Shared impact on transcriptome for the ORFs PTGIR, NFKB1, SLC39A11 and ZBTB40.

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Impact of PTGIR expression on the CP (S100A8/9 dimer) protein levels in THP1.

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Impact of IBD gene candidate ORFs on the THP-1 transcriptome.

(A) Selected example illustrating impact observed on the transcriptome of THP-1 cells following the expression of IRF5. Each dot represents a single detectable gene in the THP-1 transcriptome. The x-axis shows the log2-transformed median expression across all conditions tested (baseline). The y-axis represents the effect of transduction and expression of a given ORF, as the log2-transformed fold-induction compared to baseline. Skyblue dots represent genes with expression value within expected variation (|Z|≤2), orange dots represent genes suggestively outside the range (|Z|>2) and red dots represent genes outside expected range of variation (|Z|>4). Gray dots are genes with expression value below our detection threshold. Additive effect in log2 correspond to multiplicative effect on the original scale. The fold-change equivalent to a given effect log2-effect x is then: FC = 2x. As an example, an effect of 1 correspond to a FC = 2. (B) Correlation of effect of independent set of replicated expression of IRF5 on THP-1 transcriptome. The x-axis (inner color of dots) and y-axis (border color of dots) show the effect of two independent set of replicated ORFs on the transcriptome, as the log2-transformed fold-induction compared to baseline. Variation between sets of replicates includes effect of independent infection dates, RNA extraction, expression arrays and batches. (C) Impact of the transduction and expression of all 42 IBD gene candidate ORFs on the transcriptome of THP-1 cells. ORFs are ordered by their total number of HITS, with the number of up- and down-regulated HITS illustrated by black and gray, respectively (S2 Table & S1 Appendix). Starred ORFs are previously reported IBD candidate causal genes.</p

Proposed model of S100A8/9 induction.

PTGIR and PTGER4 are activated by their ligands (PGI2 and PGE2 respectively) turning on the AC which converts ATP into cAMP; cAMP activates PKA which in turn triggers STAT3 by phosphorylation. STAT3 binds to the promoters (S9 Fig) of S100A8 and S100A9 inducing their expression. TLR4 and when activated by its ligand LPS, activates the JAK2-STAT3 pathway which induces the expression of S100A8/9 and inhibits NF-kB. We have shown the induction of S100A8/9 genes in THP-1 activated by LPS (Fig 5). ZBTB40 could potentially bind directly to the S100A8/9 promoters inducing their expression and/or to the PLA2G1B promoter (by Encode) activating its expression. PLA2G1B encodes an enzyme that initiates the PG synthesis including PGI2 and PGE2 which bind PTGIR and PTGER4 respectively, inducing S100A8/S100A9 (see above). Based on RNAseq data, expression of NFKB1 in THP-1 induces the expression of EBI3 (one of the top 5 HITS) (S3A Table) most likely via direct binding to the EBI3 promoter by RELB subunit. EBI3 encodes the interleukine-27 subunit beta, which can bind to IL27R and activate JAK/STAT3 pathway [55,56]. In addition, NFKB1 induces the expression of PTGIR ((S10 Fig). RELB binds directly to PTGIR promoter (by Encode).</p

S4 Table -

S4A Table: gProfiler enrichment analyses of all the HITS identified in the screen. S4B Table: gProfiler enrichment analyses of the upregulated HITS identified in the screen. S4C Table: gProfiler enrichment analyses of the downregulated HITS identified in the screen. (XLSX)</p

Impact of PG receptor agonists on the expression of <i>S100A8/A9</i> in response to LPS.

Relative in mRNA expression levels of S100A8/A9 genes were evaluated after incubating THP-1 for 24 hours with or without 0.2 ug/ml of LPS in the presence or absence of 1x10-5 M of Beraprost or CAY10684, the agonists of PTGIR and PTGER4 respectively. Graph on the right represents the same data with different y-axis scale. Each bar is the mean of 3 samples from 3 different experiments ±SEM. *P P P t-test unpaired).</p

Shared impact on transcriptome for the ORFs ETS2 and ZBTB40.

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S5 Table -

S5A Table: Heatmap of gProfiler enrichment of all the HITS identified in the screen. S5B Table: Heatmap of gProfiler enrichment of the upregulated HITS identified in the screen. S5C Table: Heatmap of gProfiler enrichment of the downregulated HITS identified in the screen. (XLSX)</p

Genetic data of the 5 loci of PTGIR, ZBTB40, NFKB1, SLC39A11 and PTGER4.

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Transcription factor binding site analysis of HITS upregulated by different IBD-ORFs by PRIMA-EXPANDER.

(XLSX)</p