Project description:A major concern in common disease epigenomics is distinguishing causal from consequential epigenetic variation. One means of addressing this issue is to identify the temporal origins of epigenetic variants via longitudinal analyses. However, prospective birth-cohort studies are expensive and time-consuming. Here we report DNA methylomics of archived Guthrie cards for the retrospective longitudinal analyses of in utero-derived DNA methylation variation. We first validate two methodologies for generating comprehensive DNA methylomes from Guthrie cards. Then, using an integrated epigenomic/genomic analysis of Guthrie cards and follow-up samplings, we identify inter-individual DNA methylation variation that is present both at birth and three years later. These findings suggest that disease-relevant epigenetic variation could be detected at birth i.e. before overt clinical disease. Guthrie card methylomics offers a potentially powerful and cost-effective strategy for studying the dynamics of inter-individual epigenomic variation in a range of common human diseases. Bisulphite converted DNA was hybridised to the Illumina Infinium 450k Human Methylation Beadchip
Project description:A major concern in common disease epigenomics is distinguishing causal from consequential epigenetic variation. One means of addressing this issue is to identify the temporal origins of epigenetic variants via longitudinal analyses. However, prospective birth-cohort studies are expensive and time-consuming. Here we report DNA methylomics of archived Guthrie cards for the retrospective longitudinal analyses of in utero-derived DNA methylation variation. We first validate two methodologies for generating comprehensive DNA methylomes from Guthrie cards. Then, using an integrated epigenomic/genomic analysis of Guthrie cards and follow-up samplings, we identify inter-individual DNA methylation variation that is present both at birth and three years later. These findings suggest that disease-relevant epigenetic variation could be detected at birth i.e. before overt clinical disease. Guthrie card methylomics offers a potentially powerful and cost-effective strategy for studying the dynamics of inter-individual epigenomic variation in a range of common human diseases. Bisulphite converted DNA was sequenced
Project description:A major concern in common disease epigenomics is distinguishing causal from consequential epigenetic variation. One means of addressing this issue is to identify the temporal origins of epigenetic variants via longitudinal analyses. However, prospective birth-cohort studies are expensive and time-consuming. Here we report DNA methylomics of archived Guthrie cards for the retrospective longitudinal analyses of in utero-derived DNA methylation variation. We first validate two methodologies for generating comprehensive DNA methylomes from Guthrie cards. Then, using an integrated epigenomic/genomic analysis of Guthrie cards and follow-up samplings, we identify inter-individual DNA methylation variation that is present both at birth and three years later. These findings suggest that disease-relevant epigenetic variation could be detected at birth i.e. before overt clinical disease. Guthrie card methylomics offers a potentially powerful and cost-effective strategy for studying the dynamics of inter-individual epigenomic variation in a range of common human diseases.
Project description:A major concern in common disease epigenomics is distinguishing causal from consequential epigenetic variation. One means of addressing this issue is to identify the temporal origins of epigenetic variants via longitudinal analyses. However, prospective birth-cohort studies are expensive and time-consuming. Here we report DNA methylomics of archived Guthrie cards for the retrospective longitudinal analyses of in utero-derived DNA methylation variation. We first validate two methodologies for generating comprehensive DNA methylomes from Guthrie cards. Then, using an integrated epigenomic/genomic analysis of Guthrie cards and follow-up samplings, we identify inter-individual DNA methylation variation that is present both at birth and three years later. These findings suggest that disease-relevant epigenetic variation could be detected at birth i.e. before overt clinical disease. Guthrie card methylomics offers a potentially powerful and cost-effective strategy for studying the dynamics of inter-individual epigenomic variation in a range of common human diseases.
Project description:Molecular control of recovery after exercise in muscle is temporally dynamic. A time course of biopsies around resistance exercise (RE) combined with -omics is necessary to better comprehend the molecular choreography of skeletal muscle adaptation in humans. We collected vastus lateralis biopsies before and 30 minutes, 3-, 8-, and 24 hours after acute RE. RNA-sequencing defined the transcriptome and a time-point matched biopsy only group were controls. DNA methylomics and computational approaches complimented the transcriptome data. Moreover, we tested whether cyclic transient overexpression of transcription factor MYC for 4 weeks were sufficient to induce skeletal muscle hypertrophy in female mice.
Project description:We first demonstrate that non-genetically determined inter-individual differentially methylated regions (iiDMRs) can be temporally stable for at least two years. Then, we show that iiDMRS are associated with concomitant changes in chromatin state as measured by inter-individual differences in the levels of the histone variant H2A.Z. However, the correlation of promoter iiDMRs with gene expression is negligible and this correlation is not improved even by integrating H2A.Z information. We find that most promoter epialleles, whether genetically or non-genetically determined, are associated with low levels of transcriptional activity, depleted for house keeping genes, and either depleted for H3K4me3/enriched for H3K27me3, or lacking both these marks in human embryonic stem cells. These findings validate in an independent cohort. Interestingly, the key features of iiDMRs are reminiscent of those previously observed for promoters that undergo hyper-methylation in various cancers, in vitro cell culture, and human chronological ageing. H2A.z ChIP-seq, RNA-seq, and DNA methylation data (submitted separately) were collected for five normal individuals. T21/T22 and T31/T32 are monozygotic twins.
Project description:CarD is an essential mycobacterial protein that we had previously shown to bind the RNA polymerase (RNAP) and affect the transcriptional profile of M. smegmatis and Mycobacterium tuberculosis. For this reason, we suspected that CarD was directly regulating transcriptional complexes but we did not know at what stage of CarD was functioning and at which genes CarD interacted with the RNAP. To determine in which stage of the transcription cycle (initiation, elongation, or termination) CarD acts, we used Chromatin Immunoprecipitation sequencing (ChIP-seq) to survey the distribution of CarD throughout the M. smegmatis chromosome. Specific antibodies targeting core RNAPb, RNAPσ, or a hemagglutinin (HA) epitope fused to CarD (CarD-HA) were used to co-immunoprecipitate associated DNA. CarD-HA was immunoprecipitated from the M. smegmatis Mc2155 ΔcarD attB::tetcarD-HA strain and unfused HA was immunoprecipitated from the Mc2155 attB::pmsg431 strain with monoclonal antibodies specific for HA (Sigma). RNAP β and σ were immunoprecipitated from M. smegmatis ΔcarD attB::tetcarD-HA with monoclonal antibodies specific for these subunits (Neoclone, Madison, WI; 8RB13 for β, 2G10 for σ). Co-precipitated DNA was sequenced using a SOLiD sequencer (Life Technologies), which provided sufficient reads for 100-fold coverage of the genome. The number of sequence reads per base pair was normalized to the total number of reads and expressed as a log2 value. The reads per base pair from the HA-alone sample served as the background and was subtracted from the other datasets. We found that CarD was never present on the genome in the absence of RNAP. However, whereas RNAP core enzyme was found throughout transcribed regions of the genome, CarD was primarily associated with promoter regions and highly correlated with RNAPσ. The colocalization of σA and CarD led us to propose that in vivo, CarD associates with RNAP initiation complexes at most promoters and is therefore a global regulator of transcription initiation.