (DSBs) observed in old cells and premature aging syndromes are likely a primary source of genome instability, but the underlying cause of their formation is still unclear. DSBs might result from higher levels of damage or repair defects emerging with advancing age, but repair pathways in old organisms are still poorly understood. Here, we show that premeiotic germline cells of young and old flies have distinct differences in their ability to repair DSBs by the error-free pathway homologous recombination (HR). Repair of DSBs induced by either ionizing radiation (IR) or the endonuclease I-SceI is markedly defective in older flies. This correlates with a remarkable reduction in HR repair measured with the DR-white DSB repair reporter assay. Strikingly, most of this repair defect is already present at 8 days of age. Finally, HR defects correlate with increased expression of early HR components and increased recruitment of Rad51 to damage in older organisms. Thus, we propose that the defect in the HR pathway for germ cells in older flies occurs following Rad51 recruitment. These data reveal that DSB repair defects arise early in the aging process and suggest that HR deficiencies are a leading cause of genome instability in germ cells of older animals.