A pseudoxanthoma elasticum öröklődő betegség kialakulásáért felelős ABCC6/MRP6 gén transzkripciós szabályozásának vizsgálata = Transcriptional regulation of the pseudoxanthoma elasticum (PXE) causing ABCC6/MRP6 gene

Kísérleteimben a pseudoxanthoma elasticum öröklődő betegség kialakulásáért felelős ABCC6 gén transzkripciós szabályozását vizsgáltam és az ehhez szükséges metodikákat állítottam be és fejlesztettem tovább. Kimutattam, hogy: - a génnek 3, az evolúció során erősen konzervált nem kódoló régiója van; - a proximális promóter konzervált régió metilációja szövetspecifikus és inverz korrelációt mutat a gén kifejeződésével; - a promóterben egy aktivátor és egy represszor szekvencia található; - az aktivátor szekvencia DNS metilációra érzékeny. Beállítottam a biszulfitos genomszekvenálást, a DNáz hiperszenzibilitási esszét, illetve a gén kifejeződésének mérésére a kvantitatív PCR-t. Kifejlesztettünk egy primer-tervező és analizáló programot, mely különösen a DNS metiláció meghatározásához használatos biszulfitos genomszekvenáláshoz nyújt segítséget. A kvantitatív PCR beállítása során kialakítottunk egy általános érvénnyel alkalmazható új típusú normalizálási eljárást. Munkánk eredménye egy webszerver, öt közlemény és egy könyvfejezet. Azonosítottuk továbbá a gén 3 DNáz hiperszenzitív helyét és teszteltük számos vegyület hatását a gén kifejeződésére. Közülük többnek is kimutattuk a gátló hatását. Kísérleteink újabb iránya a gén pszeudogénjeinek kialakulását vizsgálja. Eredményeink szerint a pszeudogének non- allelikus homológ rekombinációval jöttek létre. Jelenlétük következménye pl betegségokozó mutációk kialakulása génkonverziós mechanizmussal. Kéziratunkat benyújtottuk közlésre. | The transcriptional regulation of ABCC6, the disease gene of pseudoxanthoma elasticum was investigated in the present study. I also set up and developed the techniques necessary for this work. I demonstrated: The existence of 3 evolutionarily conserved non-coding regions in the gene; The inverse correlation between the tissue-specific methylation of the proximal promoter and ABCC6 expression; The presence of an activator and a repressor in the proximal promoter; The DNA methylation dependence of the activator sequence. I set up the bisulfite genomic sequencing technique, the DNase hypersensitivity and quantitative PCR assays. We developed a primer-design and analyzer software, particularly useful in bisulfite genomic sequencing assays. We developed a novel universally applicable normalization technique for quantitative PCR. As a result of the 3-year period we developed a successful web server, published five papers, one book chapter and initiated one patenting process. We have also identified three hypersensitive sites and screened a number of molecules for their activity on the expression level of the gene. Several tested molecules had a specific inhibitory effect on ABCC6 expression Recently we studied the appearance of ABCC6 pseudogenes. Our data suggest their creation by non-allelic homologous recombination. The consequence of their presence is e.g. the development of disease-causing mutations by gene conversion events. Our manuscript describing the phenomenon is submitted

ABC transzporterek rövid és hosszútávú regulációja metabolikus stresszek hatására = Short and long term regulation of ABC transporters by metabolic sresses

Legfontosabb eredményeinket az ABCC6 gén vizsgálata során értük el. A gén mutációi a pseudoxanthoma elasticum recesszív öröklődésű betegség kialakulásához vezetnek. Megerősítettük, hogy a mutáció hordozói 10x nagyobb kockázatnak vannak kitéve a coronaria arteriabetegség kialakulása tekintetében, mint a kontroll populáció. Az ERK kaszkád aktiválódásának hatására májsejtekben megfigyelt ABCC6 expresszió-csökkenést vizsgálva azonosítottunk egy HNF4 kötőhelyet; igazoltuk, hogy az ERK1/2 aktiválása gátolja a HNF4 okozta ABCC6 expressziófokozódást; beláttuk, hogy a HNF4 szükséges a további aktivátor faktorok hatásához és meghatározza az ABCC6 szövetspecifikus expresszióját. Igazoltuk, hogy az ABCC6 gén első intronjában van egy sej- és főemlős-specifikus aktivátor szakasz, ami legalább két aktivátor fehérje kötőhelyet hordoz. Ezek egyike a C/EBP beta. Kimutattuk, hogy transzkripciós aktiváló hatásának kifejtéséhez szükség van a proximális promóterrel való együttműködésére, ami nem (közvetlenül) a HNF4 transzkripciós faktorral jön létre. Eredményeink azt sugallják, hogy az ABCC6 szerepet játszhat a hepatocyták metabolikus állapotának szabályozásában. Terveztük az ABCC6 gén epigenetikai profiljának leírását is. Beállítottunk tehát egy LC/MS-MS módszert egyedi szekvenciák és teljes genom metiláció vizsgálatára. Igazoltuk, hogy a DNS metiláció egy dinamikus egyensúly eredménye, mely a sejtek osztódásától független folyamatos demetiláció és remetiláció eredménye. | Our most important results were obtained on the ABCC6 gene. Mutations of ABCC6 lead to pseudoxanthoma elasticum, a recessive Mendelian disease. In our studies, we confirmed that mutation carriers have a 10x increased risk to develop coronary artery disease relative to controls. While studying the regulation of the gene, we observed the decrease of the expression in hepatocytes upon the activation of ERK cascade. We showed that ERK activation inhibits ABCC6 expression via HNF4, which plays a crucial role in orchestrating the transcriptional regulation of the gene and regulation of its tissue-specific expression. We also identified a primate and cell-type specific activator sequence in the 1st intron of ABCC6, which harbors at least two binding sites for activator proteins, one of which is C/EBP beta. We have shown that this activator region interacts with the proximal promoter but not directly with HNF4. Our data on the transcriptional regulation of ABCC6 suggest that ABCC6 reflect the metabolic state of hepatocytes and might play a role in influencing it. We plan to investigate the epigenetic profile of the ABCC6 gene. We set up therefore a HPLC/MS-MS method to investigate the methylation of target sequences and genomes. We showed that in contrast to previous hypotheses global methylation level is a result of a dynamic equilibrium independent of replication but depending on permanent demethylation and remethylation

Transcriptional regulation of the ABCC6 gene and the background of impaired function of missense disease-causing mutations.

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 4alpha 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

The ABCC6 Transporter: A New Player in Biomineralization

Pseudoxanthoma elasticum (PXE) is an inherited metabolic disease with autosomal recessive inheritance caused by mutations in the ABCC6 gene. Since the first description of the disease in 1896, alleging a disease involving the elastic fibers, the concept evolved with the further discoveries of the pivotal role of ectopic mineralization that is preponderant in the elastin-rich tissues of the skin, eyes and blood vessel walls. After discovery of the causative gene of the disease in 2000, the function of the ABCC6 protein remains elusive. More than 300 mutations have been now reported and the concept of a dermal disease has progressively evolved toward a metabolic disorder resulting from the remote effects caused by lack of a circulating anti-mineralization factor. Very recently, evidence has accumulated that this anti-mineralizing factor is inorganic pyrophosphate (PPi). This leads to decreased PPi/Pi (inorganic phosphate) ratio that results from the lack of extracellular ATP release by hepatocytes and probably renal cells harboring the mutant ABCC6 protein. However, the mechanism by which ABCC6 dysfunction causes diminished ATP release remains an enigma. Studies of other ABC transporters, such as ABCC7 or ABCC1 could help our understanding of what ABCC6 exact function is. Data and a hypothesis on the possible roles of ABCC6 in acquired metabolic diseases are also discussed

The BiSearch web server

BACKGROUND: A large number of PCR primer-design softwares are available online. However, only very few of them can be used for the design of primers to amplify bisulfite-treated DNA templates, necessary to determine genomic DNA methylation profiles. Indeed, the number of studies on bisulfite-treated templates exponentially increases as determining DNA methylation becomes more important in the diagnosis of cancers. Bisulfite-treated DNA is difficult to amplify since undesired PCR products are often amplified due to the increased sequence redundancy after the chemical conversion. In order to increase the efficiency of PCR primer-design, we have developed BiSearch web server, an online primer-design tool for both bisulfite-treated and native DNA templates. RESULTS: The web tool is composed of a primer-design and an electronic PCR (ePCR) algorithm. The completely reformulated ePCR module detects potential mispriming sites as well as undesired PCR products on both cDNA and native or bisulfite-treated genomic DNA libraries. Due to the new algorithm of the current version, the ePCR module became approximately hundred times faster than the previous one and gave the best performance when compared to other web based tools. This high-speed ePCR analysis made possible the development of the new option of high-throughput primer screening. BiSearch web server can be used for academic researchers at the site. CONCLUSION: BiSearch web server is a useful tool for primer-design for any DNA template and especially for bisulfite-treated genomes. The ePCR tool for fast detection of mispriming sites and alternative PCR products in cDNA libraries and native or bisulfite-treated genomes are the unique features of the new version of BiSearch software

Oral administration of pyrophosphate inhibits connective tissue calcification

Various disorders including pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI), which are caused by inactivating mutations in ABCC6 and ENPP1, respectively, present with extensive tissue calcification due to reduced plasma pyrophosphate (PPi). However, it has always been assumed that the bioavailability of orally administered PPi is negligible. Here, we demonstrate increased PPi concentration in the circulation of humans after oral PPi administration. Furthermore, in mouse models of PXE and GACI, oral PPi provided via drinking water attenuated their ectopic calcification phenotype. Noticeably, provision of drinking water with 0.3 mM PPi to mice heterozygous for inactivating mutations in Enpp1 during pregnancy robustly inhibited ectopic calcification in their Enpp1-/- offspring. Our work shows that orally administered PPi is readily absorbed in humans and mice and inhibits connective tissue calcification in mouse models of PXE and GACI PPi, which is recognized as safe by the FDA, therefore not only has great potential as an effective and extremely low-cost treatment for these currently intractable genetic disorders, but also in other conditions involving connective tissue calcification

Az ABC-fehérjék Tudományos Iskolája: a gének regulációjától a transzport-mechanizmusig = The School of ABC-proteins: From Gene Regulation to Transport Mechanism

Megállapítottuk, hogy az ABCC6 gén duplikációi a low-copy repeat 16a elemekhez kötődnek, és ilyen duplikációk több alkalommal is bekövetkeztek különböző főemlős fajokban. Populációgenetikai vizsgálatunkban kimutattuk, hogy egy inaktív ABCC6 allél növeli a koronáriás artéria betegség (CAD) kialakulásának esélyét. Az ABCC6 expressziójánk szabályozását is vizsgáltuk, megállapítottuk, hogy az ERK1/2 szignál-útvonal és a HNF4 transzkripciós faktor felelős az ABCC6 szövet-specifikus szabályozásáért. Megalkottuk az ABCC6 transzporter homológia modelljét, és tanulmányoztuk az ismert 119 misszensz PXE-t okozó mutáció eloszlását. A komplex domén-domén határfelületeken a misszensz mutációk jelentős feldúsúlását figyeltük meg, ami ezen kapcsolatok fontosságának genetikai bizonyítéka. Az ABCC6 viszgálatára alkalmas új állat modellt fejlesztettünk ki: a zebrahal (Danio rerio) modell-rendszert. Bemutattuk, hogy az ecetmuslica MRP az ortológ humán ABCC fehérjékhez hasonló biokémiai tulajdonságokkal rendelkezik, így azok magas turnover-rel rendelkező modellje. Megfigyeltük, hogy a koleszterin szelektíven módosítja az ABCG2 aktivitását. Tanulmányoztuk az ABCG2 katalitikus ciklusa során fellépő intramolekuláris átrendeződéseket. Kifejlesztettünk egy sejtes modell-rendszert az ABCA1, egy fontos koleszterin transzporter tanulmányozására. Új módszert fejlesztettünk ki a kvantitatív PCR reprodukálhatóságának javítására. 15 közleményt publikáltunk szakmailag lektorált nemzetközi folyóiratokban. | We established that duplications of ABCC6 are associated to low-copy repeat 16a and such duplications have occurred several times in different primates. Our population genetic study revealed that one inactive allele of ABCC6 increases the risk of coronary artery disease (CAD) significantly. Th signal transduction pathways leading to the modulation of ABCC6 expression have also been deciphered: the ERK1/2 pathway and HNF4 are responsible for the tissue-specific regulation of ABCC6. We have built a homology model of this transporter, and analyzed the distribution of the known 119 missense PXE-associated mutations within the structure. Significant clustering of the missense mutations has been found at domain-domain interfaces providing a genetic proof of the importance of these domain-domain interactions. A novel animal model to investigate ABCC6 has been developed: the zebrafish (Danio rerio) model system. We have demonstrated that the Drosophila MRP shares the biochemical features of its human ABCC orthologues and serves as a high turn-over model protein of human ABCC-type transporters. We have found that cholesterol selectively modulates the activity of ABCG2. We have studied the intramolecular rearrangments during the catalytic cycle of ABCG2. We have developed a cellular model system to study ABCA1, an important cholesterol transporter. We have developed a novel method to improve the quantitative PCR technique. We have published 15 papers in peer-reviewed international journals

Inhibition of DNA methyltransferase leads to increased genomic 5-hydroxymethylcytosine levels in hematopoietic cells.

5-Hydroxymethylcytosine (5hmC) is produced from 5-methylcytosine (5mC) by Ten-eleven translocation (TET) dioxygenases. The epigenetic modification 5hmC has crucial roles in both cellular development and differentiation. The 5hmC level is particularly high in the brain. While 5mC is generally associated with gene silencing/reduced expression, 5hmC is a more permissive epigenetic mark. To understand its physiological function, an easy and accurate quantification method is required. Here, we have developed a novel LC-MS/MS-based approach to quantify both genomic 5mC and 5hmC contents. The method is based on the liberation of nucleobases by formic acid. Applying this method, we characterized the levels of DNA methylation and hydroxymethylation in mouse brain and liver, primary hepatocytes, and various cell lines. Using this approach, we confirm that the treatment of different cell lines with the DNA methyltransferase inhibitor 5-aza-2\u27-deoxycytidine leads to a decrease in 5mC content. This decrease was accompanied by an increase in 5hmC levels in cell lines of hematopoietic origin. Finally, we showed that ascorbate elevates the levels of 5hmC and augments the effect of 5-aza-2\u27-deoxycytidine without significantly influencing 5mC levels