Identification of Long-Range Regulatory Elements in the Human Genome

Genome-wide association studies have shown that the majority of disease-associated genetic variants lie within non-coding regions of the human genome. Subsequently, a challenge following these discoveries is to identify how these variants modulate the risk of disease. Enhancers are non-coding regulatory elements that can be bound by proteins to activate the expression of a gene that may be linearly distant. Experimentally probing all possible enhancer–target gene pairs can be laborious. Hi-C, a technique developed by Job Dekker’s group in 2009, combines high-throughput sequencing with chromosome conformation capture to detect DNA interactions genome-wide and thereby reveals the three-dimensional architecture of chromatin in the nucleus. However, the utility of the datasets produced by this technique for discovering long-range regulatory interactions is largely unexplored. In this thesis, we develop novel approaches to identify DNA-interacting units and their interactions in Hi-C datasets with the goal of uncovering all enhancer–target gene interactions. We began by identifying significantly interacting regions in these datasets, subsequently focusing on candidate enhancer–gene pairs. We found that the identified putative enhancers are enriched for p300 binding activity, while their target promoters are likely to be cell-type-specific. Furthermore, we revealed that enhancers and target genes often interact in many-to-many relationships and the majority of enhancer–target gene interactions are intra-chromosomal and within 1 Mb of each other. Next, we refined our analytical approach to identify physically-interacting DNA regions at ~1 kb resolution and better define the boundaries of likely enhancer elements. By searching for over-represented sequences (motifs) in these putative promoter-interacting enhancers, we were then able to identify bound transcription factors. This newer approach provides the potential to identify protein complexes involved in enhancer–promoter interactions, which can be verified in future experiments. We implemented a high-throughput identification pipeline for promoter-interacting enhancer elements (HIPPIE) using both of the above described approaches. HIPPIE can be run efficiently on typical Linux servers and grid computing environments and is available as open-source software. In summary, our findings demonstrate the potential utility of Hi-C technologies for elucidating the mechanisms by which long-range enhancers regulate gene expression and ultimately result in human disease phenotypes

Analysis of Long-Range Chromosomal Interactions in \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e: A Dissertation

Long-range chromosomal interactions have been discovered in a number of organisms, suggesting that gene regulation through direct physical association with regulatory elements and/or other genes is a common and conserved phenomenon. This thesis investigates the relationship between direct physical contact of genomic loci and how these interactions may play a role in gene regulation. Analysis of such levels of chromosomal organization has been made possible in part by the emergence of Chromosome Conformation Capture (3C). This technique makes use of formaldehyde crosslinking to trap interacting chromosomal fragments, which can be detected after a number of manipulations. By adapting the 3C protocol for use in intact Saccharomyces cerevisiaecells, one can determine the structure of a chromosome or chromosomal region without introducing artifacts due to the harsh isolation of nuclei. A number of 3C-based technologies, such as 4C (Circular 3C or 3C-on-Chip) and 5C (3C Carbon Copy) have added to the knowledge of physical association of genes with regulatory elements and/or other genes. Here, we present a new non-biased technology that allows for determination of chromosomal interactions between all fragments throughout a genome. We present two-dimensional heatmaps of chromosomal interactions for all 16 chromosomes in yeast. These techniques promise to shed light onto the biochemical process by which clustering of genes and elements can result in up- or down-gene expression, which is still poorly understood. To understand how chromosomal interactions play a role in gene regulation, we study clustering of heterochromatic loci. Clustering of heterochromatic loci in silenced nuclear compartments is a phenomenon that has been observed throughout evolution. These clusters are thought to represent nuclear sub-compartments that are enriched in silencing proteins, while the rest of the nucleus is depleted in such factors. Chromosome III in Saccharomyces cerevisiae contains four heterochromatic regions: the two telomeres and the silent mating type loci, HML and HMR, located on either end of the chromosome. Our work describes a long-range interaction between the heterochromatic regions on chromosome III. We analyze the mechanism that drive these interactions and reveal roles for silencing proteins and proper nucleosome assembly in mediating heterochromatic clustering. In addition we identify a novel step in heterochromatin formation that is not essential for gene silencing but is required for long-range interactions

INSIGHTS INTO ENZYMATIC MANIPULATIONS OF NUCLEIC ACIDS

This dissertation details three studies dealing with the manipulation of nucleicacids. In the first investigation, each of the four natural nucleobases were analyzed for theability to serve as a universal template at the ligation junction of a T4 DNA ligasereaction. This resulted in the first instance of sequence-independent ligation catalyzed byany DNA ligase. Although all of the nucleobases display universal templatingcapabilities, thymidine and guanosine provided the most effective results. In addition,lowered MgCl2 and ATP concentrations, as well as the inclusion of DMSO, also aided inthe sequence-independent ligation reported here. In the course of these studies, currentmethods of removing urea from denaturing-gel purified nucleic acids provedcumbersome. Therefore, in the second study simple butanol extraction was examined as ameans to eliminate urea from nucleic acid solutions. Stepwise butanol extraction was themost effective approach to solving this problem and provided a much needed techniquefor nucleic acid purification. This type of extraction also does not result in significantlosses of nucleic acid sample. The third study exploits the molecular recognition andcatalytic properties inherent in an autocatalytic group I intron to develop a ribozyme thatcan replace the 5\u27 end of an RNA substrate with a different RNA. This 5\u27 replacementsplicing reaction can potentially repair mutations on the 5\u27 ends of RNA transcripts thatlead to a variety of genetic mutations. The model system was a common mutation in asmall model mimic of the k-ras gene in vitro, which predisposes individuals to lungcancer. This 5\u27 replacement splicing reaction occurred in vitro using this small modelsystem; the reaction was also enhanced by the alteration of the molecular interactionsinvolved. The results and implications of each of these studies are detailed in thisdissertation

Towards construction and validation of an ends-in recombination system in Escherichia coli

Homologous recombination is the primary DNA repair pathway in bacteria and it is immensely important in repairing DNA double strand breaks. Components of the homologous recombination pathway have been well conserved throughout evolution as an essential part of cell survival. Homologous recombination plays an important role in cellular processes like DNA repair as well as exchange of genetic information through chromosomal crossover. During homologous recombination, DNA strand exchange leads to formation of a heteroduplex joint between the invading and displaced DNA strands. This hetereoduplex joint is called a Holliday Junction. Resolution of the Holliday Junction proceeds via one of two pathways. In the presence of RuvC and/or RecG, Holliday Junction resolution proceeds via a “cut and paste” pathway where the invading DNA strand replaces a region of homologous DNA on the target DNA. In the absence of RuvC and RecG, Holliday Junction resolution takes place via a “copy and paste” pathway during which DNA synthesis needs to be primed at Holliday Junction intermediates formed during strand invasion. In an effort to separate this myriad of different requirements, I have attempted to develop a novel “ends-in” recombination assay system using E. coli as a model organism. This ends-in system would allow recombinant molecule formation by DNA synthesis of approximately 200 to 2000 bp size interval between the two converging ends of an invading linear dsDNA substrate oriented just like the greek letter Ù, but with the arms pointing inwards. In this study, a number of linear dsDNA assay templates were constructed and analyzed. All the constructs had two “arms” of homology to the chromosome pointing inwards i.e. in the ends-in orientation. Using this ends-in system, it was demonstrated that the presence of chi (Crossover Hotspot Initiator) sites was an important requirement for ends-in recombination in wild type E. coli cells. Our studies also showed that ends-in homologous recombination did not occur if chi sites were placed at or very near to the ends of the incoming linear dsDNA molecule, suggesting that the chi site recognition is efficient only if the incoming dsDNA has chi sites internal to the ends. Moreover, it was shown that neither RuvC nor RecG were required for successful recombinant product formation using the ends-in assay. This finding reinforces previous observations that suggest the idea that Holliday Junctions can be resolved independent of both RuvC and RecG

JUNCTION PROBES - SEQUENCE SPECIFIC DETECTION OF NUCLEIC ACIDS VIA TEMPLATE ENHANCED HYBRIDIZATION PROCESSES

As new disease biomarkers such as cancer-linked microRNAs are discovered, the need for new strategies for the detection of these disease biomarkers at isothermal conditions will increase. The junction probe (JP) technology is a restriction endonuclease-based nucleic acids detection platform that achieves isothermal amplified sensing and does not require the presence of an endonuclease recognition sequence in the target analyte. The first generation junction probe platform however suffered from long assay time (several hours). We hypothesized that the slow catalysis in the first-generation JP platform was due to an inhibition cycle. Consequently, a second generation JP platform, which is modified with phosphorothioate moieties in order to suppress the inhibition cycle, was developed. The second-generation JP platform is significantly superior to the first-generation platform and we demonstrated the potential of second-generation JP by detecting microRNA and bacterial ribosomal RNA. Importantly, second-generation JP could detect RNA in crude bacterial cell lysates without extensive sample preparation