Regulation of UGT1A1 and HNF1 transcription factor gene expression by DNA methylation in colon cancer cells

Abstract

Background: UDP-glucuronosyltransferase 1A1 (UGT1A1) is a pivotal enzyme involved in metabolism of SN-38, the active metabolite of irinotecan commonly used to treat metastatic colorectal cancer. We previously demonstrated aberrant methylation of specific CpG dinucleotides in UGT1A1-negative cells, and revealed that methylation state of the UGT1A1 5'-flanking sequence is negatively correlated with gene transcription. Interestingly, one of these CpG dinucleotides (CpG -4) is found close to a HNF1 response element (HRE), known to be involved in activation of UGT1A1 gene expression, and within an upstream stimulating factor (USF) binding site.

Results: Gel retardation assays revealed that methylation of CpG-4 directly affect the interaction of USF1/2 with its cognate sequence without altering the binding for HNF1-alpha. Luciferase assays sustained a role for USF1/2 and HNF1-alpha in UGT1A1 regulation in colon cancer cells. Based on the differential expression profiles of HNF1A gene in colon cell lines, we also assessed whether methylation affects its expression. In agreement with the presence of CpG islands in the HNF1A promoter, treatments of UGT1A1-negative HCT116 colon cancer cells with a DNA methyltransferase inhibitor restore HNF1A gene expression, as observed for UGT1A1.

Conclusions: This study reveals that basal UGT1A1 expression in colon cells is positively regulated by HNF1-alpha and USF, and negatively regulated by DNA methylation. Besides, DNA methylation of HNF1A could also play an important role in regulating additional cellular drug metabolism and transporter pathways. This process may contribute to determine local inactivation of drugs such as the anticancer agent SN-38 by glucuronidation and define tumoral response.

Figure 1

Putative transcription factor binding sites in human UGT1A1 proximal promoter. Schematic representation of the UGT1A1 gene sequence between positions -540 and +10 (relative to the transcription start site). Positions of CpG dinucleotides are shown. Sequences of the putative transcription factor binding sites are boxed. The recognition core sequence for each transcription factor is underlined.

Figure 2

Interaction of transcription factors with the UGT1A1 promoter using protein nuclear extracts of UGT1A1-expressing HT29 colon cancer cells. EMSAs were performed with 32P-end-labeled probes. Nuclear protein extracts (5 μg) from HT29 cells were pre-incubated in the presence of either no competitor or 100-fold molar excess of cold competitor oligonucleotides (C) or consensus sequence (Cs). For supershift assays, 2 μg of the indicated antibody (Ab) were added directly after the addition of the labeled probe. For USF assays, Ab1 and Ab2 are directed against USF1 and USF2, respectively.

Figure 3

Disruption of HNF1-alpha and USF recognition sequences decreases UGT1A1 basal expression in HT29 cells. Mutational analysis of the putative HNF1, OCT1, USF and NF-Y binding sites in the UGT1A1 proximal promoter. Relative luciferase activity is expressed as a fold induction over the promoterless pGL3-basic vector (set to value = 1). Columns, mean of triplicates of three independent experiments; bars, SD; *, P < 0.05

Figure 4

Incubation with in vitro translated USF1 protein demonstrates that methylation of CpG-4 impedes USF1 binding. EMSAs were performed with 32P-end-labeled probes. In vitro translated USF1 proteins were pre-incubated in the presence of either no competitor or 100-fold molar excess of cold competitor oligonucleotides (C) or consensus sequence (Cs). For supershift assay, 2 μg of the indicated antibody (Ab) were added directly after the addition of the labeled probe. Ab1 and Ab2 are directed against USF1 and USF2, respectively. In methylated probes, dC nucleotide is substituted by an internal methyl deoxyCytidine in CpG-4 dinucleotide.

Figure 5

Methylation of CpG-4 decreases USF1/2-associated complex stability. EMSAs were performed with 32P-end-labeled probes. Nuclear protein extracts (5 μg) from HT29 cells were pre-incubated in the presence of either no competitor or 10-, 50-, 100-fold molar excess of cold competitors (unmethylated and methylated) or oligonucleotides including either a consensus binding site for HNF1-alpha or USF1/2. In methylated probes, dC nucleotide in CpG dinucleotides is substituted by an internal methyl deoxyCytidine.

Figure 6

Upregulation of HNF1A expression is observed following treatment with the 5-Aza-dC demethylating agent in UGT1A1-negative HCT116 colon cancer cells. Rescue of HNF1A gene expression by 5-Aza-dC treatment of colon carcinoma cell lines. Total RNA from HT29 and HCT116 cells were randomly converted to cDNA and specific amplification for USF1, USF2, and HNF1 mRNAs were carried out. Expression of USF1 and USF2 mRNAs are not influenced by 5-Aza-dC treatment. GAPDH gene is used as internal control. Untreated; cells not exposed to 5-Aza-dC.