Epigenetic Telomere Protection by Drosophila DNA Damage Response Pathways: A Dissertation

Several aspects of Drosophila telomere biology indicate that telomere protection can be regulated by an epigenetic mechanism. First, terminally deleted chromosomes can be stably inherited and do not induce damage responses such as apoptosis or cell cycle arrest. Second, the telomere protection proteins HP1 and HOAP localize normally to these chromosomes and protect them from fusions. Third, unprotected telomeres still contain HeT-A sequences at sites of fusions. Taken together these observations support a model in which an epigenetic mechanism mediated by DNA damage response proteins protects Drosophilatelomeres from fusion. Work presented in this thesis demonstrates that the Drosophila proteins ATM and Nbs are required for the regulation of DNA damage responses similar to their yeast and mammalian counterparts. This work also establishes a role for the ATM and ATR DNA damage response pathways in the protection of both normal and terminally deleted chromosomes. Mutations that disrupt both pathways result in a severe telomere fusion phenotype, similar to HP1 and HOAP mutants. Consistent with this phenotype, HOAP localization at atm,atr double mutant telomeres is completely eliminated. Furthermore, telomeric sequences are still present, even at the sites of fusions. These results support a model in which an epigenetic mechanism mediated by DNA damage response proteins protects Drosophila telomeres from fusion

The Death Domain Kinase RIP Protects Thymocytes from Tumor Necrosis Factor Receptor Type 2–induced Cell Death

Fas and the tumor necrosis factor receptor (TNFR)1 regulate the programmed cell death of lymphocytes. The death domain kinase, receptor interacting protein (rip), is recruited to the TNFR1 upon receptor activation. In vitro, rip−/− fibroblasts are sensitive to TNF-induced cell death due to an impaired nuclear factor κB response. Because rip−/− mice die at birth, we were unable to examine the effects of a targeted rip mutation on lymphocyte survival. To address the contribution of RIP to immune homeostasis, we examined lethally irradiated mice reconstituted with rip−/− hematopoietic precursors. We observed a decrease in rip−/− thymocytes and T cells in both wild-type C57BL/6 and recombination activating gene 1−/− irradiated hosts. In contrast, the B cell and myeloid lineages are unaffected by the absence of rip. Thus, the death domain kinase rip is required for T cell development. Unlike Fas-associated death domain, rip does not regulate T cell proliferation, as rip−/− T cells respond to polyclonal activators. However, rip-deficient mice contain few viable CD4+ and CD8+ thymocytes, and rip−/− thymocytes are sensitive to TNF-induced cell death. Surprisingly, the rip-associated thymocyte apoptosis was not rescued by the absence of TNFR1, but appears to be rescued by an absence of TNFR2. Taken together, this study implicates RIP and TNFR2 in thymocyte survival

Epigenetic Telomere Protection by Drosophila DNA Damage Response Pathways

Analysis of terminal deletion chromosomes indicates that a sequence-independent mechanism regulates protection of Drosophila telomeres. Mutations in Drosophila DNA damage response genes such as atm/tefu, mre11, or rad50 disrupt telomere protection and localization of the telomere-associated proteins HP1 and HOAP, suggesting that recognition of chromosome ends contributes to telomere protection. However, the partial telomere protection phenotype of these mutations limits the ability to test if they act in the epigenetic telomere protection mechanism. We examined the roles of the Drosophila atm and atr-atrip DNA damage response pathways and the nbs homolog in DNA damage responses and telomere protection. As in other organisms, the atm and atr-atrip pathways act in parallel to promote telomere protection. Cells lacking both pathways exhibit severe defects in telomere protection and fail to localize the protection protein HOAP to telomeres. Drosophila nbs is required for both atm- and atr-dependent DNA damage responses and acts in these pathways during DNA repair. The telomere fusion phenotype of nbs is consistent with defects in each of these activities. Cells defective in both the atm and atr pathways were used to examine if DNA damage response pathways regulate telomere protection without affecting telomere specific sequences. In these cells, chromosome fusion sites retain telomere-specific sequences, demonstrating that loss of these sequences is not responsible for loss of protection. Furthermore, terminally deleted chromosomes also fuse in these cells, directly implicating DNA damage response pathways in the epigenetic protection of telomeres. We propose that recognition of chromosome ends and recruitment of HP1 and HOAP by DNA damage response proteins is essential for the epigenetic protection of Drosophila telomeres. Given the conserved roles of DNA damage response proteins in telomere function, related mechanisms may act at the telomeres of other organisms

The DNA binding activity of TAL-1 is not required to induce leukemia/lymphoma in mice

Activation of the basic helix-loop-helix (bHLH) gene TAL-1 (or SCL) is the most frequent gain-of-function mutation in pediatric T cell acute lymphoblastic leukemia (T-ALL). Similarly, mis-expression of tal-1 in the thymus of transgenic mice results in the development of clonal T cell lymphoblastic leukemia. To determine the mechanism(s) of tal-1-induced leukemogenesis, we created transgenic mice expressing a DNA binding mutant of tal-1. Surprisingly, these mice develop disease, demonstrating that the DNA binding properties of tal-1 are not required to induce leukemia/lymphoma in mice. However, wild type tal-1 and the DNA binding mutant both form stable complexes with E2A proteins. In addition, tal-1 stimulates differentiation of CD8-single positive thymocytes but inhibits the development of CD4-single positive cells: effects also observed in E2A-deficient mice. Our study suggests that the bHLH protein tal-1 contributes to leukemia by interfering with E2A protein function(s)

Drosophila atm/telomere fusion is required for telomeric localization of HP1 and telomere position effect

Terminal deletions of Drosophila chromosomes can be stably protected from end-to-end fusion despite the absence of all telomere-associated sequences. The sequence-independent protection of these telomeres suggests that recognition of chromosome ends might contribute to the epigenetic protection of telomeres. In mammals, Ataxia Telangiectasia Mutated (ATM) is activated by DNA damage and acts through an unknown, telomerase-independent mechanism to regulate telomere length and protection. We demonstrate that the Drosophila homolog of ATM is encoded by the telomere fusion (tefu) gene. In the absence of ATM, telomere fusions occur even though telomere-specific Het-A sequences are still present. High levels of spontaneous apoptosis are observed in ATM-deficient tissues, indicating that telomere dysfunction induces apoptosis in Drosophila. Suppression of this apoptosis by p53 mutations suggests that loss of ATM activates apoptosis through a DNA damage-response mechanism. Loss of ATM reduces the levels of heterochromatin protein 1 (HP1) at telomeres and suppresses telomere position effect. We propose that recognition of chromosome ends by ATM prevents telomere fusion and apoptosis by recruiting chromatin-modifying complexes to telomeres

DNA-binding-domain fusions enhance the targeting range and precision of Cas9

The CRISPR-Cas9 system is commonly used in biomedical research; however, the precision of Cas9 is suboptimal for applications that involve editing a large population of cells (for example, gene therapy). Variations on the standard Cas9 system have yielded improvements in the precision of targeted DNA cleavage, but they often restrict the range of targetable sequences. It remains unclear whether these variants can limit lesions to a single site in the human genome over a large cohort of treated cells. Here we show that by fusing a programmable DNA-binding domain (pDBD) to Cas9 and attenuating Cas9\u27s inherent DNA-binding affinity, we were able to produce a Cas9-pDBD chimera with dramatically improved precision and an increased targeting range. Because the specificity and affinity of this framework can be easily tuned, Cas9-pDBDs provide a flexible system that can be tailored to achieve extremely precise genome editing at nearly any genomic locus

The Drosophila tefu and <i>mei-41-mus304</i> Pathways Act in an Epigenetic Telomere Protection Mechanism

<p>Normal (A) and terminally deleted (B) X chromosomes are not fused in wild-type cells. Normal (C) and terminally deleted X chromosomes (D) are fused in <i>tefu mus304</i> double mutant cells. Both sister chromosome fusions (C) and non-sister fusions (D) are observed. High frequencies of X chromosome telomere fusions per cell are observed for normal and terminally deleted chromosomes in <i>tefu mus304</i> mutant cells (E). Error bars indicate the standard error of the mean. The number of cells scored for each genotype is in parenthesis. The labels X, Y, 2, 3, and 4 refer to the relevant chromosome.</p

The Drosophila tefu and <i>mei-41-mus304</i> Pathways Are Required for Telomeric Localization of HOAP

<div><p>HOAP immunostaining of mitotic chromosomes prepared from wild-type and mutant third-instar larval brains. Wild-type, <i>mus304,</i> and <i>tefu</i> mutant mitotic chromosomes exhibit HOAP localization (shown in green) at most telomeres ([A–C] arrows). <i>nbs</i> mutant cells exhibit a decreased number of telomeres with HOAP signal ([D] arrow). No HOAP was detected at the telomeres of <i>nbs mus304</i> or <i>tefu mus304</i> mutant chromosomes ([E and F] arrows). Alleles examined in these experiments include (C) <i>tefu¹/ Df(3R)PG4</i> and (E) <i>tefu^Δ356 mus304^D2</i>. The frequency of HOAP-positive telomeres is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020071#pgen-0020071-t001" target="_blank">Table 1</a>. The labels X, 2, 3, and 4 refer to the relevant chromosome.</p><p>(G) The average fluorescence intensity of anti-HOAP immunostaining at the telomeres of chromosome arms 2L, 2R, 3R, 3L, and XL was determined for wild-type, homozygous <i>mus304</i>⁻<i>, tefu¹/Df(3R)PG4,</i> or <i>nbs</i>⁻ animals. The average fluorescence intensity of the HOAP signal is higher in <i>tefu</i> and <i>nbs</i> mutant cells compared to wild-type or <i>mus304</i> mutant cells.</p><p>(H) The average fluorescence intensity of HOAP staining at all telomeres from wild-type and mutant cells. Error bars indicate the standard error of the mean.</p></div

<i>Drosophila</i> Nbs Is Required for Damage-Induced Cell Cycle Arrest

<div><p>Third-instar larval wing discs were mock treated or treated with various doses (250, 500, 1,000, and 4,000 rads) of X-rays and then stained with an antibody against phosphorylated histone H3.</p><p>(A–I) The pattern of mitotic cells in untreated and irradiated wild-type (A–C), <i>nbs</i> mutant (D–F), and <i>tefu</i> mutant (G–I) larval wing discs are shown. At 1,000 and 4,000 rads, mitosis is blocked in wild-type wing discs (B and C) whereas <i>nbs</i> mutant discs fail to arrest (E and F). <i>tefu</i> mutant wing discs have a partial mitotic arrest following treatment with 1,000 rads (H). At 4,000 rads, mitosis is completely blocked in <i>tefu</i> mutant wing discs (I).</p><p>(J) The ratio of mitotic cells in wild-type, <i>nbs, tefu,</i> and <i>mei-41</i> mutant wing discs following X-irradiation to the number of mitotic cells in untreated discs of the same genotype is shown. Error bars indicate the standard error of the mean.</p></div