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Identification and Analysis of Components that Regulate Centromere Identity and Propagation
Background: Chromosome segregation requires microtubules to attach to kinetochores in order to separate sister chromatids. The centromere is chromosomal site associated with the kinetochore. CENP-A replaces histone H3 in centromeres and determines centromere identity and propagation in an epigenetic manner. Misregulation of CENP-A causes defective segregation and genome instability, and is observed in human colorectal and breast cancers. It is important to explore how centromere identification and propagation are regulated to fully understand mechanisms underlying genome instability. However, little is known about how each CENP-A protein domains regulates centromere and how CENP-A is targeted and maintained. Objective/Hypothesis: My first objective is to study different CENP-A domains in kinetochore assembly and centromere propagation. The second is to identify proteins that physically and functionally interact with CENP-A and study their roles in regulating CENP-A assembly and centromere propagation. These studies will be performed using the advanced tools available with the Drosophila system, which will facilitate generating a detailed understanding of mechanisms of centromere regulation in vivo. Specific Aims: Cid encodes Drosophila CENP-A and provides a structural and functional foundation for kinetochore formation. Overexpression/mislocalization of Cid seeds formation of ectopic kinetochores, aneuploidy, and lethality. I will dissect Cid domain functions in centromere determination, propagation and genome stability in vivo. I will test candidates of RNA, RNAi, and DNA repair pathways for effects on Cid regulation. I will use biochemical approaches to investigate factors that interact directly with Cid and their roles in centromere regulation and genome stability. Study Design: Molecular, cell biological, and genetic studies will be carried out on Cid domain functions in regulating centromere identity and genome stability in vivo. Candidates of RNA, components of RNAi and DNA repair pathways will be studied to explore their potential roles in regulating Cid. Biochemical purification will be carried out to isolate Cid interacting proteins, especially Cid specific assembly factors. Potential Outcomes: Identification of factors regulating Cid functions will enhance our understanding of mechanisms underlying genome instability and cancer development. My results may ultimately facilitate development of new cancer diagnostic tools and therapies.
Human cancers display remarkable genome instability, and contain abnormal or heterogeneous chromosome numbers (aneuploidy) as well as rearrangements. It is becoming evident that chromosome instability plays an important role in the development of cancer, since aneuploidy is found in the earliest stages of tumorigenesis, and is required for neoplastic transformation in human cells. Normal chromosome segregation requires attachment of microtubules to kinetochore, a large multi-component complex built upon a single site on each chromosome (the centromere), to apply force to separate sister chromatids. CENP-A, a centromere specific histone H3 variant, provides a structural and functional foundation for kinetochore formation, and determines centromere identity and propagation. Loss or mislocalization of CENP-A in model organisms causes cell cycle defects and chromosome missegregation: loss leads to loss of kinetochores, and mislocalization results in chromosomes with multiple kinetochores. In fact, overexpression of CENP-A is associated with human primary colorectal and breast cancer tumors, and is a likely cause of aneuploidy in these cancers. Before considering development of cancer diagnostic and treatment tools based on CENP-A mis-regulation, we need to have a better understanding of how CENP-A protein domains contribute to kinetochore formation, and how CENP-A deposition and maintenance are regulated. I will attack these questions using Drosophila melanogaster; this model system provides powerful molecular, biochemical, cell biological, and genetic tools that allows basic mechanisms to be elucidated in greater detail compared to human studies. First, I will examine the roles of CENP-A domains in regulating centromere function and propagation, using embryo derived cell lines and flies. Second, I will biochemically identify proteins that physically interact with CENP-A, then study their roles in CENP-A deposition and propagation in vivo. Proteins and mechanisms identified by these studies will ultimately provide information essential to the development of preventions and treatments for the aneuploidy associated with human cancers.