Extremely rapid and reversible optogenetic perturbation of nuclear proteins in living embryos.

Many developmental regulators have complex and context-specific roles in different tissues and stages, making the dissection of their function extremely challenging. As regulatory processes often occur within minutes, perturbation methods that match these dynamics are needed. Here, we present the improved light-inducible nuclear export system (iLEXY), an optogenetic loss-of-function approach that triggers translocation of proteins from the nucleus to the cytoplasm. By introducing a series of mutations, we substantially increased LEXY's efficiency and generated variants with different recovery times. iLEXY enables rapid (t1/2 < 30 s), efficient, and reversible nuclear protein depletion in embryos, and is generalizable to proteins of diverse sizes and functions. Applying iLEXY to the Drosophila master regulator Twist, we phenocopy loss-of-function mutants, precisely map the Twist-sensitive embryonic stages, and investigate the effects of timed Twist depletions. Our results demonstrate the power of iLEXY to dissect the function of pleiotropic factors during embryogenesis with unprecedented temporal precision.

Checkpoint adaptation in recombination-deficient cells drives aneuploidy and resistance to genotoxic agents.

Human cancers frequently harbour mutations in DNA repair genes, rendering the use of DNA damaging agents as an effective therapeutic intervention. As therapy-resistant cells often arise, it is important to better understand the molecular pathways that drive resistance in order to facilitate the eventual targeting of such processes. We employ recombination-defective diploid yeast as a model to demonstrate that, in response to genotoxic challenges, nearly all cells eventually undergo checkpoint adaptation, resulting in the generation of aneuploid cells with whole chromosome losses that have acquired resistance to the initial genotoxic challenge. We demonstrate that adaptation inhibition, either pharmacologically, or genetically, drastically reduces the occurrence of resistant cells. Additionally, the aneuploid phenotypes of the resistant cells can be specifically targeted to induce cytotoxicity. We provide evidence that TORC1 inhibition with rapamycin, in combination with DNA damaging agents, can prevent both checkpoint adaptation and the continued growth of aneuploid resistant cells.

High nutrient levels and TORC1 activity reduce cell viability following prolonged telomere dysfunction and cell cycle arrest.

Cells challenged with DNA damage activate checkpoints to arrest the cell cycle and allow time for repair. Successful repair coupled to subsequent checkpoint inactivation is referred to as recovery. When DNA damage cannot be repaired, a choice between permanent arrest and cycling in the presence of damage (checkpoint adaptation) must be made. While permanent arrest jeopardizes future lineages, continued proliferation is associated with the risk of genome instability. We demonstrate that nutritional signaling through target of rapamycin complex 1 (TORC1) influences the outcome of this decision. Rapamycin-mediated TORC1 inhibition prevents checkpoint adaptation via both Cdc5 inactivation and autophagy induction. Preventing adaptation results in increased cell viability and hence proliferative potential. In accordance, the ability of rapamycin to increase longevity is dependent upon the DNA damage checkpoint. The crosstalk between TORC1 and the DNA damage checkpoint may have important implications in terms of therapeutic alternatives for diseases associated with genome instability.

Fly-FUCCI: A versatile tool for studying cell proliferation in complex tissues.

One promising approach for in vivo studies of cell proliferation is the FUCCI system (fluorescent ubiquitination-based cell cycle indicator). Here, we report the development of a Drosophila-specific FUCCI system (Fly-FUCCI) that allows one to distinguish G1, S, and G2 phases of interphase. Fly-FUCCI relies on fluorochrome-tagged degrons from the Cyclin B and E2F1 proteins, which are degraded by the ubiquitin E3-ligases APC/C and CRL4(Cdt2), during mitosis or the onset of S phase, respectively. These probes can track cell-cycle patterns in cultured Drosophila cells, eye and wing imaginal discs, salivary glands, the adult midgut, and probably other tissues. To support a broad range of experimental applications, we have generated a toolkit of transgenic Drosophila lines that express the Fly-FUCCI probes under control of the UASt, UASp, QUAS, and ubiquitin promoters. The Fly-FUCCI system should be a valuable tool for visualizing cell-cycle activity during development, tissue homeostasis, and neoplastic growth.

The differential processing of telomeres in response to increased telomeric transcription and RNA-DNA hybrid accumulation.

Telomeres are protective nucleoprotein structures at the ends of eukaryotic chromosomes. Despite the heterochromatic state of telomeres they are transcribed, generating non-coding telomeric repeat-containing RNA (TERRA). Strongly induced TERRA transcription has been shown to cause telomere shortening and accelerated senescence in the absence of both telomerase and homology-directed repair (HDR). Moreover, it has recently been demonstrated that TERRA forms RNA-DNA hybrids at chromosome ends. The accumulation of RNA-DNA hybrids at telomeres also leads to rapid senescence and telomere loss in the absence of telomerase and HDR. Conversely, in the presence of HDR, telomeric RNA-DNA hybrid accumulation and increased telomere transcription promote telomere recombination, and hence, delayed senescence. Here, we demonstrate that despite these similar phenotypic outcomes, telomeres that are highly transcribed are not processed in the same manner as those that accumulate RNA-DNA hybrids.

Telomeric RNA-DNA hybrids affect telomere-length dynamics and senescence.

Although telomeres are heterochromatic, they are transcribed into noncoding telomeric repeat-containing RNA (TERRA). Here we show that RNA-DNA hybrids form at telomeres and are removed by RNase H enzymes in the budding yeast, Saccharomyces cerevisiae. In recombination-competent telomerase mutants, telomeric RNA-DNA hybrids promote recombination-mediated elongation events that delay the onset of cellular senescence. Reduction of TERRA and telomeric RNA-DNA-hybrid levels diminishes rates of recombination-mediated telomere elongation in cis. Overexpression of RNase H decreases telomere recombination rates and accelerates senescence in recombination-competent but not recombination-deficient cells. In contrast, in the absence of both telomerase and homologous recombination, accumulation of telomeric RNA-DNA hybrids leads to telomere loss and accelerated rates of cellular senescence. Therefore, the regulation of TERRA transcription and telomeric RNA-DNA-hybrid formation are important determinants of both telomere-length dynamics and proliferative potential after the inactivation of telomerase.