Allison M. Cotton

Additional annotation enhances potential for biologically-relevant analysis of the Illumina Infinium HumanMethylation450 BeadChip array

BackgroundMeasurement of genome-wide DNA methylation (DNAm) has become an important avenue for investigating potential physiologically-relevant epigenetic changes. Illumina Infinium (Illumina, San Diego, CA, USA) is a commercially available microarray suite used to measure DNAm at many sites throughout the genome. However, it has been suggested that a subset of array probes may give misleading results due to issues related to probe design. To facilitate biologically significant data interpretation, we set out to enhance probe annotation of the newest Infinium array, the HumanMethylation450 BeadChip (450 k), with >485,000 probes covering 99% of Reference Sequence (RefSeq) genes (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA). Annotation that was added or expanded on includes: 1) documented SNPs in the probe target, 2) probe binding specificity, 3) CpG classification of target sites and 4) gene feature classification of target sites.ResultsProbes with documented SNPs at the target CpG (4.3% of probes) were associated with increased within-tissue variation in DNAm. An example of a probe with a SNP at the target CpG demonstrated how sample genotype can confound the measurement of DNAm. Additionally, 8.6% of probes mapped to multiple locations in silico. Measurements from these non-specific probes likely represent a combination of DNAm from multiple genomic sites. The expanded biological annotation demonstrated that based on DNAm, grouping probes by an alternative high-density and intermediate-density CpG island classification provided a distinctive pattern of DNAm. Finally, variable enrichment for differentially methylated probes was noted across CpG classes and gene feature groups, dependant on the tissues that were compared.ConclusionDNAm arrays offer a high-throughput approach for which careful consideration of probe content should be utilized to better understand the biological processes affected. Probes containing SNPs and non-specific probes may affect the assessment of DNAm using the 450 k array. Additionally, probe classification by CpG enrichment classes and to a lesser extent gene feature groups resulted in distinct patterns of DNAm. Thus, we recommend that compromised probes be removed from analyses and that the genomic context of DNAm is considered in studies deciphering the biological meaning of Illumina 450 k array data.

Translating dosage compensation to trisomy 21

Down’s syndrome is a common disorder with enormous medical and social costs, caused by trisomy for chromosome 21. We tested the concept that gene imbalance across an extra chromosome can be de facto corrected by manipulating a single gene, XIST (the X-inactivation gene). Using genome editing with zinc finger nucleases, we inserted a large, inducible XIST transgene into the DYRK1A locus on chromosome 21, in Down’s syndrome pluripotent stem cells. The XIST non-coding RNA coats chromosome 21 and triggers stable heterochromatin modifications, chromosome-wide transcriptional silencing and DNA methylation to form a ‘chromosome 21 Barr body’. This provides a model to study human chromosome inactivation and creates a system to investigate genomic expression changes and cellular pathologies of trisomy 21, free from genetic and epigenetic noise. Notably, deficits in proliferation and neural rosette formation are rapidly reversed upon silencing one chromosome 21. Successful trisomy silencing in vitro also surmounts the major first step towards potential development of ‘chromosome therapy’.

Landscape of DNA methylation on the X chromosome reflects CpG density, functional chromatin state and X-chromosome inactivation

X-chromosome inactivation (XCI) achieves dosage compensation between males and females through the silencing of the majority of genes on one of the female X chromosomes. Thus, the female X chromosomes provide a unique opportunity to study euchromatin and heterochromatin of allelic regions within the same nuclear environment. We examined the interplay of DNA methylation (DNAm) with CpG density, transcriptional activity and chromatin state at genes on the X chromosome using over 1800 female samples analysed with the Illumina Infinium Human Methylation450 BeadChip. DNAm was used to predict an inactivation status for 63 novel transcription start sites (TSSs) across 27 tissues. There was high concordance of inactivation status across tissues, with 62% of TSSs subject to XCI in all 27 tissues examined, whereas 9% escaped from XCI in all tissues, and the remainder showed variable escape from XCI between females in subsets of tissues. Inter-female and twin data supported a model of predominately cis-acting influences on inactivation status. The level of expression from the inactive X relative to the active X correlated with the amount of female promoter DNAm to a threshold of ∼30%, beyond which genes were consistently subject to inactivation. The inactive X showed lower DNAm than the active X at intragenic and intergenic regions for genes subject to XCI, but not at genes that escape from inactivation. Our categorization of genes that escape from X inactivation provides candidates for sex-specific differences in disease.

DNA Methylation Is Globally Disrupted and Associated with Expression Changes in Chronic Obstructive Pulmonary Disease Small Airways

DNA methylation is an epigenetic modification that is highly disrupted in response to cigarette smoke and involved in a wide spectrum of malignant and nonmalignant diseases, but surprisingly not previously assessed in small airways of patients with chronic obstructive pulmonary disease (COPD). Small airways are the primary sites of airflow obstruction in COPD. We sought to determine whether DNA methylation patterns are disrupted in small airway epithelia of patients with COPD, and evaluate whether changes in gene expression are associated with these disruptions. Genome-wide methylation and gene expression analysis were performed on small airway epithelial DNA and RNA obtained from the same patient during bronchoscopy, using Illuminas Infinium HM27 and Affymetrixs Genechip Human Gene 1.0 ST arrays. To control for known effects of cigarette smoking on DNA methylation, methylation and gene expression profiles were compared between former smokers with and without COPD matched for age, pack-years, and years of smoking cessation. Our results indicate that aberrant DNA methylation is (1) a genome-wide phenomenon in small airways of patients with COPD, and (2) associated with altered expression of genes and pathways important to COPD, such as the NF-E2-related factor 2 oxidative response pathway. DNA methylation is likely an important mechanism contributing to modulation of genes important to COPD pathology. Because these methylation events may underlie disease-specific gene expression changes, their characterization is a critical first step toward the development of epigenetic markers and an opportunity for developing novel epigenetic therapeutic interventions for COPD.

X-chromosome inactivation: molecular mechanisms from the human perspective

X-chromosome inactivation is an epigenetic process whereby one X chromosome is silenced in mammalian female cells. Since it was first proposed by Lyon in 1961, mouse models have been valuable tools to uncover the molecular mechanisms underlying X inactivation. However, there are also inherent differences between mouse and human X inactivation, ranging from sequence content of the X inactivation center to the phenotypic outcomes of X-chromosome abnormalities. X-linked gene dosage in males, females, and individuals with X aneuploidies and X/autosome translocations has demonstrated that many human genes escape X inactivation, implicating cis-regulatory elements in the spread of silencing. We discuss the potential nature of these elements and also review the elements in the X inactivation center involved in the early events in X-chromosome inactivation.

Inactive X chromosome-specific reduction in placental DNA methylation

Genome-wide levels of DNA methylation vary between tissues, and compared with other tissues, the placenta has been reported to demonstrate a global decrease in methylation as well as decreased methylation of X-linked promoters. Methylation is one of many features that differentiate the active and inactive X, and it is well established that CpG island promoters on the inactive X are hypermethylated. We now report a detailed analysis of methylation at different regions across the X in male and female placenta and blood. A significant (P < 0.001) placental hypomethylation of LINE1 elements was observed in both males and females. Relative to blood placental promoter hypomethylation was only observed for X-linked, not autosomal promoters, and was significant for females (P < 0.0001) not males (P = 0.9266). In blood, X-linked CpG island promoters were shown to have moderate female methylation (66% across 70 assays) and low (23%) methylation in males. A similar methylation pattern in blood was observed for ∼20% of non-island promoters as well as 50% of the intergenic or intragenic CpG islands, the latter is likely due to the presence of unannotated promoters. Both intragenic and intergenic regions showed similarly high methylation levels in male and female blood (68 and 66%) while placental methylation of these regions was lower, particularly in females. Thus placental hypomethylation relative to blood is observed globally at repetitive elements as well as across the X. The decrease in X-linked placental methylation is consistently greater in females than males and implicates an inactive X specific loss of methylation in the placenta.

Analysis of expressed SNPs identifies variable extents of expression from the human inactive X chromosome

BackgroundX-chromosome inactivation (XCI) results in the silencing of most genes on one X chromosome, yielding mono-allelic expression in individual cells. However, random XCI results in expression of both alleles in most females. Allelic imbalances have been used genome-wide to detect mono-allelically expressed genes. Analysis of X-linked allelic imbalance in females with skewed XCI offers the opportunity to identify genes that escape XCI with bi-allelic expression in contrast to those with mono-allelic expression and which are therefore subject to XCI.ResultsWe determine XCI status for 409 genes, all of which have at least five informative females in our dataset. The majority of genes are subject to XCI and genes that escape from XCI show a continuum of expression from the inactive X. Inactive X expression corresponds to differences in the level of histone modification detected by allelic imbalance after chromatin immunoprecipitation. Differences in XCI between populations and between cell lines derived from different tissues are observed.ConclusionsWe demonstrate that allelic imbalance can be used to determine an inactivation status for X-linked genes, even without completely non-random XCI. There is a range of expression from the inactive X. Genes escaping XCI, including those that do so in only a subset of females, cluster together, demonstrating that XCI and location on the X chromosome are related. In addition to revealing mechanisms involved in cis-gene regulation, determining which genes escape XCI can expand our understanding of the contributions of X-linked genes to sexual dimorphism.

Different measures of “genome-wide” DNA methylation exhibit unique properties in placental and somatic tissues

DNA methylation of CpGs located in two types of repetitive elements—LINE1 (L1) and Alu—is used to assess “global” changes in DNA methylation in studies of human disease and environmental exposure. L1 and Alu contribute close to 30% of all base pairs in the human genome and transposition of repetitive elements is repressed through DNA methylation. Few studies have investigated whether repetitive element DNA methylation is associated with DNA methylation at other genomic regions, or the biological and technical factors that influence potential associations. Here, we assess L1 and Alu DNA methylation by Pyrosequencing of consensus sequences and using subsets of probes included in the Illumina Infinium HumanMethylation27 BeadChip array. We show that evolutionary age and assay method affect the assessment of repetitive element DNA methylation. Additionally, we compare Pyrosequencing results for repetitive elements to average DNA methylation of CpG islands, as assessed by array probes classified into strong, weak and non-islands. We demonstrate that each of these dispersed sequences exhibits different patterns of tissue-specific DNA methylation. Correlation of DNA methylation suggests an association between L1 and weak CpG island DNA methylation in some of the tissues examined. We caution, however, that L1, Alu and CpG island DNA methylation are distinct measures of dispersed DNA methylation and one should not be used in lieu of another. Analysis of DNA methylation data is complex and assays may be influenced by environment and pathology in different or complementary ways.

Variable escape from X-chromosome inactivation: Identifying factors that tip the scales towards expression

In humans over 15% of X‐linked genes have been shown to ‘escape’ from X‐chromosome inactivation (XCI): they continue to be expressed to some extent from the inactive X chromosome. Mono‐allelic expression is anticipated within a cell for genes subject to XCI, but random XCI usually results in expression of both alleles in a cell population. Using a study of allelic expression from cultured lymphoblasts and fibroblasts, many of which showed substantial skewing of XCI, we recently reported that the expression of genes lies on a contiunuum between those that are subject to inactivation, and those that escape. We now review allelic expression studies from mouse, and discuss the variability in escape seen in both humans and mice in genic expression levels, between X chromosomes and between tissues. We also discuss current knowledge of the heterochromatic features, DNA elements and three‐dimensional topology of the inactive X that contribute to the balance of expression from the otherwise inactive X chromosome.

Spread of X-chromosome inactivation into autosomal sequences: role for DNA elements, chromatin features and chromosomal domains

X-chromosome inactivation results in dosage equivalence between the X chromosome in males and females; however, over 15% of human X-linked genes escape silencing and these genes are enriched on the evolutionarily younger short arm of the X chromosome. The spread of inactivation onto translocated autosomal material allows the study of inactivation without the confounding evolutionary history of the X chromosome. The heterogeneity and reduced extent of silencing on autosomes are evidence for the importance of DNA elements underlying the spread of silencing. We have assessed DNA methylation in six unbalanced X-autosome translocations using the Illumina Infinium HumanMethylation450 array. Two to 42% of translocated autosomal genes showed this mark of silencing, with the highest degree of inactivation observed for trisomic autosomal regions. Generally, the extent of silencing was greatest close to the translocation breakpoint; however, silencing was detected well over 100 kb into the autosomal DNA. Alu elements were found to be enriched at autosomal genes that escaped from inactivation while L1s were enriched at subject genes. In cells without the translocation, there was enrichment of heterochromatic features such as EZH2 and H3K27me3 for those genes that become silenced when translocated, suggesting that underlying chromatin structure predisposes genes towards silencing. Additionally, the analysis of topological domains indicated physical clustering of autosomal genes of common inactivation status. Overall, our analysis indicated a complex interaction between DNA sequence, chromatin features and the three-dimensional structure of the chromosome.