Epigenetic Inheritance of Chromatin Structure during Replication and Mitosis
Inheritance of gene transcription programs from parental cells to daughter cells requires the existence of an epigenetic mark that survives the cell cycle and re-establishes the appropriate chromatin architecture. This thesis investigates how chromatin architecture is re-established during and following DNA replication and how it is maintained throughout mitosis. Central to this thesis is the development of novel techniques that allow, for the first time, investigation of association of proteins with nascent DNA throughout the cell cycle in gastrulating Drosophila embryos. The ability to directly test the potential epigenetic roles of many proteins and modified histones during DNA replication and mitosis has a profound impact on the field. The first technique developed is Sequential Chromatin Immunoprecipitation, which allows detection of specific proteins and modified histones on replicating DNA at target genomic locations. The second newly developed technique is Chromatin Assembly Assay which allows, for the first time, investigation of the timing and order of chromatin assembly following DNA replication. Chromatin Assembly Assay followed by Proximity Ligation Assay (Olink Bioscience) detects interactions of proteins or histone modifications with nascent DNA from the time of replication all the way to mitosis. Interestingly, it is found that histone modifying enzymes, including some Trithorax and Polycomb Group proteins, but not methylated histones or chromatin remodeling enzymes, remain in close proximity with DNA replication machinery and nascent DNA during replication. Histone methylations are re-established after replication in a temporal order, mediated by complex activities of different enzymes. Contrary to recent literature, all chromatin associated proteins tested, including histone modifying enzymes and chromatin remodeling enzymes, as well as modified histones remain stably associated with DNA during all phases of mitosis. This thesis presents direct evidence in opposition to popular epigenetic theories, such as the "histone code" and "reader and writer" hypotheses, and rather suggests that proteins, not histone modifications, act as epigenetic marks through their stable association with DNA throughout the cell cycle. Additionally, chromatin structure is not severely disrupted during mitosis. Once chromatin architecture is re-established following DNA replication, the epigenetic proteins remain stable to ensure appropriate gene expression programs are inherited by daughter cells.
Molecular biology|Biochemistry|Developmental biology
Black, Kathryn L, "Epigenetic Inheritance of Chromatin Structure during Replication and Mitosis" (2014). ETD Collection for Thomas Jefferson University. AAI3611112.