Breast cancer arises from a series of acquired mutations that disrupt normal mammary epithelial homeostasis and create multi-potent cancer stem cells that can differentiate into clinically distinct breast cancer subtypes. Despite improved therapies and advances in early detection, breast cancer remains the leading diagnosed cancer in women. A predominant mechanism initiating invasion and migration for a variety of cancers including breast, is epithelial-to-mesenchymal transition (EMT). EMT— a trans-differentiation process through which mammary epithelial cells acquire a more aggressive mesenchymal phenotype—is a regulated process during early mammary gland development and involves many transcription factors involved in cell lineage commitment, proliferation, and growth. Despite accumulating evidence for a broad understanding of EMT regulation, the mechanism(s) by which mammary epithelial cells maintain their phenotype is unknown. Mitotic gene bookmarking, i.e., transcription factor binding to target genes during mitosis for post mitotic regulation, is a key epigenetic mechanism to convey regulatory information for cell proliferation, growth, and identity through successive cell divisions. Many phenotypic transcription factors, including the hematopoietic Runt Related Transcription Factor 1 (RUNX1/AML1), bookmark target genes during mitosis. Despite growing evidence, a role for mitotic gene bookmarking in maintaining mammary epithelial phenotype has not been investigated. RUNX1 has been recently identified to play key roles in breast cancer development and progression. Importantly, RUNX1 stabilizes the normal breast epithelial phenotype and prevents EMT through repression of EMT-initiating pathways. Findings reported in this thesis demonstrate that RUNX1 mitotically bookmarks both RNA Pol I and II transcribed genes involved in proliferation, growth, and mammary epithelial phenotype maintenance. Inhibition of RUNX1 DNA binding by a specific small molecule inhibitor led to phenotypic changes, apoptosis, differences in global protein synthesis, and differential expression of ribosomal RNA as well as protein coding genes and long non-coding RNA genes involved in cellular phenotype. Together these findings reveal a novel epigenetic regulatory role of RUNX1 in normal-like breast epithelial cells and strongly suggest that mitotic bookmarking of target genes by RUNX1 is required to maintain breast epithelial phenotype. Disruption of RUNX1 bookmarking results in initiation of epithelial to mesenchymal transition, an essential first step in the onset of breast cancer.

Introduction—Bullying involvement is associated with many long-term adverse outcomes. Bullied children are at risk for internalizing disorders including anxiety, depression and suicidal behavior in childhood and adulthood. Bullies are also at risk for psychiatric disorders, specifically externalizing disorders. Bully victims—children who are both bullied and bullies—have a particularly poor prognosis, with a higher risk for internalizing and externalizing disorders. The purpose of this study is to study the epidemiology, risk of psychiatric disorders, and genetic and environmental influences of being bullied, a bully, and a bully victim—in the sample and individually in males and females. Methods—Twins (N=2,844, aged 8-17) from the Virginia Twin Study of Adolescent Behavioral Development and the Young Adult Follow-Up were used to study bullying involvement. Child and mother responses from three waves of data collection were used to determine bullying involvement status and to diagnose internalizing and externalizing disorders. The epidemiology of bullying involvement was examined. The odds ratios (OR) of being involved in bullying and having a psychiatric disorder were calculated. The twin methodology was used to estimate the genetic and environmental influences of bullying involvement. Results—In the sample, 14.56% were bullied, 17.33% were bullies, and 10.69% were bully victims. Males are more often involved in bullying, but females are more severely affected by their involvement. Bullied children are at a higher risk for internalizing disorders, especially young adult depression (OR 1.29). Bullies are at a higher risk for externalizing disorders, and depression (OR 1.72). Bully victims are at a higher risk for nearly every disorder tested. Bullying involvement is heritable, and being bullied has a dominance genetic component. The heritability of being bullied, a bully, and a bully victim is 48.12%, 54.81%, and 62.62% respectively. Conclusion—Individuals involved in bullying are at risk for serious and long-lasting psychiatric disorders. Interventions need to be developed that target each category of bullying involvement, and the specific disorders that these children are at risk for, while keeping in mind that their involvement is heritable.

Programmed cell death (PCD) is an essential feature during the development of the central nervous system in Drosophila as well as in mammals. During metamorphosis, a group of peptidergic neurons (vCrz) are eliminated from the larval central nervous system (CNS) via PCD within 6-7 h after puparium formation. To better understand this process, we first characterized the development of the vCrz neurons including their lineages and birth windows using the MARCM (Mosaic Analysis with a Repressible Cell Marker) assay. Further genetic and MARCM analyses showed that not only Myoglianin (Myo) and its type I receptor Baboon is required for neuron cell death, but also this death signal is extensively regulated by endocytic trafficking in Drosophila melanogaster. We found that clathrin-mediated membrane receptor internalization and subsequent endocytic events involved in Rab5-dependent early endosome and Rab11-dependent recycling endosome differentially participate in TGF-β [beta] signaling. Two early endosome-enriched proteins, SARA and Hrs, are found to act as a cytosolic retention factor of Smad2, indicating that endocytosis mediates TGF-β [beta] signaling through regulating the dissociation of Smad2 and its cytosolic retention factor.

Thesis (Ph. D. in Human Genetics)--Vanderbilt University, May 2009. / Title from title screen. Includes bibliographical references.

The ancient continent of Sahul, encompassing Australia, New Guinea and Tasmania, contains some of the earliest archaeological evidence for humans outside of Africa, dating back to at least 50 thousand years ago (kya). New Guinea was also one of the sites were humans developed agriculture in the last 10 thousand years. Despite the importance of this part of the world to the history of humanity outside Africa, little is known about the population history of the people living here. In this thesis I present population-genetic studies using whole-genome sequencing and genotype array datasets from more than 500 indigenous individuals from Australia and New Guinea, as well as initial work on large-scale sequencing of other, worldwide, human populations in the Human Genome Diversity Project panel. Other than recent admixture after European colonization of Australia, and Southeast Asian ad- mixture in the lowlands of New Guinea in the last few millennia, the populations of Sahul appear to have been genetically independent from the rest of the world since their divergence ∼50 kya. There is no evidence for South Asian gene flow to Australia, as previously suggested, and the highlands of Papua New Guinea (PNG) have remained unaffected by non-New Guinean gene flow until the present day. Despite Sahul being a single connected landmass until ∼8 kya, different groups across Australia are nearly equally related to Papuans, and vice versa, and the two appear to have separated genetically already ∼30 kya. In PNG, all highlanders strikingly appear to form a clade relative to lowlanders, and population structure seems to have been reshaped, with major population size increases, on the same timescale as the spread of agriculture. However, present- day genetic differentiation between groups is much stronger in PNG than in other parts of the world that have also transitioned to agriculture, demonstrating that such a lifestyle change does not necessarily lead to genetic homogenization. The results presented here provide detailed insights into the population history of Sahul, and sug- gests that its history can serve as an independent source of evidence for understanding human evolutionary trajectories, including the relationships between genetics, lifestyle, languages and culture.

Non-coding small RNAs (sRNAs) regulate various cellular processes in bacteria. They bind to a chaperone protein Hfq for stability and regulate gene expression by base-pairing with target mRNAs. Although the importance of sRNAs in bacteria has been well established, the mode of origination of novel sRNA genes is still elusive, mainly because the rapid rate of evolution of sRNAs obscures their original sources. To overcome this impediment, we identified a recently formed sRNA (EcsR2) in E. coli, and show that it evolved from a degraded bacteriophage gene. Our analyses also revealed that young sRNAs such as EcsR2 are expressed at low levels and evolve at a rapid rate in comparison to older sRNAs, thereby uncovering a novel process that potentially facilitates newly emerging (and probably mildly deleterious) sRNAs to persist in bacterial genomes. We also show that even though EcsR2 is slightly deleterious to E. coli, it could bind to Hfq and mRNAs to regulate the expression of several genes. Interestingly, while EcsR2 expression is induced by glucose, the expression of its putative targets are regulated by the transcription factor CRP in response to glucose, indicating that EcsR2 has been incorporated into the carbon regulatory network in E. coli. Collectively, this work provides evidence for the emergence, evolution and functions of a novel "young" sRNA in bacteria.

No description available.

Development and application of genomic approaches based on 3C methods combined with increasingly powerful imaging approaches have enabled high-resolution genome-wide analysis of the spatial organization of chromosomes in genome function. In this thesis, I first describe an updated protocol for Hi-C (Hi-C 2.0), integrating recent improvements that significantly contribute to the efficient and high-resolution capture of chromatin interactions. Secondly, I present an assessment of the epigenetic landscape and chromosome conformation around the MYC gene in acute myeloid leukemia (AML) cells before and after small molecule, AI-10-49, treatment. MYC is up-regulated upon inhibition of the RUNX1 repressor by the fusion oncoprotein CBFβ-SMMHC. Treatment of AML cells with AI-10-49 blocks the RUNX1-CBFβ-SMMHC interaction, restoring RUNX1 at MYC regulatory elements. We demonstrate that the established loop is maintained and exchange between activating and repressive chromatin complexes at the regulatory elements, rather than altered chromatin topology, mediates disruption of target gene expression. Finally, Hi-C interaction maps represent the population-averaged steady-states. To understand the forces that promote and maintain the association of loci with specific sub-nuclear structures genome-wide, we developed liquid chromatin Hi-C. Detection of intrinsic locus-locus interaction stabilities and chromatin mobility are enabled by fragmenting chromosomes prior to fixation and Hi-C, thus removing strong polymeric constraints. Nuclear compartmentalization was found to be stable for average fragment lengths are 10-25 kb while fragmentation below 6kb led to a gradual loss of spatial genome organization. Dissolution kinetics of chromatin interactions vary widely for different domains and are analyzed in detail in the final chapter of this thesis., with lamin-associated domains being most stable, and speckle-associated loci most dynamic.

DNA replication is a fundamental process that is primarily regulated at the initiation step. In higher eukaryotes, the location and properties of replication origins are not well understood. Existing genome-wide approaches to map origins—such as nascent strand abundance mapping, Okazaki fragment mapping, or chromatin immunoprecipitation-based assays—average the behavior of a population of cells. However, due to cell-to-cell variability in origin usage, single molecule techniques are necessary to investigate the actual behavior of a cell. Here, I investigate the feasibility of using three single molecule, genome-wide technologies to map origins of replication. The Pacific Biosciences Single Molecule Real-Time (SMRT) sequencing technology, the BioNano Genomics Irys optical mapping technology, and the Oxford Nanopore Technologies MinION nanopore sequencing technology are promising approaches that can advance our understanding of DNA replication in higher eukaryotes.

The trivalent (arsenite) and pentavalent (arsenate) forms of arsenic are introduced into the environment through the use of arsenic in herbicides, pesticides, fertilizers, and the smelting of arsenic-bearing ores. Bacteria resistant to arsenic are readily isolated from surface waters, sewage, and clinical infections. Although some bacterial resistance is provided by inducible phosphate transport systems that discriminate against arsenate, marked resistance is carried on bacterial plasmids. A 6.9 kilobase fragment previously derived from one such plasmid, R45, and containing the genes for inducible resistance to arsenite and arsenate was ligated into the cloning vectors puce and pUC9 in opposite orientations and transformed into Escherichia coli JM 105. Insertion into the multiple cloning site of the pUC vectors places the inserted fragment under the inducible control of the lac operon promoter. An attempt was made to determine the direction of transcription in the fragment by growth in 10-3 M isopropyl-β-D-thiogalactoside prior to challenge with arsenite.