Differential DNA Methylation in Purified Human Blood Cells: Implications for Cell Lineage and Studies on Disease Susceptibility
Lovisa E. ReiniusNathalie AcevedoMaaike JoerinkGöran PershagenSven‐Erik DahlénDario GrecoCilla SöderhällAnnika ScheyniusJuha Kere
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Methylation of cytosines at CpG sites is a common epigenetic DNA modification that can be measured by a large number of methods, now even in a genome-wide manner for hundreds of thousands of sites. The application of DNA methylation analysis is becoming widely popular in complex disorders, for example, to understand part of the “missing heritability”. The DNA samples most readily available for methylation studies are derived from whole blood. However, blood consists of many functionally and developmentally distinct cell populations in varying proportions. We studied whether such variation might affect the interpretation of methylation studies based on whole blood DNA. We found in healthy male blood donors there is important variation in the methylation profiles of whole blood, mononuclear cells, granulocytes, and cells from seven selected purified lineages. CpG methylation between mononuclear cells and granulocytes differed for 22% of the 8252 probes covering the selected 343 genes implicated in immune-related disorders by genome-wide association studies, and at least one probe was differentially methylated for 85% of the genes, indicating that whole blood methylation results might be unintelligible. For individual genes, even if the overall methylation patterns might appear similar, a few CpG sites in the regulatory regions may have opposite methylation patterns (i.e., hypo/hyper) in the main blood cell types. We conclude that interpretation of whole blood methylation profiles should be performed with great caution and for any differences implicated in a disorder, the differences resulting from varying proportions of white blood cell types should be considered.Keywords:
CpG site
Differentially methylated regions
Blood cell
Epigenomics
Abstract Background The analysis of WGBS (whole genome bisulfite sequencing) datasets is challenging. The large number of CpG sites requires significant computing power and can lead to harsh multiple correction penalties. Typically, the number of CpG sites found in DMRs (differentially regulated regions) represent a very small proportion of the initial number of CpG sites. This is because methylation levels of the majority of CpG sites do not vary significantly between samples, and/or the CpG sites are too far dispersed to be considered a contiguous region. DMRs are like likely to be found in relatively compact CpG rich regions that vary in methylation levels. Isolating these regions could greatly reduce downstream computational and statistical challenges without any previous knowledge of sample groups. Results The proposed method was able to isolate compact CpG rich variable regions using distance, covariation, and user parameters without a priori sample information. Results were verified with EpiDISH cell deconvolution and comparable with to a complementary method DMRSeq. Isolated regions averaged just 293 bp in length yet contained an average of 29 CpG sites per region. Conclusions By defining compact CpG rich variable regions, the method hopes to provide a valid and simpler starting point for further downstream analyses. This method is applicable to any dataset containing total CpG and total CpG methylated count matrices.
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DNA methylation is a covalent modification of the nucleotide cytosine that is stably inherited at the dinucleotide CpG by somatic cells, and 70% of CpG dinucleotides in the genome are methylated. The exception to this pattern of methylation are CpG islands, CpG-rich sequences that are protected from methylation, and generally are thought to be methylated only on the inactive X-chromosome and in tumors, as well as differentially methylated regions (DMRs) in the vicinity of imprinted genes. To identify chromosomal regions that might harbor imprinted genes, we devised a strategy for isolating a library of normally methylated CpG islands. Most of the methylated CpG islands represented high copy number dispersed repeats. However, 62 unique clones in the library were characterized, all of which were methylated and GC-rich, with a GC content >50%. Of these, 43 clones also showed a CpG obs /CpG exp >0.6, of which 30 were studied in detail. These unique methylated CpG islands mapped to 23 chromosomal regions, and 12 were differentially methylated regions in uniparental tissues of germline origin, i.e., hydatidiform moles (paternal origin) and complete ovarian teratomas (maternal origin), even though many apparently were methylated in somatic tissues. We term these sequences gDMRs, for germline differentially methylated regions. At least two gDMRs mapped near imprinted genes, HYMA1 and a novel homolog of Elongin A and Elongin A2 , which we term Elongin A3 . Surprisingly, 18 of the methylated CpG islands were methylated in germline tissues of both parental origins, representing a previously uncharacterized class of normally methylated CpG islands in the genome, and which we term similarly methylated regions (SMRs). These SMRs, in contrast to the gDMRs, were significantly associated with telomeric band locations ( P = .0008), suggesting a potential role for SMRs in chromosome organization. At least 10 of the methylated CpG islands were on average 85% conserved between mouse and human. These sequences will provide a valuable resource in the search for novel imprinted genes, for defining the molecular substrates of the normal methylome, and for identifying novel targets for mammalian chromatin formation. [The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF484557 – AF484583 .]
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Abstract The epigenomic landscape of human immune cells is dynamically shaped by both genetic factors and environmental exposures. However, the relative contributions of these elements are still not fully understood. In this study, we employed single-nucleus methylation sequencing and ATAC-seq to systematically explore how pathogen and chemical exposures, along with genetic variation, influence the immune cell epigenome. We identified distinct exposure-associated differentially methylated regions (eDMRs) corresponding to each exposure, revealing how environmental factors remodel the methylome, alter immune cell states, and affect transcription factor binding. Furthermore, we observed a significant correlation between changes in DNA methylation and chromatin accessibility, underscoring the coordinated response of the epigenome. We also uncovered genotype-associated DMRs (gDMRs), demonstrating that while eDMRs are enriched in regulatory regions, gDMRs are preferentially located in gene body marks, suggesting that exposures and genetic factors exert differential regulatory control. Notably, disease-associated SNPs were frequently colocalized with meQTLs, providing new cell-type-specific insights into the genetic basis of disease. Our findings underscore the intricate interplay between genetic and environmental factors in sculpting the immune cell epigenome, offering a deeper understanding of how immune cell function is regulated in health and disease.
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Abstract Perinatally-acquired HIV has persistent effects on long-term health outcomes, even after early treatment. We hypothesize that epigenetic indicators, such as DNA methylation, may elucidate cellular processes that explain these effects. Here, we compared DNA methylation profiles in whole blood from 120 HIV-infected children on antiretroviral therapy (ART) and 60 frequency age-matched HIV-uninfected children aged 4–9 years in Johannesburg, South Africa. Using an individual CpG site approach, we found 1,309 differentially-methylated (DM) CpG sites between groups, including 1,271 CpG sites that were hyper-methylated in the HIV-infected group and 38 CpG sites that were hypo-methylated in the HIV-infected group. Six hyper-methylated CpG sites were in EBF4 , which codes for a transcription factor involved in B-cell maturation. The top hypomethylated site was in the promoter region of NLRC5 , encoding a transcription factor that regulates major histocompatibility complex (MHC) class I molecule expression. Using a differentially-methylated region (DMR) approach, we found 315 DMRs between groups, including 28 regions encompassing 686 CpG sites on chromosome 6. A large number of the genes identified in both the CpG site and DMR approaches were located in the MHC region on chromosome 6, which plays an important role in the adaptive immune system. This study provides the first evidence that changes in the epigenome are detectable in children with perinatally-acquired HIV infection on suppressive ART started at an early age.
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DNA methylation is a covalent modification of the nucleotide cytosine that is stably inherited at the dinucleotide CpG by somatic cells, and 70% of CpG dinucleotides in the genome are methylated. The exception to this pattern of methylation are CpG islands, CpG-rich sequences that are protected from methylation, and generally are thought to be methylated only on the inactive X-chromosome and in tumors, as well as differentially methylated regions (DMRs) in the vicinity of imprinted genes. To identify chromosomal regions that might harbor imprinted genes, we devised a strategy for isolating a library of normally methylated CpG islands. Most of the methylated CpG islands represented high copy number dispersed repeats. However, 62 unique clones in the library were characterized, all of which were methylated and GC-rich, with a GC content >50%. Of these, 43 clones also showed a CpG(obs)/CpG(exp) >0.6, of which 30 were studied in detail. These unique methylated CpG islands mapped to 23 chromosomal regions, and 12 were differentially methylated regions in uniparental tissues of germline origin, i.e., hydatidiform moles (paternal origin) and complete ovarian teratomas (maternal origin), even though many apparently were methylated in somatic tissues. We term these sequences gDMRs, for germline differentially methylated regions. At least two gDMRs mapped near imprinted genes, HYMA1 and a novel homolog of Elongin A and Elongin A2, which we term Elongin A3. Surprisingly, 18 of the methylated CpG islands were methylated in germline tissues of both parental origins, representing a previously uncharacterized class of normally methylated CpG islands in the genome, and which we term similarly methylated regions (SMRs). These SMRs, in contrast to the gDMRs, were significantly associated with telomeric band locations (P =.0008), suggesting a potential role for SMRs in chromosome organization. At least 10 of the methylated CpG islands were on average 85% conserved between mouse and human. These sequences will provide a valuable resource in the search for novel imprinted genes, for defining the molecular substrates of the normal methylome, and for identifying novel targets for mammalian chromatin formation. PMID: 11932239 Funding information This work was supported by: NCI NIH HHS, United States Grant ID: CA 65145 NCI NIH HHS, United States Grant ID: R01 CA065145
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CpG islands, which have higher GC content and CpG frequencies compared to the genome as a whole, are generally believed to be unmethylated in tissues except at promoters of genes undergoing X chromosome inactivation or genomic imprinting. Recent studies, however, have shown that CpG islands at promoters of a number of genes contain tissue‐dependent, differentially methylated regions (T‐DMRs). In general, the tissue‐specific methylation is restricted to a part of the promoter CpG island, with hypomethylation of the remaining sequence. In the current study, using comparison between Restriction Landmark Genomic Scanning (RLGS) and in silico RLGS, we identified ten sperm‐specific unmethylated Not I sites, T‐DMRs located in CpG islands that were hypomethylated in sperm but near‐completely methylated in the kidney and brain. Unusually, these T‐DMRs involve the whole CpG island at each of these loci. We characterized one of these genes, adenine nucleotide translocator 4 ( Ant4 ), which is expressed in germ cells. Using a promoter assay, we demonstrated that expression of Ant4 gene is controlled by DNA methylation at the CpG island sequences within the promoter region. Ant4 and other sperm‐specific hypomethylated loci represent a new class of CpG islands that become completely methylated in different cell lineages. T‐DMRs at CpG islands are functionally important gene regulatory elements that may now be categorized into two classes: T‐DMRs involving a subregion of the CpG island and those that occupy the whole CpG island.
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We recently reported the COVID-19-induced circulating leukocytes DNA methylation profile. Here, we hypothesized that some of these genes would persist differentially methylated after disease resolution. Fifteen participants previously hospitalized for SARS-CoV-2 infection were epityped one year after discharge. Of the 1505 acute illness-induced differentially methylated regions (DMRs) previously identified, we found 71 regions with persisted differentially methylated, with an average of 7 serial CpG positions per DMR. Sixty-four DMRs persisted hypermethylated, and 7 DMR persisted hypomethylated. These data are the first reported evidence that DNA methylation changes in circulating leukocytes endure long after recovery from acute illness.
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Цель. Изучение изменений в образцах злокачественных опухолей молочной железы статуса метилирования остатков цитозина в составе CpG-островков, моноаллельно метилированных в норме. Методы. Для определения CpG-динуклеотидов, моноаллельно метилированных в опухолевых и нормальных тканях молочной железы, использовали полученные авторами ранее результаты широкогеномного бисульфитного секвенирования Xmal-RRBS. Изменения моноаллельного метилирования в опухолях визуализировали с помощью гистограмм для различных метилотипов молочной железы. Результаты. В геномах умеренно метилированных опухолей нормально моноаллельно метилированные CpG-динуклеотиды имеют тенденцию к почти равновероятному изменению как в неметилированное, так и в полностью метилированное состояния. В геномах гиперметилированных опухолей нормально моноаллельно метилированные CpG-динуклеотиды чаще меняют своё состояние на полностью метилированное. Заключение. Наблюдаемый характер изменений позволяет предположить, что динамика диктуется в умеренно метилированных опухолях процессами, не определяющими её вектор, а в гиперметилированных - дополнительно - направленным процессом, повышающим уровень метилирования CpG-динуклеотидов. По аналогии с ранее опубликованными результатами исследования динамики изменений метилирования импринтированных локусов в опухолях человека, мы предполагаем, что процесс случайного определения вектора изменения характера метилирования опосредован нарушениями копийности участков моноаллельного метилирования ДНК. Причиной равновероятных разнонаправленных изменений моноаллельного метилирования может быть геномный дисбаланс, который может случайным образом приводить к делециям (или приобретенной однородительской дисомии) либо метилированного, либо неметилированного аллеля. Aim: to evaluate changes in the methylation status of cytosine residues in CpG islands, monoallelically methylated in the norm, in samples of malignant breast tumors. Methods. To determine CpG dinucleotides monoallelically methylated in tumor and normal breast tissues, we used the results of Xmal-RRBS genome-wide bisulfite sequencing obtained earlier. Changes in monoallelic methylation in tumors were visualized using histograms for different breast methylotypes. Results. In the genomes of moderately methylated tumors, normally methylated CpG dinucleotides have a tendency to an almost equiprobable methylation change to both the unmethylated and fully methylated states. In the genomes of hypermethylated tumors, normally monoallelically methylated CpG dinucleotides more often change their state to fully methylated. Conclusions. The observed nature of the changes suggests that the dynamics is dictated in moderately methylated tumors by processes that do not determine their vector, and in hypermethylated tumors, additionally, by a directed process that increases the methylation level of CpG dinucleotides. By analogy with the previously published results of a study of the dynamics of changes in methylation of imprinted loci in human tumors, we assume that the process of random determination of the vector of methylation changes is mediated by copy number aberrations at the regions of monoallelic DNA methylation. The reason for the equally probable bidirectional changes in monoallelic methylation may be a genomic imbalance that can randomly lead to deletions (or acquired uniparental disomy) of either the methylated or unmethylated allele.
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