2015 |
Kee, Younghoon |
R15Activity Code Description: Supports small-scale research projects at educational institutions that provide baccalaureate or advanced degrees for a significant number of the Nation’s research scientists but that have not been major recipients of NIH support. The goals of the program are to (1) support meritorious research, (2) expose students to research, and (3) strengthen the research environment of the institution. Awards provide limited Direct Costs, plus applicable F&A costs, for periods not to exceed 36 months. This activity code uses multi-year funding authority; however, OER approval is NOT needed prior to an IC using this activity code. |
Function and Regulation of the Fanconi Anemia Pathway in Dna Repair @ University of South Florida
? DESCRIPTION (provided by applicant): Studying the rare genetic disorder Fanconi Anemia (FA) will provide insight into a normal cellular response mechanism in response to genotoxic stress. FA, a genome instability syndrome, is characterized by congenital anomalies, bone marrow and hematopoietic stem cell maintenance failure, and heightened cellular sensitivity to DNA damaging insults. The fifteen FA proteins that have so far been identified collaborate in the cellular FA pathway to modulate DNA repair mechanisms that resolve DNA interstrand crosslinks. Conversely, heightened activity of the FA pathway and a downstream DNA homologous recombination (HR) repair may be important determinants for developing resistance to DNA damaging agents in some cancers. Thus, understanding the molecular function and regulation of the FA pathway may lead to development of novel targeting strategies for resolving FA disease, as well as for intervention against certain cancers. The central regulatory step in the FA pathway is monoubiquitination of two key FA proteins FANCD2- FANCI, by the multi-subunit nuclear E3 ubiquitin ligase, which consists of at least eight FA proteins. Deficiency in the monoubiquitination of FANCD2 and FANCI accounts for more than 90% of the FA cases, suggesting a critical role of this modification in pathogenesis. USP1 deubiquitinating enzyme and its binding partner UAF1 are also critical for the overall FA-HR repair, in part by deubiquitinating FANCD2-FANCI. In this study, we propose two lines of investigation into the regulation of the FA-HR pathway. In Aim 1, we will identify the substrate recruitment module of the FA E3 ligase complex. We will determine the mechanism how the FANCD2-FANCI heterodimer is recruited to the FA E3 ligase complex prior to their monoubiquitination. In Aim 2, we will determine the role of the USP1-UAF1 deubiquitinating enzyme complex in promoting the FA-HR repair. We will particularly focus on the role of the deubiquitinating enzyme complex in modulating a HR promoting factor RAD51AP1. Successful completion of this work will provide new mechanistic insights into regulation of the FA pathway and into broader aspects of the ubiquitin system in general.
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0.948 |
2017 — 2020 |
Kee, Younghoon |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Role of Ubr5 in the Prc1-Mediated Transcriptional Repression At Damaged Chromatin @ University of South Florida
Scientific Abstract Genome integrity is constantly threatened by various forms of genotoxic stresses, including radiation or endogenous metabolic products. Damaged DNA lesions in actively proliferating cells cause stalling of DNA replication polymerases as well as RNA polymerases, which can lead to replication fork arrest or transcriptional arrest, respectively. Understanding the cellular response to monitor and execute the polymerase stalling will give us better insight on how stressed cells, such as cancer cells, deal with the challenges. The BMI1-containing Polycomb complex (PRC1; Polycomb Repressive Complex 1) is an important regulator of epigenetic silencing programs during development, in part by inducing H2AK119 monoubiquitination (H2AK119-Ub) associated with repressed RNA Polymerase II (Pol II). Recently, the role of BMI1 in DNA damage response has gained much attention, but in what aspect and mechanism it participates in the process is unclear. In our preliminary studies, we identified UBR5 as a downstream factor whose chromatin recruitment is regulated by the BMI1-containing PRC1 complex. While transcription is repressed at UV-induced damaged sites on chromatin, depletion of the PRC1 members or UBR5 alone de-repressed transcription elongation at the damaged sites, suggesting that UBR5 functions in a linear pathway with PRC1 in inducing gene silencing at the lesions. We found that UBR5 interacts with BMI1 as well as histone chaperone complex FACT, and that UBR5 co-localizes with FACT component SPT16 to the UV-induced lesions in a BMI1-dependent manner. We further provide evidence that UBR5 directly ubiquitinates SPT16. Our evidence suggests a model that the BMI1-UBR5 axis antagonizes the FACT-dependent Pol II elongation upon UV insults. Based on these data, we propose a hypothesis that UBR5 is a key downstream effector of the PRC1- mediated gene silencing. The specific aims are to determine: 1) role of PRC1 and histone modification in UBR5 recruitment, 2) role of BMI1 and UBR5 in regulating transcriptional repression under DNA damaged or unperturbed conditions, 3) role of OTUD5 deubiquitinating enzyme in the UBR5 and PRC1-induced transcriptional repression at the lesions. The successful outcome of these studies will establish UBR5 as a key downstream mediator of the transcriptional regulation mediated by the PRC1 complex. The proposed work will fill the gap of important yet largely unknown function of the Polycomb complex in gene silencing, and will have an impact that advances the broader area of transcription, DNA damage response, and cancer development.
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0.948 |