Yeast screens identify the RNA polymerase II CTD and SPT5 as relevant targets of BRCA1 interaction.

BRCA1 has been implicated in numerous DNA repair pathways that maintain genome integrity, however the function responsible for its tumor suppressor activity in breast cancer remains obscure. To identify the most highly conserved of the many BRCA1 functions, we screened the evolutionarily distant eukaryote Saccharomyces cerevisiae for mutants that suppressed the G1 checkpoint arrest and lethality induced following heterologous BRCA1 expression. A genome-wide screen in the diploid deletion collection combined with a screen of ionizing radiation sensitive gene deletions identified mutants that permit growth in the presence of BRCA1. These genes delineate a metabolic mRNA pathway that temporally links transcription elongation (SPT4, SPT5, CTK1, DEF1) to nucleopore-mediated mRNA export (ASM4, MLP1, MLP2, NUP2, NUP53, NUP120, NUP133, NUP170, NUP188, POM34) and cytoplasmic mRNA decay at P-bodies (CCR4, DHH1). Strikingly, BRCA1 interacted with the phosphorylated RNA polymerase II (RNAPII) carboxy terminal domain (P-CTD), phosphorylated in the pattern specified by the CTDK-I kinase, to induce DEF1-dependent cleavage and accumulation of a RNAPII fragment containing the P-CTD. Significantly, breast cancer associated BRCT domain defects in BRCA1 that suppressed P-CTD cleavage and lethality in yeast also suppressed the physical interaction of BRCA1 with human SPT5 in breast epithelial cells, thus confirming SPT5 as a relevant target of BRCA1 interaction. Furthermore, enhanced P-CTD cleavage was observed in both yeast and human breast cells following UV-irradiation indicating a conserved eukaryotic damage response. Moreover, P-CTD cleavage in breast epithelial cells was BRCA1-dependent since damage-induced P-CTD cleavage was only observed in the mutant BRCA1 cell line HCC1937 following ectopic expression of wild type BRCA1. Finally, BRCA1, SPT5 and hyperphosphorylated RPB1 form a complex that was rapidly degraded following MMS treatment in wild type but not BRCA1 mutant breast cells. These results extend the mechanistic links between BRCA1 and transcriptional consequences in response to DNA damage and suggest an important role for RNAPII P-CTD cleavage in BRCA1-mediated cancer suppression.

Ins-4 and daf-28 function redundantly to regulate C. elegans L1 arrest

© 2014 The Authors.Caenorhabditis elegans larvae reversibly arrest development in the first larval stage in response to starvation (L1 arrest or L1 diapause). Insulin-like signaling is a critical regulator of L1 arrest. However, the C. elegans genome encodes 40 insulin-like peptides, and it is unknown which peptides participate in nutritional control of L1 development. Work in other contexts has revealed that insulin-like genes can promote development ("agonists") or developmental arrest ("antagonists"), suggesting that such agonists promote L1 development in response to feeding. We measured mRNA expression dynamics with high temporal resolution for all 40 insulin-like genes during entry into and recovery from L1 arrest. Nutrient availability influences expression of the majority of insulin-like genes, with variable dynamics suggesting complex regulation. We identified thirteen candidate agonists and eight candidate antagonists based on expression in response to nutrient availability. We selected ten candidate agonists (. daf-28, ins-3, ins-4, ins-5, ins-6, ins-7, ins-9, ins-26, ins-33 and ins-35) for further characterization in L1 stage larvae. We used destabilized reporter genes to determine spatial expression patterns. Expression of candidate agonists is largely overlapping in L1 stage larvae, suggesting a role of the intestine, chemosensory neurons ASI and ASJ, and the interneuron PVT in control of L1 development. Transcriptional regulation of candidate agonists is most significant in the intestine, as if internal nutrient status is a more important influence on transcription than sensory perception. Phenotypic analysis of single and compound deletion mutants did not reveal effects on L1 developmental dynamics, though simultaneous disruption of ins-4 and daf-28 increases survival of L1 arrest. Furthermore, overexpression of ins-4, ins-6 or daf-28 alone decreases survival and promotes cell division during starvation. These results suggest extensive functional overlap among insulin-like genes in nutritional control of L1 development while highlighting the role of ins-4, daf-28 and to a lesser extent ins-6.