Chromosomes are the basic unit of inheritance and the proper segregation of chromosomes to daughter cells during cell division is essential to prevent errors that can lead to diseases such as cancer or infertility. The centromere is a region of the chromosome that mediates this segregation and is determined epigenetically through the stable acquisition of the prime epigenetic mark, CENP-A, a histone H3 variant specifically found at centromeres. The Cleveland lab has previously demonstrated that CENP-A is rapidly re-localized to sites of DNA damage and to remain there transiently. I will now determine the characteristics of transient recruitment of CENP-A to DNA damage sites and determines if it facilitates overall DNA repair. By creating a site specific double strand break with engineered nucleases, I will determine how quickly CENP-A (and its partner proteins) is loaded using live cell imaging of fluorescently tagged proteins or immunofluorescence and co-staining with proteins already known to be localized to DNA damage (including 53BP1). I will use cell synchronization techniques to determine the cell cycle dependency of CENP-A localization to sites of DNA damage and the nature of this CENP-A particle (octamer, hexamer, hemisome). In addition, I will determine the extent of chromatin remodeling by CENP-A and CENP-A associated proteins using chromatin immunoprecipitation followed by quantitative real time PCR and/or DNA sequencing. Finally, by using immunofluorescence and chromatin immunoprecipiation I will determine if the recruitment of CENP-A associated proteins and DNA damage proteins is dependent on CENP-A.
Understanding the roles of components of DNA repair and centromeres has important clinical implications. Repair of DNA damage is an essential component of maintenance of genome integrity and errors in it can initiate tumorigenesis. Similarly, failure to properly segregate chromosomes, the genetic material of a cell, can lead to abnormal chromosome numbers and is one of the hallmarks of cancer. Specific Aims - The role of CENP-A and centromere components in DNA repair Specialized chromatin is associated with many aspects of chromosome function. Two of these are marked by incorporation of CENP-A, the histone H3 variant found at each functional centromere from yeast to man and which in animal cells is transiently assembled into chromatin at sites of DNA damage. At centromeres in metazoans, a crucial role of CENP-A containing chromatin has been established: unlike the genes carried on each chromosome - whose identity and information content is determined by DNA sequence - the centromere is specified epigenetically and the epigenetic mark is CENP-A-containing chromatin. A functional role has not been established for transient assembly of CENP-A into chromatin at sites of DNA damage and its later replacement by histone H3.1. Using new and innovative engineered nuclease technology in combination with well-established biochemical techniques, I propose here to identify the underlying characteristics of chromatin remodeling with CENP-A at sites of DNA damage and to determine the functional role(s) contributed by this remodeling to DNA repair.
|Rahdar, Meghdad; McMahon, Moira A; Prakash, Thazha P et al. (2015) Synthetic CRISPR RNA-Cas9-guided genome editing in human cells. Proc Natl Acad Sci U S A 112:E7110-7|