Selective binding of sterol regulatory element-binding protein isoforms and co-regulatory proteins to promoters for lipid metabolic genes in liver

Abstract

Mice were subjected to different dietary manipulations to selectively alter expression of hepatic sterol regulatory element-binding protein 1 (SREBP-1) or SREBP-2. mRNA levels for key target genes were measured and compared with the direct binding of SREBP-1 and -2 to the associated promoters using isoform specific antibodies in chromatin immunoprecipitation studies. A diet supplemented with Zetia (ezetimibe) and lovastatin increased and decreased nuclear SREBP-2 and SREBP-1, respectively, whereas a fasting/refeeding protocol dramatically altered SREBP-1 but had modest effects on SREBP-2 levels. Binding of both SREBP-1 and -2 increased on promoters for 3-hydroxy-3-methylglutaryl-CoA reductase, fatty-acid synthase, and squalene synthase in livers of Zetia/lovastatin-treated mice despite the decline in total SREBP-1 protein. In contrast, only SREBP-2 binding was increased for the low density lipoprotein receptor promoter. Decreased SREBP-1 binding during fasting and a dramatic increase upon refeeding indicates that the lipogenic "overshoot" for fatty-acid synthase gene expression known to occur during high carbohydrate refeeding can be attributed to a similar overshoot in SREBP-1 binding. SREBP co-regulatory protein recruitment was also increased/decreased in parallel with associated changes in SREBP binding, and there were clear distinctions for different promoters in response to the dietary manipulations. Taken together, these studies reveal that there are alternative molecular mechanisms for activating SREBP target genes in response to the different dietary challenges of Zetia/lovastatin versus fasting/refeeding. This underscores the mechanistic flexibility that has evolved at the individual gene/promoter level to maintain metabolic homeostasis in response to shifting nutritional states and environmental fluctuations.

FIGURE 1.

Dietary regulation expression of SREBPs in mouse liver. A, immunoblot showing nuclear expression of SREBP-1 (BP-1), SREBP-2 (BP-2), or FXR (NR1H4) across different treatment groups as described under “Materials and Methods.” Chromatin were prepared from freshly isolated pooled liver nuclei (from 4 animals/treatment group) and processed for immunoblotting as described under “Materials and Methods.” ZL, Zetia plus lovastatin; C, chow control; F, fasted; RF, fasted and refed. The immunoblot shown here is representative of the results obtained form several different gels analyzed with samples from at least four different feeding experiments. These patterns of expression are consistent with those reported by others for similar feeding protocols (13, 30, 31). B–F, total RNA from mice from the same pools from A were analyzed for expression of SREBP-1c (B), SREBP-1a (C), SREBP-2 (D), cholesterol 7 α-hydroxylase (CYP7A1)(E), or PEPCK (F) by qPCR as described under “Materials and Methods.” Expression of each mRNA indicated was normalized to the expression of ribosomal protein L32 in each sample, and the ratio in the chow sample was set at 1.0. All values are plotted relative to this value (F.I. = -fold induced). Student's t test was used to evaluate statistical significance, and values that are statistically significant (p < 0.05) are indicated by different numbers at the top of each bar. Comparisons between individual samples that resulted in p > 0.05 are labeled with the same number.

FIGURE 2.

mRNA expression for and SREBP binding to the HMG-CoA reductase gene. mRNA expression (left panel) and binding of SREBP-1 or SREBP-2 by ChIP. RNA was analyzed as described in the legend for Fig. 1. ChIP analyses were performed with antibodies to either SREBP-1 or SREBP-2 as described under “Materials and Methods.” The level of binding was assessed by qPCR following immunoprecipitation, and the value obtained for chow (C) was set at 1.0; all values are plotted relative to this value (F.I. = -fold induced). qPCR reactions were performed in triplicate, and the resulting error bars are displayed. The relative amount of PCR product generated when a control IgG fraction was used in the immunoprecipitation is also provided (IgG). Significance was evaluated as described for Fig. 1. ZL, Zetia plus lovastatin; C, chow control; F, fasted; RF, fasted and refed.

FIGURE 3.

SREBP co-regulatory protein and histone H3 acetylation at HMG-CoA reductase promoter. The binding of NF-Y (A) and CREB (B), acetylation level of histone H3 (C), and binding of CBP (D) to the HMG-CoA reductase promoter were analyzed by ChIP as detailed under “Materials and Methods.” All symbols and notations are as described in the legend for Fig. 2. Immunoblots measuring expression of the A-subunit of NF-Y and CREB are also displayed. Significance was evaluated as described for Fig. 1. There was no significant difference in any of the GFP or PEPCK samples.

FIGURE 4.

CREB is required for induction of HMG-CoA reductase in response to Zetia and lovastatin. Groups of mice (3/group) were fed a chow diet or a diet supplemented with Zetia and lovastatin as described for Figs. 1, 2, 3. Animals on each diet were infected with a control adenovirus construct expressing GFP or an adenovirus expressing A-CREB, a dominant negative version of CREB. RNA was harvested and pooled for analysis by qPCR as described for Figs. 1, 2, 3 and under “Materials and Methods.” Genes analyzed were: A, HMG-CoA reductase (Red); B, cytosolic HMG-CoA synthase (Syn); C, adenovirally encoded GFP (GFP); and D, PEPCK. The significance was evaluated as described for Fig. 1. There were no significant differences in the GFP or PEPCK control groups across all samples. F.I. = -fold induced.

FIGURE 5.

Activation of HMG-CoA reductase promoter by SREBP, NF-Y, and CREB in SL2 cells. A, transient DNA transfection. All samples received HMG-CoA reductase reporter plus an SREBP-2 expression vector. Open symbols indicate no NF-Y, and closed symbols indicate plus all three NF-Y subunits. The amount of DNA for each CREB or mutant CREB expression vector included in the transfection is noted on the x axis. Mutations M1 and L141 are in the KID; ΔQ2 denotes the deletion of the “constitutive” glutamine-rich domain. B, protein extracts from SL2 cells transfected with the wild type (Wt.) or the indicated CREB mutant were analyzed for protein expression by immunoblotting with an antibody to CREB. C, TORC2 binding to HMG-CoA reductase promoter under the different feeding conditions was analyzed by ChIP. There was no statistical difference between the different feeding groups, but the difference between all samples and the IgG control was significant at p < 0.05. Refer to the legend for Fig. 2 for symbols and notations.

FIGURE 6.

LDL receptor promoter and mRNA analysis. mRNA expression (left panel) and binding of SREBP-1 or SREBP-2 by ChIP for the LDL receptor gene was performed essentially as described in the legend for Fig. 2 but with primers specific for the LDL receptor gene mRNA and promoter. The amount of PCR product generated when a control IgG fraction was used in the immunoprecipitation is also provided (IgG). The binding and expression of Sp1 and Sp3 was also evaluated by ChIP and immunoblotting, respectively. All symbols and notations are as described in the legend for Fig. 2.

FIGURE 7.

Fatty-acid synthase mRNA and promoter analysis. mRNA expression (left panel) and binding of SREBPs and co-regulatory proteins to the proximal FAS promoter were analyzed with gene-specific primers as described under “Materials and Methods.” All symbols and notations are as described in the legend for Fig. 2.

FIGURE 8.

Squalene synthase mRNA and SREBP binding. mRNA expression (left panel) and binding of SREBPs to the squalene synthase promoter were analyzed by qPCR and ChIP, with gene-specific primers, as described under “Materials and Methods.” All symbols and notations are as described in the legend for Fig. 2.