Mammalian comparative genomics and epigenomics

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2009.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student submitted PDF version of thesis.Includes bibliographical references.The human genome sequence can be thought of as an instruction manual for our species, written and rewritten over more than a billion of years of evolution. Taking a complete inventory of our genome, dissecting its genes and their functional components, and elucidating how these genes are selectively used to establish and maintain cell types with markedly different behaviors, are key challenges of modern biology. In this thesis we present contributions to our understanding of the structure, function and evolution of the human genome. We rely on two complementary approaches. First, we study signatures of evolutionary processes that have acted on the genome using comparative sequence analysis. We generate high quality draft genome sequences of the chimpanzee, the dog and the opossum. These species share a last common ancestor with humans approximately 6 million, 80 million and 140 million years ago, respectively, and therefore provide distinct perspectives on our evolutionary history. We apply computational methods to explore the functional organization of the genome and to identify genes that contribute to shared and species-specific traits. Second, we study how the genome is bound by proteins and packaged into chromatin in distinct cell types. We develop new methods to map protein-DNA interactions and DNA methylation using single-molecule based sequencing technology. We apply these methods to identify new functional sequence elements based on characteristic chromatin signatures, and to explore the relationship between DNA sequence, chromatin and cellular state.by Tarjei Sigurd Mikkelsen.Ph.D

Socioeconomic status and duration and pattern of sickness absence. A 1-year follow-up study of 2331 hospital employees

BACKGROUND: Sickness absence increases with lower socioeconomic status. However, it is not well known how this relation depends on specific aspects of sickness absence or the degree to which socioeconomic differences in sickness absence may be explained by other factors. The purpose of the study was to examine differences in sickness absence among occupational groups in a large general hospital; how they depend on combinations of frequency and duration of sickness absence spells; and if they could be explained by self-reported general health, personal factors and work factors. METHODS: The design is a 1-year prospective cohort study of 2331 hospital employees. Baseline information include job title, work unit, perceived general health, work factors and personal factors recorded from hospital administrative files or by questionnaire (response rate 84%). Sickness absence during follow-up was divided into short (1-3 days), medium (4-14 days) and long (>14 days) spells, and into no absence, "normal" absence (1-3 absences of certain durations) and "abnormal" absence (any other absence than "normal"). Socioeconomic status was assessed by job titles grouped in six occupational groups by level of education (from doctors to cleaners/porters). Effects of occupational group on sickness absence were adjusted for significant effects of age, gender, general health, personal factors and work factors. We used Poisson or logistic regression analysis to estimate the effects of model covariates (rate ratios (RR) or odds ratios (OR)) and their 95% confidence intervals (CI). RESULTS: With a few exceptions sickness absence increased with decreasing socioeconomic status. However, the social gradient was quite different for different types of sickness absence. The gradient was strong for medium spells and "abnormal" absence, and weak for all spells, short spells, long spells and "normal" absence. For cleaners compared to doctors the adjusted risk estimates increased 4.2 (95% CI 2.8-6.2) and 7.4 (95% CI 3.3-16) times for medium spells and "abnormal" absence, respectively, while the similar changes varied from 0.79 to 2.8 for the other absence outcomes. General health explained some of the social gradient. Work factors and personal factors did not. CONCLUSIONS: The social gradient in sickness absence was different for absences of different duration and patterns. It was strongest for absences of medium length and "abnormal" absence. The social gradient was not explained by other factors

A unique regulatory phase of DNA methylation in the early mammalian embryo

Summary DNA methylation is highly dynamic during mammalian embryogenesis. It is broadly accepted that the paternal genome is actively depleted of 5-methyl cytosine at fertilization, followed by passive loss that reaches a minimum at the blastocyst stage. However, this model is based on limited data, and to date no base-resolution maps exist to support and refine it. Here, we generated genome-scale DNA methylation maps in mouse gametes and through post-implantation embryogenesis. We find that the oocyte already exhibits global hypomethylation, most prominently at specific families of long interspersed element-1 and long terminal repeat retro-elements, which are disparate between gametes and resolve to lower methylation values in zygote. Surprisingly, the oocyte contributes a unique set of Differentially Methylated Regions (DMRs), including many CpG Island promoter regions, that are maintained in the early embryo but are lost upon specification and absent from somatic cells. In contrast, sperm-contributed DMRs are largely intergenic and resolve to hypermethylation after the blastocyst stage. Our data provide a complete genome-scale, base-resolution timeline of DNA methylation in the pre-specified embryo, when this epigenetic modification is most dynamic, before returning to the canonical somatic pattern.Stem Cell and Regenerative Biolog

A unique regulatory phase of DNA methylation in the early mammalian embryo

DNA methylation is highly dynamic during mammalian embryogenesis. It is broadly accepted that the paternal genome is actively depleted of 5-methylcytosine at fertilization, followed by passive loss that reaches a minimum at the blastocyst stage. However, this model is based on limited data, and so far no base-resolution maps exist to support and refine it. Here we generate genome-scale DNA methylation maps in mouse gametes and from the zygote through post-implantation. We find that the oocyte already exhibits global hypomethylation, particularly at specific families of long interspersed element 1 and long terminal repeat retroelements, which are disparately methylated between gametes and have lower methylation values in the zygote than in sperm. Surprisingly, the oocyte contributes a unique set of differentially methylated regions (DMRs)—including many CpG island promoters—that are maintained in the early embryo but are lost upon specification and absent from somatic cells. In contrast, sperm-contributed DMRs are largely intergenic and become hypermethylated after the blastocyst stage. Our data provide a genome-scale, base-resolution timeline of DNA methylation in the pre-specified embryo, when this epigenetic modification is most dynamic, before returning to the canonical somatic pattern.Burroughs Wellcome (Career Award)National Institutes of Health (U.S.) (5RC1AA019317)National Institutes of Health (U.S.) (U01ES017155)National Institutes of Health (U.S.) (P01GM099117)National Human Genome Research Institute (U.S.) (1P50HG006193-01

Systematic dissection of regulatory motifs in 2000 predicted human enhancers using a massively parallel reporter assay

Genome-wide chromatin annotations have permitted the mapping of putative regulatory elements across multiple human cell types. However, their experimental dissection by directed regulatory motif disruption has remained unfeasible at the genome scale. Here, we use a massively parallel reporter assay (MPRA) to measure the transcriptional levels induced by 145-bp DNA segments centered on evolutionarily conserved regulatory motif instances within enhancer chromatin states. We select five predicted activators (HNF1, HNF4, FOXA, GATA, NFE2L2) and two predicted repressors (GFI1, ZFP161) and measure reporter expression in erythroleukemia (K562) and liver carcinoma (HepG2) cell lines. We test 2104 wild-type sequences and 3314 engineered enhancer variants containing targeted motif disruptions, each using 10 barcode tags and two replicates. The resulting data strongly confirm the enhancer activity and cell-type specificity of enhancer chromatin states, the ability of 145-bp segments to recapitulate both, the necessary role of regulatory motifs in enhancer function, and the complementary roles of activator and repressor motifs. We find statistically robust evidence that (1) disrupting the predicted activator motifs abolishes enhancer function, while silent or motif-improving changes maintain enhancer activity; (2) evolutionary conservation, nucleosome exclusion, binding of other factors, and strength of the motif match are predictive of enhancer activity; (3) scrambling repressor motifs leads to aberrant reporter expression in cell lines where the enhancers are usually inactive. Our results suggest a general strategy for deciphering cis-regulatory elements by systematic large-scale manipulation and provide quantitative enhancer activity measurements across thousands of constructs that can be mined to develop predictive models of gene expression.National Institutes of Health (U.S.) (Grant HG004037)National Institutes of Health (U.S.) (Grant HG004037-S1

Does computer use pose a hazard for future long-term sickness absence?

The aim of the study was to investigate if weekly duration of computer use predicted sickness absence for more than two weeks at a later time

A Cohort Study on Meniscal Lesions among Airport Baggage Handlers

Meniscal lesions are common and may contribute to the development of knee arthrosis. A few case-control and cross-sectional studies have identified knee-straining work as risk factors for meniscal lesions, but exposure-response relations and the role of specific exposures are uncertain, and previous results may be sensitive to reporting and selection bias. We examined the relation between meniscal lesions and cumulative exposure to heavy lifting in a prospective register-based study with complete follow-up and independent information on exposure and outcome. We established a cohort of unskilled men employed at Copenhagen Airport or in other companies in the metropolitan Copenhagen area from 1990 to 2012 (the Copenhagen Airport Cohort). The cohort at risk included 3,307 airport baggage handlers with heavy lifting and kneeling or squatting work tasks and 63,934 referents with a similar socioeconomic background and less knee-straining work. Baggage handlers lifted suitcases with an average weight of approximately 15 kg, in total approximately five tonnes during a 9-hour workday. The cohort was followed in the National Patient Register and Civil Registration System. The outcome was a first time hospital diagnosis or surgery of a meniscal lesion. Baggage handlers had a higher incidence of meniscal lesions than the referents. Within baggage handlers spline regression showed that the incidence rate ratio was 1.91 (95% confidence interval: 1.29-2.84) after five years as a baggage handler and then decreased slowly to reach unity after approximately 30 years, adjusted for effects of potential confounders. This relation between baggage handling and meniscal lesions was present for work on the apron which involves lifting in a kneeling or squatting position, but not in the baggage hall, which only involves lifting in standing positions. The results support that long-term heavy lifting in a kneeling or squatting position is a risk factor for the development of symptomatic meniscal lesions

Baggage handler seniority and musculoskeletal symptoms:is heavy lifting in awkward positions associated with the risk of pain?

OBJECTIVES: Heavy lifting is associated with musculoskeletal disorders but it is unclear whether it is related to acute reversible effects or to chronic effects from cumulated exposure. The aim of this study was to examine whether musculoskeletal symptoms in Danish airport baggage handlers were associated with their seniority as baggage handler, indicating chronic effects from cumulated workload. METHODS: We established a group of baggage handlers employed at Copenhagen Airport during the period 1983–2012 (n=3092) and a reference group of men in other unskilled occupations with less heavy work (n=2478). Data regarding work history, lifestyle and musculoskeletal symptoms were collected using a self-administered questionnaire (response rate 70.1% among baggage handlers and 68.8% among the reference group). RESULTS: The ORs of self-reported musculoskeletal symptoms during the last 12 months in the neck/upper back, lower back, shoulders, elbows, wrists, hips and knees were significantly higher in baggage handlers than in the reference group. These differences were explained by significant linear effects of baggage handler seniority for six anatomical regions. Adjustment for age, body mass index, smoking and leisure-time physical activity did not change these results. The findings were stable over age strata and among present and former baggage handlers. CONCLUSIONS: The risk of musculoskeletal symptoms in six anatomical regions increased with increasing seniority as a baggage handler. This is consistent with the assumption that cumulated heavy lifting may cause chronic or long-lasting musculoskeletal symptoms. However, we cannot exclude that other factors related to baggage handler seniority may explain some of the associations