DNA methylation and Sp1 binding determine the tissue-specific transcriptional activity of the mouse Abcc6 promoter

Abstract

SummaryThe gene encoding the ABCC6 protein, an ABC transporter of the multidrug resistance-associated protein (MRP), is mainly expressed in liver and kidney. Mutations in ABCC6 are responsible for the development of the pseudoxanthoma elasticum (PXE) phenotype. PXE is a recessive disease characterized by the calcification of elastic fibers resulting in dermal, vascular and ocular clinical manifestations. The physiological function of ABCC6 and the rodent orthologs Abcc6 is unknown and their precise relationship to elastic fibers is only a matter of speculation. Despite several studies focused on the transcriptional regulation of ABCC6/Abcc6, the molecular signals conferring the tissue-specificity to the ABCC6/Abcc6 expression are not well defined. In this report, we determined the level of the mouse Abcc6 promoter methylation in tissues with low level of expression (tail extremity and skin), intermediate (kidney) and high level of expression (liver). We observed that high and moderate levels of methylation correlated with low levels of Abcc6 expression. Moreover, we determined that CpG methylation of the Abcc6 proximal promoter region was interfering with the binding of the Sp1 transcription factor thereby inhibiting Sp1-dependent transactivation. Thus, our data provides the first direct evidence that an epigenetic mechanism regulates the binding of the transcription factor Sp1 to the proximal promoter and participates in the tissue-specific expression control of the mouse Abcc6 gene.

A Mouse Model of β-Thalassemia Shows a Liver-Specific Down-Regulation of Abcc6 Expression

“…In WT liver extracts, we found that the relative amount of DNA pulled down with antibodies to NF-E2, Sp1, and HNF4␣ were 1.5-to 2.5-fold greater than those in negative controls, indicating an association of these TFs with the Abcc6 proximal promoter ( Figure 6). As expected, 12,23 pull-down results obtained with HNF1␣ and MTF-1 showed no major association. When chromatin immunoprecipitation assay was carried out with Hbb th3/ϩ liver extracts, we found similar results for HNF1␣, Sp1, and HNF4␣ when compared to WT levels ( Figure 6).…”

“…At 10 months, Abcc6 expression had dropped to 55% of the wild-type levels and at 14 months, the mRNA expression had further decreased to 44%. Despite levels much lower than in the liver, 12 the kidney expression profile in Hbb th3/ϩ mice presented no major difference when compared to WT mice ( Figure 1B) (P Ͼ 0.2). We also evaluated the level of Abcc6 expression in the small intestine at 10 months and found no change between WT and Hbb th3/ϩ mice ( Figure 1C).…”

The ERK1/2-Hepatocyte Nuclear Factor 4α Axis Regulates Human ABCC6 Gene Expression in Hepatocytes

“…We have also identified by luciferase assay a silencer and a DNA methylation-dependent activator sequence in this conserved region. Several transcription factors have been implicated later in the regulation of the human and mouse ABCC6 genes based on luciferase reporter gene experiments (15)(16)(17)(18)(19). More recently, it has been proposed, that the ABCC6 promoter polymorphisms may be functionally linked to PXE (20) and our results suggested that these polymorphisms are modulating the binding capacity of previously identified transcription factors (21).…”

“…In this initial study on the regulation of the gene we reported an inverse correlation between the CpG island hypermethylation located in the conserved proximal promoter and the expression of the gene in various cell lines (14). This finding was confirmed in mouse tissues (15). We have also identified by luciferase assay a silencer and a DNA methylation-dependent activator sequence in this conserved region.…”

Finding the fifth base: Genome-wide sequencing of cytosine methylation

“…However, numerous studies have demonstrated the critical importance of knowing the methylation status of individual CpG sites, which can vary even when in very close proximity to apparently invariable methylcytosines (Prendergast and Ziff 1991;Weaver et al 2004Weaver et al , 2007. Inhibition of binding of the SP1 transcription factor and the insulator protein CTCF by cytosine DNA methylation within their binding elements has been extensively documented (Clark et al 1997;Kitazawa et al 1999;Mancini et al 1999;Bell and Felsenfeld 2000;Hark et al 2000;Inoue and Oishi 2005;Douet et al 2007). It was recently demonstrated that RNA-directed DNA methylation of a single CpG located within a putative conserved intronic cis element of the Petunia floral homeotic gene pMADS3 caused ectopic expression of pMADS3 (Shibuya et al 2009).…”