Hu, Yuzhao (August 2023) Role of cryptochromes in chromatin remodeling and DNA damage repair. PhD thesis, Cold Spring Harbor Laboratory.
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Abstract
Cryptochromes (CRYs) are UVA/blue light receptors found in both plants and animals. In animals, CRYs are involved in the entrainment of the circadian clock. In plants, CRYs play essential roles to regulate various aspects of plant growth and development. Originating from photolyases responsible for directly repairing UV-induced DNA damage, CRYs have undergone evolutionary changes and lost their enzymatic activity for DNA repair. Nevertheless, studies have shown that mammalian CRYs are involved in regulating the DNA damage response (DDR), but the detailed mechanism remains inadequately elucidated. Thus, my research focused on investigating the involvement of plant CRYs in the DDR. My study demonstrates that CRY1 and CRY2 positively regulate plant resistance to UVC-induced DNA damage and enhance DNA damage repair. Surprisingly, examination of the CRY2 protein under UVC exposure reveals the induction of CRY2 nuclear speckles, indicating the activation of CRY2 photoreceptor by UVC. Furthermore, a time course transcriptomic experiment reveals that CRYs promote plant’s transcriptional response to UVC. Notably, my study identified CAMTA transcription factors as potential downstream regulators of CRYs, mediating the DNA damage-induced transcriptional response. Together, these results characterize the positive roles of CRYs in regulating plant DDR and provide mechanistic insights into how CRYs mediate the DDR. In a recent study conducted in the Pedmale laboratory, two deubiquitinases, UBP12 and UBP13, were identified as negative regulators of CRY2-mediated hypocotyl growth. Intriguingly, UBP12/13 have also been implicated in plant resistance to UVC, but their underlying mechanism remains unexplored. To address this gap in knowledge, my thesis research aimed to investigate whether UBP12/13 function within the same pathway as CRYs to regulate plant DDR. Through genetic experiments, I discovered that UBP12/13 act as negative regulators of DDR and operate in the same genetic pathway as CRYs to modulate plant resistance against DNA damage. Remarkably, UBP12/13 exhibit antagonistic effects on several aspects of CRYs' function in DDR. Specifically, UBP12/13 inhibits DNA damage repair and dampens the transcriptional response mediated by the CAMTA transcription factors under UVC exposure. Additionally, at the molecular level, UBP12/13 demonstrates a stronger interaction with the CRY2 protein upon UVC exposure, facilitating the destabilization of CRY2 and subsequent impairment of the role of CRYs in DDR. Collectively, these findings characterize UBP12/13 as crucial negative regulators in the CRY-mediated DDR pathway to alleviate the detrimental effects of DDR on normal cellular functions, such as cell cycle progression. CRYs have been implicated in the regulation of large-scale chromatin condensation and decondensation. However, the precise mechanism by which CRYs govern these chromatin changes remains poorly understood. Notably, using affinity purification of CRY2 and mass spectrometry to identify interacting proteins, a novel group of CRY2 interactors was revealed: the ISWI chromatin remodeling complex, consisting of CHR11, CHR17, RLT1, RLT2, and ARID5. The homologs of ISWI chromatin remodelers in Drosophila and mammals have been known to actively participate in large-scale chromatin condensation and decondensation processes. Thus, the identification of the ISWI complex as CRY2 interactors positions them as promising candidates for mediating the large-scale chromatin changes associated with CRY function. To validate the interaction between CRY2 and the ISWI complex, co-immunoprecipitation experiments were conducted, confirming the physical interaction between CRY2 and three different components of the ISWI complex: CHR11, RLT1, and ARID5. Intriguingly, the interaction between CRY2 and the ATPase subunit of the ISWI complex, CHR11, was found to be independent of light conditions. Moreover, my investigations revealed that the ISWI complex functions downstream of CRY2 in regulating blue light-mediated processes such as hypocotyl growth inhibition and floral transition. Notably, my findings demonstrate that the ISWI complex acts as a negative regulator of blue light-induced heterochromatin condensation during seedling development, opposing the role of CRY2 in this process. Collectively, these results identify the ISWI complex as novel downstream regulators within the CRY2-mediated blue light signaling pathway, presenting ISWI as promising candidates for mediating large-scale chromatin changes downstream of CRYs. In summary, this thesis provides comprehensive insights into the pivotal roles of plant cryptochromes in DNA damage response and chromatin remodeling. The research establishes the critical involvement of CRYs and UBP12/13 deubiquitinases in plant DNA damage response, shedding light on their significant contributions to UVC-induced DNA damage repair and transcriptional regulation. Furthermore, the study identifies the ISWI chromatin remodeling complex as a novel interacting partner of CRY2, uncovering its participation in large-scale chromatin changes alongside CRYs. These findings greatly enhance our understanding of the molecular mechanisms underlying CRY-mediated chromatin regulation and hold implications for future investigations into DNA repair mechanisms and chromatin dynamics in plants.
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