Generation of an iPSC line of a patient with Angelman syndrome due to an imprinting defect.

Angelman syndrome (AS) is a neurodevelopmental disorder with leading symptoms of happy demeanor, intellectual disability, ataxia and seizures. AS can be caused by genetic and epigenetic aberrations, resulting in the absence of functional UBE3A protein in the brain. UBE3A is an imprinted gene, which is, in neurons of the brain, expressed exclusively from maternal chromosome 15. The generated iPSC line was derived from skin fibroblasts of a patient with AS, who, due to an imprinting defect, lacked DNA methylation at the chromosome 15 imprinting center, which controls maternal-specific expression of UBE3A. Resource table.

Corrigendum: Angelman syndrome-derived neurons display late onset of paternal UBE3A silencing.

This corrects the article DOI: 10.1038/srep30792.

Angelman syndrome-derived neurons display late onset of paternal UBE3A silencing.

Genomic imprinting is an epigenetic phenomenon resulting in parent-of-origin-specific gene expression that is regulated by a differentially methylated region. Gene mutations or failures in the imprinting process lead to the development of imprinting disorders, such as Angelman syndrome. The symptoms of Angelman syndrome are caused by the absence of functional UBE3A protein in neurons of the brain. To create a human neuronal model for Angelman syndrome, we reprogrammed dermal fibroblasts of a patient carrying a defined three-base pair deletion in UBE3A into induced pluripotent stem cells (iPSCs). In these iPSCs, both parental alleles are present, distinguishable by the mutation, and express UBE3A. Detailed characterization of these iPSCs demonstrated their pluripotency and exceptional stability of the differentially methylated region regulating imprinted UBE3A expression. We observed strong induction of SNHG14 and silencing of paternal UBE3A expression only late during neuronal differentiation, in vitro. This new Angelman syndrome iPSC line allows to study imprinted gene regulation on both parental alleles and to dissect molecular pathways affected by the absence of UBE3A protein.

A Mouse Model for Imprinting of the Human Retinoblastoma Gene.

The human RB1 gene is imprinted due to integration of the PPP1R26P1 pseudogene into intron 2. PPP1R26P1 harbors the gametic differentially methylated region of the RB1 gene, CpG85, which is methylated in the female germ line. The paternally unmethylated CpG85 acts as promoter for the alternative transcript 2B of RB1, which interferes with expression of full-length RB1 in cis. In mice, PPP1R26P1 is not present in the Rb1 gene and Rb1 is not imprinted. Assuming that the mechanisms responsible for genomic imprinting are conserved, we investigated if imprinting of mouse Rb1 can be induced by transferring human PPP1R26P1 into mouse Rb1. We generated humanized Rb1_PPP1R26P1 knock-in mice that pass human PPP1R26P1 through the mouse germ line. We found that the function of unmethylated CpG85 as promoter for an alternative Rb1 transcript and as cis-repressor of the main Rb1 transcript is maintained in mouse tissues. However, CpG85 is not recognized as a gametic differentially methylated region in the mouse germ line. DNA methylation at CpG85 is acquired only in tissues of neuroectodermal origin, independent of parental transmission of PPP1R26P1. Absence of CpG85 methylation in oocytes and sperm implies a failure of imprint methylation establishment in the germ line. Our results indicate that site-specific integration of a proven human gametic differentially methylated region is not sufficient for acquisition of DNA methylation in the mouse germ line, even if promoter function of the element is maintained. This suggests a considerable dependency of DNA methylation induction on the surrounding sequence. However, our model is suited to determine the cellular function of the alternative Rb1 transcript.

Human PPP1R26P1 functions as cis-repressive element in mouse Rb1.

The human retinoblastoma gene (RB1) is imprinted; the mouse Rb1 gene is not. Imprinted expression of RB1 is due to differential methylation of a CpG island (CpG85), which is located in the pseudogene PPP1R26P1 in intron 2 of RB1. CpG85 serves as promoter for an alternative RB1 transcript, which is expressed from the unmethylated paternal allele only and is thought to suppress expression of the full-length RB1 transcript in cis. PPP1R26P1 contains another CpG island (CpG42), which is biallelically methylated. To determine the influence of PPP1R26P1 on RB1 expression, we generated an in vitro murine embryonic stem cell model by introducing human PPP1R26P1 into mouse Rb1. Next generation bisulfite sequencing of CpG85 and CpG42 revealed differences in their susceptibility to DNA methylation, gaining methylation at a median level of 4% and 18%, respectively. We showed binding of RNA polymerase II at and transcription from the unmethylated CpG85 in PPP1R26P1 and observed reduced expression of full-length Rb1 from the targeted allele. Our results identify human PPP1R26P1 as a cis-repressive element and support a connection between retrotransposition of PPP1R26P1 into human RB1 and the reduced expression of RB1 on the paternal allele.

Hematopoietically expressed homeobox is a target gene of farnesoid X receptor in chenodeoxycholic acid-induced liver hypertrophy.

UNLABELLED: Farnesoid X receptor (FXR/Fxr) is a bile acid-regulated nuclear receptor that promotes hepatic bile acid metabolism, detoxification, and liver regeneration. However, the adaptive pathways under conditions of bile acid stress are not fully elucidated. We found that wild-type but not Fxr knockout mice on diets enriched with chenodeoxycholic acid (CDCA) increase their liver/body weight ratios by 50% due to hepatocellular hypertrophy. Microarray analysis identified Hex (Hematopoietically expressed homeobox), a central transcription factor in vertebrate embryogenesis and liver development, as a novel CDCA- and Fxr-regulated gene. HEX/Hex was also regulated by FXR/Fxr and CDCA in primary mouse hepatocytes and human HepG2 cells. Comparative genomic analysis identified a conserved inverted repeat-1-like DNA sequence within a 300 base pair enhancer element of intron-1 in the human and mouse HEX/Hex gene. A combination of chromatin immunoprecipitation, electromobility shift assay, and transcriptional reporter assays demonstrated that FXR/Fxr binds to this element and mediates HEX/Hex transcriptional activation. CONCLUSION: HEX/Hex is a novel bile acid-induced FXR/Fxr target gene during adaptation of hepatocytes to chronic bile acid exposure.

Differential expression and function of caveolin-1 in human gastric cancer progression.

Caveolin-1 is a scaffold protein of caveolae that acts as a tumor modulator by interacting with cell adhesion molecules and signaling receptors. The role of caveolin-1 in the pathogenesis of gastric cancer (GC) is currently unknown. We show by confocal immunofluorescence microscopy and immunohistochemistry of biopsies from GC patients (n = 41) that the nonneoplastic mucosa expressed caveolin-1 in foveolar epithelial cells and adjacent connective tissue. GC cells of only 3 of 41 (7%) patients expressed caveolin-1 and were all of the intestinal type. Quantitative PCR and Western blotting confirmed that, compared with nonneoplastic tissue, the overall caveolin-1 mRNA was decreased in 14 of 19 (74%) GC patients and protein in 7 of 13 (54%), respectively. Strong caveolin-1 reactivity was found in the nonepithelial compartment (myocytes, fibroblasts, perineural, and endothelial cells) in both tumor-free and GC samples. In a series of human GC cell lines, caveolin-1 expression was low in cells derived from a primary tumor (AGS and SNU-1) but was increased in cell lines originating from distant metastases (MKN-7, MKN-45, NCI-N87, KATO-III, and SNU-5). Ectopic expression of caveolin-1 in AGS cells decreased proliferation but promoted anchorage-independent growth and survival. RNAi-mediated knockdown of endogenous caveolin-1 in MKN-45 cells accelerated cell growth. These data indicate that caveolin-1 exhibits a stage-dependent differential expression and function in GC and may thereby contribute to its pathogenesis.