Marcin Ratajewski

Polish Academy of Sciences, Warsaw, Masovian Voivodeship, Poland

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Publications (18)64.66 Total impact

  • Iwona Karwaciak, Lukasz Pulaski, Marcin Ratajewski
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    ABSTRACT: Here, we report for the first time a functional study of transcriptional regulation of the human ABCB10 gene. We cloned a functional promoter sequence, and then showed that E2F2, E2F3 and E2F4 can activate transcription from the ABCB10 promoter. We identified sites responsible for this activation and confirmed direct binding of E2F4 to these sites in EMSA and ChIP assays. Finally, by silencing the expression of E2F factors we demonstrated their importance in maintenance of the basal ABCB10 expression. This study provides important atypical examples of E2F4 being a transcriptional activator rather than repressor as well as directly binding to a promoter and regulating it through an alternative and classical DNA consensus response element sequences. It also provides a mechanistic link between E2F4 and ABCB10, both of which are involved in the same physiological phenomena: erythroid lineage differentiation and maturation as well as protection against cardiomyocyte cell death.
    Genomics 01/2014; · 3.01 Impact Factor
  • Toxicology Letters 01/2014; · 3.15 Impact Factor
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    ABSTRACT: Mast cells differentiate from circulating pluripotent hematopoietic progenitors. During this differentiation, the progenitor cells are exposed to changes in oxygen availability. HIF1A is the major sensor of oxygen concentration in mammalian cells. We investigated the expression of HIF1A during the in vitro differentiation of peripheral blood-derived progenitors into human mast cells. In a series of experiments, we determined the changes in CD34 expression, selected mast cell markers, and HIF1A in human mast cell cultures. While the expression of CD34 dramatically decreased, the expression of mast cell-specific genes, including FCER1A, MS4A2, TPSB2, and CMA1, steadily increased. HIF1A expression similarly increased during mast cell differentiation, reaching its maximum level at five weeks of culture. The analysis of the promoter methylation status showed decreasing levels of methylation at the HIF1A promoter, increasing levels of methylation at the CD34 promoter, and no significant changes in other genes. In silico analysis of the promoter regions of these genes revealed large CpG islands in close proximity to the HIF1A and CD34 transcription initiation sites, but not in other investigated genes. In conclusion, in vitro mast cell differentiation was associated with decreased CD34 expression and increased HIF1A expression. These changes were paralleled with changes in the methylation status of the respective promoters, suggesting that DNA methylation-dependent epigenetic regulation mediates the gene expression changes involved in maintaining the phenotype of hematopoietic stem cells and mature mast cells. Therefore, the baseline expression of HIF1A is epigenetically regulated in a cell type- and differentiation stage-specific fashion.
    Immunogenetics 03/2013; · 2.89 Impact Factor
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    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.
    Frontiers in Genetics 01/2013; 4:27.
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    ABSTRACT: Retinoic acid-related orphan receptor γT (RORγT) is the orphan nuclear receptor that regulates the development of Th17 cells and the expression of IL-17. The differentiation of Th17 cells is associated with the upregulation of RORγT mRNA, and the mechanisms regulating that process in human cells are not well understood. We investigated the transcriptional regulation of RORγT in a human lymphocytic cell line and Th17 differentiated from naive CD4(+) cells from human peripheral blood. A series of experiments, including 5' deletion and in situ mutagenesis analysis of the human RORγT promoter, chromatin immunoprecipitation, and overexpression of selected transcription factors, revealed that the transcription factors upstream stimulatory factor 1 (USF-1) and USF-2 are indispensable for the transcription of RORγT in human lymphocytes. There was also upregulation of USF-1 and USF-2 during the differentiation of Th17 cells from naive CD4(+) cells. In this article, we report the first analysis, to our knowledge, of the human RORγT promoter and demonstrate the role of the USF-1 and USF-2 transcription factors in regulating the expression of RORγT in human lymphocytes. Thus, USFs are important for the molecular mechanisms of Th17 differentiation, and possible changes in the expression of USFs might be of interest for inflammatory conditions with a Th17 component. Furthermore, these observations suggest a possible link between metabolic disorders in which the role of glucose-induced USF expression has already been established and autoimmune diseases in which the upregulation of RORγT is frequently detected.
    The Journal of Immunology 08/2012; 189(6):3034-42. · 5.52 Impact Factor
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    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
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    ABSTRACT: Pregnane X receptor (PXR) is a member of the nuclear hormone receptor (NHR) superfamily, which regulates xenobiotic and endobiotic metabolism in the liver. This transcription factor is activated by structurally diverse ligands, including drugs and environmental pollutants. PXR regulates the expression of numerous genes that function in biotransformation and the disposition of xenobiotics upon binding to an AG(G/T)TCA DNA motif in target promoter regions. We performed a screen of mycotoxins that pose a known environmental threat to human and animal health for the ability to activate PXR function in a human hepatocyte cell line, HepG2. We found that aflatoxins B1, M1, and G1 activated PXR. This activation was associated with upregulation of CYP3A4 expression and increased occupancy of PXR protein on the CYP3A4 promoter. Using a microarray approach, we also found that aflatoxin B1 upregulated the expression of multiple genes involved in xenobiotic metabolism, including genes known to be regulated in a PXR-dependent fashion. We also observed an effect of aflatoxin B1 on the expression in other functional groups of genes, including the downregulation of genes involved in cholesterologenesis. The results of this study indicate that aflatoxin B1 is able to activate PXR, a known regulator of liver xenobiotic metabolism, in human hepatocytes, and it can upregulate the expression of PXR-dependent genes responsible for aflatoxin B1 biotransformation, including CYP3A4.
    Toxicology Letters 05/2011; 205(2):146-53. · 3.15 Impact Factor
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    ABSTRACT: Mutations in the DHCR24 gene, which encodes the cholesterol biosynthesis enzyme 3ß-hydroxysterol-∆24 reductase, result in an autosomal recessive disease called desmosterolosis. Further, reduced expression of DHCR24 is found in the temporal cortex of Alzheimer's disease patients. This suggests that variability in the regulatory regions of DHCR24 may contribute to the development of this neurodegenerative disease. In this work, we functionally characterised the proximal fragment of the human DHCR24 gene, for the first time. We show that the transcription of DHCR24 is initiated from a single CpG-rich promoter that is regulated by DNA methylation in some cell types. An activator sequence was also uncovered in the -1203/-665 bp region by reporter gene assays. Furthermore, sodium butyrate (a well-known HDAC inhibitor) increased DHCR24 expression in SH-SY5Y cells by recruiting acetylated core histones H3 and H4 to the enhancer region, as demonstrated by transient transfection and chromatin immunoprecipitation assays. Understanding the regulation of the DHCR24 gene may lead to alternative therapeutic strategies in at least some Alzheimer's patients.
    Molecular Biology Reports 02/2011; 38(2):1091-101. · 2.51 Impact Factor
  • Iwona Sachrajda, Marcin Ratajewski
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    ABSTRACT: The role of the ABCB8 gene in human cells is poorly understood, although it has been suggested to be involved in multidrug resistance in some types of cancers (e.g., melanomas). In this study, the main mechanism of transcriptional regulation of the ABCB8 gene was characterized. EMSA and ChIP assays revealed that the transcription factor Sp1 binds to the ABCB8 core promoter region, and Sp1 consensus elements were crucial for promoter activity in a luciferase reporter gene assay. Mithramycin A, an inhibitor of Sp1 binding, downregulated the expression of ABCB8 (and other ABC genes) in a concentration-dependent manner and sensitized a melanoma cell line to doxorubicin treatment. These findings may have therapeutic applications in at least a subset of melanoma patients.
    MGG Molecular & General Genetics 11/2010; 285(1):57-65. · 2.58 Impact Factor
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    ABSTRACT: ABCC6 mutations are responsible for the development of pseudoxanthoma elasticum, a rare recessive disease characterized by calcification of elastic fibers. Although ABCC6 is mainly expressed in the liver the disease has dermatologic, ocular, and cardiovascular symptoms. We investigated the transcriptional regulation of the gene and observed that hepatocyte growth factor (HGF) inhibits its expression in HepG2 cells via the activation of ERK1/2. Similarly, other factors activating the cascade also inhibited ABCC6 expression. We identified the ERK1/2 response element in the proximal promoter by luciferase reporter gene assays. This site overlapped with a region conferring the tissue-specific expression pattern to the gene and with a putative hepatocyte nuclear factor 4alpha (HNF4alpha) binding site. We demonstrated that HNF4alpha regulates the expression of ABCC6, acts through the putative binding site, and determines its cell type-specific expression. We also showed that HNF4alpha is inhibited by the activation of the ERK1/2 cascade. In conclusion we describe here the first regulatory pathway of ABCC6 expression showing that the ERK1/2-HNF4alpha axis has an important role in regulation of the gene.
    Journal of Biological Chemistry 05/2010; 285(30):22800-8. · 4.65 Impact Factor
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    ABSTRACT: The HMC-1 cell line represents the phenotype of immature mast cells. The HIF1A gene product HIF-1alpha plays key roles in maintaining oxygen homeostasis in eukaryotic organisms and is involved in many processes, including immune response and hematopoiesis. In this study we investigated HIF1A expression in HMC-1 immature hematopoietic cells and CD34+ hematopoietic progenitors. HMC-1 cells exhibited exceptionally low levels of HIF1A expression compared to other cell lines as determined by real-time PCR, and multipotent CD34+ hematopoietic progenitors in bone marrow exhibited significantly lower levels of HIF1A mRNA compared to mature blood cells in peripheral blood. We searched for the mechanisms responsible for suppression of HIF1A expression in HMC-1 cells and obtained evidence for a DNA methylation-dependent process. In vitro methylation of the HIF1A promoter resulted in a decrease in its transcriptional activity and the level of DNA methylation in the HIF1A promoter region in analyzed cell lines was negatively correlated with HIF1A expression. Furthermore, the DNA demethylating agent 5'-azacytidine increased HIF1A expression, and MeCP2 protein was preferentially associated with the HIF1A promoter in vivo. In conclusion, we report that the HIF1A gene in HMC-1 immature hematopoietic cells is suppressed by a process dependent on DNA methylation, and we present evidence indicating downregulation of HIF1A expression in multipotent CD34+ hematopoietic progenitors.
    Biochemical and Biophysical Research Communications 12/2009; 391(1):1028-32. · 2.28 Impact Factor
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    Marcin Ratajewski, Lukasz Pulaski
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    ABSTRACT: Charcot-Marie-Tooth disease (CMT) is a heritable neurodegenerative condition, some types of which (notably CMT4A) are caused by mutations in the GDAP1 gene that encodes a protein of unknown molecular function implicated in regulation of mitochondrial fission. Here we present for the first time a functional analysis of the GDAP1 gene promoter which we found to be transcriptionally regulated by YY1, a broadly studied factor that seems to be involved in regulating many of the same cellular phenomena as GDAP1. We show that GDAP1 is broadly expressed in cancer cell lines of different tissue origin, contrasting with the restricted neuronal distribution reported by some authors. There is a consensus YY1 binding site in the GDAP1 core promoter which we show to be functional in both in vitro binding assays and in living cells. Overexpression of YY1 activated the GDAP1 promoter in a reporter gene system as well as increased the level of endogenous mRNA. RNAi-mediated knockdown of YY1 in HEK293 cells led to decreased GDAP1 expression. While YY1 is known to exert both positive and negative regulatory influences on nuclear-encoded mitochondrial proteins, as well as on neurodegeneration-related genes, in all cell lines we studied (including neuroblastoma) the effect of YY1 on GDAP1 expression is activatory. This leads to interesting conclusions about the possible clinical role of this interaction and suggests a broader regulatory network.
    Genomics 09/2009; 94(6):407-13. · 3.01 Impact Factor
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    Marcin Ratajewski, Hugues de Boussac, Lukasz Pulaski
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    ABSTRACT: Pseudoxanthoma elasticum (PXE) is a heritable connective tissue disease caused by mutations in the ABCC6 gene that encodes a transmembrane transporter of unknown function, expressed mainly in the liver. It has been suggested that some PXE patients for whom no mutations can be found in the coding region of ABCC6 probably suffer from insufficient level of active protein due to lowered gene expression. Here we report the functional analysis of previously reported natural polymorphisms found in the ABCC6 gene promoter. The only polymorphism known to be significantly more common in PXE patients was located within one of the PLAG transcription factor binding sites located by us previously. This mutation negatively influenced PLAG-mediated induction of ABCC6 promoter in a reporter gene system. Moreover, site-directed mutagenesis of an analogous sequence within another PLAG-binding site in the promoter both depressed PLAG binding and specifically repressed ABCC6 promoter activity in cells of liver origin. Thus, we have identified novel sequence determinants of liver-specific transcription of the ABCC6 gene with direct relevance for at least some PXE patients.
    Biochemical and Biophysical Research Communications 04/2009; 383(1):73-7. · 2.28 Impact Factor
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    ABSTRACT: Mutations in the ABCC6 gene are known as causative factors of pseudoxanthoma elasticum (PXE), a connective tissue calcification disorder, but the molecular mechanism of pathogenesis or the physiological function of ABCC6 protein is the subject of intense debate. The ABCC6 gene expression is tightly regulated at the transcriptional level and its tissue-specific distribution is consistent with PXE being a metabolic disease caused by failure of ABCC6 function in organs distant from the diseased sites. In an effort to provide clues to its role by elucidating the mechanisms of its regulation, we identified ABCC6 as a target gene for transcriptional induction by PLAG1 and PLAGL1, transcription factors from the PLAG family of cell cycle progression-related DNA-binding proteins. Both these factors are shown to bind to the same single consensus-binding element in the ABCC6 proximal promoter in cell lines of hepatic and renal origin by reporter gene assay, electrophoretic mobility shift assay and chromatin immunoprecipitation. PLAG-mediated ABCC6 transactivation may play an important role in determining the level of tissue-specific expression of this gene. The described mechanism can also find potential application in therapeutic interventions in forms of PXE related to impaired ABCC6 expression.
    Human Genetics 11/2008; 124(5):451-63. · 4.63 Impact Factor
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    ABSTRACT: Mast cells play important roles in many pathological conditions where local hypoxia is observed, including asthma, rheumatic diseases, and certain types of cancer. Here, we investigated how expression of the hypoxia-inducible factor 1, alpha subunit gene (HIF1A), is regulated in mast cells. The product of HIF1A is hypoxia-inducible factor 1alpha (HIF-1alpha), is a major nuclear transcription factor modulating gene expression in response to hypoxic conditions. We observed that under hypoxic conditions, exposure of mast cells to ionomycin and substance P resulted in significant up-regulation of HIF1A expression as compared with resting mast cells incubated under identical conditions. The ionomycin-mediated increase in HIF-1alpha protein levels was sensitive to the transcription inhibitor actinomycin D and to inhibitors of calcineurin, cyclosporin A (CsA), and FK506. The increased HIF-1alpha protein level was paralleled by a severalfold increase in HIF-1alpha mRNA that could be also inhibited with actinomycin D and CsA. The HIF1A promoter activity was significantly increased in ionomycin-activated mast cells, and the promoter activity could be inhibited by CsA and FK506. Furthermore, in situ mutagenesis experiments showed that the ionomycin-mediated HIF1A promoter activity depends on a conservative NFAT-binding site. Thus, accumulation of HIF-1alpha in activated mast cells requires up-regulation of HIF1A gene transcription and depends on the calcineurin-NFAT signaling pathway.
    The Journal of Immunology 08/2008; 181(3):1665-72. · 5.52 Impact Factor
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    ABSTRACT: Mast cells play important roles in many pathological conditions where local hypoxia is observed, including asthma, rheumatic diseases, and certain types of cancer. Here, we investigated how expression of the hypoxia-inducible factor 1, α subunit gene (HIF1A), is regulated in mast cells. The product of HIF1A is hypoxia-inducible factor 1α (HIF-1α), is a major nuclear transcription factor modulating gene expression in response to hypoxic conditions. We observed that under hypoxic conditions, exposure of mast cells to ionomycin and substance P resulted in significant up-regulation of HIF1A expression as compared with resting mast cells incubated under identical conditions. The ionomycin-mediated increase in HIF-1α protein levels was sensitive to the transcription inhibitor actinomycin D and to inhibitors of calcineurin, cyclosporin A (CsA), and FK506. The increased HIF-1α protein level was paralleled by a severalfold increase in HIF-1α mRNA that could be also inhibited with actinomycin D and CsA. The HIF1A promoter activity was significantly increased in ionomycin-activated mast cells, and the promoter activity could be inhibited by CsA and FK506. Furthermore, in situ mutagenesis experiments showed that the ionomycin-mediated HIF1A promoter activity depends on a conservative NFAT-binding site. Thus, accumulation of HIF-1α in activated mast cells requires up-regulation of HIF1A gene transcription and depends on the calcineurin-NFAT signaling pathway.
    The Journal of Immunology 08/2008; 181(3):1665-1672. · 5.52 Impact Factor
  • Marcin Ratajewski, Grzegorz Bartosz, Lukasz Pulaski
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    ABSTRACT: Mutations in the human ABCC6 gene are responsible for the disease pseudoxanthoma elasticum, although the physiological function or substrate of the gene product (an ABC transporter known also as MRP6) is not known. We found that the expression of this gene in cells of hepatic origin (where this gene is predominantly expressed in the body) is significantly upregulated by retinoids, acting as agonists of the retinoid X receptor (RXR) rather than the retinoid A receptor (RAR). The direct involvement of this nuclear receptor in the transcriptional regulation of ABCC6 gene expression was confirmed by transient transfection and chromatin immunoprecipitation assays. This constitutes the first direct proof of previously suggested involvement of nuclear hormone receptors in ABCC6 gene expression and the first identification of a transcription factor which may be relevant to regulation of ABCC6 level in tissues and in some PXE patients.
    Biochemical and Biophysical Research Communications 01/2007; 350(4):1082-7. · 2.28 Impact Factor
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    ABSTRACT: Multidrug resistance proteins, which catalyse the detoxification of xenobiotics and excretion of metabolites, are very often controlled at the transcriptional level by interaction of exogenous compounds or hormones with nuclear receptors. Since synthetic glucocorticoids have found extensive use as anti-inflammatory drugs, also in the inhaled form in the treatment of asthma, lung cancer is potentially highly prone to transcriptional induction of multidrug resistance proteins by these steroids. MRP3 and MRP2 are major active anionic conjugate transporters in human cells and play a significant role in clinical multidrug resistance in cancer. A549 cells (non-small-cell lung cancer cell line) were challenged with glucocorticoids (dexamethasone, hydrocortisone and prednisone) at physiologically and therapeutically relevant concentrations for 24h and changes in MRP2 and MRP3 expression were followed on four levels: promoter regulation (luciferase reporter constructs), mRNA level (semi-quantitative real-time PCR), protein level (Western blotting) and activity (drug resistance and cellular transport of the model substrate calcein). DEX and HCT in the submicromolar concentration range caused a 2-fold induction of transcriptional activity at the MRP3 promoter construct, while MRP2 expression was not activated. All investigated glucocorticoids caused a modest stimulation of organic anion transport activity. We conclude that glucocorticoids used in clinical practice have the ability to transcriptionally upregulate human MRP3 gene expression in lung-derived cells where this protein is a major component of the organic anion extrusion system. This phenomenon has to be taken into account when designing treatments for lung cancer, especially for patients treated simultaneously with glucocorticoids against inflammatory symptoms.
    The Journal of Steroid Biochemistry and Molecular Biology 09/2005; 96(3-4):229-34. · 3.98 Impact Factor
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    ABSTRACT: ABCC6 encodes MRP6, a member of the ABC protein family with an unknown physiological role. The human ABCC6 and its two pseudogenes share 99% identical DNA sequence. Loss-of-function mutations of ABCC6 are associated with the development of pseudoxanthoma elasticum (PXE), a recessive hereditary disorder affecting the elastic tissues. Various disease-causing mutations were found in the coding region; however, the mutation detection rate in the ABCC6 coding region of bona fide PXE patients is only approximately 80%. This suggests that polymorphisms or mutations in the regulatory regions may contribute to the development of the disease. Here, we report the first characterization of the ABCC6 gene promoter. Phylogenetic in silico analysis of the 5' regulatory regions revealed the presence of two evolutionarily conserved sequence elements embedded in CpG islands. The study of DNA methylation of ABCC6 and the pseudogenes identified a correlation between the methylation of the CpG island in the proximal promoter and the ABCC6 expression level in cell lines. Both activator and repressor sequences were uncovered in the proximal promoter by reporter gene assays. The most potent activator sequence was one of the conserved elements protected by DNA methylation on the endogenous gene in non-expressing cells. Finally, in vitro methylation of this sequence inhibits the transcriptional activity of the luciferase promoter constructs. Altogether these results identify a DNA methylation-dependent activator sequence in the ABCC6 promoter.
    Journal of Biological Chemistry 06/2005; 280(19):18643-50. · 4.65 Impact Factor

Publication Stats

145 Citations
64.66 Total Impact Points

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Institutions

  • 2008–2013
    • Polish Academy of Sciences
      • Institute of Medical Biology
      Warsaw, Masovian Voivodeship, Poland
  • 2011
    • University of Lodz
      • Department of Molecular Biophysics
      Łódź, Lodz Voivodeship, Poland