Computational Approaches for the Analysis of Chromosome Conformation Capture Data and Their Application to Study Long-Range Gene Regulation: A Dissertation

Over the last decade, development and application of a set of molecular genomic approaches based on the chromosome conformation capture method (3C), combined with increasingly powerful imaging approaches have enabled high resolution and genome-wide analysis of the spatial organization of chromosomes. The aim of this thesis is two-fold; 1), to provide guidelines for analyzing and interpreting data obtained from genome-wide 3C methods such as Hi-C and 3C-seq and 2), to leverage the 3C technology to solve genome function, structure, assembly, development and dosage problems across a broad range of organisms and disease models. First, through the introduction of cWorld, a toolkit for manipulating genome structure data, I accelerate the pace at which *C experiments can be performed, analyzed and biological insights inferred. Next I discuss a set of practical guidelines one should consider while planning an experiment to study the structure of the genome, a simple workflow for data processing unique to *C data and a set of considerations one should be aware of while attempting to gain insights from the data. Next, I apply these guidelines and leverage the cWorld toolkit in the context of two dosage compensation systems. The first is a worm condensin mutant which shows a reduction in dosage compensation in the hermaphrodite X chromosomes. The second is an allele-specific study consisting of genome wide Hi-C, RNA-Seq and ATAC-Seq which can measure the state of the active (Xa) and inactive (Xi) X chromosome. Finally I turn to studying specific gene – enhancer looping interactions across a panel of ENCODE cell-lines. These studies, when taken together, further our understanding of how genome structure relates to genome function

The long-range interaction landscape of gene promoters

The vast non-coding portion of the human genome is full of functional elements and disease-causing regulatory variants. The principles defining the relationships between these elements and distal target genes remain unknown. Promoters and distal elements can engage in looping interactions that have been implicated in gene regulation. Here we have applied chromosome conformation capture carbon copy (5C) to interrogate comprehensively interactions between transcription start sites (TSSs) and distal elements in 1% of the human genome representing the ENCODE pilot project regions. 5C maps were generated for GM12878, K562 and HeLa-S3 cells and results were integrated with data from the ENCODE consortium. In each cell line we discovered \u3e1,000 long-range interactions between promoters and distal sites that include elements resembling enhancers, promoters and CTCF-bound sites. We observed significant correlations between gene expression, promoter-enhancer interactions and the presence of enhancer RNAs. Long-range interactions show marked asymmetry with a bias for interactions with elements located approximately 120 kilobases upstream of the TSS. Long-range interactions are often not blocked by sites bound by CTCF and cohesin, indicating that many of these sites do not demarcate physically insulated gene domains. Furthermore, only approximately 7% of looping interactions are with the nearest gene, indicating that genomic proximity is not a simple predictor for long-range interactions. Finally, promoters and distal elements are engaged in multiple long-range interactions to form complex networks. Our results start to place genes and regulatory elements in three-dimensional context, revealing their functional relationships

Affordable In-Space Transportation

Current and proposed launch systems will provide access to low-Earth orbit (LEO), and destinations beyond LEO, but the cost of delivering payloads will preclude the use of these services by many users. To develop and encourage revolutionary commercial utilization of geosynchronous orbit (GEO) and to provide an affordable means to continue NASA space science and exploration missions, the transportation costs to in-space destinations must be reduced. The principal objective of this study was to conceptually define three to four promising approaches to in-space transportation for delivery of satellites and other payloads, 3,000- to 10,000-lb class, to GEO destinations. This study established a methodology for evaluating in-space transportation systems based on life-cycle cost. The reusable concepts seemed to fare better in the evaluation than expendable, since a major driver in the life-cycle cost was the stage production cost

Influences of state anxiety on gaze behavior and stepping accuracy in older adults during adaptive locomotion

This article is available open access through the publisher’s website at the link below. Copyright © The Authors 2011.OBJECTIVES: Older adults deemed to be at a high risk of falling transfer their gaze from a stepping target earlier than their low-risk counterparts. The extent of premature gaze transfer increases with task complexity and is associated with a decline in stepping accuracy. This study tests the hypothesis that increased anxiety about upcoming obstacles is associated with (a) premature transfers of gaze toward obstacles (i.e., looking away from a target box prior to completing the step on it in order to fixate future constraints in the walkway) and (b) reduced stepping accuracy on the target in older adults. METHODS: High-risk (9) and low-risk (8) older adult participants walked a 10-m pathway containing a stepping target area followed by various arrangements of obstacles, which varied with each trial. Anxiety, eye movements, and movement kinematics were measured. RESULTS: Progressively increasing task complexity resulted in associated statistically significant increases in measures of anxiety, extent of early gaze transfer, and stepping inaccuracies in the high-risk group. DISCUSSION: These results provide evidence that increased anxiety about environmental hazards is related to suboptimal visual sampling behavior which, in turn, negatively influences stepping performance, potentially contributing to increased falls risk in older adults.Biotechnology and Biological Sciences Research Counci

Endoplasmic Reticulum Stress Coping Mechanisms and Lifespan Regulation in Health and Diseases

Multiple factors lead to proteostatic perturbations, often resulting in the aberrant accumulation of toxic misfolded proteins. Cells, from yeast to humans, can respond to sudden accumulation of secretory proteins within the endoplasmic reticulum (ER) through pathways such as the Unfolded Protein Response (UPR). The ability of cells to adapt the ER folding environment to the misfolded protein burden ultimately dictates cell fate. The aging process is a particularly important modifier of the proteostasis network; as cells age, both their ability to maintain this balance in protein folding/degradation and their ability to respond to insults in these pathways can break down, a common element of age-related diseases (including neurodegenerative diseases). ER stress coping mechanisms are central to lifespan regulation under both normal and disease states. In this review, we give a brief overview of the role of ER stress response pathways in age-dependent neurodegeneration

Glaucoma-Related Differences in Gaze Behavior When Negotiating Obstacles

Purpose: Safe navigation requires avoiding objects. Visual field loss may affect how one visually samples the environment, and may thus contribute to bumping into objects and falls. We tested the hypothesis that gaze strategies and the number of collisions differ between people with glaucoma and normally sighted controls when navigating around obstacles, particularly under multitasking situations. Methods: Twenty persons with moderate–severe glaucoma and 20 normally sighted controls walked around a series of irregularly spaced vertical obstacles under the following three conditions: walking with obstacles only, walking and counting backward to simulate a conversation, and walking while performing a concurrent visual search task to simulate locating a landmark. We quantified gaze patterns and the number of obstacle contacts. Results: Compared with controls, people with glaucoma directed gaze closer to their current position (P < 0.05). They also directed a larger proportion of fixations (in terms of number and duration) to obstacles (P < 0.05). Despite this finding, considerably more people with glaucoma contacted an obstacle (P < 0.05). Multitasking led to changes in gaze behavior in both groups, and this was accompanied by a large increase in obstacle contacts among those with glaucoma (P < 0.05). Conclusions: Glaucoma alters gaze patterns when negotiating a series of obstacles and increases the likelihood of collisions. Multitasking in this situation exacerbates these changes. Translational Relevance: Understanding glaucoma-related changes in gaze behavior during walking in cluttered environments may provide critical insight for orientation and mobility specialists and guide the design of gaze training interventions to improve mobility

Understanding the mechanisms of IGF2 gene regulation in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. HCC has a very well studied etiology, and is associated with chronic hepatic viral infections (hepatitis viruses B and C), alcohol abuse, or other causes of chronic liver damage. Currently, tumor resection and liver transplantation are the only potentially curative treatments available for HCC. However, the presence of extra-hepatic invasion and metastasis makes patients ineligible for these treatments. High IGF2 levels are associated with metastatic HCC, and we recently showed that IGF2-induced signaling through Igf1R stimulates the invasiveness and metastatic phenotype of HCC cells. However, the precise mechanisms by which IGF2 expression is enhanced in HCC are not well understood. IGF2 is an imprinted gene normally expressed from the paternal allele. Loss of imprinting, which activates the normally silent maternal allele, has been implicated as an epigenetic marker for the enhanced risk of human cancer. However, many HCCs that display elevated IGF2 expression levels retain a normal imprinting pattern. Therefore, additional gene regulation mechanisms must also influence IGF2 expression in HCC. Hypothesis: Long-range genomic interactions are important for the regulation of IGF2 gene expression, and alterations in these long-range interactions lead to elevated IGF2 gene expression in HCC. To address this hypothesis I have utilized chromosome conformation capture carbon copy (5C) technology to elucidate long-range interactions involving the IGF2 promoters in a normal hepatocyte cell line, THLE-2, and an HCC cell line HepG2

Measuring the reproducibility and quality of Hi-C data

BACKGROUND: Hi-C is currently the most widely used assay to investigate the 3D organization of the genome and to study its role in gene regulation, DNA replication, and disease. However, Hi-C experiments are costly to perform and involve multiple complex experimental steps; thus, accurate methods for measuring the quality and reproducibility of Hi-C data are essential to determine whether the output should be used further in a study. RESULTS: Using real and simulated data, we profile the performance of several recently proposed methods for assessing reproducibility of population Hi-C data, including HiCRep, GenomeDISCO, HiC-Spector, and QuASAR-Rep. By explicitly controlling noise and sparsity through simulations, we demonstrate the deficiencies of performing simple correlation analysis on pairs of matrices, and we show that methods developed specifically for Hi-C data produce better measures of reproducibility. We also show how to use established measures, such as the ratio of intra- to interchromosomal interactions, and novel ones, such as QuASAR-QC, to identify low-quality experiments. CONCLUSIONS: In this work, we assess reproducibility and quality measures by varying sequencing depth, resolution and noise levels in Hi-C data from 13 cell lines, with two biological replicates each, as well as 176 simulated matrices. Through this extensive validation and benchmarking of Hi-C data, we describe best practices for reproducibility and quality assessment of Hi-C experiments. We make all software publicly available at http://github.com/kundajelab/3DChromatin_ReplicateQC to facilitate adoption in the community