(A) Summary of ChIP-seq analysis by GLITR. The results were obtained using an FDR1.5% and a fold change in peak intensity of 10 relative to control IgG as cut off values. (B) Genome-wide mapping of SREBP-2 binding regions relative to RefSeq mouse genes. The ‘promoter’ and ‘downstream’ are defined as 2 kb of 5′ or 3′ flanking sequence. Intergenic region refers to all locations other than ‘promoter’, ‘5′ UTR’, ‘exon’, ‘intron’, ‘3′UTR’ or ‘downstream’. FDR: false discovery rate; FC: fold change. The total number of SREBP2 peaks was 1800 (=100%). These were distributed in the genome as follows: ‘promoter’- 1398 (= 77.6%); ‘exon’- 44 (= 2.4%); ‘downstream’- 18 (= 1.0%): ‘3utr’- 11 (= 0.61%); ‘5utr’- 146 (= 8.1%); ‘intergenic’- 124 (= 6.9%); ‘intron’- 59 (= 3.3%).

(A) 293 cells were transfected with an siRNA (10 nM) for a negative control siRNA (lanes 1 and 2) or siRNA targeting hSREBP-2 (lanes 3 and 4). Twenty-four hours after transfection, cells were cultured in 5% lipoprotein deficient media (LPDS) lanes 2 and 4 or LPDS plus sterols (12 μg/ml cholesterol and 1 μg/ml 25-hydroxycholesterol) for 24 h. and harvested for RNA or total protein (Sanchez et al., 1995). (A) mRNA levels for LC3B, ATG4B, ATG4D, HMGCR and GAPDH were analyzed by RT-qPCR and relative expression is plotted. (B) Whole cell lysates were analyzed for SREBP-2 or LC3 protein levels by immunoblotting. Data are representative of 3 separate experiments. Data are mean ± SEM; n = 3 for 3 separate experiments. HMGCR, HMG Co-A reductase; BP2i, SREBP-2 siRNA; NCi, negative control siRNA.

(A–D) HeLa cells in 8-well chamber slides were transfected with siRNA for hSREBP-2 or negative control siRNA for 24h, dishes were refed serum-depleted media with oleic acid (0.1 mM) for 24 h. (A–C): Co-localization of LDs (BODIPY 493/503, green) and autophagosome (LC3, red). (D) Quantitation of co-localization of LDs with autophagosomes. 1, control; 2, BP2i (SREBP-2 siRNA); 3, NCi (Negative control siRNA). Data are mean ± SEM; n = 3 for 3 separate experiments. *p-value <0.001. Scale bar = 2 μm. In all experiments, approximately 30 cells per sample were counted, and triplicate samples were counted per experimental condition. Additional information is provided in Experimental Procedures and additional cell images are provided in Figure S7. (E) HeLa cells were transfected with hSREBP-2 or negative control siRNA for 36h, and then cells were cultured with serum-depleted media for indicated time. Triglyceride levels were measured from total cell lysates and normalized with protein concentration. The p-value was calculated using t test (n = 3, Nci vs BP2i).

Immunofluorescence of SREBP-2 in mouse liver from normal chow (A) or chow supplemented with lovastatin and ezetimibe (LE) for one week (B). Livers pieces were embedded in paraformaldehyde, sectioned, and stained with a rabbit anti-SREBP-2 polyclonal antibody (affinity purified), followed by fluorescein-labeled goat anti-rabbit IgG. Magnification, 400× bar shows scale for 10 μm. (C) Hepatic nuclear extracts were prepared from mice fed chow and LE diets. (D) Gene-specific ChIP analysis using chromatin from the LE group that was immuno-enriched with an antibody to SREBP-2 (Filled bars) or a control IgG (open bars). Gene specific primers for the indicated promoter regions were used in the qPCR. Data are mean ± SEM; n = 3 for 3 separate experiments.

(A) Gene Set Enrichment Analysis: KS plot. The gene list for the ChIP-seq peaks that were located within 20 kb of a known gene was compared for their correlation to expression of all mouse genes rank ordered for degree of differential expression in RNA isolated from livers of mice fed chow supplemented with LE versus chow supplemented with cholesterol as described in Experimental Procedures (Subramanian et al., 2005). All genes that were expressed based on the microarray (~2.5 × 10⁴) were ranked by fold difference and p value (x-axis) and the graph plots the running enrichment score on the ordinate. Here, many of the genes from the ChIP-seq data set were located in the highly differentially expressed portion of the micorarray data set indicating the genes identified by the ChIP-seq analysis are differentially expressed in the liver of LE vs cholesterol fed mice (p-value =2×e⁻¹⁴). The red vertical dotted line is drawn at the microarray gene index corresponding to the maximum enrichment score (ES). Genes with a corresponding adjacent peak have a tick mark above the main plot. To indicate relative enrichment of the peak regions, peak region density is mirrored around the tick marks as indicated. (B) Web Logos for Motifs present in SREBP-2 ChIP-Seq Peaks. Four motifs were identified by running MEME on the 1800 peak sequences. Motif 1 occurred in 892 of the peaks (E-value = 1.4×10⁻²⁷⁸), motif 2 occurred in 193 of the peaks (E-value = 2.1×10⁻⁰⁷⁰), motif 3 occurred in 231 of the peaks (E-value = 7.8×10⁻⁰⁴⁷), and motif 4 occurred in 148 of the peaks (E-value = 1.4×10⁻⁰³¹)

HeLa cells in 8-well chamber slides or 6-well plates were transfected with hSREBP-2 or negative control siRNA for 24h, and then cells were incubated with media containing 5% LPDS in the absence (−St) or presence (+St) of sterols (12 μg/ml cholesterol and 1 μg/ml 25-hydroxycholesterol) for 24 h. (A) Representative immunofluorescence images with LC3 antibody staining. (B) Quantitation of the percentage of cells with autophagosomes. (C) Immunoblotting with hSREBP-2 (IgG ID2) antibody. Data are mean ± SEM; n = 3 for 3 separate experiments. *p-value <0.05. Scale bar = 10 μm.