Role of Ucp1 enhancer methylation and chromatin remodelling in the control of Ucp1 expression in murine adipose tissue

Abstract

Aims/hypothesis: Increasing the expression of the brown adipose tissue-specific gene uncoupling protein-1 (Ucp1) is a potential target for treating obesity. We investigated the role of DNA methylation and histone modification in Ucp1 expression in adipose cell lines and ex vivo murine adipose tissues.

Methods: Methylation state of the Ucp1 enhancer was studied using bisulphite mapping in murine adipose cell lines, and tissue taken from cold-stressed mice, coupled with functional assays of the effects of methylation and demethylation of the Ucp1 promoter on gene expression and nuclear protein binding.

Results: We show that demethylation of the Ucp1 promoter by 5-aza-deoxycytidine increases Ucp1 expression while methylation of Ucp1 promoter-reporter constructs decreases expression. Brown adipose tissue-specific Ucp1 expression is associated with decreased CpG dinucleotide methylation of the Ucp1 enhancer. The lowest CpG dinucleotide methylation state was found in two cyclic AMP response elements (CRE3, CRE2) in the Ucp1 promoter and methylation of the CpG in CRE2, but not CRE3 decreased nuclear protein binding. Chromatin immunoprecipitation assays revealed the presence of the silencing DiMethH3K9 modification on the Ucp1 enhancer in white adipose tissue and the appearance of the active TriMethH3K4 mark at the Ucp1 promoter in brown adipose tissue in response to a cold environment.

Conclusions/interpretation: The results demonstrate that CpG dinucleotide methylation of the Ucp1 enhancer exhibits tissue-specific patterns in murine tissue and cell lines and suggest that adipose tissue-specific Ucp1 expression involves demethylation of CpG dinucleotides found in regulatory CREs in the Ucp1 enhancer, as well as modification of histone tails.

Fig. 1

Location of CpGs and transcription factor binding sites on the Ucp1 enhancer. a Selected CpGs of interest indicated by black flags and numbered 1 to 6 relative to cyclic AMP response element (CRE) sites, CRE2 and CRE4. A selection of transcription factors whose binding has been previously described, are shown in their relative positions. CREB, PPAR, PGC-1α, retinoic acid (RA), retinoid X receptor (RXR), thyroid hormone receptor (TR), brown fat response element (BRE) and nuclear factor erythroid derived 212 (NFE-212). b Alignment of mouse, rat and human 350 bp of the Ucp1 enhancer. These sequences identified by BLAST are aligned here using Vector NTI. Highlighted grey regions are the core sequences (CGTCA) of the CREs in the mouse promoter with CRE3 positioned upstream of CRE2. CpGs chosen for analysis are underlined and in bold, these are coded 1–6 (5′ to 3′) in superscript

Fig. 2

Ucp1 mRNA expression and CpG dinucleotide methylation of the Ucp1 enhancer in differentiated HIB-1B and 3T3-L1 cells. a Effect of vehicle (white bar) and 10 µmol/l forskolin (black bar) on Ucp1 mRNA abundance normalised to 18S rRNA. b DNA was extracted from differentiated HIB-1B (white bars) and 3T3-L1 cells (black bars), bisulphite modified, amplified by PCR and pyrosequenced to determine CpG methylation over positions 1–6 of the Ucp1 enhancer. Bars represent mean ± SEM (n = 3 experiments). Significantly different from control, **p < 0.01; significant difference between adipose tissues types, *p < 0.05

Fig. 3

Effect of 5-aza-deoxycytidine on CpG dinucleotide methylation of the Ucp1 enhancer and Ucp1 mRNA expression in HIB-1B and 3T3-L1 cells. a CpG methylation over positions 1 to 6 of the Ucp1 enhancer in 3T3-L1 cells grown to 80% confluence and treated with vehicle alone (white bars) or 1 μmol/l 5-aza-deoxycytidine (black bar) for 48 h. b Ucp1 mRNA transcription normalised to 18S rRNA in 3T3-L1 cells and HIB-1B cells treated with 1 μmol/l 5-aza-deoxycytidine or vehicle alone for 48 h before treatment with 10 µmol/l forskolin (black bars) or vehicle (white bars). The data are presented as fold increase in Ucp1 mRNA over vehicle only 3T3-L1 cells. Bars represent mean ± SEM (n = 3 experiments)

Fig. 4

Effect of methylation of pGL3 luciferase reporter constructs containing various fragments of the Ucp1 promoter and transfected into HIB1B and 3T3-L1 cells. a Fold expression of SSSI methylated (Meth) and mock-methylated (Mock) reporter constructs in HIB-1B cells treated with vehicle (white bars) and 10 μmol/l forskolin (black bars). Black circles represent CREs 1, 3, 2 and 4, in order left to right, and the dashed line indicates missing promoter sequences between enhancer and proximal promoter. b Fold expression of SssI methylated (Meth) and mock-methylated (Mock) reporter constructs in 3T3-L1 cells treated with vehicle (white bars) and 10 mol/l forskolin (black bars). Bars represent mean ± SEM (n = 3 experiments)

Fig. 5

Effect of cold stress on expression of Ucp1 mRNA and methylation of CpG dinucleotides in the Ucp1 enhancer in different tissues from mice. a Ucp1 mRNA abundance normalised to 18S rRNA in iBAT, iWAT, gWAT and liver from mice housed in either warm (22 ± 2°C, white bars) or cold (8 ± 2°C, black bars) conditions for 24 h before sampling. b Methylation of CpG dinucleotides in positions 1 to 6 of the Ucp1 enhancer in tissues from mice housed in a warm environment. c Methylation of CpG dinucleotides in positions 1 to 6 of the Ucp1 enhancer in tissues from mice housed in a cold environment: iBAT (black bars), iWAT (white bars), gWAT (bars with horizontal stripes) and liver (bars with vertical stripes). Bars represent mean ± SEM (n = 4 mice). Significant difference between warm and cold, **p < 0.01

Fig. 6

Effect of methylation of the CpG in CRE2 and CRE3 sequences on binding to nuclear proteins. End-labelled oligonucleotide probes containing sequences flanking either CRE2 or CRE3 were incubated in the absence (free) and presence (bound) of nuclear proteins prepared from HIB-1B cells. Competition of each probe with oligonucleotide (EO) or excess methylated oligonucleotide (MEO) was examined at 10×, 25× and 50× the probe concentration, as indicated, in the presence of nuclear proteins

Fig. 7

Chromatin immunoprecipitation analysis of the association of the Ucp1 promoter with active (TriMethH3K4) and repressed (DiMethH3K9) histone marks. a Immunoprecipitation by TriMethH3K4 of the Ucp1 promoter close to the transcription start site, relative to the promoter of the expressed reference gene Prgp2. b Immunoprecipitation by DiMethH3K9 of the Ucp1 promoter close to the transcription start site, relative to the promoter of the suppressed gene Afp. c Immunoprecipitation by DiMethH3K9 of the Ucp1 enhancer region, relative to the promoter of the suppressed reference gene Afp. Adipose tissue nuclei were extracted and cross-linked and the DNA was immunoprecipitated using anti-TriMethH3K4 or anti-DiMethH3K9. Ucp1 start site and Prgp2, Afp start site DNA in the bound and input fractions were quantified by qRTPCR. iBAT and gWAT were taken from mice housed in either warm (22 ± 2°C, white bars) or cold, (8 ± 2°C, black bars) conditions for 24 h before sampling. Bars represent the mean ± SEM of triplicate assays