View a list of abstracts for the Bridging Genes and Enviroment: How Epigenetics Remembers the Past to Shape Your Future poster session on April 28th (listed alphabetically by name).
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Thus, targeting lncRNAs for methylation assessment seems essential. The challenge is that most lncRNA genes are poorly characterized and not known to produce RNA.
As a first step to prioritize the lncRNA genes to interrogate for DNA methylation, we will evaluate each lncRNA site of interest and identify which are functional.
We are developing a plug-in for the Galaxy web-based platform for data intensive biomedical research using user-friendly graphical interfaces. The program queries common NCBI Gene Expression Omnibus (GEO) microarray platforms for the existence of expression probes interrogating lncRNA genes of interest via chromosomal location. The query input is a selection of public lncRNA databases or a custom BED file with identifiers and start and end coordinates.
When a chromosomal location match is found, expression data relevant to the input lncRNAs will be extracted from GEO pre-normalized dataset. We also plan on implementing functionality to search GEO RNA-seq datasets.
To broaden utility, the plug-in scripts will also be packaged as a stand-alone graphical tool and are inherently command-line friendly.
This tool will facilitate comprehensive investigations into the role of lncRNA genes in genomic and epigenetic regulation in complex diseases and will enable using the most up-to-date gene expression datasets.
Here, we present our recent work indicating that transcription emanating from MT LTRs is likely responsible for transcription-coupled de novo DNA methylation and deposition of H3K36me3 at a significant fraction of genomic regions showing orphan/“intergenic” methylation in oocytes, encompassing CGI DMRs and a subset of gametic imprinted DMRs. Furthermore, we identified dozens of genes that are driven by chimaeric transcripts initiating in solo MT LTRs, including many gene previously shown to play central roles in oocyte function. Taken together, these observations reveal that retrotransposons can have a profound effect on both the methylome and transcriptome in the mammalian germline.
We first identified transcription start sites of escapees (escTSSs) based on higher transcription levels in female cells using FANTOM5 Cap Analysis of Gene Expression data. Greater similarity of DNA methylation levels between the sexes was found to be consistent with bi-allelic activity at these escTSSs. The significant over-representations of YY1 transcription factor binding motif and ChIP-seq peaks around escTSSs highlighted its positive association with escapees. Furthermore, YY1 occupancy is significantly biased towards the inactive X (Xi) at long non-coding RNA loci that are frequent contacts of previously reported Xi-specific superloops in female GM12878 cells. While revealing the unique properties of the X as reflected by genomic datasets, our study elucidated the importance of YY1 on transcriptional activity on Xi in general through sequence-specific binding, and its involvement at anchor regions of Xi-specific chromatin superloops.
Hypothesis: Specific genomic imprinted regions are associated with asthma and related phenotypes (atopy and airway hyperresponsiveness (AHR)).
Methods: To identify candidate genomic regions for imprinting we used GWAS data from two family-based studies: the Canadian Asthma Primary Prevention Study (CAPPS) and the Study of Asthma Genes and Environment (SAGE). We used a likelihood-based variant of the Transmission Disequilibrium Test. Parent-of-origin effects were tested by including a modifier (the sex of parents) in the analysis.
Results: In the joint analysis with 148 asthmatic trios, 13 SNPs showed significant parent-of-origin effects with p<10-5. Three SNPs remained significant after 100,000 permutations. Notably, we showed a parent-of-origin effect at a known imprinted gene, CTNNA3. Six SNPs were in or near Long non-coding RNA genes. The analyses for atopy (237 trios) and AHR (231 trios) yielded 3 and 2 significant SNPs after permutation, respectively.
Work in progress: To increase statistical power and confirm our results, we will perform a meta-analysis using 3 family studies: CAPPS, SAGE and the Saguenay-Lac-Saint-Jean study.
Low SES increases the likelihood of chronic stress from living in unfavourable conditions. There is evidence that chronic psychological stress contributes to the development and progression of pathologies. Stressed individuals are prone to viral infections, arthritis and multiple sclerosis and tend to develop premature coronary disease.
The plasticity of dynamic DNAm suggests a mechanism where persistent environmental factors, like chronic stress, cause chromatin modifications that affect developmental programming, and remain detectable in adult life. In this way, variability of environmental exposures across individuals is reflected in age-related DNAm trajectories.
"Epigenetic age" constructs measure deviations from the average age-related DNA methylation trajectory in a population, and quantify inter-individual variability of age-related DNAm.
We develop a novel bioinformatic approach to construct a monocyte-specific (MS) epigenetic clock that incorporates prior knowledge about DNAm sites associated with age. We use this MS epigenetic clock to asses epigenetic age acceleration, and find that it is associated with SES.
Global transcriptome analysis of CD34+ chronic-phase CML cells
Colin A. Hammond1,2, Davide Pellacani1, David J.H.F. Knapp1,2, Xiaoyan Jiang1,2,4, Martin Hirst3, Connie J. Eaves1,2,4.
1Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada. 2Department of Medicine, University of British Columbia, Vancouver, BC, Canada. 3Centre for High-Throughput Biology, University of British Columbia, Vancouver, BC, Canada. 4Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada.
Introduction: Chronic myeloid leukemia (CML) is a clonal multi-lineage leukemia in which all cells express a BCR-ABL1 fusion oncogene. The effects of this constitutively activated tyrosine kinase on gene-expression are believed to contribute to the malignant phenotype and competitive advantage of CML cells over the co-existing normal hematopoietic cells. While previous comparative investigations of CML-associated changes in gene-expression have mainly relied on microarrays to capture a subset of total mRNAs, we performed global gene expression profiling in primary samples of CML CD34+ and normal CD34+ hematopoietic cells using RNA-Seq.
Methods: Strand-specific RNA-Seq libraries were created from RNA extracted from highly purified (>90%) CD34+ blood cells isolated from adult patients with chronic phase CML, normal adult bone marrow (BM) donors, and from a large pool of normal cord blood (CB) samples. Differentially expressed genes were identified using the DESeq tool and custom R scripts.
Results: The majority of differentially expressed genes identified had lower expression in CD34+ cells isolated from CML patients as compared with the CD34+ cells from normal adult BM or CB. Differentially expressed genes found in both analyses included genes previously associated with CML malignancy including IRF8, GAS2, and GLI2. Interestingly, a number of the genes differentially expressed between the CML and CB samples are consistent with age-related transcriptomic differences observed between primitive hematopoietic cells in fetal and adult mice.
Conclusion: These results set the stage for further analyses of the intrinsic epigenomic mechanisms underpinning CML-associated changes in gene expression within CD34+ stem and progenitor cells.
Introduction: Genomic instability within cancer cells often leads to profound genomic heterogeneity, clonal evolution, and drug resistance. We hypothesize that epigenomic heterogeneity should exist within tumour cell populations to at least the same degree. Whole-genome bisulfite sequencing is a technique used to profile DNA methylation; however, this methodology is limited by high input DNA requirements (at least 100s of thousands of cells). We therefore aimed to develop a bisulfite sequencing method that can profile single-cells for the purpose of characterizing the epigenomic heterogeneity within tumour cell populations.
Methods: We developed a method, termed Post-Bisulfite Adapter Ligation (PBAL), which combines automated bisulfite conversion of DNA with traditional whole-genome shotgun library construction; this protocol can be scaled with an Agilent Bravo liquid handling robot, allowing for interrogation of many individual single-cells in parallel. Completed libraries are indexed, pooled, and sequenced on the Illumina massively parallel sequencing platform. Reads are then aligned to the human genome using a specialized bisulfite sequencing aligner (Novoalign).
Results: To date, we have sequenced 889 PBAL libraries on the Illumina MiSeq and 148 on the Illumina HiSeq. Using a filter within Novoalign to remove contaminants, we averaged <1% alignment from negative controls, 30% alignment from single-cells, and 85% alignment from control populations of 100 cells. The conversion efficiency of unmethylated cytosines measured, using a T7 control genome, was 98.5%. Analyses of 168 breast epithelial single cells (184-hTert) revealed an average of ~20% difference in CpG methylation between pairwise common CpG’s. Finally, we were able to detect copy-number variations and are currently working to associate these with epigenetic changes.
Conclusions: We have successfully developed a protocol for whole-genome DNA methylation using single-cells as input. We applied this protocol to assess epigenetic heterogeneity within 184-hTert immortalized breast epithelial cells and found ~20% heterogeneity at a single CpG resolution.
Rebecca A.G. De Souza§, Sumaiya A. Islam§, Lisa M. McEwen, Anthony Mathelier,
Austin Hill, Sarah M. Mah, Wyeth W. Wasserman, Michael S. Kobor, Blair R. Leavitt
Department of Medical Genetics, Centre for Molecular Medicine and Therapeutics, Child and
Family Research Institute, University of British Columbia, Vancouver, BC, Canada, V5Z
§These authors contributed equally to this work
Despite extensive progress in Huntington’s Disease (HD) research, very little is
known about the association of epigenetic variation and HD pathogenesis in human brain
tissues. Moreover, its contribution to the tissue-specific transcriptional regulation of the
huntingtin gene (HTT), in which HTT expression levels are highest in brain and testes, is
currently unknown. To investigate the role of DNA methylation in HD pathogenesis and
tissue-specific expression of HTT, we utilized the Illumina HumanMethylation450K
BeadChip array to measure DNA methylation in a cohort of age-matched HD and control
human cortex and liver tissues. In cortex samples, we found minimal evidence of HD associated
DNA methylation at probed sites after correction for cell heterogeneity but did
observe an association to age of disease onset. By contrast, comparison of matched cortex
and liver samples revealed tissue-specific DNA methylation of the HTT gene region at 38
sites (FDR < 0.05). Importantly, we identified a novel differentially methylated binding site
in the HTT proximal promoter for the transcription factor CTCF. This CTCF site displayed
increased occupancy in cortex, where HTT expression is higher, compared to liver.
Additionally, CTCF silencing reduced the activity of a HTT promoter-reporter construct,
suggesting that CTCF plays a role in regulating HTT promoter function. Overall, although we
were unable to detect HD-associated DNA methylation alterations at queried sites, we found
that DNA methylation may be correlated to age of disease onset in cortex tissues. Moreover,
our data suggest that DNA methylation may, in part, contribute to tissue-specific HTT
transcription through differential CTCF occupancy.
Identification of DNA methylation signatures in chorioamnionitis associated placentas
Chaini R. Konwar1,2, Magda E. Price1,2,3, Wendy P. Robinson1,2,3
1Dept. Medical Genetics, University of British Columbia, Vancouver BC; 2Child and Family Research Institute, Vancouver BC; 3Dept. of Obstetrics and Gynecology, University of British Columbia, Vancouver BC
Introduction: One of the major causes of spontaneous preterm birth is inflammation of the placenta and associated membranes, known as chorioamnionitis (CA). Identification of DNA methylation (DNAm) signatures in the placenta that reflect in utero conditions may improve our understanding of the causes of preterm birth and provide candidate markers for earlier diagnosis of inflammation. Inflammation in placenta is associated with an increase in placental-specific macrophages, Hofbauer cells (HCs). DNAm signatures vary vastly between different cell lineages and change with i) gestational age; and ii) placental pathology, hence it may be possible to detect differences in HC number by measuring placental DNAm.
Methods: Placental samples (chorionic villi) from 12 CA cases and 12 preterm births without other pathology (controls) were collected previously by Robinson lab and run on the Illumina HumanMethylation450 BeadChip array (450K array). Additionally, independent control term samples were used to profile DNAm using the 450k array in 5 HCs samples and matched 5 chorionic villous samples. DNAm data was filtered and normalized, and then differential methylation was assessed using R as the statistical tool.
Results: We identified 18 differentially methylated sites associated with CA status based on i) statistical significance (False Discovery Rate, FDR <5%); and ii) biological significance (magnitude of methylation difference, Δβ>0.1). Majority of these sites were associated with immune-system genes including Orosomucoid 1 which is known to exhibit increased expression in response to infection. Of the 18 CA associated DNAm sites, 3 DNAm sites were also identified as differentially methylated in comparison of chorionic villi with HCs.
Conclusion: CA-associated placentas showed altered DNAm signatures which are not observed in the absence of inflammation. The changes are consistent with changes in innate immune function and may reflect both increased numbers of immune cells as well as altered function.
Lisa M. McEwen (1), Meaghan J. Jones (1), Julia L. MacIsaac (1), William H. Dow (2), Lluis Rosero-Bixby (2), Michael S. Kobor (1), David H. Rehkopf (3)
1. Department of Medical Genetics, Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, University of British Columbia, 950 West 28th Avenue, Vancouver, BC, Canada
2. Centro Centroamericano de Población, Universidad de Costa Rica, San José 2060
3. Division of General Medical Disciplines, School of Medicine, Stanford University, Stanford, CA
Aging is an inevitable biological process, consisting of a variety of physiological and molecular changes, which occur at different rates across individuals. The Nicoya Peninsula in Costa Rica represents a unique geographical area enriched for healthy centenarians, and hosts a community with an estimated mortality rate ratio of 0.80 (0.69-0.93). The increased lifespan has been hypothesized to be a result of lifestyle factors such as nutrition, exercise, and social status; however, it remains to be scientifically explained and thus represents an exciting cohort for exploration, especially at the molecular level. DNA methylation (DNAm) is a process that is not only responsive to environmental factors but also has the potential to regulate gene expression. Accordingly, this epigenetic modification is a great candidate as a possible mediator between Nicoyan environmental factors and the observed longevity phenotype. DNA methylation of whole blood was assessed in 95 Costa Ricans (48 Nicoyans, 47 non-Nicoyans), aged between 70-110 years old, using the Illumina 450k Methylation Array. We identified a significant difference in the DNA methylation-based predicted proportion of two age-associated cell types: CD8+ naïve and CD8+ memory T cells. Whereas older people normally have lower CD8+ naïve T cells and higher CD8T+ memory cells, the Nicoyan Costa Ricans showed higher CD8+T naïve and lower CD8+ memory T cells in contrast to non-Nicoyan (naïve: p-value=0.01, memory: p-value=0.05). In summary, by DNA methylation profiling, we were able to detect a biological distinction of cellular abundance that may potentially represent a slow-aging marker in an ethnically novel centenarian cohort.
We hypothesize that a significant portion of the complement of differentially expressed genes identified between two phenotypically and genetically distinct lab mouse strains can be explained by sequence variation in cis-regulatory regions. We chose Mus musculus castaneus and domesticus for our study because 1) they differ by ~20 million SNPs and 2) produce fertile F1 offspring i.e. the two genomes are dissimilar and compatible. By generating transcriptome data on F1 primary cells, (cells carrying both parental genomes), confounding experimental and biological variation (trans effects) are minimized or eliminated. Using allele-specific expression analysis (Younesy 2013), I identified the complement of genes showing strain-specific expression bias in embryonic day 13.5 primordial germ cells extracted from F1 embryos generated by reciprocally crossing castaneus and domesticus.
Ongoing ChIP-seq experiments for enhancer, promoter and repressive histone marks in the same F1 cells will yield a set of candidate regulatory sequence variants near the genes showing strain-specific expression bias which we will validate via reciprocal CRISPR-mediated genome editing.
INTRODUCTION: The majority of asthmatics trace the onset of symptoms to early childhood including the development of atopy and a decline in lung function by school-age. The Canadian Healthy Infant Longitudinal Development (CHILD) birth cohort aims to determine the role of a wide range of environmental and genetic factors in the development of asthma and allergy. This poster shows early findings from epigenetic analyses of environmental exposures.
METHODS: From 2008-2012, 3,624 pregnant women were recruited in four Canadian cities and one rural populations. Environmental exposure data include repeated detailed questionnaires and home assessments, estimates of outdoor pollution and biomarkers. Children were seen at 3 months of age, and again at 1, 3 and 5 years including health history, allergy testing and biomarkers. A sub-sample of children had peripheral blood mononuclear cell (PBMC) DNA methylation profiles by Illumina 450k array at birth (n=91) and 12 months (n=138) assessed with principal component analysis.
RESULTS: Preliminary results from adjusted principal component analysis show our index of indoor exposures at 3 months explained 13% of the adjusted variance in cord blood but is confounded by wheeze and pre-natal stress in the mother. Estimates of traffic related air pollution (TRAP) explained 10% of the adjusted variance in the 12 month sample but the indoor exposure index, wheeze and maternal stress measured at 3 months were no longer significant.
CONCLUSION: In the CHILD birth cohort, early analyses of PBMC DNA methylation patterns in cord blood were associated with oxidizing home exposures at 3 months and with estimates of TRAP exposure at birth in the 12 month sample.