ABSTRACT
Neuronatin (Nnat) is an imprinted gene that is expressed exclusively from the paternal allele while the maternal allele is silent and methylated. The Nnat locus exhibits some unique features compared with other imprinted domains. Unlike the majority of imprinted genes, which are organised in clusters and coordinately regulated, Nnat does not appear to be closely linked to other imprinted genes. Also unusually, Nnat is located within an 8-kb intron of the Bc10 gene, which generates a biallelically expressed, antisense transcript. A similar organisation is conserved at the human NNAT locus on chromosome 20. Nnat expression is first detected at E8.5 in rhombomeres 3 and 5, and subsequently, expression is widespread within postmitotic neuronal tissues. Using modified BAC transgenes, we show that imprinted expression of Nnat at ectopic sites requires, at most, an 80-kb region around the gene. Furthermore, reporter transgenes reveal distinct and dispersed cis-regulatory elements that direct tissue-specific expression and these are predominantly upstream of the region that confers allele-specific expression.
Subject(s)
Chromosomes, Artificial, Bacterial/genetics , Enhancer Elements, Genetic/genetics , Gene Expression Regulation , Genomic Imprinting/genetics , Membrane Proteins/genetics , Nerve Tissue Proteins/genetics , Transgenes/genetics , Alleles , Animals , Choristoma/genetics , Cloning, Molecular , Female , In Situ Hybridization , Introns/genetics , Male , Mice , Mice, Transgenic , Plasmids/genetics , Promoter Regions, Genetic/genetics , RNA, Messenger/analysis , RNA, Messenger/geneticsABSTRACT
The H19 imprinted gene is silenced when paternally inherited and active only when inherited maternally. This is thought to involve a cis-acting control region upstream of H19 that is responsible for regulating a number of functions including DNA methylation, asynchronous replication of parental chromosomes and an insulator. Here we report on the function of a 1.2 kb upstream element in the mouse, which was previously shown to function as a bi-directional silencer in Drosophila. The cre-loxP-mediated targeted deletion of the 1.2 kb region had no effect on the maternal allele. However, there was loss of silencing of the paternal allele in many endodermal and other tissues. The pattern of expression was very similar to the expression pattern conferred by the enhancer elements downstream of H19. We could not detect an effect on the expression of the neighbouring imprinted Igf2 gene, suggesting that the proposed boundary element insulating this gene from the downstream enhancers was unaffected. Despite derepression of the paternal H19 allele, the deletion surprisingly did not affect the differential DNA methylation of the locus, which displayed an appropriate epigenetic switch in the parental germlines. Furthermore, the characteristic asynchronous pattern of DNA replication at H19 was also not disrupted by the deletion, suggesting that the sequences that mediate this were also intact. The silencer is therefore part of a complex cis-regulatory region upstream of the H19 gene and acts specifically to ensure the repression of the paternal allele, without a predominant effect on the epigenetic switch in the germline.
Subject(s)
DNA Methylation , Gene Silencing , Genomic Imprinting , Muscle Proteins/genetics , RNA, Untranslated , Animals , DNA Replication , Female , Gene Deletion , Gene Expression , Gene Targeting , Insulin-Like Growth Factor II/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred CBA , RNA, Long NoncodingABSTRACT
The H19 gene is subject to genomic imprinting because it is methylated and repressed after paternal inheritance and is unmethylated and expressed after maternal inheritance. We recently identified a 1.1-kb control element in the upstream region of the H19 gene that functions as a cis-acting silencer element in Drosophila. Here we investigate the function of this element in mice. We demonstrate that both H19-lacZ and H19-PLAP reporter transgenes can undergo imprinting with repression and hypermethylation after paternal transmission at many integration sites. However, transgenes that were deleted for the 1.1-kb silencer element showed loss of paternal repression, but they did not show marked changes in the paternal methylation of the remaining upstream region. This study demonstrates that the 1.1-kb control element identified in Drosophila is required to silence paternally transmitted H19 minitransgenes in mice.
Subject(s)
Alkaline Phosphatase/genetics , Drosophila/genetics , Genes, Tumor Suppressor , Genomic Imprinting , Isoenzymes/genetics , Muscle Proteins/genetics , RNA, Untranslated , Animals , Chromosome Mapping , DNA Methylation , Female , Gene Library , Genes, Reporter , Genetic Techniques , Male , Mice , Mice, Inbred Strains , Mice, Transgenic , RNA, Long Noncoding , Testis/enzymology , beta-Galactosidase/geneticsABSTRACT
A restricted energy intake in the immediate postnatal period has been found to result in a significant increase in the maximal expression of lactase by enterocytes from 2-week-old pigs. Although villus size was significantly reduced on a low compared with a high food intake, total villus lactase activity was unaffected because of the compensatory increase in lactase expression by individual enterocytes.
