Cse4p is a centromere-specific chromatin protein with a histone-fold domain that is greater than 60% identical to histone H3 and the mammalian centromere protein, CENP-A. Cse4p has similar biochemical properties to H3 and is believed to replace H3 in centromere-specific nucleosomes in yeast. To identify residues in the histone-fold domain of Cse4p that function in specifying centromere structure and function, amino adds that differ between Cse4p and H3 were systematically changed to analogous H3 residues. Extensive substitution of contiguous Cse4p residues with H3 counterparts resulted in cell lethality. However, all large lethal substitution alleles could be subdivided into smaller viable alleles, many of which caused elevated rates of mitotic chromosome loss. The severity of the phenotypes exhibited by the histone-fold domain mutants correlates directly with the number of putative DNA contact sites changed or alterations in regions implicated in histone interactions. These results indicate that the histone-fold domain of Cse4p functions through a cooperative mechanism in which residues throughout the histone-fold domain recognize centromere DNA. To investigate the relationship between the histone-fold domain of Cse4p and centromere DNA, chromosome loss rates were measured in double mutants carrying both a mutation in Cse4p and chromosomes with mutant centromere DNA. The Cse4p mutants had conserved changes in which analogously positioned residues in H3 were substituted for Cse4p residues at sites in the histone-fold domain that are in close proximity to the DNA. Mutations throughout the histone-fold domain caused significant increases in chromosome loss rates of chromosomes carrying conserved centromere DNA elements CDE I and II but showed no effect with CDE III mutant centromeres. This genetic evidence strongly supports direct interactions between Cse4p and CDE I and CDE II centromere DNA elements. The results are discussed in the context of the known structure of H3 and models are proposed which best describe the path of the centromere DNA around a Cse4p variant nucleosome that relies on critical contacts between CDE I and II, but not CDE III.

During the course of DNA replication, replication forks often stall or collapse as they proceed from oriC to the terminus due to housekeeping types of DNA damage or proteins bound to DNA. If the DNA is not repaired or if the replication forks do not restart, viability of the cell then becomes compromised. In Escherichia coli, if DNA damage is detected, approximately 40 genes are expressed to repair the offending DNA lesion. This is known the SOS response. Two proteins RecA and LexA regulate the SOS response, where RecA (when bound to ssDNA), serves as the sensor for DNA damage and LexA serves the repressor for SOS expression. When the RecA nucleoprotein filament forms, this complex will accelerate autocleavage of LexA inducing the response. Recently it has been observed that in a population of cells approximately 15% of the population had RecA bound to DNA, however at any given time approximately 0.3% of the population is induced for SOS expression suggesting that the cell can decide whether induce the SOS response or not. The aim of this work is to understand how the cell decides whether or not to express the SOS response at housekeeping types of DNA damage. Regulation is important because the cell would not want to express the SOS response every time replication forks encounter housekeeping types of DNA damage. The first component of this work looks at SOS expression in populations of cells during log phase growth using the fluorecense microscopy and the transcriptional fusion sulA-gfp. Results show that a SOS expression is stochastic and occurs in a small population of wild type cells. The second component of my work focuses on how the cell decides when to express the SOS response by using recA constitutive mutants that are defective in this regulation. Results show that the concentration and conformation of the RecA nucleoprotein filament is crucial for this to occur. Lastly novel recA mutants were created and examined for their role in suppressing constitutive SOS expression. It is observed that suppression of constitutive SOS expression could be seen when these mutations were supplied in cis and in trans, suggesting multiple levels of SOS regulation.

p53 protein is considered a major player in maintaining the genomic integrity. It regulates cell cycle, recognizes damaged DNA, and promotes apoptosis of cells that are defective or developmentally programmed for removal. Most cancers, including breast cancer, lose genomic integrity and have impaired p53. Li-Fraumeni patients and BALB/c mice bearing germline mutations in one allele of the p53 tumor suppressor gene develop mammary tumors. The p53 pathway is also required for the protective effect of pregnancy on breast cancer. Therefore, identifying the cellular pathways that regulate p53 will provide improvements in assessing breast cancer risk in individuals as well as therapeutic targets. The function of p53 could be regulated directly by Mdm2 and Mdm4. While Mdm2 is clearly a negative regulator of p53, the roles of Mdm4 on p53 are still not fully defined. Transgenic mice which over-express Mdm4 specifically in mammary gland were used to investigate the effects of Mdm4 on p53 function. Ovarian hormones also regulate p53 activity, but through indirect mechanisms. Oligonucleotide-based transcriptional profiling was conducted to identify mechanisms, by which estrogen and progesterone enhance p53 activity in mammary epithelial cells. The results from these studies showed that Mdm4 is unlikely to be a primary regulator of p53 functions in mammary epithelium. In contrast, expression profiling revealed groups of genes that are associated with the sensitization of p53 by estrogen and progesterone as well as tamoxifen and progesterone. These results implicate a common pathway used to sensitize p53 which involves proteins in the extracellular matrix.

Loss of p53 function can lead to a variety of cancers, including breast cancer. Mice heterozygous for the p53 gene (designated Trp53 +/−) develop spontaneous mammary tumors, but this depends on the strain background and has been linked to a locus on chromosome 7 (designated SuprMam1). Mammary tumors are common in BALB/c-Trp53 +/−females, but are rare in C57BL/6-Trp53 +/− mice. Prevalence of genomic instability appears to contribute to the phenotype as loss of heterozygosity (LOH) is significantly more common among tumors arising in BALB/c-Trp53+/− mice compared to C57BL/6J-Trp53+/− mice. This increased LOH in BALB/c-Trp53+/− tumors was shown to be due to recombination events. The BALB/c strain has been shown to have a deficiency in non-homologous end joining (NHEJ) of DNA double strand breaks (dsb), however, this does not account for the increase of LOH events in tumors. Our hypothesis was that BALB/c-Trp53 +/− mice are more susceptible to mammary tumors due to impaired Homologous Recombination Repair (HRR) leading to LOH. Using the COMET assay, we demonstrate that dsbs persist longer in BALB/c-Trp53 +/− mouse embryonic fibroblasts (MEFs) compared to C57BL/6J- Trp53+/− MEFs. Similarly, co-localization of H2AX and the homologous recombination protein RAD51 remain at dsbs longer in BALB/c-Trp53+/− MEFs compared to C57BL/6-Trp53+/− MEFs. Palb2 , a gene that lies within the SuprMam1 interval and has been shown to contribute to heritable breast cancer, was chosen as an initial candidate gene. No coding SNPs or expression differences of Palb2 were found in the mammary glands between the two strains. Additional fine mapping and use of a filtering criteria in the SuprMam1 region yielded an additional 34 candidate genes. We demonstrate no significant differences in any of these genes in whole mammary glands and primary mammary epithelial cells between the two strains. Finally, using a congenic mouse strain, we demonstrate the lack of irradiation (IR) sensitivity alleles within the SuprMam1 region. These results suggest a possible defect in HRR in the BALB/c strain that is unlikely related to Palb2. The gene or genes responsible for increased mammary tumor incidence in the BALB/c-Trp53+/− remain to be identified.

Transcription regulation in eukaryotes is a complex process governed by the concerted action of different factors. The work in this thesis is focused on transcriptional regulation in Saccharomyces cerevisiae. I analyzed the regulation of the phospholipid biosynthetic gene INO1 , which has been a model gene for transcription studies for over three decades. Some major questions that I have addressed are: what kinds of cis regulatory sequences and trans factors are important for regulation of INO1? What is the sequence of events in this regulation? How is the recruitment of these trans factors consequential for INO1 transcription? I present my results here for the role of the basic helix loop helix transcription factor (bHLH) family in coordinated regulation of INO1 transcription. I report that the centromeric binding factor 1 (Cbf1p) together with two other members of the bHLH protein family, Ino2p and Ino4p, are required for efficient derepression of INO1 transcription. Together these bHLH transcription factors recruit the ISW2 chromatin-remodeling complex onto the INO1 promoter to drive productive transcription from the INO1 locus. My efforts in studying the regulation of INO1 led me to study the regulation of SNA3, a gene found in tandem upstream (→→) to the INO1 gene and regulated by the same environmental conditions as INO1. Studies on the mechanism of coregulation of adjacent genes in budding yeast have been largely speculative. I provide evidence that the same bHLH proteins which regulate INO1 also regulate SNA3, albeit differentially. Significantly, my results also show that the regulation of both SNA3 and INO1 is dictated from the intergenic region between the two genes. This is a novel mechanism of transcription regulation in yeast as regulation from downstream of ORF is unknown in yeast. Thus, my results with both SNA3 and INO1 provide novel details on how the process of transcription is regulated in response to an environmental cue.

A-kinase anchoring protein AKAP95 is implicated in mitotic chromosome condensation by recruiting the condensin complex. Here, we report a differential regulation of condensation of maternal and paternal chromosomes mediated by AKAP95 and chromatin composition in mitotic mouse zygotes. AKAP95 is synthesized upon oocyte activation, targeted to the female pronucleus and specifically associates with maternal chromosomes at mitosis. Peptide competition and rescue experiments show that AKAP95 is required for recruitment of the mCAP-D2 condensin subunit to, and condensation of, maternal chromosomes. In contrast, AKAP95 is dispensable for mCAP-D2 targeting and condensation of paternal chromosomes. In vitro nuclear reconstitution and disassembly assays indicate that human hCAP-D2 targets protamine-containing chromatin independently of AKAP95, but requires AKAP95 for association with histone-containing chromosomes. We propose a concept whereby (1) recruitment of condensins to chromatin is affected by chromatin composition and (2) AKAP95 renders histone-containing chromatin permissive to condensin targeting.

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