Fig 1: Regulation of IFNG expression by WNT10B in TALL-104 cells.To confirm that WNT10B regulated IFNG as a result of the role played by LINC00926 in T cells, TALL-104, a human lymphoblastic cell line known to express IFNG was used. LINC00926 was knocked down in these cells using siRNA and WNT10B and IFNG were measured by qRT-PCR at the indicated time points. Induction of IFNG in activated TALL-104 (A). Effect of knocking down of LINC00926 (B 48 h, and E 72 h) on WNT10B at different times (C 48 h and F 72 h) and on IFNG levels (D 48 h and G 72 h), as measured by qRT-PCR. KD2: 2nd Knockdown, representing the siRNA used.
Fig 2: WNT10B promotor engagement by MLL1, via LINC00926, plays a role in its expression.ChIP-qPCR was performed on DNA samples obtained after co-IP with anti-human MLL1 on whole cell lysate from fixed and crosslinked PBMCs. Fifteen primer pairs which covered ~2 Kb upstream and ~1Kb downstream from WNT10B transcription start site were used for qPCR to detect enrichment of the DNA after co-IP. In another experiment, either MLL1 or KDM5B was knocked down with siRNA to see if it affected the expression of WNT10B and hence IFNG. Of the 15 primer sets only 4 (WNT10Bc-f) primers showed enrichment of the WNT10B promotor region (A). Histone writer (MLL1) and eraser (KDM5B) were knocked down with siRNA and its effect on the expression of WNT10B and IFNG shown as measured by qRT-PCR. B–D Similarly, the effect of reduction in KDM5B on WNT10B in THP1 cells is shown, as measured by qRT-PCR (E, F).
Fig 3: Regulation of inflammatory gene expression by WNT10B.In order to evaluate the effect of increased WNT10B on the expression of inflammatory genes, pre-activated PBMCs were exposed to recombinant human WNT10B from outside. Levels of IFNG and IL17A transcripts were then measured after the indicated duration of culture. In another experiment, inhibitor (ICG001) of WNT signaling pathway was used to see whether WNT signaling was responsible for the increased expression of IFNG and IL17A. Next, in order to show that LINC00926 was responsible for the increased expression of IFNG, LINC00926 was knocked down in PBMCs and the levels of WNT10B and IFNG was measured by qRT-PCR. Effect of enhanced WNT10B signaling on IFNG and IL17A in pre-stimulated PBMCs (A, B respectively) and upon inhibition of WNT signaling, on IFNG and IL17A as measured by qRT-PCR (C, D respectively). Effect of LINC00926 knock down on WNT10B and IFNG expression as measured by qRT-PCR in PBMCs (E–G) and by ELISA of culture supernatant for IFNG (H).
Fig 4: Schematic explaining the role of LINC00926 in orchestrating the regulation of WNT10B in order to regulate IFNG expression.In order to introduce H3K4me3 methylation around WNT10B promotor, LINC00926 associates with MLL1 and brings it to the promotor of WNT10B. This presence of MLL1/LINC00926 complex around WNT10B promotor upstream of TSS, introduces H3K4me3 in this region in PTSD. This results in easy access of the transcription complex and thereby increased transcription of WNT10B leading to increased WNT signaling in PTSD. Consequently, the expression of inflammatory genes like IFNG and IL17A are elevated due to increased WNT signaling, leading to inflammatory state in PTSD patients. On the contrary, in controls, WNT10B expression is not altered and therefore it is present at physiologically favorable levels, and therefore, the expression of inflammatory genes is not affected.
Fig 5: LINC00926-MLL1 interaction in order to regulate WNT10B expression.We predicted that LINC00926 interacted physically with MLL1 to introduce H3K4me3 around WNT10B promotor. Thus, we first analyzed the RNA–protein interaction by online prediction tools, performed ChIP-qPCR and siRNA-based knockdown to see the effect on WNT10B expression. A provides the predicted interaction scores for LINC00926-MLL1 and HOTAIR-EZH2 from the analysis tools. Following this, four separate co-immunoprecipitation using Ab against MLL1 to pull down LINC00926 and qRT-PCR to detect its enrichment was performed on whole cell extract from fixed and crosslinked PBMCs from healthy control to show that these two molecules are physically associated (B). C provides the mean of the four co-immunoprecipitations. Then, in order to show that LINC00926 influenced the expression of WNT10B, we knocked down LINC00926 in PBMCs by employing siRNA and measured the WNT10B transcripts by qRT-PCR (D, E). Each dot represents a human participant.
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