In order to evaluate SREBP-2 binding on a genome-wide scale we first prepared an antibody to a region of the mouse SREBP-2 within its amino-terminal nuclear targeted domain that bears no similarity to the corresponding region of SREBP-1 (Jeon et al., 2008
). Before embarking on the ChIP-seq analysis, we first evaluated the specificity and quality of the antibody (). Nuclear levels of hepatic SREBP-2 are significantly elevated in mice fed a chow-diet supplemented with lovastatin plus ezetimibe (LE) to inhibit cholesterol production in the liver and simultaneously limit cholesterol uptake from the diet (Bennett et al., 2008
; Jeon et al., 2008
). Sections cut from control and LE treated liver were stained with the SREBP-2 antibody and shows strong nuclear reaction with the LE sample relative to chow fed control, which shows diffuse non-nuclear staining.
Validation for SREBP-2 antibody and chromatin supplemented by chow and chow with LE
SREBP nuclear localization results from proteolytic maturation that cleaves the ~120 kDa membrane bound precursor roughly in half. An immunoblot analysis confirmed that mature SREBP-2 was increased dramatically in the LE sample (). Next, chromatin from the LE sample was used in gene-specific chromatin immunoprecipitation studies with primers that flank known SREBP-2 binding sites within key target genes Hmgcr, Ldlr, and Fasn. These gene-specific ChIP reactions showed significant enrichment by the SREBP-2 antibody whereas a genomic fragment from the negative control Yy1 gene was not enriched (). Based on these results we were encouraged to proceed with the genome-wide binding analysis.
A portion of the DNA used in the confirmation ChIPs in was used in the ChIP-seq procedure for parallel sequencing on the Solexa Genome Analyzer II. The reads returned from the sequencer were then mapped onto the mouse genome, and putative SREBP-2 binding peaks were identified using the global identifier of target regions (GLITR) program (Tuteja et al., 2009
). GLITR calculated a false discovery rate (FDR) for each peak, and we identified all binding peaks that have a FDR less than 1.5% and a fold change in peak intensity greater than 10 (relative to the control IgG). The total number of identified peaks and the number contained within 20 kb of a known gene are shown in . The sequencing reads were also overlayed as a track in the UCSC genome browser and the areas close to peaks associated with 4 different genes are displayed in Figure S1
as an example of the data set.
ChIP-seq analysis for SREBP-2 distribution on mouse liver chromatin supplemented with LE
The genome-wide distribution of the peaks relative to the position of known genes is shown in . It is striking to note that 77% of the binding sites are localized to a region located within 2 kb of the 5′ end of known genes. By contrast, if the peaks were randomly sampled from the genome only 38 sites would be expected to be present in this region (Figure S2A
), demonstrating a strong enrichment of SREBP-2 sites in proximal promoter regions (p
-value < 10−6
). Note that this preference of binding for proximal promoter regions is highly unusual, since most transcription factor binding sites studied by ChIP-seq tend to show more widely distributed binding throughout the entire genome (Seo et al., 2009
). The gene-proximal location of the binding sites was also revealed by a plot of the distance of each peak relative to a transcription start site for a known gene (Figure S2B
Because of the very high number of short sequence reads, Johnson et al (Johnson et al., 2007
) suggested that the ChIP-seq procedure would identify binding sites very accurately. When we evaluated the sequence reads at the Hmgcr
promoter relative to our previously published DNAse I footprinting analysis using recombinant SREBP and 32
P labeled DNA from this region in vitro
(Millinder-Vallett et al., 1996
), the enrichment of the ChIP seq reads correlated very precisely with the three regions of DNA protected by recombinant SREBP in vitro
) providing a compelling example of the congruence of the in vitro
and in vivo
DNA binding studies.
To evaluate how the genome-wide binding analysis correlates with global regulation of gene expression by SREBP-2, we also compared gene expression patterns using RNA from livers of mice fed chow supplemented with either LE where SREBP-2 nuclear levels are elevated () or cholesterol where nuclear levels of SREBP-2 are dramatically lower. A gene set enrichment analysis (GSEA) for this comparison is plotted in ; the X-axis presents a rank order of all genes according to their differential expression (fold change and p value) in LE vs. cholesterol fed samples. The tick-marks above the plot indicate the presence of a ChIP-identified binding peak near the gene sampled in the expression microarray and the relative peak density within the ranked gene list is also displayed around the tick marks. The high peak density within the high differentially expressed region of the GSEA revealed a very high correlation between the two data sets (p =2e−14).
The motif finding program MEME (Bailey, 2002
) was next applied to the sequences covered by the SREBP-2 peaks (). We found four motifs showing strong enrichment in the SREBP-2 peak regions (with E-values < 1.4×10−31
). Interestingly, the highest scoring motif corresponds to an SREBP binding element that was defined by our previous studies using sequence alignment, in vitro
mutagenesis, and chemical probing studies for recombinant SREBPs (Millinder-Vallett et al., 1996
). The second highest scoring motif corresponds to an E-box that is prototypical of bHLH-LZ transcription factors. The generic E-box reads 5′-CANNTG-3′ and different bHLH proteins have a preference for the bases in the middle (Murre et al., 1994
). It is interesting that the E-box identified by our global analysis 5′-CACGTG-3′ is identical to the preferred E-box identified for SREBPs by in vitro
DNA site selection and mutagenesis (Kim et al., 1995
). The additional over-represented motifs correspond to consensus sites for NF-Y and Sp1, two transcription factors known to function synergistically with SREBPs in transcriptional activation (Osborne, 1995
Because the SRE motif (motif 1 above) is an atypical binding site for bHLHLZ proteins, we used site-specific manual ChIP to confirm SREBP-2 binding to several of the predicted SRE motif peaks. We designed primers to amplify the regions overlapping 12 SRE containing peaks and there was significant enrichment in all 12 with the SREBP-2 precipitated chromatin ().
Table I Primer pairs were designed to amplify the predicted SREBP-2 binding region from 12 genes predicted to harbor the SRE element and two separate sets of chromatin prepared from mice fed the LE diet and enriched with the SREBP-2 antibody versus control IgG (more ...)
Next, we used DAVID Gene Ontology software (Dennis et al., 2003
) to categorize the SREBP-2 ChIP-seq genes by apparent functional annotation. In this analysis, broad biological categories that were significantly represented were mostly metabolic processes, and more specific categories included lipid metabolic processes and cholesterol metabolism as expected (Figure S4A
). Several other categories were significantly associated with the SREBP-2 ChIP-seq data set, including regulation of apoptosis and autophagy (Figure S4B
). SREBPs are known to be substrates for the apoptotic Caspase 3 in vitro
and SREBP cleavage in cells can be induced by signals that initiate apoptosis (Wang et al., 1996
). However, there was no prior suggestion that SREBPs might be associated with autophagy.
Autophagy is a catabolic response cells utilize to adapt to limiting nutrients in order to recapture and recycle cellular building blocks as well as to degrade damaged proteins and whole organelles and to attack intracellular pathogens (Mizushima and Klionsky, 2007
; Mizushima and Levine, 2010
; Sumpter and Levine, 2010
). SREBP-2 is key to the cells response to low levels of cellular sterols, a key class of cellular nutrients. Cellular reserves of cholesterol are stored as cholesterol-esters within cytoplasmic lipid droplets along with triglycerides (TG) and recent observations suggest that liberation of stored lipids from within these droplets may be mobilized by an autophagy related process (Singh et al., 2009
). When autophagy was blocked by knockdown of a critical autophagy protein, a significant fraction of total cell TG and cholesterol accumulated within autophagosomes (Singh et al, 2009
). Additionally, sterol depletion has been shown to induce autophagy directly (Cheng et al., 2006
). Thus, we reasoned that the activation of SREBP-2 during autophagy might be an important part of the cellular nutrient adaptation response to low sterols.
To analyze SREBP-2’s putative role in activation of genes involved in autophagy during sterol depletion, we subjected 293 cells to a standard sterol depletion protocol known to induce nuclear accumulation of SREBP-2 (). The autophagy genes LC3B, ATG4B,
were predicted as SREBP-2 targets from the ChIP-seq analysis (Figure S4B
) and expression of all three were induced by the sterol depletion similar to Hmgcr
analyzed as a control. The induction was dependent on SREBP-2 because the inclusion of an siRNA targeting SREBP-2 dramatically reduced the level of induction of all of these genes while having no effect on expression of the control Gapdh
SREBP-2 regulates autophagy-related gene expression
We also cultured 293 cells overnight in the presence and absence of serum and monitored the expression and maturation of SREBP-2 as well as the conversion of LC3 into the lipidated LC3-II form that occurs when autophagy is activated (Mizushima et al., 2010
). Serum starvation induced LC3-II formation and this was also significantly reduced by the siRNA targeting SREBP-2 ().
The LC3-II form accumulates within the autophagosomal membrane prior to lysosomal fusion and this can be evaluated by the localization of LC3 into punctate immuno-staining foci (Mizushima et al., 2010
). The LC3 staining in autophagosomes was significantly increased after serum starvation and this was severely blunted by inclusion of the SREBP-2 siRNA (). LC3-II also accumulates within lipid droplet membranes targeted for lipoautophagy (Shibata et al., 2010
; Singh et al., 2009
) and our hypothesis predicts that LC3-II association with lipid droplets should also be regulated by SREBP-2 levels. When we monitored the co-localization of LC3 with lipid droplets using immunofluorescence for LC3 coupled with the fluorescent lipohilic dye bodipy to stain lipid droplets, we observed and increase in LC3 association with lipid droplets after serum starvation that was also reduced by the SREBP-2 siRNA ( and S5
). The degree of co-localization is unlikely to be influenced by a potential artifact resulting in red emission when the bodipy green dye stains lipid droplets (Ohsaki et al., 2010
) because incubation with bodipy alone did not result in any evidence of false co-localization (Figure S5D
SREBP-2 is required for autophagosome formation
SREBP-2 deficiency decreases co-localization of lipid droplets (LDs) with autophagosome and triglyceride mobilization
If SREBP-2 is essential for mobilization of lipid droplet contents through lipoautophagy then a knockdown of SREBP-2 during nutrient depletion conditions should delay the disappearance of cellular triglyceride levels similar to the delay observed when the autophagy pathway component ATG5 was targeted for knockdown (Singh et al., 2009
). Consistent with this idea, cellular triglyceride mobilization after nutrient depletion was delayed when SREBP-2 was depleted ().