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. 2018 Mar;24(3):304-312.
doi: 10.1038/nm.4479. Epub 2018 Feb 12.

Transcriptional Regulation of Macrophage Cholesterol Efflux and Atherogenesis by a Long Noncoding RNA

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Free PMC article

Transcriptional Regulation of Macrophage Cholesterol Efflux and Atherogenesis by a Long Noncoding RNA

Tamer Sallam et al. Nat Med. .
Free PMC article

Abstract

Nuclear receptors regulate gene expression in response to environmental cues, but the molecular events governing the cell type specificity of nuclear receptors remain poorly understood. Here we outline a role for a long noncoding RNA (lncRNA) in modulating the cell type-specific actions of liver X receptors (LXRs), sterol-activated nuclear receptors that regulate the expression of genes involved in cholesterol homeostasis and that have been causally linked to the pathogenesis of atherosclerosis. We identify the lncRNA MeXis as an amplifier of LXR-dependent transcription of the gene Abca1, which is critical for regulation of cholesterol efflux. Mice lacking the MeXis gene show reduced Abca1 expression in a tissue-selective manner. Furthermore, loss of MeXis in mouse bone marrow cells alters chromosome architecture at the Abca1 locus, impairs cellular responses to cholesterol overload, and accelerates the development of atherosclerosis. Mechanistic studies reveal that MeXis interacts with and guides promoter binding of the transcriptional coactivator DDX17. The identification of MeXis as a lncRNA modulator of LXR-dependent gene expression expands understanding of the mechanisms underlying cell type-selective actions of nuclear receptors in physiology and disease.

Conflict of interest statement

Competing Financial Interest Statement:

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1. Regulation of the non-coding RNA MeXis by LXR
A. Schematic representation of the MeXis gene locus on the Integrative Genome Viewer (IGV) (top) and histone marks from LICR ENCODE data in the immediate region of the MeXis gene (bottom). B. Real-time PCR analysis of MeXis and Abca1 expression in primary mouse macrophages treated with vehicle (Ctrl), GW3965 (GW, 0.5 μM) and/or the RXR ligand LG268 (LG, 50 nM). Results are representative of four independent experiments. Values are means ± SD. **** P<0.0001 by Two-way ANOVA followed by multiple comparisons test (Dunnett’s). C. Real-time PCR analysis of MeXis expression in primary mouse macrophages treated with vehicle (Ctrl), GW3965 (GW, 0.5 μM) , oxidized LDL (oxLDL, 50 μg/ml), or acetylated LDL (acLDL, 50 μg/ml). Results are representative of four independent experiments. Values are means ± SD. **** P<0.0001 by Two-way ANOVA followed by multiple comparisons test (Dunnett’s). D. Real-time PCR analysis of MeXis and LeXis expression in primary mouse macrophages treated with vehicle or GW3965 (GW, 0.5 μM) (n = 3/group) or in liver harvested from WT mice treated with vehicle or GW3965 (40 mg/kg, by gavage) for 3 consecutive days (n = 8/group). Values are means ± SD. E. Chip-Seq analysis of LXR binding at the MeXis gene locus.Chip for LXRα and LXRβ in 3xFLAG-LXRα and 3xFLAG-LXRβ expressing immortalized bone marrow macrophage cell using FLAG, RXR and H3K27ac antibody. Cells treated with LXR agonist (GW3965, 1uM) and antagonist (GW2033, 1uM) shown. Blue shaded bar highlighting binding of LXR /RXR at MeXis.F. Real-time PCR analysis of MeXis expression in primary mouse macrophages from mice of the indicated genotypes treated with vehicle or GW3965 (GW, 0.5 μM). N=(4 for WT, αKO, βKO vehicle and 3 for DKO & βKO GW). Experiment repeated once with similar results. Values are means ± SEM. * P<0.05 by two-sided student’s t-test. G. Prediction of coding potential of the indicated lncRNAs using Coding-Non-Coding Index (CNCI) software. A negative value indicates low coding potential.
Figure 2
Figure 2. MeXis regulates Abca1 expression and function
A. Real-time PCR analysis of MeXis and Abca1 expression from primary macrophages treated with the indicated siRNAs (50 nM) followed by either vehicle (Ctrl) or a combination of GW3965 (GW, 0.5 μM) and the RXR ligand LG268 (LG, 50 nM) for 36 h. Results are representative of four independent experiments. Values are means ± SD.**** P<0.0001 by Two-way ANOVA followed by multiple comparisons test (Sidak’s). B. Real-time PCR analysis of MeXis and Abca1 expression 10 days after stable overexpression of control vector (Vect) or MeXis in RAW cells treated with vehicle (Ctrl) or GW3965 (GW, 0.5 μM). Results are representative of three independent experiments. Values are means ± SD.*** P < 0.001 by two-sided student’s t-test. C. Cholesterol efflux in the presence of ApoA-I from RAW macrophages loaded with [3H]cholesterol (1.0 μCi/ml) and treated with the acyl-CoA:cholesterol O-acyltransferase inhibitor (2 μg/ml) and either with DMSO or LXR ligand (1 μM GW3965). ApoA-I-specific efflux represents percent radiolabelled cholesterol efflux in the presence of ApoA-I normalized to DMSO. Experiments were conducted in triplicate. Data are expressed as mean ± SD.* P<0.05 by two-sided student’s t-test.D. Real-time PCR analysis of MeXis and Abca1 expression in primary mouse macrophages (results are representative of four independent experiments; values are means ± SD) and of Abca1 expression in heart, kidney and liver of mice fed a western diet for 3 weeks (N = 8/group; values are means ± SEM).* P<0.05; ** P < 0.01 by two-sided student’s t-test.E. Western blot analysis of Abca1 levels in primary mouse macrophages of WT and MeXis−/− mice treated with GW (0.5 μM for 16 hours). Actin was used as a loading control. The experiment repeated twice with similar results. F. Cholesterol efflux in the presence of ApoA-I or HDL from WT or MeXis−/− macrophages loaded with [3H]cholesterol (1.0 μCi/ml) and treated with the acyl-CoA:cholesterol O-acyltransferase inhibitor (2 μg/ml) and either with DMSO or LXR ligand (1 μM GW3965). Experiments were conducted in triplicate. Data are expressed as mean ± SD. * P<0.05 by two-sided student’s t-test.G. Cholesterol content measured in peritoneal macrophages isolated from mice on western diet for 12 weeks (N = 3/group). ** P < 0.01 by two-sided student’s t-test. H. Oil-red-O staining of peritoneal macrophages isolated from WT or MeXis−/− mice and treated with oxidized LDL (100 μg/ml) for 72 h.The experiment was repeated 3 times with similar results.Scale bars, 50 μm. I. Total serum cholesterol levels in 10-week-old chow-fed male C57BL/6 mice transduced with adenoviral vectors encoding GFP control (Ad-GFP) or MeXis (Ad-MeXis) for 6 days (n = 8 per group). ** P < 0.01 by two-sided student’s t-test. J. GFP and MeXis expression in liver 6 days after transduction of mice with Ad-GFP or Ad-MeXis, respectively (n = 8 per group except Abcg1 n=7 per group).* P<0.05; *** P < 0.001 by two-sided student’s t-test. Data are expressed as mean ± SEM. K. Left, western blot analysis of Abca1 levels in liver from the mice in I (n = 4 per group). Right, quantification of protein levels normalized to actin. Data expressed as mean ± SD. *** P < 0.001 by two-sided student’s t-test.
Figure 3
Figure 3. Loss of MeXis impairs macrophage Abca1 expression and accelerates atherosclerosis
Ldlr−/− mice were transplanted with WT or MeXis−/− bone marrow and maintained on a Western diet for 17 weeks. A. Percentage of aorta surface area with atherosclerotic plaque by en face analysis. Data are mean ± SEM. (N = 16 WT, 15 MeXis). ** P < 0.01 by two-sided student’s t-test. B. Representative photographs (from 16 WT and 15 MeXis−/−) from en face analysis of aortas. Scale bars, 5 mm. C. Quantification of lesion area from oil-red O-stained aortic root sections. Mean ± SEM. (N = 10/ group). * P<0.05 by two-sided student’s t-test. D. Oil Red-O staining of frozen sections from the aortic roots. Representative of 10 per group. Scale bars, 200 μm. E. Representative histology of the aortic root stained with the macrophage marker CD68 and H & E. Representative of 8 per group. Scale bars, 200 μm.F. Representative images of an aortic lesion before and after laser capture microdissection of CD68-positive cells from MeXis−/− mice. Scale bar, 200 μm. G. Abca1 and MeXis expression in laser capture samples as determined by realtime PCR. Samples taken from 6 animals WT and 4 MeXis−/−. Number of samples for Abca1expression 7 per group and MeXis expression 7WT & 11MeXis−/−. Data are mean ± SEM. * P<0.05 by two-sided student’s t-test.
Figure 4
Figure 4. MeXis alters chromosome architecture at the Abca1 locus
A. Gene expression of enhancer RNAs (short noncoding RNAs transcribed from enhancer elements)at the Abca1 locus in primary mouse macrophages of WT or MeXis−/− mice treated with GW3965 (1 μM). The x-axis indicates the location at which gene expression was measured relative to the Abca1 transcription start site. N= 2WT, 3 MeXis−/−. Mean ± SD. B. Expression from the Abca1FL allele in primary macrophages from mice of the indicated genotypes (n=3/group). Mean ± SD.** P < 0.01 by two-sided student’s t-test. C. Genome browser view of ATAC-seq data from primary mouse macrophages of WT or MeXis−/− mice treated with DMSO control or GW3965 (1 μM) for 3 h. Reads were from 4 individual samples per group. D. ATAC seq analysis showing accessibility at peaks around the Abca1 and Tlr4 genepromoters in WT or MeXis−/− macrophages with GW stimulation. Peak position arbitrary numbers the accessibility peaks shown in C at the Abca1 locus in relationship to promoter TSS. E. Heat map of accessibility regions in WT and MeXis−/− macrophages with or without GW3965 treatment.Top of panel shows genome-wide accessibility sites significantly induced by GW3965 in both WT &MeXis−/− macrophages. Bottom panel shows accessibility sites significantly induced by GW3965 only WT macrophages.
Figure 5
Figure 5. Identification of DDX17 as a binding partner of MeXis
A. RNA immunoprecipitation analysis of DDX17. Following immunoprecipitation using IgG control or DDX17 antibody from mouse peritoneal macrophages that were untreated or treated with the cross-linking agent formaldehyde,expression of MeXis, 36B4, cyclophilin (cyloph) and Neat1 was determined by qPCR analysis. N=3/group. Data are mean ± SD. B. Recruitment of DDX17 and LXR at the Abca1 gene locus in mouse macrophages from WT or MeXis−/− mice as determined by ChIP qPCR analysis. Site 1, 2, and 3 are regions containing LXR binding elements known to bound LXR from Chip studies. Data are expressed as percent input retrieved normalized to an upstream control site (region a). N=4/group. Data are mean ± SD. * P<0.05; ** P < 0.01; **** P < 0.0001 by two-sided student’s t-test. C. ChIRP-qPCR analysis of a series of ATAC-seq sites at the Abca1 locus. Sites 1-6 are accessibility sites shown in figure 4e induced with LXR activation in WT but not MeXis−/− macrophages (n=4/group). Chirp probes designed against LacZ or MeXis. Mean ± SD. * P<0.05; ** P < 0.01; *** P < 0.001 by two-sided student’s t-test. D. Western blot analysis of DDX17 levels in lentivirus-transduced immortalized iBMDM (pool of selection-positive cells). Lentiviruses contained either a GFP control or the indicated guide RNAs targeting the DDX17 locus. Actin was used as a loading control.Representative of two independent western blots. E. Abca1 expression in iBMDMs transduced with the indicated lentiviruses and treated with DMSO (Ctrl) or GW3965 (1 μM) for 12 hours (n= 3 per group). Data are mean ± SD. ** P < 0.01; *** P < 0.001 by Two-way ANOVA followed by two-sided student’s t-test. F. MeXis or Abca1 expression in iBMDMs (n= 3 per group). Ctrl is GFP, DDX is DDX17KO, DDXMeXKO is DDX17/MeXis double knockout. Data are mean ± SD. NS= Not significant at P < 0.05 by two-sided student’s t-test. G. Western blot analysis of Abca1 and DDX17 levels in iBMDMs from F.Numbers in blot are quantitative Abca1 protein normalized to control (actin).
Figure 6
Figure 6. Functional conservation of the LXR-MeXis axis in humans
A. Sequence similarity across species at the MeXis gene locus. B. ABCA1 and TCONS00016111 expression in differentiated THP-1 monocytes treated with DMSO (Ctrl) or GW3965 (0.5 μM) for 16 hours (N=4 per group). Mean ± SD. * P < 0.05 by two-sided student’s t-test. C. ABCA1 and TCONS00016111 expression in differentiated THP-1 cells treated with the indicated ASOs (50 nM) and GW3965 (0.5 μM) (N=3 per group). Data are mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001 by one-way ANOVA followed by multiple comparisons test (Dunnett’s).D. Cholesterol efflux in the presence of ApoA-I of THP-1 cells treated with the indicated ASO, loaded with [3H]cholesterol (1.0 μCi/ml), and treated with acyl-CoA:cholesterol O-acyltransferase inhibitor (2 μg/ml) and with either DMSO or LXR ligand (1 μM GW3965) (n=3/group). Data are mean ± SD. * P < 0.05 by two-sided student’s t-test. E. Abca1 gene expression in MeXis−/− BMDM macrophages treated with control (antisense MeXis) or MeXis lentivirus (n=3 per group). Data are mean ± SD. * P < 0.05 by two-sided student’s t-test. F. Abca1 expression in THP-1 cells treated with control or MeXis lentivirus (n=3 per group).Mean ± Two-sided student’s t-test. Not significant at P<0.05. 95% Confidence Interval (-0.1025 to 1.338). G. Regional association plot of variants at TCONS-0016111 and the risk of coronary artery disease in humans from the CARDIoGRAMplusC4D Consortium.

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