Journal: Journal of Lipid Research

Article Title: High-fructose diet downregulates long-chain acyl-CoA synthetase 3 expression in liver of hamsters via impairing LXR/RXR signaling pathway

doi: 10.1194/jlr.m032599

Figure Lengend Snippet: Fig. 4. Detection of fructose diet-induced changes in the expres- sion of LXR/RXR isoforms in hamster liver. (A) Real-time qPCR assays were conducted to examine LXR , LXR , RXR , and RXR mRNA levels of ND and FD liver samples. (B) Six individual liver nuclear protein extracts from the ND group and from the FD group were analyzed for LXR / and RXR / protein expression by Western blotting. The membrane was reprobed with anti-HDAC1 antibody as a control of equal nuclear protein loading. (C) The protein abundances of LXR/RXR were quantifi ed with Alpha View software with normalization by signals of HDAC1. Values are mean ± SEM of six samples per group. *P < 0.05 and *** P < 0.001 com- pared with the ND group.

Article Snippet: Nuclear extracts were isolated from hamster livers, and LXR/RXR isoforms were detected with individual antibodies against LXR / (PP-K8607-00, PPK8917-00; R and D systems); RXR (D-20, Santa Cruz Biotechnology); and RXR (#8517, Cell Signaling).

Techniques: Expressing, Western Blot, Membrane, Control, Software

Journal: Journal of Lipid Research

Article Title: High-fructose diet downregulates long-chain acyl-CoA synthetase 3 expression in liver of hamsters via impairing LXR/RXR signaling pathway

doi: 10.1194/jlr.m032599

Figure Lengend Snippet: Fig. 7. LXR-dependent induction of ACSL3 gene expression in hamster primary hepatocytes and HepG2 cells. (A) Hamster primary hepatocytes were cultured in HepatoZYME-SFM medium overnight. GW3965 at indicated concentrations were added to the cells for 24 h before the isolation of total RNA. Real-time qPCR assays were conducted to examine ACSL3 and ABCA1mRNA levels of untreated and treated samples. (B) Specifi c siRNAs and scrambled siRNA were individually transfected into HepG2 cells in 6-well plates. Two days after transfection, total RNA was isolated, and cDNA was synthesized from total RNA. LXR , LXR , and GAPDH mRNA levels were measured by q-PCR, and relative mRNA levels are presented. (C and D) After siRNA transfections, cells were treated with 5 M of GW3965 for 24 h before cell lysis for total RNA isolations and qPCR analysis of mRNA levels of ACSL3 (C) and ABCA1 (D). Data shown are representative of two sepa- rate siRNA transfections that had similar results.

Article Snippet: Nuclear extracts were isolated from hamster livers, and LXR/RXR isoforms were detected with individual antibodies against LXR / (PP-K8607-00, PPK8917-00; R and D systems); RXR (D-20, Santa Cruz Biotechnology); and RXR (#8517, Cell Signaling).

Techniques: Gene Expression, Cell Culture, Isolation, Transfection, Synthesized, Lysis

Journal: bioRxiv

Article Title: Open the LID: LXRα regulates ChREBPα transactivity in a target gene-specific manner through an agonist-modulated LBD-LID interaction

doi: 10.1101/2019.12.20.869974

Figure Lengend Snippet: A. Schematic representation of the synthetic luciferase reporters constructs. ChoRE, carbohydrate response element; LXRE, LXR response element. B. Huh7 cells cultured in 25 mM glucose were transfected with synthetic luciferase reporters containing ChoRE+LXRE, ChoRE-only, or LXRE-only, and plasmids expressing ChREBPα, the activated quadruple mutant ChREBP-Q, or ChREBPβ, together with Mlxγ, with or without LXRα/RXRα. The Renilla luciferase reporter pRL-CMV was used as internal control. Dual luciferase reporter assays were performed 24 hours post transfection. C. Huh7 cells cultured in 25 mM glucose were transfected with a Chrebpβ or Lpk -driven luciferase reporter luciferase reporter, and plasmids expressing ChREBPα, ChREBP-Q, ChREBPβ together with Mlxγ, with or without LXRα/RXRα. The Renilla luciferase reporter pRL-CMV was used as internal control. Dual luciferase reporter assays were performed 24 hours post transfection. Data are presented as mean ± SEM (n=3-7). Significant differences are shown as *p < 0.05, **p < 0.01, ***p < 0.001 compared to control within the same ChREBP isoform transfection. ns, not significant.

Article Snippet: The lysates were then thawed on ice, cleared by centrifugation at 17000 g , 10 min, and immunoprecipitated with 2 μg ChREBP (NB400-135, Novus Biologicals), LXRα (PP-PPZ0412; R&D Systems, Minneapolis, MN, USA), or LXRβ (PP-K8917; R&D Systems) antibodies bound to protein A Dynabeads (Invitrogen) for 2 hours at 4°C.

Techniques: Luciferase, Construct, Cell Culture, Transfection, Expressing, Mutagenesis, Control

Journal: bioRxiv

Article Title: Open the LID: LXRα regulates ChREBPα transactivity in a target gene-specific manner through an agonist-modulated LBD-LID interaction

doi: 10.1101/2019.12.20.869974

Figure Lengend Snippet: A. Co-immunoprecipitation (CoIP) of LXRα and ChREBPα expressed in COS-1 cells cultured in 25 mM glucose. Lysates were immunoprecipitated with ChREBP and LXRα antibodies, and input and immunoprecipitated proteins immunoblotted the same antibodies (n=3). One representative western blot is shown. B. Distribution of ChREBP-LXR peak pairs. ChREBP ChIP-seq data from fasted and high-carbohydrate refed mouse liver ( Poungvarin et al , 2015 ) and LXR ChIP-seq data from T0901317-treated mouse liver ( Boergesen et al , 2012 ) were reanalyzed to generate a genome-wide map of ChREBP and LXR binding sites. The top 10,000 peaks from each dataset were used to calculate the Peak(ChREBP)-to-Peak(LXR) distance, and all peak pairs with a peak-to-peak distance < 1,000 bp were plotted against the number of peak pairs. C. Localization of ChREBP-LXR peak pairs. The Peak(ChREBP)-to-Peak(LXR) distance were plotted against the position relative to transcription start site (TSS) of the closest gene. Blue dots, verified LXR/ChREBP target genes, Acaca, Mlxipl (Chrebpα and β), Pklr, Scd1, Srebf1 and Txnip . Orange curve, moving average (window: 100 bp) of peak-to-peak distance as a function of distance to TSS. D. Global landscape of LXR-ChREBP co-occupancy. Browser view of LXR and ChREBP tracks on Chromatin 3. Square brackets indicate the scale maxima of log2(ChIP/input) ratios. E. Local pattern of LXR-ChREBP co-occupancy. Browser view of LXR and ChREBP tracks on the promoter regions of the ChREBP target genes Mlxipl ( Chrebpβ ) and Pklr ( Lpk ). Square brackets indicate the scale maxima of ChIP/input ratios. F. Genomic positions of the ChREBP-LXR peak pairs. The peak pairs were mapped to genomic elements using Rgmatch. The elements shown are grouped as follows: 5’ far, 25 kb to 5 kb upstream of TSS; Promoter, < 5 kb upstream of TSS; TSS, -200 bp to + 200 bp; E1, first exon; I1, first intron; 3’ near, < 5 kb downstream of TSS; 3’ far, 5 kb to 25 kb downstream of TSS. G. Genome-wide co-occurrence of mouse hepatic transcription factors (TFs). Comparison of LXR and ChREBP with published binding profiles of PPARα ( Boergesen et al , 2012 ) and FXR ( Ijssennagger et al , 2016 ). Forbes coefficients (FC) of genomic co-occurrence between ChREBP, LXR, FXR and PPARα were calculated using the Genomic HyperBrowser.

Article Snippet: The lysates were then thawed on ice, cleared by centrifugation at 17000 g , 10 min, and immunoprecipitated with 2 μg ChREBP (NB400-135, Novus Biologicals), LXRα (PP-PPZ0412; R&D Systems, Minneapolis, MN, USA), or LXRβ (PP-K8917; R&D Systems) antibodies bound to protein A Dynabeads (Invitrogen) for 2 hours at 4°C.

Techniques: Immunoprecipitation, Cell Culture, Western Blot, ChIP-sequencing, Genome Wide, Binding Assay, Comparison

Journal: bioRxiv

Article Title: Open the LID: LXRα regulates ChREBPα transactivity in a target gene-specific manner through an agonist-modulated LBD-LID interaction

doi: 10.1101/2019.12.20.869974

Figure Lengend Snippet: A. Huh7 cells cultured in 25 mM glucose were transfected with a Chrebpβ (n=3) or Lpk -driven luciferase reporter (n=6), and plasmids expressing ChREBPα/Mlxγ, with or without LXRα/RXRα. The Renilla luciferase reporter pRL-CMV was used as internal control. Six hours post transfection, cells were treated with 2.5 mM or 25 mM glucose for 18 hours. Dual luciferase reporter assays were performed 24 hours post transfection. B. LXRαβ wild type (WT), LXRα -/- , LXRβ -/- and LXRα -/- β -/- mice were fasted for 24 hours (white bars) or fasted for 24 hours and refed for 12 hours (black bars) (n=5-8 mice per group). Hepatic gene expression of Lpk ( Pklr ), Chrebpβ and Chrebpα was analyzed by quantitative RT-PCR and normalized to Tbp . Data are presented as mean ± standard error of the mean (SEM). Significant differences are shown as *p < 0.05, **p < 0.01, ***p < 0.001 compared to control within the same treatment, and # p < 0.05, ## p < 0.01, ### p < 0.001 between indicated groups.

Article Snippet: The lysates were then thawed on ice, cleared by centrifugation at 17000 g , 10 min, and immunoprecipitated with 2 μg ChREBP (NB400-135, Novus Biologicals), LXRα (PP-PPZ0412; R&D Systems, Minneapolis, MN, USA), or LXRβ (PP-K8917; R&D Systems) antibodies bound to protein A Dynabeads (Invitrogen) for 2 hours at 4°C.

Techniques: Cell Culture, Transfection, Luciferase, Expressing, Control, Gene Expression, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Open the LID: LXRα regulates ChREBPα transactivity in a target gene-specific manner through an agonist-modulated LBD-LID interaction

doi: 10.1101/2019.12.20.869974

Figure Lengend Snippet: A. Top panel: Schematic representation of the ChREBPα full-length (FL), ChREBPβ, and the LID protein. Bottom panel: CoIP of LXRα and ChREBPα, ChREBPβ, or LID, expressed in COS-1 cells cultured in 25 mM glucose. The ChREBP expression plasmids were transfected with a DNA ratio of ChREBPα:ChREBPβ:LID = 1:6:1, to obtain comparable protein levels. Lysates were immunoprecipitated with ChREBP, FLAG (for LID), or LXRα antibodies, and input and immunoprecipitated proteins immunoblotted with the same antibodies (n=3). One representative western blot is shown. LID, low-glucose inhibitory domain; GRACE, glucose-response activation conserved element; bHLH, basic helix-loop-helix domain: ZIP, leucine zipper. B. Top panel: Schematic representation of the LXRα FL and truncations. Bottom panel: CoIP of ChREBPα and LXRα FL or truncations expressed in COS-1 cells cultured in 25 mM glucose. Lysates were immunoprecipitated with ChREBP antibody (n=3). Input and immunoprecipitated proteins were immunoblotted with ChREBP or FLAG (for LXRα FL and truncations) antibodies. One representative western blot is shown. NTD, N-terminal domain; DBD, DNA-binding domain; LBD, ligand-binding domain.

Article Snippet: The lysates were then thawed on ice, cleared by centrifugation at 17000 g , 10 min, and immunoprecipitated with 2 μg ChREBP (NB400-135, Novus Biologicals), LXRα (PP-PPZ0412; R&D Systems, Minneapolis, MN, USA), or LXRβ (PP-K8917; R&D Systems) antibodies bound to protein A Dynabeads (Invitrogen) for 2 hours at 4°C.

Techniques: Cell Culture, Expressing, Transfection, Immunoprecipitation, Western Blot, Activation Assay, Binding Assay, Ligand Binding Assay

Journal: bioRxiv

Article Title: Open the LID: LXRα regulates ChREBPα transactivity in a target gene-specific manner through an agonist-modulated LBD-LID interaction

doi: 10.1101/2019.12.20.869974

Figure Lengend Snippet: A. Left panels: Local pattern of LXR-ChREBP co-occupancy. Browser view of LXR and ChREBP tracks in the promoter region of Lpk ( Pklr ), Txnip, Fasn and Scd1 . Square brackets indicate the scale maxima of ChIP/input ratios. Arrows indicate the genomic locations of quantitative RT-PCR primers. Right panel: AML12 cells transfected with ChREBPα/Mlxγ and LXRα/RXRα were treated with DMSO (0.1%) or GW3965 (10 µM) for 18 hours. ChREBP or LXR binding to genomic location indicated in the right panels were detected by ChIP using antibodies against ChREBP, LXR or IgG as negative control. Data are presented as mean ± SEM (n=3-5). Significant differences are shown as **p < 0.01, ***p < 0.001 compared to ChIP-IgG, and # p < 0.05, ## p < 0.01, ### p < 0.001 between DMSO and GW3965 groups. ns, not significant. B. CoIP of LXRα and ChREBPα, expressed in COS-1 cells cultured in 25 mM glucose, treated with DMSO (0.1%) or GW3965 (1 µM) for 18 hours. Lysates were immunoprecipitated with ChREBP antibody (n=3). Input and immunoprecipitated proteins were immunoblotted with ChREBP or LXRα antibodies. One representative western blot is shown.

Article Snippet: The lysates were then thawed on ice, cleared by centrifugation at 17000 g , 10 min, and immunoprecipitated with 2 μg ChREBP (NB400-135, Novus Biologicals), LXRα (PP-PPZ0412; R&D Systems, Minneapolis, MN, USA), or LXRβ (PP-K8917; R&D Systems) antibodies bound to protein A Dynabeads (Invitrogen) for 2 hours at 4°C.

Techniques: Quantitative RT-PCR, Transfection, Binding Assay, Negative Control, Cell Culture, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: Open the LID: LXRα regulates ChREBPα transactivity in a target gene-specific manner through an agonist-modulated LBD-LID interaction

doi: 10.1101/2019.12.20.869974

Figure Lengend Snippet: A. Schematic representation of the synthetic luciferase reporters constructs. ChoRE, carbohydrate response element; LXRE, LXR response element. B. Huh7 cells cultured in 25 mM glucose were transfected with synthetic luciferase reporters containing ChoRE+LXRE, ChoRE-only, or LXRE-only, and plasmids expressing ChREBPα, the activated quadruple mutant ChREBP-Q, or ChREBPβ, together with Mlxγ, with or without LXRα/RXRα. The Renilla luciferase reporter pRL-CMV was used as internal control. Dual luciferase reporter assays were performed 24 hours post transfection. C. Huh7 cells cultured in 25 mM glucose were transfected with a Chrebpβ or Lpk -driven luciferase reporter luciferase reporter, and plasmids expressing ChREBPα, ChREBP-Q, ChREBPβ together with Mlxγ, with or without LXRα/RXRα. The Renilla luciferase reporter pRL-CMV was used as internal control. Dual luciferase reporter assays were performed 24 hours post transfection. Data are presented as mean ± SEM (n=3-7). Significant differences are shown as *p < 0.05, **p < 0.01, ***p < 0.001 compared to control within the same ChREBP isoform transfection. ns, not significant.

Article Snippet: The lysates were then thawed on ice, cleared by centrifugation at 17000 g , 10 min, and immunoprecipitated with 2 μg ChREBP (NB400-135, Novus Biologicals), LXRα (PP-PPZ0412; R&D Systems, Minneapolis, MN, USA), or LXRβ (PP-K8917; R&D Systems) antibodies bound to protein A Dynabeads (Invitrogen) for 2 hours at 4°C.

Techniques: Luciferase, Construct, Cell Culture, Transfection, Expressing, Mutagenesis, Control

Journal: bioRxiv

Article Title: Open the LID: LXRα regulates ChREBPα transactivity in a target gene-specific manner through an agonist-modulated LBD-LID interaction

doi: 10.1101/2019.12.20.869974

Figure Lengend Snippet: A. Co-immunoprecipitation (CoIP) of LXRα and ChREBPα expressed in COS-1 cells cultured in 25 mM glucose. Lysates were immunoprecipitated with ChREBP and LXRα antibodies, and input and immunoprecipitated proteins immunoblotted the same antibodies (n=3). One representative western blot is shown. B. Distribution of ChREBP-LXR peak pairs. ChREBP ChIP-seq data from fasted and high-carbohydrate refed mouse liver ( Poungvarin et al , 2015 ) and LXR ChIP-seq data from T0901317-treated mouse liver ( Boergesen et al , 2012 ) were reanalyzed to generate a genome-wide map of ChREBP and LXR binding sites. The top 10,000 peaks from each dataset were used to calculate the Peak(ChREBP)-to-Peak(LXR) distance, and all peak pairs with a peak-to-peak distance < 1,000 bp were plotted against the number of peak pairs. C. Localization of ChREBP-LXR peak pairs. The Peak(ChREBP)-to-Peak(LXR) distance were plotted against the position relative to transcription start site (TSS) of the closest gene. Blue dots, verified LXR/ChREBP target genes, Acaca, Mlxipl (Chrebpα and β), Pklr, Scd1, Srebf1 and Txnip . Orange curve, moving average (window: 100 bp) of peak-to-peak distance as a function of distance to TSS. D. Global landscape of LXR-ChREBP co-occupancy. Browser view of LXR and ChREBP tracks on Chromatin 3. Square brackets indicate the scale maxima of log2(ChIP/input) ratios. E. Local pattern of LXR-ChREBP co-occupancy. Browser view of LXR and ChREBP tracks on the promoter regions of the ChREBP target genes Mlxipl ( Chrebpβ ) and Pklr ( Lpk ). Square brackets indicate the scale maxima of ChIP/input ratios. F. Genomic positions of the ChREBP-LXR peak pairs. The peak pairs were mapped to genomic elements using Rgmatch. The elements shown are grouped as follows: 5’ far, 25 kb to 5 kb upstream of TSS; Promoter, < 5 kb upstream of TSS; TSS, -200 bp to + 200 bp; E1, first exon; I1, first intron; 3’ near, < 5 kb downstream of TSS; 3’ far, 5 kb to 25 kb downstream of TSS. G. Genome-wide co-occurrence of mouse hepatic transcription factors (TFs). Comparison of LXR and ChREBP with published binding profiles of PPARα ( Boergesen et al , 2012 ) and FXR ( Ijssennagger et al , 2016 ). Forbes coefficients (FC) of genomic co-occurrence between ChREBP, LXR, FXR and PPARα were calculated using the Genomic HyperBrowser.

Article Snippet: The lysates were then thawed on ice, cleared by centrifugation at 17000 g , 10 min, and immunoprecipitated with 2 μg ChREBP (NB400-135, Novus Biologicals), LXRα (PP-PPZ0412; R&D Systems, Minneapolis, MN, USA), or LXRβ (PP-K8917; R&D Systems) antibodies bound to protein A Dynabeads (Invitrogen) for 2 hours at 4°C.

Techniques: Immunoprecipitation, Cell Culture, Western Blot, ChIP-sequencing, Genome Wide, Binding Assay, Comparison

Journal: bioRxiv

Article Title: Open the LID: LXRα regulates ChREBPα transactivity in a target gene-specific manner through an agonist-modulated LBD-LID interaction

doi: 10.1101/2019.12.20.869974

Figure Lengend Snippet: A. Huh7 cells cultured in 25 mM glucose were transfected with a Chrebpβ (n=3) or Lpk -driven luciferase reporter (n=6), and plasmids expressing ChREBPα/Mlxγ, with or without LXRα/RXRα. The Renilla luciferase reporter pRL-CMV was used as internal control. Six hours post transfection, cells were treated with 2.5 mM or 25 mM glucose for 18 hours. Dual luciferase reporter assays were performed 24 hours post transfection. B. LXRαβ wild type (WT), LXRα -/- , LXRβ -/- and LXRα -/- β -/- mice were fasted for 24 hours (white bars) or fasted for 24 hours and refed for 12 hours (black bars) (n=5-8 mice per group). Hepatic gene expression of Lpk ( Pklr ), Chrebpβ and Chrebpα was analyzed by quantitative RT-PCR and normalized to Tbp . Data are presented as mean ± standard error of the mean (SEM). Significant differences are shown as *p < 0.05, **p < 0.01, ***p < 0.001 compared to control within the same treatment, and # p < 0.05, ## p < 0.01, ### p < 0.001 between indicated groups.

Article Snippet: The lysates were then thawed on ice, cleared by centrifugation at 17000 g , 10 min, and immunoprecipitated with 2 μg ChREBP (NB400-135, Novus Biologicals), LXRα (PP-PPZ0412; R&D Systems, Minneapolis, MN, USA), or LXRβ (PP-K8917; R&D Systems) antibodies bound to protein A Dynabeads (Invitrogen) for 2 hours at 4°C.

Techniques: Cell Culture, Transfection, Luciferase, Expressing, Control, Gene Expression, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Open the LID: LXRα regulates ChREBPα transactivity in a target gene-specific manner through an agonist-modulated LBD-LID interaction

doi: 10.1101/2019.12.20.869974

Figure Lengend Snippet: A. Left panels: Local pattern of LXR-ChREBP co-occupancy. Browser view of LXR and ChREBP tracks in the promoter region of Lpk ( Pklr ), Txnip, Fasn and Scd1 . Square brackets indicate the scale maxima of ChIP/input ratios. Arrows indicate the genomic locations of quantitative RT-PCR primers. Right panel: AML12 cells transfected with ChREBPα/Mlxγ and LXRα/RXRα were treated with DMSO (0.1%) or GW3965 (10 µM) for 18 hours. ChREBP or LXR binding to genomic location indicated in the right panels were detected by ChIP using antibodies against ChREBP, LXR or IgG as negative control. Data are presented as mean ± SEM (n=3-5). Significant differences are shown as **p < 0.01, ***p < 0.001 compared to ChIP-IgG, and # p < 0.05, ## p < 0.01, ### p < 0.001 between DMSO and GW3965 groups. ns, not significant. B. CoIP of LXRα and ChREBPα, expressed in COS-1 cells cultured in 25 mM glucose, treated with DMSO (0.1%) or GW3965 (1 µM) for 18 hours. Lysates were immunoprecipitated with ChREBP antibody (n=3). Input and immunoprecipitated proteins were immunoblotted with ChREBP or LXRα antibodies. One representative western blot is shown.

Article Snippet: The lysates were then thawed on ice, cleared by centrifugation at 17000 g , 10 min, and immunoprecipitated with 2 μg ChREBP (NB400-135, Novus Biologicals), LXRα (PP-PPZ0412; R&D Systems, Minneapolis, MN, USA), or LXRβ (PP-K8917; R&D Systems) antibodies bound to protein A Dynabeads (Invitrogen) for 2 hours at 4°C.

Techniques: Quantitative RT-PCR, Transfection, Binding Assay, Negative Control, Cell Culture, Immunoprecipitation, Western Blot