Genome Replication Progression and Epigenetic Regulation in Mammalian Cells

Among all mammalian cellular organelles, the nucleus gets special attention as it contains the genetic information of the cell, the DNA. This is also the site where all DNA-dependent functions, such as genome duplication prior to cell division, DNA transcription, and damage repair upon various DNA breakages undergo. The nucleus also contains the subcompartment, an assembly site for the ribosome, the nucleolus. Among all the DNA-templated events, genome duplication is the most eventful, involving a large array of proteins simultaneously unwinding and synthesizing a new pair of DNA at thousands of locations of the same long polymer, yet tightly regulated in time and space. It is an event on a scale hitherto undreamt of. While the faithful reconstitution of cellular identity involves the accurate inheritance of epigenetics, the information that sits atop the genetic information, the dynamic nature of epigenetics also influences the chromatin structure and regulates various aspects of DNA-templated events, including genome replication programs. The progression of development also triggers changes in the epigenetic landscape, thereby influencing the replication program. During genome replication, the chromatin decondenses, the DNA unwinds, and new DNA is synthesized and rewrapped around the histone core. The spatio-temporal regulation of the replication program also ensures the faithful inheritance of the epigenetic nature, specifically the histone post-translational modifications. However, the intricate interplay between epigenetics and the genome replication program remains a mystery, awaiting further exploration. The present work first sheds light on the developmental changes in genome replication programs, features of replication forks, and underlying chromatin dynamics in human cells. It also uncovered the change in replication time of less-studied tandem and interspersed repeats, which constitute a large chunk of the genome, using repli-FISH. As a prerequisite, a novel approach to analyze the repli-FISH was developed. A switch in the replication timing of ribosomal DNA and differential replication program of Alu, LINE1, and Centromere were observed. Interactions between genome replication machinery and RNA polymerase I responsible for rDNA transcription were observed in pluripotent stem cells. Overall, the study also complements and expands our understanding of the developmentally regulated genome replication program in human cells. High-throughput image analysis was used to quantify the histone modification marks in different cell cycle phases to characterize the relationship between genome replication and epigenetics. Furthermore, the statistical tool nucim was used to map the dynamic localization of histone marks to different compaction classes with cell cycle and sub-S phase progression in pluripotent stem cells. Dynamic localization in constitutive heterochromatin and its replication timing regulation by H3K36me3 was unveiled. Furthermore, genome-wide dynamics of H3K27me3 and H3K4me3, which also mark the poised/bivalent chromatin in embryonic stem cells, unveiled a peculiar pattern. Using synthetic biology, the replication program was disrupted by localizing the constitutive heterochromatin next to nuclear lamin, and its effect on epigenetics was studied. Furthermore, the earliest replication origins and their (epi)genetic/chromatin characteristics are explored to validate the Domino model of replication progression. The approach to finding the cells with the earliest origins is developed in living and fixed cells was established for a detailed exploration. The ribosomal DNA tandem repeat was discovered as one of the preferred locations to start the replication across mammalian cell lines/species. To investigate the genetic nature of these origins, methods to identify, collect, and amplify single-cell genomes linearly were established to measure the micro-copy number gain. Finally, a model was presented describing the genome replication progression and interaction of epigenetic and replication programs.