No description available.

No description available.

No description available.

Chromatin remodelers alter DNA-histone interactions in eukaryotic organisms, and have been well characterized in yeast and Arabidopsis. While there are maize proteins with similar domains as known remodelers, the ability of the maize proteins to alter nucleosome position has not been reported. Mutant alleles of genes encoding several maize proteins (RMR1, CHR101, CHR106, CHR127, CHR156, CHB102, and CHR120) with similar functional domains to known chromatin remodelers were identified. Altered expression of Chr101, Chr106, Chr127, Chr156, Chb102, and Chr120 was demonstrated in plants homozygous for the mutant alleles. These mutant genotypes were subjected to nucleosome position analysis to determine if misregulation of putative maize chromatin proteins would lead to altered DNA-histone interactions. Nucleosome position changes were observed in plants homozygous for chr101, chr106, chr127, chr156, chb102, and chr120 mutant alleles, suggesting that CHR101, CHR106, CHR127, CHR156, CHB102, and CHR120 may affect chromatin structure. The role of RNA polymerases in altering DNA-histone interactions was also tested. Changes in nucleosome position were demonstrated in homozygous mop2-1 individuals. These changes were demonstrated at the b1 tandem repeats and at newly identified loci. While the α-amanitin-inhibited RNA polymerase II demonstrated reduced expression of an RNA polymerase II transcribed gene, no changes in nucleosome position were detected in the α-amanitin-treated plants. Additionally, differential DNA-histone interactions and altered expression of putative chromatin remodelers in different maize haplotypes suggest a role for differentially expressed chromatin proteins in haplotype-specific variation. / 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 14, 2017. / chromatin, chromatin remodelers, nucleosome position, transcription start site, Zea mays / Includes bibliographical references. / Karen M. McGinnis, Professor Directing Dissertation; Myra M. Hurt, University Representative; Hank W. Bass, Committee Member; Brian P. Chadwick, Committee Member; Jonathan H. Dennis, Committee Member.

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.

This project was concerned with the functional components of mammalian DNA replication origins and how the misuse of a start site for DNA replication at the FMR1 locus might contribute to human Fragile X Syndrome. In the first part of this dissertation, I identified a novel origin of DNA replication near the CGG repeats at the human Fragile X Mental Retardation (FMR1) gene promoter. Expansion of these repeats leads to the epigenetic chromosome modifications that cause Fragile X Syndrome. The experiments described in this dissertation suggest that the position of the FMR1 origin favors contraction of the CGG repeats, thus providing a mechanism to avoid repeat expansion. This model predicts that a change in origin usage accompanies repeat expansion, and I discussed how this could occur. In the second part of this dissertation, I examined the requirement of DNA sequence elements in a mammalian origin to direct DNA replication to start at specific chromosomal sites. In particular, I studied the role of a dinucleotide repeat (DNR) sequence element in the activity of the Chinese hamster dihydrofolate reductase origin beta. The DNR element could be functionally replaced with two different transcriptional elements. This result suggests that DNR shares a functional role with these elements, and we speculate that this role may be to create the proper chromatin environment for recruitment and action of other replication factors to initiate replication.

DJ-1, A NOVEL ANDROGEN RECEPTOR BINDING PROTEIN, ACTIVATES RECEPTOR SIGNALING IN PROSTATE CANCER AND CORRELATES WITH THE DEVELOPMENT OF ANDROGEN-INDEPENDENT DISEASE Jennifer Erin Tillman Dissertation completed under the direction of Dr. Susan Kasper, PhD This research projects focused on investigation of the role of DJ-1 in prostate cancer. In this dissertation, the mechanisms regulating the transition from hormone responsive to hormone refractory prostate cancer were investigated by analyzing androgen and anti-androgen treatment on endogenous AR activity in primary human prostate epithelial cells and established prostate cancer cell lines. We determined that flutamide treatment exhibited agonist activities in cells derived from tumor and non-tumor specimens which contained wild-type AR. After proteomic comparison of these cells to those where flutamide functioned normally as an antagonist, we identified DJ-1, an oncogene and positive regulator of AR. DJ-1 expression increased following flutamide treatment as a result of DJ-1 protein stabilization. To address the function of DJ-1 in prostate, we performed a yeast two-hybrid screen to identify novel DJ-1 binding proteins. The androgen receptor (AR) was identified as a putative DJ-1 binding protein, which was confirmed in the LAPC4 and LNCaP human prostate cancer cell lines. This is the first evidence that DJ-1 directly interacts with AR. We also demonstrate that modulation of DJ-1 expression regulates AR transcriptional activity. Importantly, both the subcellular localization of DJ-1 and the interaction with AR is regulated by androgens and anti-androgens. Additionally, we performed immunohistochemical staining on two human prostate cancer tissue arrays providing the first large scale expression analysis of DJ-1 in prostate. DJ-1 expression does not change with Gleason pattern, but increases after androgen deprivation therapy, indicating that it may be involved in the development of androgen independence. Taken together, we demonstrate that DJ-1 directly interacts with AR, and that this interaction is hormonally regulated. These data provide a novel mechanism for DJ-1 mediated regulation of AR in the progression of prostate cancer to androgen-independence.

The temperate mycobacteriophage Bxb1 infects and forms lysogens of Mycobacterium smegmatis, a fast-growing relative of and model for M. tuberculosis. In Bxb1, as in other bacteriophages, switching between the lysogenic cycle and lytic cycle depends on a site-specific DNA recombinase called an integrase. In Bxb1 the directionality of the DNA recombination process depends on the phage-encoded gp47 protein, which acts as the Recombination Directionality Factor (RDF). A number of lines of evidence suggest that Bxb1 gp47 has some additional biological role as well, however. First, very close homologues are found in 15 other mycobacteriophages, including phage L5, whose system for phage DNA integration / excision does not include the Bxb1 gp47 homologue. Second, Bxb1 gp47 and homologues are found clustered with genes for phage DNA replication. Here we present our investigation into the putative multi-functionality of Bxb1 gp47. To begin, we performed a bioinformatic analysis which predicts Bxb1 gp47 and its homologues to contain a calcineurin-like phosphoesterase domain; this domain is predicted to confer a metal-dependent phosphatase activity on these proteins. Further, we analyzed the phenotypic repercussions of altering the Bxb1 gp47 gene by site-specific phage mutagenesis, performed using Bacteriophage Recombineering by Electroporation of DNA (BRED). Results from this work were consistent with the hypothesis that Bxb1 gp47 has an essential function in the lytic-cycle replication of Bxb1, and also showed that gp47 RDF activity is separable from the lytic-cycle function inferred for gp47. Finally, a number of variants of Bxb1 gp47 protein were overexpressed and purified for studies of RDF and phosphatase activity. RDF activity assays suggested that Motif I of the calcineurin-like phosphoesterase domain has no role in RDF activity, but that Motif V may be involved in both RDF and phosphatase activities. The phosphatase activity assays we have performed provide support for the hypothesis that Bxb1 gp47 is a manganese-dependent phosphatase enzyme. If this result is confirmed, Bxb1 gp47 will be revealed as a highly novel RDF with a secondary phosphatase activity.

Thesis (Ph. D.)--University OF MICHIGAN.