Long non-coding RNA Gm15441 attenuates hepatic inflammasome activation in response to metabolic stress

Chad N. Brocker; Donghwan Kim; Tisha Melia; Kritika Karri; Thomas J. Velenosi; Shogo Takahashi; Jessica A. Bonzo; David J. Waxman; Frank J. Gonzalez

These six Gm15441 upstream sequences were synthesized and cloned into the pGL4.11 reporter, and luciferase assays were performed to assay the functionality of PPARA binding to the Gm15441 promoter. A PPRE-luciferase construct containing an Acox1 PPRE repeat was used as a positive . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/675785 doi: bioRxiv preprint control, and an empty pGL4.11 plasmid was used as a negative control. Luciferase activity was significantly elevated in primary mouse hepatocytes transfected with five of the six pGL4.11 constructs, consistent with direct regulation by PPARA at multiple loci ( Figure 3C) . PPARA binding was also assessed by ChIP assays using a polyclonal PPARA antibody and chromatin isolated from livers of wild-type and Ppara -/mice fed either control diet or a diet-containing WY-14643. Enrichment of PPARA binding to PPREs of known PPARA target genes, namely Acot1 and Acoxt1, was determined by comparing binding to liver chromatin from wild-type mice fed control diet vs. WY-14643-containing diet ( Figure 3D ). Ppara -/livers were used as a negative control to identify non-specific binding. Fscn2 primers were used as a non-target gene promoter and negative control. Enrichment of PPARA binding was found using primer sets covering Gm15441 regions B, C, D, and F, while no binding was seen with Gm15441 region E (Figure 3D) , which was transcriptionally inactive ( Figure 3C) . Enrichment was strongest with region C, in agreement with the luciferase reporter gene data. Enrichment at sites C and D was significantly increased by WY-14643 treatment, indicating increased PPARA recruitment at these sites. No PPARA binding was seen with chromatin from Ppara -/mice on the control diet. Together, these data indicate that PPARA regulates Gm15441 transcription by direct binding of PPARA to multiple PPRE sites within 10 kb of the Gm15441 TSS.