Additional file 5 of MeDeCom: discovery and quantification of latent components of heterogeneous methylomes

Supplementary Text. PDF document with supplementary notes. (PDF 158 kb

Additional file 3 of MeDeCom: discovery and quantification of latent components of heterogeneous methylomes

CpGs used for the analysis of memory and naive B cells. A comma-separated value table file. (CSV 35 kb

Image_7_The nudibranch Berghia stephanieae (Valdés, 2005) is not able to initiate a functional symbiosome-like environment to maintain Breviolum minutum (J.E.Parkinson & LaJeunesse 2018).png

Photosymbiosis is found in different animal lineages and is best understood in cnidarians. A successful initiation and maintenance of the symbiosis between the animal hosts and the photosymbiotic partners is based on a recognition by specific host receptors. This triggers signaling cascades that promote the photobiont tolerance by the host, including an interpartner nutrient exchange and the ability of the host to cope with increased levels of reactive oxygen species (ROS) generated by the photobiont. Key to the successful symbiosis is the inhibition of the phagosomal maturation resulting in the formation of the symbiosome. In animals other than cnidarians, little is known about the photosymbiosis initiation and maintenance, for instance in sea slugs belonging to the Nudibranchia. Here, we investigated the gene expression profile of Berghia stephanieae, which is able to incorporate Breviolum minutum from its cnidarian prey Exaiptasia diaphana (Rapp, 1829) but is not able to maintain the algae for more than a couple of days during starvation. We show that the recognition of the algae is based on similar mechanisms present in cnidarians, and we identified some additional candidate genes that might be molluscan specific for photobiont recognition. Downstream, B. stephanieae responds to increased levels of ROS but is not able to stop the phagosomal maturation or decrease the immune response against B. minutum, which seem to be the key factors missing in B. stephanieae that accounts for the unstable symbiosis in this slug. Hence, B. stephanieae can be considered a transitional state toward a stable photosymbiosis and can help to elucidate general aspects of the evolutionary processes involved in establishing photosymbioses in animals.</p

Human Cytomegalovirus Drives Epigenetic Imprinting of the <i>IFNG</i> Locus in NKG2C^hi Natural Killer Cells

<div><p>Memory type 1 T helper (T_H1) cells are characterized by the stable expression of interferon (IFN)-γ as well as by the epigenetic imprinting of the <i>IFNG</i> locus. Among innate cells, NK cells play a crucial role in the defense against cytomegalovirus (CMV) and represent the main source of IFN-γ. Recently, it was shown that memory-like features can be observed in NK cell subsets after CMV infection. However, the molecular mechanisms underlying NK cell adaptive properties have not been completely defined. In the present study, we demonstrated that only NKG2C^hi NK cells expanded in human CMV (HCMV) seropositive individuals underwent epigenetic remodeling of the <i>IFNG</i> conserved non-coding sequence (CNS) 1, similar to memory CD8⁺ T cells or T_H1 cells. The accessibility of the CNS1 was required to enhance IFN-γ transcriptional activity in response to NKG2C and 2B4 engagement, which led to consistent IFN-γ production in NKG2C^hi NK cells. Thus, our data identify epigenetic imprinting of the <i>IFNG</i> locus as selective hallmark and crucial mechanism driving strong and stable IFN-γ expression in HCMV-specific NK cell expansions, providing a molecular basis for the regulation of adaptive features in innate cells.</p></div

CNS1 demethylation occurs independent of CD57/sKIR expression and is stably imprinted in NKG2C^hi NK cells.

<p>(A–C) Phenotype and methylation status of the CNS1 was analyzed in ex vivo NKG2C^hi NK cell expansions from representative HCMV⁺ donors (n = 4), as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004441#ppat-1004441-g001" target="_blank">Figure 1</a>. (A and B) CD56 and NKG2C expression was analyzed by FC on PBMC, after gating on viable CD3⁻ CD56⁺ NK cells, while CD57 and sKIR (KIR2DL3 in HLA-C1⁺ donor) expression was analyzed after gating on CD56^dim NKG2C^hi NK cells. CpG methylation of the <i>IFNG</i> CNS1 was analyzed in FACS sorted CD56^dim NKG2C⁺ NK cell subsets as indicated and is depicted as mean percentage of methylation at each CpG site. (C) The same HCMV⁺ individual displaying an expanded NKG2C^hi population was analyzed twice with an interval of one year between the two measurements. NKG2C and CD57 expression was analyzed by FC on PBMC, after gating on viable CD3⁻ CD56^dim NK cells, while sKIR expression was analyzed after gating on CD56^dim CD57⁺ NKG2C^+/− NK cells. CpG methylation of the <i>IFNG</i> CNS1 was analyzed in FACS sorted CD56^dim CD57⁺ NKG2C⁻ (gray bars) and in CD56^dim CD57⁺ NKG2C⁺ NK cells (black bars) and is depicted as mean percentage of methylation at each CpG site.</p

NKG2C⁺ NK cells express IFN-γ in response to NKG2C engagement.

<p>(A and B) Viable CD3⁻ CD56^dim NK cells were FACS sorted and stimulated by cross-linking of NKG2C and/or 2B4 for 16 hours. Analysis of intracellular IFN-γ expression was performed by FC after gating on CD56^dim CD57⁺ NKG2A⁻ sKIR⁺ cells from HCMV⁺ donors displaying or not an expansion of NKG2C^hi cells. Gating strategy is depicted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004441#ppat.1004441.s003" target="_blank">Figure S3D</a>. One representative experiment (A) and mean percentage of IFN-γ producing cells ± SEM (B) are depicted (n≥8). Percentage of IFN-γ producing cells was calculated after subtracting the value observed in unstimulated cells. ***<i>p</i><0.001 calculated with Mann-Whitney test. (C) Surface expression of CD48 and HLA-E was analyzed by FC on 221 (dashed line) or 221-AEH cells (black line), with isotype control or secondary staining only (solid grey histogram). One representative staining out of two is depicted. (D and E) Viable CD3⁻ CD56^dim NK cells were FACS sorted and stimulated by co-culture with 221 or 221-AEH for 6 hours. Analysis of intracellular IFN-γ expression was performed by FC after gating on CD56^dim CD57⁺ NKG2A⁻ sKIR⁺ NKG2C^hi/+/− cells from HCMV^+/− donors. One representative experiment (D) and mean percentage of IFN-γ producing cells ± SEM (E) are depicted (n = 6). *<i>p</i><0.05, **<i>p</i><0.01, calculated with Mann-Whitney test.</p

RRBS-based global methylation analysis of NK cell and T cell subsets.

<p>Genomic DNA of ex vivo FACS sorted NK and T cell subsets was analyzed for CpG methylation by RRBS. (A–C) Beta-values for each methylation site identified by RRBS were averaged from two donors for each T or NK cell subset as indicated. Sites with coverages below 5 were removed from further analysis. (A) Clustering analysis of differentially methylated CpG sites found by RRBS. Clustering was performed on 1,000 most variant methylation sites out of 30,000 randomly chosen sites, localized within gene bodies and promoter regions. The indicated cell subsets cluster according to the similarities of their methylation profile. Beta-values depicted from dark-red to yellow represent level of methylation of the individual CpG sites. (B) Similarities between the cell subsets as judged by the Euclidian distance. (C) Principle component analysis (PCA) of mean methylation levels from two donors for indicated cell subsets. The PCA revealed a clear separation of CD4⁺ T_H1 and CD8⁺ memory cells, naïve CD4⁺ and CD8⁺ cells, and NK cells by the first two components. (D) CpG methylation sites of T and NK cell subsets of one representative donor identified by RRBS in gene bodies of <i>TXB21</i>, <i>EOMES</i>, <i>PRF1</i>, <i>TNF</i>, <i>PRDM1</i> and <i>ZBTB32</i>. Line diagram represents sequence conservation within vertebrates. Blue and violet bars indicate the methylation level (0–100%) and coverage for each identification, respectively. Coverages of 5 and more reads are represented as a full violet bar.</p

Expanded NKG2C^hi NK cells display complete demethylation of the <i>IFNG</i> CNS1.

<p>Analysis of surface marker expression and methylation status of the <i>IFNG</i> promoter and CNS1 was analyzed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004441#ppat-1004441-g001" target="_blank">Figure 1</a> in ex vivo NK cell subsets derived from representative HCMV⁻ (n = 2) (A) or HCMV⁺ (n = 4) donors (B and C), without (B) or with expansion of NKG2C^hi NK cells (C). NKG2C and CD57 expression was analyzed by FC on PBMC, after gating on viable CD3⁻ CD56^dim NK cells, while sKIR (KIR2DL3 in HLA-C1⁺ donor) expression was analyzed after gating on viable CD3⁻ CD56^dim CD57⁺ NKG2C^+/− NK cells. CpG methylation of the <i>IFNG</i> promoter and CNS1 was analyzed in FACS sorted CD56^dim CD57⁺ NKG2C⁻ (gray bars) and in CD56^dim CD57⁺ NKG2C^+/hi NK cells (black bars) from each donor and is depicted as mean percentage of methylation at each CpG site.</p

CNS1 accessibility regulates <i>IFNG</i> transcriptional activity induced by NKG2C engagement.

<p>(A) Surface expression of NKG2C and 2B4 on NKL (black line) or isotype control (solid grey histogram) was determined by FC. (B) Intracellular expression of IFN-γ by NKL was detected by FC after crosslinking of NKG2C and/or 2B4 for 16 hours. One representative experiment out of four is depicted. (C and D) Luciferase reporter assay of <i>IFNG</i> sequences transfected in NKL. (C) Construct containing the <i>IFNG</i> promoter (<i>IFNG</i>p) region was cloned into the Luciferase reporter vector pGL3 upstream of the Firefly luciferase gene (<i>Luc</i>). pGL3 reporter vectors were transfected into NKL cells along with Renilla reporter vector pRL-TK as internal control and luciferase activity was measured after stimulation of NKL, as indicated. Relative luciferase units (RLU) were calculated in relation to the activity of the <i>IFNG</i>p (−571 to +71) stimulated with aNKG2C+a2B4, after normalization to Renilla luciferase and basic pGL3 activity. Mean RLU ± SEM (n = 3) are depicted. (D) Constructs containing the <i>IFNG</i>p (−49 to +71) region with or without the CNS1 were cloned into the CpG-free vector pCpGL. Luciferase activity of untreated (unmethylated, open circles) and of CpG-methyltransferase (M.SssI)-treated vectors (methylated, black circles) was measured after transfection into NKL cells. RLU were calculated relative to the activity displayed by the unmethylated CNS1+<i>IFNG</i>p (−49 to +71) vector stimulated with aNKG2C+a2B4, after normalization to Renilla luciferase and basic pCpGL activity. One representative experiment out of three is depicted.</p

Cytokine-primed NK cells undergo epigenetic remodeling of the <i>IFNG</i> CNS1.

<p>(A) Alignment of the human <i>IFNG</i> and mouse <i>Ifng</i> locus as presented by VISTA browser, DNA sequence identity >50% over at least 100 bp. Conserved regions with >70% sequence identity are marked in red and are depicted relative to the <i>IFNG</i>/<i>Ifng</i> transcriptional start site (TSS). Arrow indicates transcription direction and exon/UTR regions are indicated in blue. (B–D) Methylation status of the <i>IFNG</i> promoter and/or CNS1 was analyzed by determining CpG methylation of isolated DNA by bisulfite pyrosequencing. Five CpGs from −53 to +171 bp located in the <i>IFNG</i> promoter and six CpGs from −4399 to −4278 bp in the CNS1 region were analyzed and mean percentage of methylation at each individual CpG is depicted. (B and C) CpG methylation of naïve CD4⁺ T cells, T_H1 cells and NK cells, FACS sorted ex vivo as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004441#s4" target="_blank">Materials and Methods</a>. One representative experiment out of two (T cells) or out of three (NK cells) is shown. (D) FACS sorted total NK cells were labeled with 500 nM CFSE and cultured in the presence of the indicated cytokines. After five days, viable CFSE^lo NK cells, which have undergone proliferation, were FACS sorted and the methylation status of the <i>IFNG</i> CNS1 was analyzed. Mean percentage of methylation ± SEM at each individual CpG is depicted (n = 3).</p