Alu repeats as transcriptional regulatory platforms in macrophage responses to M. tuberculosis infection

Abstract

To understand the epigenetic regulation of transcriptional response of macrophages during early-stage M. tuberculosis (Mtb) infection, we performed ChIPseq analysis of H3K4 monomethylation (H3K4me1), a marker of poised or active enhancers. De novo H3K4me1 peaks in infected cells were associated with genes implicated in host defenses and apoptosis. Our analysis revealed that 40% of de novo regions contained human/primate-specific Alu transposable elements, enriched in the AluJ and S subtypes. These contained several transcription factor binding sites, including those for members of the MEF2 and ATF families, and LXR and RAR nuclear receptors, all of which have been implicated in macrophage differentiation, survival, and responses to stress and infection. Combining bioinformatics, molecular genetics, and biochemical approaches, we linked genes adjacent to H3K4me1-associated Alu repeats to macrophage metabolic responses against Mtb infection. In particular, we show that LXRα signaling, which reduced Mtb viability 18-fold by altering cholesterol metabolism and enhancing macrophage apoptosis, can be initiated at response elements present in Alu repeats. These studies decipher the mechanism of early macrophage transcriptional responses to Mtb, highlighting the role of Alu element transposition in shaping human transcription programs during innate immunity.

Figure 1.

Analysis of H3K4me1 peaks in uninfected and H37Rv-infected THP-1 cells. (A) Venn diagram representing the number of H3K4me1 peaks in cells infected with H37Rv (V) and uninfected controls (N). (B) Representation of the genomic distribution of the H3K4me1 peaks present in infected but not uninfected cells (VnotN). (C) Gene Ontology analysis of H3K4me1 peaks in VnotN performed with GREAT software. (D) Panther pathway analysis for H3K4me1 VnotN peaks performed with GREAT software.

Figure 2.

Enrichment for SINEs (AluJ and AluS) in H3K4me1 peaks. (A) GAT simulation showing 1.4-fold more AluJ and AluS in H3K4me1 regions with P-value of 0.0001. (B) Genomic distribution of the H3K4me1 peaks containing Alu repeats present in infected but not uninfected cells (VnotN). (C) Gene ontology analysis on genes associated with VnotN peaks that contain a TE. (D) Scatter Plot of the Homer Analysis showing that the analysis including transposable elements (TEs) (y-axis) highlighted new TF (blue) not present in the mask analysis only (red) (x-axis), TF present in both analysis are represented in purple.

Figure 3.

De novo H3K4 monomethylation regions within Alu repeats are enriched for specific transcription factor motifs in Mtb-infected cells. (A) Positions of the centers of ATF, -DR2, –DR4 and MEF2 motifs in Alu repeats located in H3K4me1 V regions. The positions of the sites within Alu were calculated from the genomic coordinates of sites and Alu in the intersectBed results. (B) Distribution of distances of ATF, -DR2, -DR4 and MEF2 motifs located Alu repeats in VnotN peaks relative to TSS (kb). The distribution of regions with ATF/DR2/DR4/MEF2 sites within 100 kb of TSS of T09 target genes were calculated from the distance in GREAT region-gene association tables with a custom Perl program. (C) Venn Diagrams representing the number of Alu repeats containing DR1, DR4, MEF2 and/or ATF motifs located in VnotN (left) or V (right) H3K4me1 regions. (D) In depth Panther analysis on Metabolic Processes (GO:0008152) enriched in Alu/DR4, /DR2, /MEF2, /ATF motifs found in H3K4me1 V peaks (Cf. Supplementary Figure S2). The left-hand pie chart corresponds to level 1 analysis of Metabolic Processes and its distribution in different pathways. The pie chart at right corresponds to level 2 analysis of primary metabolic processes from the left-hand panel and its detailed distribution into different pathways, notably in lipid metabolism.

Figure 4.

LXRα binding to cognate motifs in Alu repeats is enhanced by Mtb infection. (A) RT-qPCR of LXRA in THP-1 cells or primary human macrophages uninfected (Ni) and infected with H37Ra or H37Rv, as indicated. Expression was normalized to reference genes (18S, GAPDH, b-actin; n is a minimum of 3). (B) Western blots showing LXRα expression in uninfected and H37Ra-infected THP-1 cells. (C) ChIP-qPCR analysis of the effect of H37Ra infection of LXRα binding to previously characterized DR4 elements in the LXRA, ABCA1, ABCG1, NR4A3 genes. (D) ChIP-qPCR analysis of LXRα binding to newly identified DR4 elements in Alu repeats in VnotN peaks corresponding to genes TMEM156, PPARG, APOC1, SCD and ACACA. (E) RT/qPCR analysis of regulation by LXR agonist TO901317 of genes analyzed by ChIP-qPCR in (D). Results are average of 3 experimental replicates performed in biological triplicates, ***P-values < 0.005.

Figure 5.

Function of DR4 motifs in Alu repeats as enhancer elements. (A) LXR agonist TO901317 induces recruitment of coactivators NCoA1 and p300 to Alu repeats containing DR4 elements in the regions of the PPARG and SCD genes, as assessed by ChIP assay. (B) ChIP assays reveal that H3K27 acetylation is enhanced in regions of Alu repeats of the PPARG and SCD genes by H37Ra (Ra) infection and TO901317 (TO9). (C) Left: Schematic representation of the SCD gene and its downstream region including sequences upstream of the Alu repeat screened for eRNAs. Right: Results of screening for eRNAs in non-infected (Ni) or H37Ra-infected (Ra) THP-1 cells in the absence or presence of TO901317. (D) Analysis of association of the downstream Alu repeat with the TSS of the SCD gene by ChIP-chromatin conformation capture assay. Left: Schematic representation of ChIP-3C assay. Center: ChIP-3C signal (Blue) obtained by amplification across the junction of the ApaI 3C ligation product in non-infected (Ni) or H37Ra (Ra)-infected THP-1 cells treated with vehicle or TO901317, as indicated. An amplification (orange) across the ApaI site in the region of the TSS of the SCD gene is also shown to control for digestion (see Supplementary Table S11 for primers). Right: A control experiment showing the amount of ChIP-3C product generated in non-infected or H37Ra-infected cells in the presence or absence of ligase.

Figure 6.

Analysis of the effects of LXR agonist TO901317 on LXRα target gene regulation in infected cells. (A) Pictures of representative results of CFU analysis of DMSO- or TO901317- (20 nM) treated THP-1 cells infected with H37Ra for 5 days: dilution 10⁻⁵ of H37Ra spread on 7H10 plates and incubated for 3 weeks. (B) Quantification of CFU assay results at day 0 and day 5 after infection and treatment, n = 3. (C) Assessment of Mtb replication after DNA and RNA extraction was performed by (RT-)qPCR analysis of the ratio of 16SRNA mycobacterial RNA to genomic DNA. (D) Results of IPA (Ingenuity®) pathway enrichment analysis for genes regulated at least 2-fold by treatment with LXR agonist TO901317. Green is for downregulated genes, and red is for upregulated genes. (E) RT-qPCR analysis of expression of genes encoding cholesterol transporters ABCA1, ABCG1, ABCG4 and ABCG5 in uninfected (Ni) or H37Ra (Ra)-infected THP-1 cells treated 24 h with DMSO- or TO901317 (20nM). (F) Imaging of Lipid Droplets (LD) in infected (H37Ra) vs uninfected (NI) THP-1 cells treated with DMSO (veh.) or TO901317, and stained with Bodipy 495/503 and DAPI (Axiovert microscope, X100 objective). (G) Quantification of LD, by size and intensity, number of LD per cell and the total lipid droplet content in H37Ra-infected THP-1 cells treated with DMSO or TO901317. Measurement was done on 20 different images for each condition and on at least 10 cells per image. Measurement and quantification were done using Imaris. **P < 0.05, *P < 0.005. (H) Cholesterol efflux inhibitors Ritonavir (30μM) or Nelfinavir (10 μM) reverse the antimycobacterial effects of TO901317. Quantification of Mtb replication after DNA and RNA extraction was performed by qPCR. Results are represented as expression of 16SRNA normalized to genomic DNA.