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Analysis of human peripheral blood T cells and single-cell-derived T cell clones uncovers extensive clonal CpG island methylation heterogeneity throughout the genome.

ABSTRACT: Methylation of cytosine residues in CpG dinucleotides is generally associated with silencing of gene expression. DNA methylation, as a somatic event, has the potential of diversifying gene expression in individual cells of the same lineage. There is little quantitative data available concerning the extent of methylation heterogeneity in individual cells across the genome. T cells from the peripheral blood can be grown as single-cell-derived clones and can be analyzed with respect to their DNA methylation patterns by restriction landmark genomic scanning. The use of the methylation-sensitive enzyme NotI to cut and end-label DNA fragments before their separation in two dimensions provides a quantitative assessment of methylation at NotI sites that characteristically occur in CpG islands. We have undertaken quantitative analysis of two-dimensional DNA patterns to determine the extent of methylation heterogeneity at NotI sites between peripheral blood single-cell-derived T cell clones from the same individual. A total of 1,068 NotI-tagged fragments were analyzed. A subset of 156 fragments exhibited marked methylation heterogeneity at NotI sites between clones. Their average intensity among clones correlated with their intensity in uncultured, whole-blood-derived T cells, indicating that the methylation heterogeneity observed in clones was largely attributable to methylation heterogeneity between the individual cells from which the clones were derived. We have cloned one fragment that exhibited variable NotI-site methylation between clones. This fragment contained a novel CpG island for a gene that we mapped to chromosome 4. The methylation status of the NotI site of this fragment correlated with expression of the corresponding gene. Our data suggest extensive diversity in vivo in the methylation and expression profiles of individual T cells at multiple unrelated loci across the genome.

SUBMITTER: Zhu X

PROVIDER: S-EPMC22187 | biostudies-literature | 1999 Jul

REPOSITORIES: biostudies-literature

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Analysis of human peripheral blood T cells and single-cell-derived T cell clones uncovers extensive clonal CpG island methylation heterogeneity throughout the genome.

Zhu X X Deng C C Kuick R R Yung R R Lamb B B Neel J V JV Richardson B B Hanash S S

Proceedings of the National Academy of Sciences of the United States of America 19990701 14

Methylation of cytosine residues in CpG dinucleotides is generally associated with silencing of gene expression. DNA methylation, as a somatic event, has the potential of diversifying gene expression in individual cells of the same lineage. There is little quantitative data available concerning the extent of methylation heterogeneity in individual cells across the genome. T cells from the peripheral blood can be grown as single-cell-derived clones and can be analyzed with respect to their DNA me ...[more]

PMID: 10393947

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Project description:IntroductionB cells are acknowledged as crucial players in the pathogenesis of multiple sclerosis (MS). Several disease modifying drugs including cladribine have been shown to exert differential effects on peripheral blood B cell subsets. However, little is known regarding functional changes within the peripheral B cell populations. In this study, we obtained a detailed picture of B cell repertoire changes under cladribine treatment on a combined immunoglobulin (Ig) transcriptome and proteome level.MethodsWe performed next-generation sequencing of Ig heavy chain (IGH) transcripts and Ig mass spectrometry in cladribine-treated patients with relapsing-remitting multiple sclerosis (n = 8) at baseline and after 6 and 12 months of treatment in order to generate Ig transcriptome and Ig peptide libraries. Ig peptides were overlapped with the corresponding IGH transcriptome in order to analyze B cell clones on a combined transcriptome and proteome level.ResultsThe analysis of peripheral blood B cell percentages pointed towards a significant decrease of memory B cells and an increase of naive B cells following cladribine therapy. While basic IGH repertoire parameters (e.g. variable heavy chain family usage and Ig subclasses) were only slightly affected by cladribine treatment, a significantly decreased number of clones and significantly lower diversity in the memory subset was noticeable at 6 months following treatment which was sustained at 12 months. When looking at B-cell clones comprising sequences from the different time-points, clones spanning between all three time-points were significantly more frequent than clones including sequences from two time-points. Furthermore, Ig proteome analyses showed that Ig transcriptome specific peptides could mostly be equally aligned to all three time-points pointing towards a proportion of B-cell clones that are maintained during treatment.DiscussionOur findings suggest that peripheral B cell related treatment effects of cladribine tablets might be exerted through a reduction of possibly disease relevant clones in the memory B cell subset without disrupting the overall clonal composition of B cells. Our results -at least partially- might explain the relatively mild side effects regarding infections and the sustained immune response after vaccinations during treatment. However, exact disease driving B cell subsets and their effects remain unknown and should be addressed in future studies.

Project description:CpG islands were originally identified by epigenetic and functional properties, namely, absence of DNA methylation and frequent promoter association. However, this concept was quickly replaced by simple DNA sequence criteria, which allowed for genome-wide annotation of CpG islands in the absence of large-scale epigenetic datasets. Although widely used, the current CpG island criteria incur significant disadvantages: (1) reliance on arbitrary threshold parameters that bear little biological justification, (2) failure to account for widespread heterogeneity among CpG islands, and (3) apparent lack of specificity when applied to the human genome. This study is driven by the idea that a quantitative score of "CpG island strength" that incorporates epigenetic and functional aspects can help resolve these issues. We construct an epigenome prediction pipeline that links the DNA sequence of CpG islands to their epigenetic states, including DNA methylation, histone modifications, and chromatin accessibility. By training support vector machines on epigenetic data for CpG islands on human Chromosomes 21 and 22, we identify informative DNA attributes that correlate with open versus compact chromatin structures. These DNA attributes are used to predict the epigenetic states of all CpG islands genome-wide. Combining predictions for multiple epigenetic features, we estimate the inherent CpG island strength for each CpG island in the human genome, i.e., its inherent tendency to exhibit an open and transcriptionally competent chromatin structure. We extensively validate our results on independent datasets, showing that the CpG island strength predictions are applicable and informative across different tissues and cell types, and we derive improved maps of predicted "bona fide" CpG islands. The mapping of CpG islands by epigenome prediction is conceptually superior to identifying CpG islands by widely used sequence criteria since it links CpG island detection to their characteristic epigenetic and functional states. And it is superior to purely experimental epigenome mapping for CpG island detection since it abstracts from specific properties that are limited to a single cell type or tissue. In addition, using computational epigenetics methods we could identify high correlation between the epigenome and characteristics of the DNA sequence, a finding which emphasizes the need for a better understanding of the mechanistic links between genome and epigenome.

Dataset Information

Analysis of human peripheral blood T cells and single-cell-derived T cell clones uncovers extensive clonal CpG island methylation heterogeneity throughout the genome.

Publications

Analysis of human peripheral blood T cells and single-cell-derived T cell clones uncovers extensive clonal CpG island methylation heterogeneity throughout the genome.

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