The catalytic domain of JMJD2 and JARID- JmjC family demethylases.

<p><b>A</b>. Sequence alignment of JmjC domains of JMJD2 and JARID demethylases from human (Hs), <i>A</i>. <i>nidulans</i> (An) and <i>S</i>. <i>cerevisiae</i> (Sc). Conserved residues responsible for the catalytic activity are marked in yellow (α-ketoglutarate binding site) and blue (Fe²⁺ binding site). Specificity-determining residues for H3K9K36me2/3 are marked in green [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006222#pgen.1006222.ref061" target="_blank">61</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006222#pgen.1006222.ref062" target="_blank">62</a>]. <b>B</b>. Domain composition of JARID group H3K4me2/3 demethylases from human, <i>A</i>. <i>nidulans</i> and <i>S</i>. <i>cerevisiae</i>. KdmB possesses conserved histidine and glutamate as well as phenylalanine, asparagine and lysine residues responsible for Fe²⁺ ion chelating and α-ketoglutarate binding respectively, which are found in all catalytically active JmjC demethylases.</p

KdmB influences transcriptional activity and H3K4me3 levels.

<p>The Figure shows global correlation analysis of H3K4me3 levels and KdmB-dependent transcription in nutrient-rich culture cells (primary metabolism). All genes were categorized according to two criteria, i.e. at least 4-fold differential expression in WT and <i>kdmB</i>Δ as well as the degree of H3K4 trimethylation. This resulted in four categories, low (log₂ RPKM ≤ 5, panel <b>A</b>) and high (log₂ RPKM > 5, panel <b>B</b>) H3K4me3 levels and transcriptional up-/or downregulation in the <i>kdmB</i> mutant. For each open reading frame in these categories, the coverage in CPM (counts per million of reads) was calculated within a 2kb window around the predicted ATG (-500 to +1500) and represents the average enrichment level of this mark. Details on the bioinformatic procedure used to determine CPM values over all points in all genes are given in Materials and Methods. Red lines in the meta-plots indicate CPM values for the WT, while green lines indicate values obtained for <i>kdmB</i>Δ. The number of individual genes in each category and their level of de-regulation are shown in the bar-graph between the meta-plots. Each bar in the graph represents the differential expression value of an individual gene in this group. With this procedure four different correlation groups (G1 –G4) emerged, i.e. genes with low and high H3K4m3 levels requiring KdmB for normal transcription (WT-up/G1 and WT-up/G3, respectively) are expressed stronger in the wild type. Genes with low and high H3K4m3 levels under negative KdmB influence (<i>kdmB</i>Δ-up/G2 and <i>kdmB</i>Δ-up/G4, respectively) are stronger expressed in the <i>kdmB</i>Δ mutant. For all values, p<0.005 was set as threshold. <b>C.</b> Genome viewer image of one representative gene (locus AN6321) within the low H3K4me3 category in which <i>kdmB</i> deletion leads to gain of H3K4me3 (green boxed area) and higher transcription (<i>kdmB</i>Δ-up/G2 gene).</p

Genome viewer image presenting the chromatin landscape of chromosome IV in wild type (WT) and <i>kdmB</i> deletion (<i>kdmB</i>Δ) cells grown in liquid shake cultures for 17 hours (primary metabolism).

<p>RNA-seq represents transcriptional activity of the locus. A control ChIP (No Antibody) was performed to detect non-specific ChIP-seq signals.</p

Chromatin landscape of the sterigmatocystin gene cluster.

<p>The ST cluster is indicated within the red box, the colour key is in the black box at the bottom of the figure. <b>A</b>. In both WT and <i>kdmBΔ</i> the ST cluster in 17 h cultures (PM) remains silent and lacks the investigated histone marks with the exception of H3K9me3 at <i>stcC</i>). <b>B.</b> The ST cluster is strongly induced at 48h (SM) in the wild type and the cluster genes gain low levels of H3K4me3, H3K36me3 and H3Ac. Levels of H3K9me3 at <i>stcC</i> are decreased in comparison to the flanking H3K9me3 domains under these SM conditions. In <i>kdmBΔ</i> the ST cluster remains almost silent. Unlike in the WT, besides low levels of H3K36me3, other activating histone marks are not detected at the ST locus in <i>kdmBΔ</i>.</p

H3K4me3 localizes to actively transcribed genes.

<p><b>A.</b> The metaplot depicts the enrichment pattern of H3K4 trimethylation (H3K4me3), H3K36 trimethylation (H3K36me3), and H3K9/K14 acetylation (H3Ac) for all genes on chromosome 4 in wild type actively growing cells (17h cultures). All genes are aligned to the predicted ATG (position 0) and analysed for a 2 kb window starting with 500 bp of their 5´UTR and promoter sequences (-500) followed by 1500 bp of their coding region (indicated positions within coding region 500, 1000 and 1500 bp). Counts were binned in 10-bp windows and averaged. <b>B.</b> The scatter plots shows the relationship between the average expression level and H3K4me3 enrichment. Values on the x-axis represent the average expression level of each gene determined by RNA-seq from both 17 and 48h cultures (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006222#sec012" target="_blank">methods</a>; log₂ RPKM). On the y axis, normalized and H3K4me3 levels averaged over the whole gene (log₂ RPKM) are shown for each of these genes. Grey dots represent constitutively expressed genes), and genes differentially expressed in the two tested conditions (with a p-value of p ≤0.001) are represented as black or red dots where the latter indicate differentially expressed genes involved in SM biosynthesis.</p

KdmB is required for normal induction of SMB genes.

<p><b>A.</b> Upper panel: percentage of de-regulated genes in <i>kdmBΔ</i> during PM (17 h) and SM (48 h) with the division into SM cluster genes as annotated in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006222#pgen.1006222.ref072" target="_blank">72</a>] and basic metabolism genes (cell structure and function). Differential expression cut off was set to higher than 4-fold difference (log₂ ≥ 2, p<0.05). The lower graph depicts the total number of de-regulated genes in <i>kdmBΔ</i> during PM (17 h) and SM (48 h) cultures,. <b>B.</b> HPLC-chromatograms of supernatant extracts from wild type and kdmBΔ cells growing for 48 hours in conventional AMM or in the special SM-promoting “ZM” medium. a) wild type extract (supernatant, AMM), b) wild type extract (supernatant, ZM), c) <i>kdmBΔ</i> extract (supernatant, AMM), d) <i>kdmBΔ</i> extract (supernatant, ZM). Peaks are assigned to compounds according to standards running in parallel analyses. (1) Austinol, (2) Dehydroaustinol, (3)-Sterigmatocystin, (4) Emericellamide C, (5) Emericellamide D, (6) Orsellinic acid, (7) 2,ω-Dihydroxyemodin, (8) ω-Hydroxyemodin, (9) 2-Hydroxyemodin, (10) Emodin.</p

Levels of H3K4me3 are increased in <i>kdmBΔ</i> histones.

<p>MS/MS Base Peak Chromatograms (BPC) of tryptic histone digests analysing peptides T₃-R₈ of histone H3 show the different variants of methylated K4 and their ratios in the wild type (WT) and the <i>kdmBΔ</i> strain.</p

Boxplots of ChIP-seq results for Wild Type (WT) and <i>kdmBΔ</i> strains analysed for the two growth-phase dependent conditions, i.e. the two time points of harvesting after 17h (primary metabolism, PM) and 48h (secondary metabolism, SM).

<p>The [log₂ (RPKM)] values at the two time points are given for the analysed chromatin modifications (H3K4me3, H3Ac or H3K36me3) and the gene set is divided into functional categories related to “Cell structure and function” (5676 genes) and to genes belonging to “SM clusters” (149 genes). H3K4me3 median of the log₂ (RPKM) values for cell structure and function is higher than for SM clusters in all strains and conditions. The differences in H3 acetylation (H3Ac) and H3K36 trimethylation (H3K36me) are not significant between the categories and time points.</p

FcγR and C1q binding and antibody-dependent enhancement of pHu-E16 variants.

<p><b>A</b>. SPR analysis of FcγR and C1q binding. FcγRs including CD16A (CD16V¹⁵⁸, CD16F¹⁵⁸), CD32A (CD32A-R¹³¹ and CD32A-H¹³¹ fused to an aglycosylated Fc region of IgG2 (CD32A-R¹³¹-G2agl and CD32A-H¹³¹-G2agl)), CD32B (CD32B fused to an aglycosylated Fc region of IgG2 (CD32B-G2agl)) and CD64, and C1q were injected at the same concentration over the surfaces with Hu-E16 variants captured on immobilized WNV E protein. Buffer injection was subtracted as blank and responses were normalized to same level of captured antibodies. <b>B</b>. ADE of WNV infection with antibody variants. Serial dilutions of Hu-E16 variants were mixed with WNV RVP and added to CD32A-expressing K562 cells. Forty-eight hours later, cells were analyzed by flow cytometry for GFP expression.</p

Time course of pHu-E16 expression in <i>N. benthamiana</i> plants.

<p>Total proteins from leaves infiltrated or co-infiltrated with pHu-E16scFv-C_H^1-3, pHu-E16 HC/pHu-E16scFv-C_L, pHu-E16scFv-C_H^1-3/pHu-E16scFv-C_L (Tetra pHu-E16), or pHu-E16scFv-C_H^1-3/pHu-E16 LC were extracted on days 5 -10 post infiltration and analyzed by an ELISA that detects the assembled form of pHu-E16 MAb variants, Mean ± SD of samples from three independent infiltrations are presented.</p