Two uniquely arranged thyroid hormone response elements in the far upstream 5' flanking region confer direct thyroid hormone regulation to the murine cholesterol 7alpha hydroxylase gene

Abstract

Cholesterol 7alpha hydroxlyase (CYP7A1) is a key enzyme in cholesterol catabolism to bile acids and its activity is important for maintaining appropriate cholesterol levels. The murine CYP7A1 gene is highly inducible by thyroid hormone in vivo and there is an inverse relationship between thyroid hormone and serum cholesterol. Eventhough gene expression has been shown to be upregulated, whether the induction was mediated through a direct effect of thyroid hormone on the CYP7A1 promoter has never been established. Using gene targeted mice, we show that either of the two TR isoforms are sufficient to maintain normal hepatic CYP7A1 expression but a loss of both results in a significant decrease in expression. We also identified two new functional thyroid hormone receptor-binding sites in the CYP7A1 5' flanking sequence located 3 kb upstream from the transcription start site. One site is a DR-0, which is an unusual type of TR response element, and the other consists of only a single recognizable half site that is required for TR/retinoid X receptor (RXR) binding. These two independent TR-binding sites are closely spaced and both are required for full induction of the CYP7A1 promoter by thyroid hormone, although the DR-0 site was more crucial.

Figure 1

Thyroid hormone regulation of the CYP7A1 gene. (A) Total RNA was isolated from livers of mice that were fed a normal chow (C), an iodine-deficient diet supplemented with PTU (P) or an iodine-deficient diet supplemented with PTU and injected with T3 (P+T3). Equal amounts (20 µg) of total RNA from individual animals was loaded in separate lanes and analyzed by northern analysis to measure mRNA levels for CYP7A1 and 5′DI. Signals were quantified using Quantity One software from Bio-Rad using densitometric scans from autoradiograms, and the intensities were normalized relative to the control ribosomal protein L32 mRNA for each lane. Results are expressed as a fold change relative to the value from the control chow-fed animals. The mean values obtained from individual measurements from six animals in each group are shown with error bars. (B) Total RNA was isolated from livers of WT, TRα(0/0), TRβ(−/−) and TRα(0/0)/TRβ(−/−) that were fed a normal chow.

Figure 2

CYP7A1 promoter is activated by TR in response to T3. (A) HepG2 cells were transfected with the indicated promoter–luciferase fusion construct and where indicated expression vectors were added for TRβ and RXRα. Cells were cultured in serum-free minimal medium and 1 µM T3 was added as indicated and described in Materials and Methods. Results are expressed as corrected luciferase light units divided by the internal control signal for β-galactosidase activity. (B) Similar experiments were performed in HepG2 cells with the indicated promoter–luciferase fusion construct along with expression vectors for TRβ and RXRα. The fold (x) change in the promoter activity by T3 relative to cells transfected with each luciferase reporter alone is shown beside each bar. The data from (A) and (B) represent the mean of duplicates for three individual experiments and include error bars (SEM). RLU, relative light units.

Figure 3

Identification of TR/RXR-binding sites in the CYP7A1 promoter. (A) The 5′-flanking sequence alignment of the rat and mouse CYP7A1 promoter is shown. Arrows indicate putative TREs. (B) In the upper panel, the sequence of the wild-type TRE1 is presented as TRE1/WT. The sequence of mutations in TRE1 is indicated with lower case lettering with mTRE1. The full sequences for the oligonucleotide probes are detailed in Materials and Methods. An autoradiogram from a representative gel shift assay is shown in the lower panel. ³²P-labeled probes were incubated with in vitro translated TRα, TRβ and RXRα, as indicated. Where indicated, a 100-fold molar excess of the indicated unlabeled probe (Comp.) was included in the binding reactions with the labeled probe. D4 denotes the consensus DR-4. WT and M1 denote the wild-type TRE1 and mutant TRE1, respectively. The arrow denotes the position of specific protein–DNA complexes. (C) Similar experiments were performed for TRE2 as described in (B).

Figure 4

TRE1 and TRE2 are responsible for the TR response of CYP7A1. (A) Transfection assays with wild-type and the indicated mutant luciferase reporter constructs were performed in cultured HepG2 cells as described in the legend to Figure 3. The data represent the mean of duplicate samples for three individual experiments and include error bars. M1 and M2 denote mutants of TRE1 and TRE2, respectively, where the identical nucleotide substitutions used to disrupt TR binding in Figure 3B and C were introduced into TRE1 or/and TRE2 of the luciferase reporter constructs. (B) The sequence from −3132 to −3008 was fused to the −342 sequence of the truncated CYP7A1 promoter reporter construct and compared to the full-length and −342 truncated promoter construct for T3 responsiveness as described in (A).

Figure 5

The TRE1/TRE2 do not mediate an LXR response. HepG2 cells were transfected with the indicated promoter–luciferase fusion construct and where indicated expression vectors for TRβ/RXRα or LXRα/RXRα were also included. Cells were cultured in serum-free minimal medium in the presence or absence of 1 µM T3 or 5 µM GW3695. Results are expressed as normalized luciferase light units divided by β-galactosidase activity. The 25-fold activation by T3 for the pGL3R7α-3132/WT construct was set to 100%. The magnitude of the T3 response of pGL3R7α-3132/mTRE1,2 and pGL3R7α-3008/WT is presented relative to that of pGL3R7α-3132/WT. The 30-fold activation of pGL3R7α-3132/WT by GW3695 relative to cells transfected with luciferase reporter alone was set to 100%. The magnitude of the GW3695 response of pGL3R7α-3132/mTRE1,2 and pGL3R7α-3008/WT was presented relative to that of pGL3R7α-3132/WT. The data represent the mean of duplicates for three individual experiments and include error bars.

Figure 6

Recruitment of TRβ to the CYP7A1 TREs in H4IIE cells. Chromatin was prepared from H4IIE cells grown under normal conditions. The ChIP assays were performed with anti-TRβ as described in Materials and Methods. Immunoprecipitates were analyzed by quantitative PCR using primers that flanked the distal TREs or proximal DR-4 (LXRE) as indicated at the top of the figure. Results are expressed as a fold change in comparing the level of DNA amplification specifically precipitated by the TR-β antibody relative to that precipitated by a normal mouse IgG as control. The recruitment of TRβ to a non-relevant region of the genome in the YY1 locus is shown as an additional negative control. The data represent the mean of triplicates for two individual experiments and include error bars.