Eukaryotic genomes replicate via the synchronous firing of clusters of origins that together produce multi-replicon domains, each of which replicates at a defined time during S-phase. This temporal program is termed the DNA replication-timing program. Replication Timing (RT) is a stable epigenetic property that is cell type specific and is extensively regulated during differentiation in units that range from 400-800kb called replication domains. DNA that replicates at distinct times during S-phase is also spatially separated in the nucleus. Consistent with this, the binary nuclear compartments defined by chromatin spatial proximity maps, align precisely with the replication-timing program. But the dynamics of this relationship during differentiation and cell cycle have been poorly understood. To this end, we first showed that there is a coordinated switch in nuclear compartment along with a switch in replication timing during differentiation. It was also observed that regions of the genome that switch replication timing and nuclear compartment continue to maintain their structural boundaries. Genome-wide analysis of replication domains revealed that they are indeed stable structural units corresponding to Topologically-Associating Domains (TADs) defined by Hi-C. Next we showed that the interphase chromatin structure consisting of TADs and their long-range contacts are established during early G1 coincident with the establishment of the replication-timing program. We also show that developmentally regulated regions of the genome have fundamentally different higher order structure. In G2 phase, the replication timing-program is lost while inter-phase chromatin structure acquired in early G1 was retained. This shows that interphase chromatin structure is not sufficient to dictate RT and lead us to hypothesize that the chromatin structure set-up during early G1 may act as a scaffold to seed the assembly of some factor capable of setting replication initiation thresholds. The de-coupling of chromatin structure and RT could then be due to the removal of this factor during S-phase. Consistent with this hypothesis, we discovered a protein Rif1 that enters the nucleus right after mitosis and its knockout has a profound disruptive effect on RT in both mouse and human cells. Lastly, we explored the conservation of replication timing at single cell level that revealed a highly conserved yet stochastic regulation of replication timing. Surprisingly, the intrinsic (within cell) stochasticity and the extrinsic (cell-to-cell) stochasticity were similar. This is consistent with a model of replication timing regulation where the timing is the outcome of stochastic origin firing and is not affected by the precise environment within a cell. In summary, the work descried in this thesis uncovers a model where replication-timing is regulated at the unit of chromatin structure called TADs, which are generally stable across cell-types, but the compartment that they reside in corresponds to the time of their replication. Interphase chromatin structure is established along with the establishment of RT and may act as scaffold for replication regulation factors like Rif1. Finally, replication timing and its association with chromatin structure are highly conserved and are observed even at the single chromosome level. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester 2017. / July 12, 2017. / Chromatin Conformation Capture, Chromatin Structure, DNA Replication Timing, Epigenetics, Genomics, Single-cell / Includes bibliographical references. / David M. Gilbert, Professor Directing Dissertation; Alan R. Lemmon, University Representative; Hank W. Bass, Committee Member; Brian P. Chadwick, Committee Member; Jonathan H. Dennis, Committee Member.

Apicomplexans are responsible for major human diseases such as toxoplasmosis caused by Toxoplasma gondii (T. gondii) and the deadliest form of malaria caused by Plasmodium falciparum (P. falciparum). The genomes of these pathogens are now sequenced ushering in a new era of drug development. A major hurdle to exploiting this genome resource is that a large number of the encoded genes are "hypotheticals" and have yet to be characterized. Hypothetical proteins comprise roughly half of the predicted gene complement of T. gondii and P. falciparum and represent the largest class of uniquely functioning proteins in these parasites. Following the idea that functional relationships can be informed by the timing of gene expression, we devised a strategy to identify the core set of apicomplexan cell division cycling genes with important roles in parasite division, which includes many uncharacterized proteins. We assembled an expanded list of orthologs from the T. gondii and P. falciparum genome sequences (2781 putative orthologs), compared their mRNA profiles during synchronous replication, and sorted the resulting set of dual cell cycle regulated orthologs (744 total) into protein pairs conserved across many eukaryotic families versus those unique to the Apicomplexa. The analysis identified more than 100 ortholog gene pairs with unknown function in T. gondii and P. falciparum that displayed co-conserved mRNA abundance, dynamics of cyclical expression and similar peak timing that spanned the complete division cycle in each parasite. The unknown cyclical mRNAs encoded a diverse set of proteins with a wide range of mass and showed a remarkable conservation in the internal organization of ordered versus disordered structural domains. A representative sample of cyclical unknown genes (16 total) was epitope tagged in T. gondii tachyzoites yielding the discovery of new protein constituents of the parasite inner membrane complex, key mitotic structures and invasion organelles. These results demonstrate the utility of using gene expression timing and dynamic profile to identify proteins with unique roles in Apicomplexa biology. Additionally, we selected one of these newly identified membrane proteins to further characterize in both T. gondii and P. falciparum. We named the protein inner membrane complex protein 16 (IMC16) due to its IMC localization however; this protein uniquely preferentially targets the developing daughter IMC early in budding and is completely absent from the mother IMC in dividing parasites. IMC16's membrane association cannot be attributed to an alveolin domain and its partial solubility suggests this protein may need more than post-translational modifications to anchor it into the membrane. Proteomic work to determine possible protein interactions highlight a possible phosphorylation by cyclin dependent protein kinase 1 (CDPK1) in the cytoplasm and dephosphorylation by IMC2a to allow it to associate with the IMC similar to the phosphorylation/dephosphorylation mechanisms used by glideosome associated protein 45 (GAP45) to help associate and anchor the glideosome to the IMC.

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