Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Epigenetic and post-transcriptional regulation of CD16 expression during human natural killer cell development 1

doi: 10.4049/jimmunol.1701128

Figure Lengend Snippet: DNA methylation and isoform-specific expression within the promoter regions of FCGR3A and FCGR3B. A) Representation of the FCGR3 locus spanning approximately 100 kb of chromosome 1q23.3 displaying transcripts annotated in the RefSeq database (adapted from the UCSC Genome Browser). Below, promoter regions of FCGR3A and FCGR3B are enlarged displaying the various transcript variants annotated in the RefSeq database that differ in the 5′ region. MassARRAY amplicons (green bars) used are shown along with the positions of CpG dinucleotides (green markers). TSS, transcriptional start site. B) The MassARRAY EpiTYPER assay was used to interrogate the DNA methylation levels of CpGs across the promoter region of FCGR3A and FCGR3B in a gene-specific manner in sorted NK CD16a+ and CD16a- fractions. Methylation plots are aligned to enlargements of gene promoters (in B) according to the hg19 genome assembly. C, D) DNA methylation levels in NK cell lines and neutrophils, respectively. E) The expression ratio of FCGR3A to FCGR3B in CD16a+ NK cells and neutrophils as determined by the MassARRAY iPLEX assay. F) Quantitative RT-PCR analysis of 5′ transcript variants using primers specific for variant 1 or variants 3 and 2 indicating promoter usage in NK cells, neutrophils, and NK cell lines YT and NKL (both primer pairs do not distinguish FCGR3A vs. FCGR3B). D1-4 represents donor numbers 1–4. Expression values expressed relative to the average of four housekeeping (HK) genes.

Article Snippet: Antibodies and flow cytometric analysis The following antibodies were used to stain human peripheral blood cells: CD3 (SK7, BD Biosciences), CD14 (TÜK4, Miltenyi), CD15 (VIMC6, Miltenyi), CD16 (VEP13, Miltenyi), CD16 (3G8, BD Biosciences), and CD56 (N901, Beckman Coulter).

Techniques: DNA Methylation Assay, Expressing, MassARRAY EpiTYPER assay, Methylation, Quantitative RT-PCR, Variant Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Epigenetic and post-transcriptional regulation of CD16 expression during human natural killer cell development 1

doi: 10.4049/jimmunol.1701128

Figure Lengend Snippet: Analysis of DNA methylation- and lineage-specific activity of FCGR3 promoter sequences. A) Illustration of FCGR3A and FCGR3B sequences cloned into luciferase constructs (black bars). B) Four separate promoter sequence fragments were cloned from either FCGR3A (left) or FCGR3B (right) and transfected in various cell lines. Luciferase assays showing sequence- and gene-specific activity (relative to empty-vector control). Pmed1-A and Pmed1-B were additionally methylated in vitro prior to transfection. Error bars represent SEM of n=3 individual experiments; ND, not done. C) Sequence alignment of Pmed1-A and Pmed1-B with numbered CpGs. All sequence variants are enlarged below; an 8 bp repeat occurring in the vicinity of CpG#6 is highlighted in blue, asterisks below the sequence indicate homology. D) Luciferase activity following site-directed mutagenesis of CpG#s 1&2 of Pmed1-A compared to unaltered FCGR3A and FCGR3B sequences in YT and K562 cells.

Article Snippet: Antibodies and flow cytometric analysis The following antibodies were used to stain human peripheral blood cells: CD3 (SK7, BD Biosciences), CD14 (TÜK4, Miltenyi), CD15 (VIMC6, Miltenyi), CD16 (VEP13, Miltenyi), CD16 (3G8, BD Biosciences), and CD56 (N901, Beckman Coulter).

Techniques: DNA Methylation Assay, Activity Assay, Clone Assay, Luciferase, Construct, Sequencing, Transfection, Plasmid Preparation, Control, Methylation, In Vitro, Mutagenesis

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Epigenetic and post-transcriptional regulation of CD16 expression during human natural killer cell development 1

doi: 10.4049/jimmunol.1701128

Figure Lengend Snippet: Identification of miR-218 as a potential regulator of FCGR3A. (A) Expression ratio of predicted miRNAs that were present in the miRNA expression array comparing CD16a+ and CD16a- NK cells freshly isolated from adult peripheral blood. A ratio <1 indicates low expression in CD16a+ NK cells while a ratio >1 indicates high expression in CD16a+ NK cells. (B) Predicted miRNA regulators of FCGR3A have putative binding sites in the FCGR3A 3′ UTR. (C) Validation of expression of predicted miRNA regulators of FCGR3A by qPCR (n=3). (D) Luciferase expression as a ratio of firefly/renilla for each expression vector (n=2). Data are presented as mean±SD, * indicates p<0.05.

Article Snippet: Antibodies and flow cytometric analysis The following antibodies were used to stain human peripheral blood cells: CD3 (SK7, BD Biosciences), CD14 (TÜK4, Miltenyi), CD15 (VIMC6, Miltenyi), CD16 (VEP13, Miltenyi), CD16 (3G8, BD Biosciences), and CD56 (N901, Beckman Coulter).

Techniques: Expressing, Isolation, Binding Assay, Biomarker Discovery, Luciferase, Plasmid Preparation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Epigenetic and post-transcriptional regulation of CD16 expression during human natural killer cell development 1

doi: 10.4049/jimmunol.1701128

Figure Lengend Snippet: MiR-218 negatively regulates CD16a in primary human NK cells. Primary human NK cells were enriched by magnetic selection to >70% purity and infected with lentivirus containing either miR-218 or empty vector. 48hr after infection, NK cells were sorted as live/CD3-/CD56+/GFP+ lymphocytes. (A) Representative histogram plot (of one of six donors) of CD16a expression in live/CD3-/CD56+/GFP+ primary human NK cells infected with miR-218 or empty vector virus. (B) CD16a expression in primary human NK cells infected with miR-218 or empty vector virus (* indicates p=0.05). (C) Validation of miR-218 over-expression by real-time PCR (** indicates p<0.01). (D) FCGR3A mRNA expression assessed by real-time RT-PCR in sorted NK cells infected with either miR-218 or empty vector (*** indicates p<0.005). (B–D) Data are presented as mean±SD, n=6.

Article Snippet: Antibodies and flow cytometric analysis The following antibodies were used to stain human peripheral blood cells: CD3 (SK7, BD Biosciences), CD14 (TÜK4, Miltenyi), CD15 (VIMC6, Miltenyi), CD16 (VEP13, Miltenyi), CD16 (3G8, BD Biosciences), and CD56 (N901, Beckman Coulter).

Techniques: Selection, Infection, Plasmid Preparation, Expressing, Virus, Biomarker Discovery, Over Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: BMC Genomics

Article Title: Evidence for widespread changes in promoter methylation profile in human placenta in response to increasing gestational age and environmental/stochastic factors

doi: 10.1186/1471-2164-12-529

Figure Lengend Snippet: Cluster dendrogram based on all autosomal Infinium probes distinguishes placentas of different gestational age . Dendrogram showing the relationship between placental samples from three gestational ages based on DNA methylation levels (β-values) of all analysable Infinium probes. All samples clustered within their gestational age group, with no overlap between gestations, suggesting there are consistent genome-scale DNA methylation patterns associated with each gestational age. First trimester samples clustered away from second and third trimester samples, indicating that overall Infinium methylation patterns are more similar in second and third trimester compared to first trimester.

Article Snippet: Sequenom MassARRAY EpiTYPING was performed to validate Infinium methylation, as previously described [ ].

Techniques: DNA Methylation Assay, Methylation

Journal: BMC Genomics

Article Title: Evidence for widespread changes in promoter methylation profile in human placenta in response to increasing gestational age and environmental/stochastic factors

doi: 10.1186/1471-2164-12-529

Figure Lengend Snippet: Number of probes showing differential methylation between first, second and third trimester placental tissue.

Article Snippet: Sequenom MassARRAY EpiTYPING was performed to validate Infinium methylation, as previously described [ ].

Techniques: Methylation

Journal: BMC Genomics

Article Title: Evidence for widespread changes in promoter methylation profile in human placenta in response to increasing gestational age and environmental/stochastic factors

doi: 10.1186/1471-2164-12-529

Figure Lengend Snippet: Unsupervised clustering based on probes with Δβ > 0.2 between First and Third trimester . HeatMap showing unsupervised clustering of all placenta samples (x-axis) based on 954 probes with a Δβ > 0.2 between First and Third trimester (y-axis). The majority of differentially methylated probes show higher methylation in third trimester (883 probes) compared to only 71 probes with lower methylation in third trimester. Second trimester placentas cluster as a separate group, and show a methylation profile that is an intermediate of first and third trimesters. Green corresponds to low methylation and Red to high methylation.

Article Snippet: Sequenom MassARRAY EpiTYPING was performed to validate Infinium methylation, as previously described [ ].

Techniques: Methylation

Journal: BMC Genomics

Article Title: Evidence for widespread changes in promoter methylation profile in human placenta in response to increasing gestational age and environmental/stochastic factors

doi: 10.1186/1471-2164-12-529

Figure Lengend Snippet: Variance levels of probes in the third compared to the first trimester . Scatter plot of probe variance ( s 2 ) at first trimester (x-axis) and third trimester (y-axis). Dots represent individual probes and the vertical red dotted line marks s 2 = 0.02 for first trimester, while the horizontal red dotted line marks s 2 = 0.02 for third trimester. Probes on the outside of the red line are deemed 'variable'. This analysis revealed that there are 73 probes (A) which are highly variable in both first and third trimester. Only 33 probes (B) were variable in first, but not third trimester, while 279 probes (C) were variable in third, but not first trimester. This analysis suggests that most of the variable probes become so throughout pregnancy, supporting the hypothesis that accumulating environmental factors contribute to inter-individual variation in DNA methylation, in term placenta.

Article Snippet: Sequenom MassARRAY EpiTYPING was performed to validate Infinium methylation, as previously described [ ].

Techniques: DNA Methylation Assay

Journal: BMC Genomics

Article Title: Evidence for widespread changes in promoter methylation profile in human placenta in response to increasing gestational age and environmental/stochastic factors

doi: 10.1186/1471-2164-12-529

Figure Lengend Snippet: Correlation between methylation and gene expression change between first and third trimester . Methylation difference (Δβ) between first and third trimester (x-axis) was plotted against gene expression log fold change (y-axis) between first and third trimester. A positive change in log fold expression indicates higher expression in first trimester, while a positive change in methylation indicates higher methylation in the third trimester. Therefore, the top left panel includes genes which showed lower methylation and higher expression in first compared to third trimester. The three highlighted genes (CCR7, GNLY and CCL21) ranked highly in IPA analysis. Grey dots represent Infinium probes. Black dots represent specific genes of interest.

Article Snippet: Sequenom MassARRAY EpiTYPING was performed to validate Infinium methylation, as previously described [ ].

Techniques: Methylation, Gene Expression, Expressing

Journal: BMC Genomics

Article Title: Evidence for widespread changes in promoter methylation profile in human placenta in response to increasing gestational age and environmental/stochastic factors

doi: 10.1186/1471-2164-12-529

Figure Lengend Snippet: Average methylation of all samples for first, second and third trimester . Methylation Index (MI) was calculated for each sample by calculating the mean of all analysable Infinium β-values (26, 162 probes) for that sample. The MIs were then grouped by gestation and shown as box and whisker plots. First and second trimester placentas show a similar overall level of methylation (p = 0.46) with median MIs of 0.238 and 0.241, respectively. Third trimester samples show significantly elevated average MI values (median = 0.256) relative to both first and second trimester, indicating that there is a significant increase in methylation level from second to third trimester.

Article Snippet: Sequenom MassARRAY EpiTYPING was performed to validate Infinium methylation, as previously described [ ].

Techniques: Methylation, Whisker Assay