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Pseudoxanthoma elasticum (PXE, OMIM 264800) is a rare autosomal recessive disorder with ectopic mineralization and fragmentation of elastin fibers. It is caused by mutations of the ABCC6 gene that leads to decreased serum levels of inorganic pyrophosphate (PPi) anti-mineralization factor. The occurrence of severe complications among PXE patients highlights the importance of early diagnosis so that prompt multidisciplinary care can be provided to patients. We aimed to examine dermal connective tissue with nonlinear optical (NLO) techniques, as collagen emits second-harmonic generation (SHG) signal, while elastin can be excited by two-photon excitation fluorescence (TPF). We performed molecular genetic analysis, ophthalmological and cardiovascular assessment, plasma PPi measurement, conventional histopathological examination, and ex vivo SHG and TPF imaging in five patients with PXE and five age- and gender-matched healthy controls. Pathological mutations including one new variant were found in the ABCC6 gene in all PXE patients and their plasma PPi level was significantly lower compared with controls. Degradation and mineralization of elastin fibers and extensive calcium deposition in the mid-dermis was visualized and quantified together with the alterations of the collagen structure in PXE. Our data suggests that NLO provides high-resolution imaging of the specific histopathological features of PXE-affected skin. In vivo NLO may be a promising tool in the assessment of PXE, promoting early diagnosis and follow-up.

Összefoglaló. A pseudoxanthoma elasticum (PXE, OMIM # 264800) egy autoszomális recesszív módon öröklődő multiszisztémás érintettséggel járó kórkép, melynek háttérében az ABCC6 gén mutációi állnak. A tünetek kialakulásának oka az ektópiás mineralizáció. Kalcium-só kristályok rakódnak le elsősorban a bőrben, a szem Bruch-membránjában és az erek endotheliumában, így a bőrelváltozások mellett a látás csökkenése és cardiovascularis eltérések is jelentkezhetnek. A klinikai tünetek változó súlyosságúak lehetnek, heterogén megjelenésűek. A betegek fenotípusának azonosítása, valamint gondozása multidiszciplináris feladat, bőrgyógyász, szemész, kardiológus és klinikai genetikus együttműködésén alapul. Célunk, hogy bemutassuk a betegségben előforduló tüneteket, melyek ismerete megkönnyíti a kórkép felismerését, illetve hogy felhívjuk a figyelmet a korai diagnózis fontosságára és ismertessük a korszerű diagnosztikai módszereket. A súlyos szisztémás tünetek kialakulása miatt rendkívüli jelentőséggel bír a társszakmák együttműködése, hogy a korai diagnózis által időben megfelelő gondozásban és terápiában részesülhessenek a betegek. Orv Hetil. 2022; 163(18): 702–711. Summary. Pseudoxanthoma elasticum (PXE, OMIM # 264800) is an autosomal recessive, multisystemic disorder, associated with mutations of the ABCC6 gene. Ectopic mineralization is in the background of the clinical manifestations of the disease. Calcium-salt crystals are deposited primarily in the skin, in the Bruch membrane of the eyes, and in the vascular endothelium. Thus, in addition to the skin lesions, visual impairment and cardiovascular involvement also occur. Clinical symptoms show varying severity and display heterogeneous appearance. The identification of the phenotype and care of the patients require a multidisciplinary perspective based on the collaboration of a dermatologist, ophthalmologist, cardiologist, and clinical geneticist. The aim of our work is to describe the development of symptoms of the disease, in order to facilitate the diagnosis. In addition, we aim to draw attention to the importance of early diagnosis of pseudoxanthoma elasticum, and to present modern diagnostic methods. Considering the development of severe systemic complications, the early diagnosis with the collaboration between related specialists is crucial to provide optimal clinical care and management of the patients. Orv Hetil. 2022; 163(18): 702–711.

Determining the epigenetic program that generates unique cell types in the kidney is critical for understanding cell-type heterogeneity during tissue homeostasis and injury response. Here, we profile open chromatin and gene expression in developing and adult mouse kidneys at single cell resolution. We show critical reliance of gene expression on distal regulatory elements (enhancers). We reveal key cell type-specific transcription factors and major gene-regulatory circuits for kidney cells. Dynamic chromatin and expression changes during nephron progenitor differentiation demonstrates that podocyte commitment occurs early and is associated with sustained Foxl1 expression. Renal tubule cells follow a more complex differentiation, where Hfn4a is associated with proximal and Tfap2b with distal fate. Mapping single nucleotide variants associated with human kidney disease implicates critical cell types, developmental stages, genes, and regulatory mechanisms. The single cell multi-omics atlas reveals key chromatin remodeling events and gene expression dynamics associated with kidney development., Epigenetic and transcriptional dynamics are critical for both tissue homeostasis and injury response in the kidney. Leveraging a single cell multiomics atlas of the developing mouse kidney, the authors reveal key events in chromatin regulation and gene expression dynamics during postnatal development.

Determining the epigenetic program that generates unique cell types in the kidney is critical for understanding cell-type heterogeneity during tissue homeostasis and injury response.Here, we profiled open chromatin and gene expression in developing and adult mouse kidneys at single cell resolution. We show critical reliance of gene expression on distal regulatory elements (enhancers). We define key cell type-specific transcription factors and major gene-regulatory circuits for kidney cells. Dynamic chromatin and expression changes during nephron progenitor differentiation demonstrated that podocyte commitment occurs early and is associated with sustainedFoxl1expression. Renal tubule cells followed a more complex differentiation, whereHfn4awas associated with proximal andTfap2bwith distal fate. Mapping single nucleotide variants associated with human kidney disease identified critical cell types, developmental stages, genes, and regulatory mechanisms.We provide a global single cell resolution view of chromatin accessibility of kidney development. The dataset is available via interactive public websites.

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′‐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′‐deoxycytidine without significantly influencing 5mC levels.

Kidney disease is the major cause of morbidity and mortality in patients with diabetes. Poor glycemic control shows the strongest correlation with diabetic kidney disease (DKD) development. A period of poor glycemia increases kidney disease risk even after an extended period of improved glucose control—a phenomenon called metabolic memory. Changes in the epigenome have been proposed to mediate the metabolic memory effect, as epigenome editing enzymes are regulated by substrates of intermediate metabolism and changes in the epigenome can be maintained after cell division. Epigenome-wide association studies (EWAS) have reported differentially methylated cytosines in blood and kidney samples of DKD subjects when compared with controls. Differentially methylated cytosines were enriched on regulatory regions and some correlated with gene expression. Methylation changes predicted the speed of kidney function decline. Site-specific methylome editing tools now can be used to interrogate the functional role of differentially methylated regions. Methylome changes can be detected in blood and kidneys of patients with DKD. Methylation changes can predict future kidney function changes. Future studies shall determine their role in disease development.

Heritable ectopic mineralization disorders represent a phenotypically diverse group of conditions characterized by deposition of calcium phosphate complexes in soft connective tissues. The prototype of such conditions is pseudoxanthoma elasticum (PXE), and related conditions with overlapping clinical features include generalized arterial calcification of infancy (GACI) and arterial calcification due to CD73 deficiency (ACDC). Molecular genetic investigations have revealed mutations in the genes physiologically involved in generation of inorganic pyrophosphate (PPi) and phosphate (Pi), and the findings suggest a unifying pathomechanism relating to reduced PPi/Pi ratio. This hypothesis is based on the notion that PPi serves as a powerful inhibitor of mineralization while Pi is a pro-mineralization factor, and an appropriate PPi/Pi ratio is critical for prevention of ectopic mineralization under homeostatic conditions. PXE International, the premiere patient support organization, advocating on behalf of patients and families with PXE, sponsors regular research meetings evaluating the progress in this and related ectopic mineralization disorders. The latest meetings were held in September 2014 in Bethesda, MD and in September 2015 in Budapest, Hungary. This report summarizes the latest progress in research on PXE and related ectopic mineralization disorders, based on presentations and discussions in these meetings, with pharmacologic implications for currently intractable disorders.

Little is known about the molecular mechanism including microRNAs (miRNA) in hypercholesterolemia-induced cardiac dysfunction. We aimed to explore novel hypercholesterolemia-induced pathway alterations in the heart by an unbiased approach based on miRNA omics, target prediction and validation. With miRNA microarray we identified forty-seven upregulated and ten downregulated miRNAs in hypercholesterolemic rat hearts compared to the normocholesterolemic group. Eleven mRNAs with at least 4 interacting upregulated miRNAs were selected by a network theoretical approach, out of which 3 mRNAs (beta-2 adrenergic receptor [Adrb2], calcineurin B type 1 [Ppp3r1] and calcium/calmodulin-dependent serine protein kinase [Cask]) were validated with qRT-PCR and Western blot. In hypercholesterolemic hearts, the expression of Adrb2 mRNA was significantly decreased. ADRB2 and PPP3R1 protein were significantly downregulated in hypercholesterolemic hearts. The direct interaction of Adrb2 with upregulated miRNAs was demonstrated by luciferase reporter assay. Gene ontology analysis revealed that the majority of the predicted mRNA changes may contribute to the hypercholesterolemia-induced cardiac dysfunction. In summary, the present unbiased target prediction approach based on global cardiac miRNA expression profiling revealed for the first time in the literature that both the mRNA and protein product of Adrb2 and PPP3R1 protein are decreased in the hypercholesterolemic heart.

Little is known about the molecular mechanism including microRNAs (miRNA) in hypercholesterolemia-induced cardiac dysfunction. We aimed to explore novel hypercholesterolemia-induced pathway alterations in the heart by an unbiased approach based on miRNA omics, target prediction and validation. With miRNA microarray we identified forty-seven upregulated and ten downregulated miRNAs in hypercholesterolemic rat hearts compared to the normocholesterolemic group. Eleven mRNAs with at least 4 interacting upregulated miRNAs were selected by a network theoretical approach, out of which 3 mRNAs (beta-2 adrenergic receptor [Adrb2], calcineurin B type 1 [Ppp3r1] and calcium/calmodulin-dependent serine protein kinase [Cask]) were validated with qRT-PCR and Western blot. In hypercholesterolemic hearts, the expression of Adrb2 mRNA was significantly decreased. ADRB2 and PPP3R1 protein were significantly downregulated in hypercholesterolemic hearts. The direct interaction of Adrb2 with upregulated miRNAs was demonstrated by luciferase reporter assay. Gene ontology analysis revealed that the majority of the predicted mRNA changes may contribute to the hypercholesterolemia-induced cardiac dysfunction. In summary, the present unbiased target prediction approach based on global cardiac miRNA expression profiling revealed for the first time in the literature that both the mRNA and protein product of Adrb2 and PPP3R1 protein are decreased in the hypercholesterolemic heart.

A fascinating aspect of sexual dimorphism in various animal species is that the two sexes differ substantially in lifespan. In humans, for example, women's life expectancy exceeds that of men by 3-7 years. Whether this trait can be attributed to dissimilar lifestyles or genetic (regulatory) factors remains to be elucidated. Herein, we demonstrate that in the nematode Caenorhabditis elegans, the significantly longer lifespan of hermaphrodites-which are essentially females capable of sperm production-over males is established by TRA-1, the terminal effector of the sex-determination pathway. This transcription factor directly controls the expression of daf-16/FOXO, which functions as a major target of insulin/IGF-1 signaling (IIS) and key modulator of aging across diverse animal phyla. TRA-1 extends hermaphrodite lifespan through promoting daf-16 activity. Furthermore, TRA-1 also influences reproductive growth in a DAF-16-dependent manner. Thus, the sex-determination machinery is an important regulator of IIS in this organism. These findings provide a mechanistic insight into how longevity and development are specified unequally in the two genders. As TRA-1 is orthologous to mammalian GLI (glioma-associated) proteins, a similar sex-specific mechanism may also operate in humans to determine lifespan. © 2018 The Anatomical Society and John Wiley & Sons Ltd.

Background Tourette Syndrome (TS) is a neurodevelopmental disorder characterized by motor and vocal tics. The cause of TS remains elusive but dopamine (DA) has a central role within the cortico-striatal pathway. TS has high heritability and a complex genetic background, but environmental factors also play an important role in the development of the disorder. These factors often operate via epigenetic mechanisms (i.e. covalent chromatin modifications). Methods We used a rodent tic model to test the epigenetic alterations in the striatum. Juvenile male Wistar Kyoto rats were injected with 6-OH-dopamine in the left medial forebrain bundle resulting in degeneration of nigrostriatal dopaminergic neurons. Chronic application of L-DOPA after consolidation of the lesion leads to motor tics due to the striatal hypersensitivity to DA. We studied genome-wide DNA methylation by Reduced Representation Bisulfite Sequencing technique (RRBS) in 3 groups: animals undergoing lesion only, animals treated with L-DOPA after the lesion and animals co-treated with L-DOPA and riluzole (medication used in TS) after the lesion. Results Surprisingly, by investigating the striata we detected only a few DNA methylation changes between the lesioned and control sides of the brain in each group. However, when we compared the control or the lesioned striata of the three groups of animals we more abundant methylation differences. L-DOPA treatment brought about statistically significant DNA hypermethylation at over 1000 CpG sites and hypomethylation at around 300 sites genome-wide. Several dozens of genes were identified in the vicinity of hypermethylated sites, while considerably less were hypomethylated. Some of the relevant hits include genes coding for neurotransmitter transporters, such as SLC6A2 (Norepinephrine transporter), Cacna1h (Calcium channel alpha 1 h subunit), Tnfrsf8 (TNF receptor superfamily member 8), HAT1 (histone acetyltransferase 1), plus several microRNA genes. Discussion This project is a methylome study on a Tourette Syndrome animal model that may provide novel candidate genes and a better understanding of the molecular mechanisms behind the pathophysiology of TS.

ATP binding cassette type-C (ABCC) transporters move molecules across cell membranes upon hydrolysis of ATP; however, their coupling of ATP hydrolysis to substrate transport remains elusive. Drosophila multidrug resistance-associated protein (DMRP) is the functional ortholog of human long ABCC transporters, with similar substrate and inhibitor specificity, but higher activity. Exploiting its high activity, we kinetically dissected the catalytic mechanism of DMRP by using E2-d-glucuronide (E2G), the physiologic substrate of human ABCC. We examined the DMRP-mediated interdependence of ATP and E2G in biochemical assays. We observed E2G-dependent ATPase activity to be biphasic at subsaturating ATP concentrations, which implies at least 2 E2G binding sites on DMRP. Furthermore, transport measurements indicated strong nonreciprocal cooperativity between ATP and E2G. In addition to confirming these findings, our kinetic modeling with the Complex Pathway Simulator indicated a 10-fold decrease in the E2G-mediated activation of ATP hydrolysis upon saturation of the second E2G binding site. Surprisingly, the binding of the second E2G allowed for substrate transport with a constant rate, which tightly coupled ATP hydrolysis to transport. In summary, we show that the second E2G binding-similar to human ABCC2-allosterically stimulates transport activity of DMRP. Our data suggest that this is achieved by a significant increase in the coupling of ATP hydrolysis to transport.-Karasik, A., Ledwitch, K. V., Aranyi, T., Varadi, A., Roberts, A., Szeri, F. Boosted coupling of ATP hydrolysis to substrate transport upon cooperative estradiol-17-β-D-glucuronide binding in a Drosophila ATP binding cassette type-C transporter.

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.

Summary Cardiomyocytes from human embryonic stem cells (hESC-CMs) and induced pluripotent stem cells (hiPSC-CMs) represent new models for drug discovery. Although hypertrophy is a high-priority target, we found that hiPSC-CMs were systematically unresponsive to hypertrophic signals such as the α-adrenoceptor (αAR) agonist phenylephrine (PE) compared to hESC-CMs. We investigated signaling at multiple levels to understand the underlying mechanism of this differential responsiveness. The expression of the normal α1AR gene, ADRA1A, was reversibly silenced during differentiation, accompanied by ADRA1B upregulation in either cell type. ADRA1B signaling was intact in hESC-CMs, but not in hiPSC-CMs. We observed an increased tonic activity of inhibitory kinase pathways in hiPSC-CMs, and inhibition of antihypertrophic kinases revealed hypertrophic increases. There is tonic suppression of cell growth in hiPSC-CMs, but not hESC-CMs, limiting their use in investigation of hypertrophic signaling. These data raise questions regarding the hiPSC-CM as a valid model for certain aspects of cardiac disease., Highlights • Human iPSC-CMs are unresponsive to α-adrenergic hypertrophic signals • Silenced ADRA1A is accompanied by ADRA1B upregulation during differentiation • ADRA1B signal supports hypertrophy in hESC-CMs but is inhibited in hiPSC-CMs • Similar phenotype of hESC-CMs and hiPSC-CMs may mask differences in signaling, In this article, Földes and colleagues show that hiPSC-derived cardiomyocytes are relatively unresponsive to α-adrenergic hypertrophic signals compared to hESC cardiomyocytes. The main difference in hiPSC-CMs that accounts for the defective response is the suppression of growth by tonic antihypertrophic pathways. Superficial similarities in phenotype between cardiomyocytes derived from hESCs or hiPSCs may mask complex differences in signaling.

Background Lentiviral vectors (LV) are widely used for various gene transfer or gene therapy applications. The effects of LV on target cells are expected to be limited to gene delivery. Yet, human hematopoietic CD34+ cells respond to functional LVs as well as several types of non-integrating LVs by genome-wide DNA methylation changes. Results A new algorithm for the analysis of 450K Illumina data showed that these changes were marked by de novo methylation. The same 4126 cytosines located in islands corresponding to 1059 genes were systematically methylated. This effect required cellular entry of the viral particle in the cells but not the genomic integration of the vector cassette. Some LV preparations induced only mild sporadic changes while others had strong effects suggesting that LV batch heterogeneity may be related to the extent of the epigenetic response. Conclusion These findings identify a previously uncharacterized but consistent cellular response to viral components and provide a novel example of environmentally modified epigenome. Electronic supplementary material The online version of this article (doi:10.1186/s13072-016-0077-1) contains supplementary material, which is available to authorized users.

Gilles de la Tourette Syndrome (TS) is a neurodevelopmental disorder marked by the appearance of multiple involuntary motor and vocal tics. TS presents high comorbidity rates with other disorders such as attention deficit hyperactivity disorder (ADHD) and obsessive compulsive disorder (OCD). TS is highly heritable and has a complex polygenic background. However, environmental factors also play a role in the manifestation of symptoms. Different epigenetic mechanisms may represent the link between these two causalities. Epigenetic regulation has been shown to have an impact in the development of many neuropsychiatric disorders, however very little is known about its effects on Tourette Syndrome. This review provides a summary of the recent findings in the genetic background of TS, followed by an overview on different epigenetic mechanisms, such as DNA methylation, histone modifications and non-coding RNAs in the regulation of gene expression. Epigenetic studies in other neurological and psychiatric disorders are discussed along with the TS-related epigenetic findings available in the literature to date. Moreover, we are proposing that some general epigenetic mechanisms seen in other neuropsychiatric disorders may also play a role in the pathogenesis of TS.

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.

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.

Vitamin K is a cofactor required for gamma-glutamyl carboxylation of several proteins regulating blood clotting, bone formation and soft tissue mineralization. Vitamin K3 is an important intermediate during conversion of the dietary vitamin K1 to the most abundant vitamin K2 form. It has been suggested that ABCC6 may have a role in transporting vitamin K or its derivatives from the liver to the periphery. This activity is missing in pseudoxanthoma elasticum, a genetic disorder caused by mutations in ABCC6 characterized by abnormal soft tissue mineralization. Here we examined the efflux of the glutathione conjugate of vitamin K3 (VK3GS) from the liver in wild type and Abcc6(-/-) mice, and in transport assays in vitro. We found in liver perfusion experiments that VK3GS is secreted into the inferior vena cava, but we observed no significant difference between wild type and Abcc6(-/-) animals. We overexpressed the human ABCC6 transporter in Sf9 insect and MDCKII cells and assayed its vitamin K3-conjugate transport activity in vitro. We found no measurable transport of VK3GS by ABCC6, whereas ABCC1 transported this compound at high rate in these assays. These results show that VK3GS is not the essential metabolite transported by ABCC6 from the liver and preventing the symptoms of pseudoxanthoma elasticum.

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.

Abnormalities in DNA methylation of CpG islands that play a role in gene regulation affect gene expression and hence play a role in disease, including cancer. Bisulfite-based DNA methylation analysis methods such as methylation-specific PCR (MSP) and bisulfite sequencing (BiSeq) are most commonly used to study gene-specific DNA methylation. Assessing specificity and visualizing the position of PCR primers in their genomic context is a laborious and tedious task, primarily due to the sequence changes induced during the bisulfite conversion. For this purpose, we developed methGraph, a web application for easy, fast and flexible visualization and accurate in silico quality evaluation of PCR-based methylation assays. The visualization process starts by submitting PCR primer sequences for specificity assessment and mapping on the genome using the BiSearch ePCR primer-search algorithm. The next step comprises the selection of relevant UCSC genome annotation tracks for display in the final graph. A custom track showing all individual CpG dinucleotides, representing their distribution in the CpG island is also provided. Finally, methGraph creates a BED file that is automatically uploaded to the UCSC genome browser, after which the resulting image files are extracted and made available for visualization and download. The generated high-quality figures can easily be customized and exported for use in publications or presentations. methGraph is available at http://mellfire.ugent.be/methgraph/.

Loss-of-function mutations of ABCC6 cause pseudoxanthoma elasticum (PXE). This Mendelian disorder is characterized by elastic calcification leading to dermal, ocular, and cardiovascular symptoms like coronary artery disease (CAD) and stroke. Although PXE is a recessive disease, microscopic dermal lesions, serum alterations, and higher anecdotal incidence of stroke or CAD among carriers were reported. Here we investigated the association of the c.3421C>T loss-of-function mutation of ABCC6 and CAD and stroke. A previous study demonstrated the association of the c.3421C>T mutation with CAD; however, the frequency found in the control population was unexpectedly high, contradicting, thus, the prevalence of PXE. In the present study, genomic DNA from 749 healthy blood donors was used as control, while 363 and 361 patients suffering from stroke and CAD were investigated, respectively. One carrier was found in our control group, which is in accordance with the reported prevalence of this mutation. No significant association was found between carrier status and stroke in our cohort. In contrast, a significant association of carrier status and CAD was observed (5/361 carriers: p = 0.016, odds ratio [OR] = 10.5). We propose that carriers of ABCC6 loss-of-function mutations benefit from CAD prevention therapy.

The methylation status of CpG dinucleotides located in or near regulatory elements affects gene expression. The CpG-rich sequence located outside the 5' promoter region of the human Tyrosine Hydroxylase (TH) gene appears to influence the functional effect of the adjacent intronic HUMTH01 microsatellite. In order to identify new regulatory elements in this region acting on gene expression, the methylation profile of the TH CpG island was investigated using the bisulfite sequencing method. The overall methylation level of this region is correlated to TH-expressing and non-expressing status in cell lines and DNA demethylation treatment with 5-azacytidine increased TH expression. Moreover, in a homogeneous background of methylated CpGs, a single CpG in the first exon of the gene is constantly either unmethylated or methylated in, respectively, TH-expressing or non-expressing cell lines, tissues and single cells. Further analysis ascertained that this CpG is contained in a sequence characterized by putative binding sites for the AP2, Sp1 and KAISO factors. Characterization of this sequence shows that these factors specifically bind their respective sites. Finally, the binding of KAISO, a transcriptional repressor, is conditioned by the methylation of this sequence, which may, thus, participate in the regulation of TH gene expression according to its methylation pattern.

Hepatocyte nuclear factor 4 alpha (HNF4α) nuclear receptor is a master regulator of hepatocyte development, nutrient transport and metabolism. HNF4α is regulated both at the transcriptional and post-transcriptional levels by different mechanisms. Several kinases (PKA, PKC, AMPK) were shown to phosphorylate and decrease the activity of HNF4α. Activation of the ERK1/2 signalling pathway, inducing proliferation and survival, inhibits the expression of HNF4α. However, based on our previous results we hypothesized that HNF4α is also regulated at the post-transcriptional level by ERK1/2. Here we show that ERK1/2 is capable of directly phosphorylating HNF4α in vitro at several phosphorylation sites including residues previously shown to be targeted by other kinases, as well. Furthermore, we also demonstrate that phosphorylation of HNF4α leads to a reduced trans-activational capacity of the nuclear receptor in luciferase reporter gene assay. We confirm the functional relevance of these findings by demonstrating with ChIP-qPCR experiments that 30-minute activation of ERK1/2 leads to reduced chromatin binding of HNF4α. Accordingly, we have observed decreasing but not disappearing binding of HNF4α to the target genes. In addition, 24-hour activation of the pathway further decreased HNF4α chromatin binding to specific loci in ChIP-qPCR experiments, which confirms the previous reports on the decreased expression of the HNF4a gene due to ERK1/2 activation. Our data suggest that the ERK1/2 pathway plays an important role in the regulation of HNF4α-dependent hepatic gene expression.

Background One of the main epigenetic mechanisms is DNA methylation. It mostly occurs in CpG islands in the promoters by addition of a methyl group to a cytosine that may inhibit gene expression. It is known that many developmental disorders are characterized by alterations in epigenetic mechanisms. Moreover, studies in animal models of neuropsychiatric disorders have shown that epigenetics may play an important role in pathogenesis and therapy. The aim of this study is to investigate the effect of drugs used in different psychiatric diseases including Tourette Syndrome (TS) on DNA methylation to find epigenetic targets that may influence the efficacy of treatment. Methods Treatment: Wistar Kyoto rats were chronically treated from post-natal day (PND) 35 to PND50 by intraperitoneal injection of either aripiprazole or riluzole or vehicle. Metabolite changes during the treatment were quantified by magnetic resonance spectroscopy in the striatum and prefrontal cortex. Samples: DNA isolated from striatum and prefrontal cortex was used for methylation studies. Reduced Representation Bisulfite Sequencing (RRBS): RRBS is a high-throughput technique used to analyze genome-wide methylation profiles at the single nucleotide level. It provides information of the most relevant genomic regions which are likely to influence gene expression. We analyzed striatum and prefrontal cortex of 2 rats in each group treated with aripiprazole (a dopaminergic neuroleptic) or riluzole (a glutamatergic anxiolytic) or vehicle in order to identify DNA methylation changes due to these drug treatments. Mass-Spectrometry: The analysis was performed by LC-MS/MS on striatum and prefrontal cortex in order to quantify the amount of 5-methyl-Cytosine (mC) and 5-HydroxyMethylCytosine (5hmC) after drug treatment. Results Mass-Spectrometry: LC-MS/MS showed significant differences in 5hmC levels in prefrontal cortex after treatment with aripiprazole and riluzole. The same trend was also observed for mC levels. We found no differences in methylation or hydroxy-methylation levels in striatal samples. RRBS: we observed differences in overall methylation levels between treatment using the two drugs and the vehicle. We identified differentially methylated CpG sites (DMS) and characterized their distribution across the genome (eg. promoter, intron, exon, UTR, etc.). Treatment with riluzole led to identification of 156 DMS in striatum and 346 DMS in prefrontal cortex. Treatment with aripiprazole yielded 117 DMS in striatum and 466 DMS in prefrontal cortex. The identified CpG sites were annotated, a candidate gene list was created and pathway analysis was also performed on these genes. Interestingly, most of the changes occurred in intergenic regions far from TSS suggesting that enhancers are the regions most prone to undergo methylation changes. Moreover, several genes (eg. OXTR, DRD2, etc.) have been associated with brain disorders or functions. Discussion The known functions of implicated genes suggest that some of the observed epigenetic changes might underlie the amelioration of symptoms by these drugs and some may account for certain adverse effects. Data on differential DNA methylation will be compared with imaging data on the respective brain regions. The results give insights into the mechanism of action of aripiprazole and riluzole as well as the side effects, not just in TS, but also in a broader context regarding these psychiatric medications.

Predicting the functional consequence of a given amino acid replacement, that is, the difference between the wild type protein and its nonsynonymous single nucleotide polymorph variants (nsSNPs) is a great challenge. This is especially true in the case of large membrane proteins, like ABC transporters. Kelly et al. has published such an ambitious study in a recent issue of Protein Science focusing on nsSNP in structurally conserved segments within the nucleotide binding domains (NBDs) of ABC transporters supposed to be involved in interdomain communication.1 With the aid of structural rationalization the authors predicted the impact of 40 nsSNPs found in seven clinically important human ABC transporters using a bivalent scoring: disease or neutral. We have performed a deep search of the available literature to find published data on the 40 nsSNPs to delineate them with the predictions. We have incorporated only data from original peer-reviewed publications into our dataset, and our hits were checked in various gene-specific databases too [BioBase and ABCC6 Database]. Based on the available information, we categorized the published data on a given nsSNP as (i) “disease-associated” when the publication clearly demonstrates that the variant segregates with the disease but is absent from at least 200 alleles of unrelated, nonaffected individuals; or (ii) in vitro experimental evidence when the specific variant was heterologously expressed and its (transport) function and/or folding-stability was investigated (Table I). We considered functional differences only if the level of expression of the given nsSNP variant was comparable to the wild type expressed in the same system. Our results, in comparison with the predictions of Kelly et al. are summarized in Table I. The left five columns of the table is taken from the publication of Kelly et al. On the right we list our findings and give the related primary reference. In several cases more than one independent studies were found showing the same result. To keep the list of references short, we indicated only one reference, if more publications were found we added +n (where n is the number of additional independent publications). Table I Predicted and Published Phenotype of nsSNPs in Human ABC Transporters We found the published data on 19 of the 40 ABC nsSNPs. Of these, 16 nsSNPs had been tested in in vitro experiments and in seven examples the given nsSNP resulted in impaired function and/or folding. In one case the published nsSNP generates a new splice site (exon skipping), what affects the mRNA maturation therefore the function or folding of the protein with the given nsSNP can not be concluded (ABCB11, E1186K). One of the nsSNPs was characterized solely by its association to a genetic disease (ABCC6, A1291T associated to pseudoxanthoma elasticum). One additional nsSNP was found as homozygous nsSNP in several healthy individuals therefore it was categorized as neutral, on the basis of this human genetic data (ABCC6, R1268Q). There are two nsSNPs with both published genetic association and in vitro experimental characterization in our data set (ABCB11 V444A associated to intrahepatic cholestasis of pregnancy; ABCG2, Q141K associated to gout). Our conclusions on the nsSNPs are listed in column “Published phenotype” of Table I.; for the sake of simplicity we kept the bivalent terminology of Kelly et al.: disease or neutral. Comparing the predicted phenotype of the Kelly's paper with the phenotype extracted from the published data reveals misprediction in roughly half of the cases (10/19) thus demonstrating the inherent difficulties of rationalizing and predicting the functional impact of snSNPs.

Introduction Le pseudoxanthome elastique (PXE) est une affection hereditaire du tissu conjonctif caracterisee par des manifestations cutanees, retiniennes et cardiovasculaires (CV). Aucune etude n’a precise a ce jour l’existence d’une possible correlation entre la severite des differentes atteintes. Pourtant cet element est important a considerer en pratique clinique pour etablir le suivi des patients. A partir des donnees d’une importante cohorte de patients PXE, nous avons recherche une association entre ces differentes atteintes. Materiel et methodes La totalite des dossiers des patients enregistres dans une base de donnees (2008–2015) a ete revue retrospectivement. Une atteinte cutanee severe etait definie par la presence de lesions de PXE sur au moins 8 zones bastions (differentes faces du cou et grands plis) et/ou debordant les zones bastions et/ou en zones non bastions (paroi abdominale, muqueuses etc.). Une atteinte ophtalmologique severe etait definie par une cecite uni- ou bilaterale liee a une complication specifique du PXE. Une atteinte CV etait definie par la survenue d’un evenement CV (infarctus du myocarde et/ou AVC et/ou revascularisation de membre). Les associations entre les signes cliniques ont ete recherchees par un Chi2 et un test de Fisher. Resultats Au total, 133 patients (89 femmes ; 44 ± 17 ans) ont ete inclus dans l’etude. Quatre-vingt-douze (70 %) sujets avaient une atteinte cutanee severe, 35 (26 %) avaient une cecite uni- ou bilaterale et 18 (14 %) avaient eu un evenement CV. Une association significative etait observee entre la severite cutanee et les evenements CV (p = 0,013) et etait dependante de l’âge. Il existait une association entre atteinte cutanee severe et cecite de pres (p = 0,04). Il existait une association (p = 0,022) entre evenements CV et cecite, independante de l’âge. Conclusion Cette analyse retrospective issue d’une grande cohorte de patients PXE montre une association entre l’etendue des lesions cutanees et des evenements CV d’une part, et d’une cecite de pres d’autre part. Le caractere transversal de l’etude ne permet pas d’affirmer la valeur predictive d’une atteinte cutanee severe et precoce dans l’apparition d’evenements CV ou la cecite. Cependant, il parait raisonnable en pratique dermatologique de considerer qu’un patient PXE ayant une atteinte dermatologique etendue, mais asymptomatique sur les plans CV et ophtalmologique, beneficie d’un bilan initial puis d’un suivi cardiovasculaire et ophtalmologique reguliers pour depister une insuffisance coronarienne, des lesions silencieuses d’AVC, une arteriopathie des membres inferieurs et des complications des stries angioides. La nature de l’association reste a determiner sur le plan physiopathologique.

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.

International audience; 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.

Loss-of-function mutations in ABCC6 can cause chronic or acute forms of dystrophic mineralization described in disease models such as pseudoxanthoma elasticum (OMIM 26480) in human and dystrophic cardiac calcification in mice. The ABCC6 protein is a large membrane-embedded organic anion transporter primarily found in the plasma membrane of hepatocytes. We have established a complex experimental strategy to determine the structural and functional consequences of disease-causing mutations in the human ABCC6. The major aim of our study was to identify mutants with preserved transport activity but failure in intracellular targeting. Five missense mutations were investigated: R1138Q, V1298F, R1314W, G1321S and R1339C. Using in vitro assays, we have identified two variants; R1138Q and R1314W that retained significant transport activity. All mutants were transiently expressed in vivo, in mouse liver via hydrodynamic tail vein injections. The inactive V1298F was the only mutant that showed normal cellular localization in liver hepatocytes while the other mutants showed mostly intracellular accumulation indicating abnormal trafficking. As both R1138Q and R1314W displayed endoplasmic reticulum localization, we tested whether 4-phenylbutyrate (4-PBA), a drug approved for clinical use, could restore their intracellular trafficking to the plasma membrane in MDCKII and mouse liver. The cellular localization of R1314W was significantly improved by 4-PBA treatment, thus potentially rescuing its physiological function. Our work demonstrates the feasibility of the in vivo rescue of cellular maturation of some ABCC6 mutants in physiological conditions very similar to the biology of the fully differentiated human liver and could have future human therapeutic application.

The ABCC6 gene encodes an organic anion transporter protein, ABCC6/MRP6. Mutations in the gene cause a rare, recessive genetic disease, pseudoxanthoma elasticum, while the loss of one ABCC6 allele is a genetic risk factor in coronary artery disease. We review here the information available on gene structure, evolution as well as the present knowledge on its transcriptional regulation. We give a detailed description of the characteristics of the protein, and analyze the relationship between the distributions of missense disease – causing mutations in the predicted threedimensional structure of the transporter, which suggests functional importance of the domain-domain interactions. Though neither the physiological function of the protein nor its role in the pathobiology of the diseases are known, a current hypothesis that ABCC6 may be involved in the efflux of one form of Vitamin K from the liver is discussed. Finally, we analyze potential strategies how the gene can be targeted on the transcriptional level to increase protein expression in order to compensate for reduced activity. In addition, pharmacologic correction of trafficking-defect mutants or suppression of stop codon mutations as potential future therapeutic interventions are also reviewed.

The completion of the Human Genome Project has brought the understanding that our genome contains an unexpectedly large proportion of segmental duplications. This poses the challenge of elucidating the consequences of recent duplications on physiology. We have conducted an in-depth study of a subset of segmental duplications on chromosome 16. We focused on PKD1 and ABCC6 duplications because mutations affecting these genes are responsible for the Mendelian disorders autosomal dominant polycystic kidney disease and pseudoxanthoma elasticum, respectively. We establish that duplications of PKD1 and ABCC6 are associated to low-copy repeat 16a and show that such duplications have occurred several times independently in different primate species. We demonstrate that partial duplication of PKD1 and ABCC6 has numerous consequences: the pseudogenes give rise to new transcripts and mediate gene conversion, which not only results in disease-causing mutations but also serves as a reservoir for sequence variation. The duplicated segments are also involved in submicroscopic and microscopic genomic rearrangements, contributing to structural variation in human and chromosomal break points in the gibbon. In conclusion, our data shed light on the recent and ongoing evolution of chromosome 16 mediated by segmental duplication and deepen our understanding of the history of two Mendelian disorder genes.

The design of adequate primers for polymerase chain reaction (PCR) is sometimes a difficult task. This is the case when either the target sequence harbors unusual nucleotide motifs or the template is complex. Unusual nucleotide motifs can be repeat elements, whereas complex templates are targets for mispriming and alternative amplification products. Such examples are GC-rich native or bisulfite-treated genomic DNA sequences. Bisulfite treatment leads to the specific conversion of non-methylated cytosines to uracyls. This is the key step of bisulfite genomic sequencing, widely used to determine DNA methylation of a sequence. Here, we describe BiSearch Web server (http://bisearch.enzim.hu), a primer design software created for designing primers to amplify such target sequences. Furthermore, we developed a unique post-design primer analysis module, to carry out genome wide searches to identify genomic mispriming sites and to test by electronic (in silico) PCR (ePCR) for alternative PCR products. This option is currently available on four native or bisulfite-treated mammalian genomes.

The design of adequate primers for polymerase chain reaction (PCR) is sometimes a difficult task. This is the case when either the target sequence harbors unusual nucleotide motifs or the template is complex. Unusual nucleotide motifs can be repeat elements, whereas complex templates are targets for mispriming and alternative amplification products. Such examples are GC-rich native or bisulfite-treated genomic DNA sequences. Bisulfite treatment leads to the specific conversion of non-methylated cytosines to uracyls. This is the key step of bisulfite genomic sequencing, widely used to determine DNA methylation of a sequence. Here, we describe BiSearch Web server (http://bisearch.enzim.hu), a primer design software created for designing primers to amplify such target sequences. Furthermore, we developed a unique post-design primer analysis module, to carry out genome wide searches to identify genomic mispriming sites and to test by electronic (in silico) PCR (ePCR) for alternative PCR products. This option is currently available on four native or bisulfite-treated mammalian genomes.

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.

Bisulfite genomic sequencing is the most widely used technique to analyze the 5-methylation of cytosines, the prevalent covalent DNA modification in mammals. The process is based on the selective transformation of unmethylated cytosines to uridines. Then, the investigated genomic regions are PCR amplified, subcloned and sequenced. During sequencing, the initially unmethylated cytosines are detected as thymines. The efficacy of bisulfite PCR is generally low; mispriming and non-specific amplification often occurs due to the T richness of the target sequences. In order to ameliorate the efficiency of PCR, we developed a new primer-design software called BiSearch, available on the World Wide Web. It has the unique property of analyzing the primer pairs for mispriming sites on the bisulfite-treated genome and determines potential non-specific amplification products with a new search algorithm. The options of primer-design and analysis for mispriming sites can be used sequentially or separately, both on bisulfite-treated and untreated sequences. In silico and in vitro tests of the software suggest that new PCR strategies may increase the efficiency of the amplification.

The mouse tyrosine hydroxylase (Th) gene is located in an evolutionarily conserved imprinted gene cluster on distal chromosome 7. It is associated with a CpG island that spans the promoter of the gene. Using a bisulfite sequencing method we show that the Th promoter is fully methylated in both male and female mouse germ cells and in human spermatozoa, suggesting that it belongs to the newly identified category of CpG islands, the similarly methylated regions (SMRs). Contrary to other tissue-specific gene sequences, the mouse Th promoter escapes the initial wave of genome demethylation during the first few cell cycles, but becomes demethylated between the morula and the blastocyst stages. This unusual methylation ontogeny may be a characteristic of the SMRs and/or related to the localization of the Th gene in an imprinted gene cluster.