Unknown,Transcriptomics,Genomics,Proteomics

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Age-related methylation changes are associated with altered transcriptional circuitry [Methyl-seq]

ABSTRACT: Altered DNA methylation patterns represent an attractive mechanism for the phenotypic changes associated with human aging. Several studies have described age-related methylation changes to various extents, but their functional significance has remained largely unclear. We have now used an integrated methylome and transcriptome sequencing approach to characterize age-related methylation changes in the human epidermis and to analyze their impact on gene expression. Our results show limited and localized methylation differences between young and old methylomes at single-base resolution. Similarly, the comparison of transcriptomes from young and old samples revealed a highly defined set of differentially expressed genes with functional annotations in skin homeostasis. Further data analysis showed a robust correlation between age-related promoter hypermethylation and gene silencing, particularly at promoters that were pre-marked with stem cell-specific chromatin features. In addition, we also observed age-related methylation changes at transcription factor binding sites, with a significant enrichment of stem cell regulatory networks. Our results provide a high-resolution analysis of age-related methylation changes and suggest that they result in highly defined alterations in the transcriptional programme of the human epidermis. Interestingly, several of our findings can be interpreted to reflect epigenetic changes in aging stem cells, thus supporting a critical role of stem cells in human aging. Whole genome methylation analysis of H. sapiens. Two samples were analyzed, one sample containing DNA from young, one sample containing DNA from old human skin.

ORGANISM(S): Homo sapiens

SUBMITTER: Guenter Raddatz

PROVIDER: E-GEOD-46450 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Aging is associated with highly defined epigenetic changes in the human epidermis.

Raddatz Günter G Hagemann Sabine S Aran Dvir D Söhle Jörn J Kulkarni Pranav P PP Kaderali Lars L Hellman Asaf A Winnefeld Marc M Lyko Frank F

Epigenetics & chromatin 20131031 1

<h4>Background</h4>Altered DNA methylation patterns represent an attractive mechanism for understanding the phenotypic changes associated with human aging. Several studies have described global and complex age-related methylation changes, but their structural and functional significance has remained largely unclear.<h4>Results</h4>We have used transcriptome sequencing to characterize age-related gene expression changes in the human epidermis. The results revealed a significant set of 75 differen ...[more]

PMID: 24279375

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Project description:Gold nanoparticles (GNPs) have been used in the development of novel therapies as a way of delivery of both stimulatory and tolerogenic peptide cargoes. Here we report that intradermal injection of GNPs loaded with the proinsulin peptide C19-A3, in patients with type 1 diabetes, results in recruitment and retention of immune cells in the skin. These include large numbers of clonally expanded T-cells sharing the same paired T-cell receptors (TCRs) with activated phenotypes, half of which, when the TCRs were re-expressed in a cell-based system, were confirmed to be specific for either GNP or proinsulin. All the identified gold-specific clones were CD8+, whilst proinsulin-specific clones were both CD8+ and CD4+. Proinsulin-specific CD8+ clones had a distinctive cytotoxic phenotype with overexpression of granulysin (GNLY) and KIR receptors. Clonally expanded antigen-specific T cells remained in situ for months to years, with a spectrum of tissue resident memory and effector memory phenotypes. As the T-cell response is divided between targeting the gold core and the antigenic cargo, this offers a route to improving resident memory T-cells formation in response to vaccines. In addition, our scRNAseq data indicate that focusing on clonally expanded skin infiltrating T-cells recruited to intradermally injected antigen is a highly efficient method to enrich and identify antigen-specific cells. This approach has the potential to be used to monitor the intradermal delivery of antigens and nanoparticles for immune modulation in humans the Enhanced Epidermal Antigen Specific Immunotherapy Trial -1 (EEASI) study was a two-centre, open-label, uncontrolled, single-group first-in-human Phase 1A safety study of C19-A3 GNP peptide in individuals with T1D (https://clinicaltrials.gov/ct2/show/NCT02837094). The investigational medicinal product (IMP) was C19-A3 GNP (Midacore™), which comprises Midacore™ GNPs (Midatech Pharma Plc, Cardiff, UK)(1, 20) of a size of less than 5 nm, covalently coupled to an 18-amino acid human peptide, the sequence of which is identical to the residues from position 19 in the C-peptide of proinsulin through to position 3 on the A-chain of the same molecule (GSLQPLALEGSLQKRGIV). The peptide is synthesised with a linker to facilitate binding to the GNPs: 3-mercaptopropionyl-SLQPLALEGSLQKRGIV 2 acetate salt (disulfide bond). The chemical composition of the IMP contained a ratio of 4 C19-A3 peptides: 11 glucose C2: 29 glutathione ligands as determined by 1H-NMR (proton nuclear magnetic resonance). A typical batch contained: [C19-A3 peptide] = 1.33 mg/ml; [gold] = 5.5 mg/ml; [glucose linker] = 0.6 mg/ml; [glutathione linker] = 1.79 mg/ml. As the drug substance was diluted 1:7 to 1:10, depending on the content of C19-A3 peptide per particle, and as 50 μl of the diluted solution was administered to the study participants, this corresponded to C19-A3 peptide: 10 μg; gold: 39 μg; glucose linker: 4.3 μg; glutathione: 12.7 μg. Participants diagnosed with T1D and confirmed to possess the HLA-DRB1∗0401 genotype, were given three doses of C19-A3 GNP at 4-weekly intervals (weeks 0, 4, and 8) in the deltoid region of alternate arms (2 doses in one arm and 1 dose in the other arm) via CE-marked 600 μm length MicronJet600™ hollow microneedles (NanoPass Technologies Ltd. Israel) attached to a standard Luer-lock syringe. The single-dose given in 50 μl volume was equivalent to 10 μg of C19-A3 peptide Skin suction blisters were raised: Briefly, suction cups were applied to the deltoid region of participants’ arms, at the site of previous injection. Skin suction blisters were performed by gradually applying negative pressure (up to 60 kPa) from a suction pump machine VP25 (Eschmann, Lancing, UK) through a suction blister cup with a 15-mm hole in the base (UHBristol NHS Foundation Trust Medical Engineering Department, Bristol, UK) for 2–4 hr until a unilocular blister had formed within the cup. The cup was left in place for a further 30-60 minutes to encourage migration of lymphocytes into the blister fluid. The cup was then removed, and the blister fluid aspirated using a needle and syringe. Blister fluid was immediately suspended in 10ml heparinised RPMI (Gibco)+ 5% foetal calf serum Cells were counted then washed once and resuspended in an appropriate volume for scRNAseq. Samples were used fresh on the day of the collection. Samples were processed using a 10xGenomics V1 human 5’GEX kit and libraries were constructed following standard 10xGenomics protocols. Samples were pooled and sequenced on Illumina NextSeq and MiSeq scRNAseq libraries were sequenced aiming for 20,000 reads per cell for GEX and 5,000 reads/cell for VDJ.

Dataset Information

Age-related methylation changes are associated with altered transcriptional circuitry [Methyl-seq]

Publications

Aging is associated with highly defined epigenetic changes in the human epidermis.

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