| My work has been focused on a cellular structure that is essential for accurate genome inheritance: the centromere. Centromeres are chromosomal domains that do not rely on the presence of any specific DNA sequence. Rather, they are determined by the presence of a histone variant called CENP-A. Stable transmission of CENP-A containing chromatin is accomplished through 1) an unusually high level of protein stability, 2) selfdirected recruitment of nascent CENP-A near existing molecules, and 3) strict cell cycle regulation of assembly. Together, these features lead to a self-sustaining loop that allows for epigenetic maintenance of centromeres.;My own contributions to the understanding of epigenetic centromere inheritance are of a quantitative nature. To put my work in context, I will start with an extensive INTRODUCTION of epigenetics, centromeres, and quantitative biology. Next, in CHAPTER 2, I will detail two of the main methodologies that have allowed for the quantitative analysis of centromere inheritance in subsequent chapters. These are, firstly, fluorescent SNAPbased pulse-labeling, used to distinguish between old and new protein pools; and secondly, a macro for ImageJ that I have developed, allowing for the accurate and unbiased quantification of fluorescence signals at centromeres. In CHAPTER 3, the cis requirements for assembly and extreme stability of centromeric nucleosomes are analyzed. I demonstrate that both G1 phase loading and long-term centromeric retention are unique features of the (CENP-A/H4)2 subnucleosomal core, and are self-directed through a CENP-A encoded targeting domain. CHAPTER 4 provides a quantitative analysis of centromeric chromatin. The absolute number of CENP-A molecules at centromeres has been determined in addition to its quantitative regulatory mechanism and distribution. Finally, an overarching DISCUSSION of my results is presented, providing an outlook on how my findings can guide future centromere research. |
