[Show abstract][Hide abstract] ABSTRACT: The human ATP-binding cassette family C member 6 (ABCC6) gene encodes an ABC transporter protein expressed primarily in the liver and to a lesser extent in the kidneys and the intestines. We review here the mechanisms of this restricted tissue-specific expression and the role of hepatocyte nuclear factor 4α which is responsible for the expression pattern. Detailed analyses uncovered further regulators of the expression of the gene pointing to an intronic primate-specific regulator region, an activator of the expression of the gene by binding CCAAT/enhancer-binding protein beta, which interacts with other proteins acting in the proximal promoter. This regulatory network is affected by various environmental stimuli including oxidative stress and the extracellular signal-regulated protein kinases 1 and 2 pathway. We also review here the structural and functional consequences of disease-causing missense mutations of ABCC6. A significant clustering of the missense disease-causing mutations was found at the domain-domain interfaces. This clustering means that the domain contacts are much less permissive to amino acid replacements than the rest of the protein. We summarize the experimental methods resulting in the identification of mutants with preserved transport activity but failure in intracellular targeting. These mutants are candidates for functional rescue by chemical chaperons. The results of such research can provide the basis of future allele-specific therapy of ABCC6-mediated disorders like pseudoxanthoma elasticum or the generalized arterial calcification in infancy.
[Show abstract][Hide abstract] ABSTRACT: The ATP-binding cassette G subfamily member ABCG2 protein is involved in drug resistance of various types of cancer including hepatocellular carcinoma (HCC). The transcriptional regulation of the ABCG2 gene was shown to depend on various transcription factors, and three alternative promoters were described. Here we aimed to decipher the role of hepatocyte growth factor (HGF) and the related kinase cascades on the expression of ABCG2 and the role of the different promoters in this process in the HepG2 human HCC cell line. We observed that HGF treatment increased the amount of ABCG2 on the cell surface in parallel with an increased ABCG2 transcription. ABCG2 mRNA expression was also increased by EGF, oxidative stress or activation of the aryl hydrocarbon receptor, while decreased by TGFb. Treatment with U0126, a specific inhibitor of the ERK1/2 cascade, prevented the HGF and the oxidative stress induced ABCG2 upregulation. We also show that the regulation of ABCG2 by various modulators involve specific alternative promoters. In conclusion, we demonstrate a unique role of the ERK1/2 cascade on ABCG2 modulation in HepG2, and the differential use of the alternative ABCG2 promoters in this cell line. This study reveals the molecular participants of ABCG2 overexpression as new potential treatment targets in HCC.
Biochemical and Biophysical Research Communications 08/2012; 426(2):172-6. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pseudoxanthoma elasticum (PXE), a rare recessive genetic disease causing skin, eye, and cardiovascular lesions, is characterized by the calcification of elastic fibers. The disorder is due to loss-of-function mutations of the ABCC6 gene, but the pathophysiology of the disease is still not understood. Here we investigated the transcriptional regulation of the gene, using DNase I hypersensitivity assay followed by luciferase reporter gene assay. We identified three DNase I hypersensitive sites (HSs) specific to cell lines expressing ABCC6. These HSs are located in the proximal promoter and in the first intron of the gene. We further characterized the role of the HSs by luciferase assay and demonstrated the transcriptional activity of the intronic HS. We identified the CCAAT/enhancer-binding protein β (C/EBPβ) as a factor binding the second intronic HS by chromatin immunoprecipitation and corroborated this finding by luciferase assays. We also showed that C/EBPβ interacts with the proximal promoter of the gene. We propose that C/EBPβ forms a complex with other regulatory proteins including the previously identified regulatory factor hepatocyte nuclear factor 4α (HNF4α). This complex would account for the tissue-specific expression of the gene and might serve as a metabolic sensor. Our results point toward a better understanding of the physiological role of ABCC6.Journal of Investigative Dermatology advance online publication, 5 July 2012; doi:10.1038/jid.2012.218.
Journal of Investigative Dermatology 07/2012; · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent studies demonstrated that cytosine methylation in the genome can be reversed without DNA replication by enzymatic mechanisms based on base excision-repair pathways. Both enzymatic methylation and demethylation mechanisms are active in the cell nucleus at the same time. One can hypothesize that the actual level of CpG methylation could be the result of a balance between the two antagonistic processes with a rapid turnover. In the present study, we used mass spectrometry to measure the total methyl-cytosine content of the genome in cultured human cells after short incubation with the known methyltransferase inhibitor 5-deoxy-azacytidine. A significant decrease of the DNA methylation was observed. Indeed, the inhibition of the methylation can only result in a rapid reduction of the overall methyl-cytosine level if the process of demethylation is simultaneous. These observations suggest that the enzymatic mechanisms responsible of the opposing reactions of DNA methylation and demethylation act simultaneously and may result in a continuous and rapid turnover of methylated cytosines. This conclusion is supported by the observation that 5-deoxy-azacytidine was incorporated in the genomic DNA of non-dividing cells and could be detected as soon as after two hours of incubation, hence providing a mechanistic explanation to the inhibition of methyltransferases. The observations are compatible with the idea that the enzymatic mechanisms that bring together of the opposing reactions of DNA methylation and demethylation act simultaneously and may result in a continuous and unsuspected rapid turnover of DNA methylation. This conclusion is at odds with the generally accepted view of high stability of cytosine methylation where the role of enzymatic demethylation is considered as limited to some special situations such as transcription. It places DNA methylation in the same category as other epigenetic modifications with covalent modifications dynamically added to and removed from the chromatin with high turnover rate.
Epigenetics: official journal of the DNA Methylation Society 02/2012; 7(2):141-5. · 4.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Polyploidy has played a most important role in speciation and evolution of plants and animals. It is thought that low frequency
of polyploidy in mammals is due to a dosage imbalance that would interfere with proper development in mammalian polyploids.
The first tetraploid mammal, Tympanoctomys barrerae (Octodontidae), appears to be an exception to this rule. In this study we investigated X chromosome inactivation (XCI) and
genomic imprinting in T. barrerae, two epigenetic processes usually involved in dosage control in mammalian genomes. The imprinting status of the Peg1 gene was determined by Peg1 allelic expression studies. The inactive X chromosome was identified on interphase nuclei by immunofluorescence using specific
antisera raised against Met3H3K27 and macroH2A1. Quantitative PCR was used to compare the Peg1/Dmd ratio in T. barrerae and in its most closely related diploid species, Octomys mimax. Our data demonstrate that parental-specific silencing of at least one gene and normal X chromosomal dosage mechanism are
conserved in the tetraploid genome. We hypothesize a concerted action of genetic and epigenetic mechanisms during the process
of functional diploidization of this tetraploid genome.