Dosage compensation is a process that many multicellular organisms employ to equalize the expression of X-linked genes between males and females. In Drosophila melanogaster, it is achieved by a two-fold transcriptional activation of the single X chromosome in males. This is mediated by the male-specific lethal (MSL) complex, which is composed of at least five proteins (MSL-1, MSL-2, MSL-3, MOF, MLE) and two non-coding roXRNAs (RNA on X). In a two-step model, MSL complex targets the male X first by binding to chromatin entry sites in a sequence-dependent manner, and then spreads to bind all active genes in a sequence-independent mechanism. In order to biochemically characterize the MSL complex, we applied an approach that combines chromatin immunoprecipitation with mass spectrometry to preserve and analyze protein-protein interactions on the chromatin template. This approach enabled us to capture interacting proteins identified through genetics, but previously not detected in mass spectrometry of soluble complexes. We also identified enriched combinations of associated histone tail modifications by mass spectrometry rather than relying on antibody-based recognition. In addition to this proof-of-principle for the ChIP-MS approach, we identified novel candidates for MSL interaction, including CG4747, a putative H3K36me3 binding protein associated with transcribed bodies of active genes. We observed that CG4747 colocalizes with H3K36me3 and when the SET2 H3K36me3 methyl-transferase is disrupted, this colocalization is lost. CG4747 acts synergistically with SET2 for robust MSL-targeting on the male X chromosome at chromatin entry sites and active genes. Taken together, we successfully adapted ChIP-MS for the study of Drosophila chromatin proteins, and characterized CG4747 as a protein that interacts with the MSL dosage compensation complex. We propose that ChIP-MS is a powerful general method that may prove particularly useful for comprehensive analyses of chromatin-bound regulatory complexes.

Much of the eukaryotic transcriptional machinery is conserved from yeast to human. However, the distance over which transcriptional activation can occur differs between Saccharomyces cerevisiae and metazoans. In S. cerevisiae, the upstream activating sequence (UAS) is generally found within 300 base pairs of the transcription start site; when the UAS is moved too far away, activation no longer occurs. In contrast, metazoan enhancers can activate from as far as 100 kilobases from the start site. In past work, our lab identified five genes that, when mutant, allow transcription activation to occur at a greater-than-normal distance from the GAL1 UAS. As this long-distance activation phenotype was weak, we have now studied long-distance activation as a polygenic trait, isolating strains with multiple mutations that together confer a strong phenotype. To do this, we constructed strains containing two reporters, HIS3 and URA3. For each reporter, the GAL1 UAS was placed approximately 800 base pairs upstream of the transcription start sites. By iterative selection for stronger and stronger expression of HIS3, followed by screening for stronger expression of URA3, we isolated three strains, each containing multiple mutations that contribute to the strength of the long distance activation phenotype. Causative mutations were identified in MOT3, GRR1, MIT1, PTR3, YOR019W, and MSN2 that contribute to the long distance activation phenotype. Strains containing multiple mutations were found to activate the reporter construct at distances up to 2 kilobases. Microarray analysis revealed genome wide transcriptional changes in the mutant strains. Statistical analysis of the microarray results suggests other potential sites of long distance activation throughout out the genome. These results have extended our understanding of mutations that allow long distance activation and have demonstrated the value of studying a phenotype as a polygenic trait.

BRCA1 is a breast and ovarian tumor suppressor. The role of BRCA1 in the repair of double strand DNA breaks by homologous recombination [HR] is its best understood function and the function most often implicated in BRCA1 breast cancer suppression. However, BRCA1 has less well defined roles in multiple other molecular processes.

The impact of ionizing radiation exposure on the immune system is dramatic and potentially lethal. The mechanism behind the radiosensitivity of immune subsets has been the concern of researchers for decades, while the long-term effects of radiation exposure in youth have been scarcely explored. The life-long impact of very prevalent latent human viruses, such as Cytomegalovirus (CMV), have recently been brought to the forefront of aging research. However, most short and long-term studies of radiation on the immune system have been performed with animals devoid of latent viruses. Here we describe an innate cause for immune subset radiosensitivity differences, and introduce a post-exposure intervention. We also describe the effects of latent CMV on acute immune sensitivity. Further, we discover that the long-term impact of radiation exposure on the immune system is dependent on whether or not latent CMV was present at the time of exposure. These findings intersect with the research fields of DNA repair, radiobiology, virology, aging, basic immunity, and stand to inform future radiation policy and research.

When cells are challenged by genotoxic stress, the tumor suppressor protein p53 promotes adaptive responses, including cell cycle arrest, DNA repair, or apoptosis. How these distinct fates are specified through an action of p53 protein is not known. It has been suggested that p53 levels, its post-translational modifications or the cell’s genetic background aid p53 in deciding cells’ fate. As a transcription factor, additional evidence indicates that differences in the DNA-binding ability of p53 protein to its response elements are also important determinants of whether p53 will activate growth arrest genes or apoptotic genes. Here we utilized a set of mutant cell lines which, unlike the parental A1-5 cell line which expresses a mouse tsp53 and becomes growth arrested at 32°C, are capable of growth at this same incubation temperature. We found that the tsp53 in the two mutant cell lines, ALTR-17 and ALTR-24, could induce expression of p21, the principle growth arrest gene induced by p53, although to a lesser extent than in parental cells. Interestingly, evaluation of the conformation of tsp53 using conformation-specific antibodies showed that the protein, which we showed could induce expression of p21 reporter construct, existed in different forms which were found to bind DNA using ChIP assays. We also showed, using microarray technology, that the different forms of p53 are capable of inducing/repressing various sets p53-inducible genes. We conclude that the tsp53 may stably exist in various forms capable of binding DNA and decide cell fate through a p53 transcription-dependent pathway.

Immune responses to extracellular pathogens are mediated by the Th2 cytokines Interleukin (IL)-4, IL-5 and IL-13, which are typically expressed by T helper type-2 (Th2) cells. The genes for IL4, IL5 and IL13 are grouped together within 150 kb of human chromosome 5q31, and their expression is regulated at multiple levels to ensure these proteins are rapidly secreted together in response to antigenic stimulation. Indeed, co-expression of these cytokines is vital for effective Th2 immunity and pathogen expulsion. This dissertation combines comparative approaches with molecular experimentation to provide novel insights into the mechanisms that govern the co-expression of Th2 cytokine genes.Conserved non-coding sequence (CNS)-1 has been shown to coordinately regulate the expression of all three Th2 cytokines genes. Using reporter assays with CNS-1 deletion mutants, we mapped a potent, 68 bp T cell activation-dependent enhancer core within CNS-1, which drives transcription of both human IL13 and IL4. The CNS-1 core contained three CREB binding sites to which CREB and the coactivators CREB binding protein and p300 were recruited in vivo upon T cell activation. Furthermore, CBP and p300 were required for CNS-1 core activity. These data define the region within CNS-1 responsible for enhancement of both IL4 and IL13 transcription in response to T cell receptor signaling.Coordinated expression of Th2 cytokine genes is rooted in an extensive array of cis-regulatory regions. We performed a multi-species comparative analysis of the Th2 cytokine gene cluster to locate CNSs, which may represent cis-regulatory elements, and identify transcription factor binding sites shared among them, which may mediate coregulated expression. Sites for GATA transcription factors were the most prevalent and widely distributed throughout the Th2 cytokine locus, consistent with the known role of GATA3 as a Th2 master switch. Notably, binding motifs for ETS proteins were also predicted within several Th2 CNSs. The majority of these sites bound Ets-1 both in vitro and in vivo in murine Th2 cells. Importantly, IL-4, IL-5, and IL-13 expression was markedly decreased in Th2 cells from Ets-1-/-mice. These data suggest an important and novel role for Ets-1 in the concerted expression of Th2 cytokine genes.

My dissertation is focused on the genetics analyses of a mouse mutation, p100H. This mutation is caused by a radiation-induced chromosomal inversion that disrupts both the p gene and Sox6. The human counterparts of these two murine genes are either known to cause human disease (human P gene) or have potential implications in developing a new strategy to treat human disease (SOX6).In the first part of my dissertation (Chapter One, Part I) on the human P gene, I found that all Navajos with albinism carry a homozygous deletion of 122.5 kb of genomic DNA, including exons 10 20 of the P gene, indicating that albinism among the Navajos is OCA2. This deletion of P gene is Navajo-specific, since I did not find this deletion allele in 34 other individuals with albinism who list various other Native American origins. The Navajo-specific P gene deletion came from a founder mutation. The estimated age of the deletion is 400 1,000 years. In addition, as part of my dissertation (Chapter one, Part II), I found that many patients with Hermansky-Pudlak syndrome-like clinical presentations actually carry P gene mutation. This highlights the importance of molecular analysis in clinical diagnosis.In the second part of my dissertation (Chapter Two), I characterized a novel function of the transcription factor Sox6 in red cell development and in the silencing of epsilon globin, an embryonic globin gene. This finding bears significance in the field of globin gene regulation and has an important potential in the development of new therapeutic strategies for treating sickle cell anemia and beta thalassemia.

Birdsfoot trefoil (Lotus corniculatus L.) is one of our leguminous forage crops. It has gained some importance in North America since 1934, when Professor Johnstone-Wallace of Cornell University found a type adapted to New York State. It appears to be especially suited to long term pasture seedings and to have a very definite place in modern grassland farming and soil conservation practices. [...]

The present investigation was undertaken to study the etfect X-irradiation on the sex ratio of the offspring to irradiated male mice. It also appeared worthwhile to find out whether nitrogen mustard would exert etfects similar to those suspected of X-rays on sex ratio (Kalmus et al, 1952), since many of the effects nitrogen mustard exerts on the living organism are strikingly similar to the effects of radiations(Boyland, 1952; Bastrup-Madsen, 1951).

The rapidly growing field of teratology has recorded an amazing variety of developmental aberrations, either "spontaneous", or with some more or less clear-cut genetic or environmental cause. This preliminary descriptive stage is certainly necessary as a guide to the further, more detailed, study of the pathogenesis of individual abnormalities. However no matter how many more abnormalities are added to the list this method will add very little to knowledge of the basic causes of developmental defects. [...]