The novel imprinted carboxypeptidase A4 gene ( CPA4) in the 7q32 imprinting domain

By a search for novel human imprinted genes in the vicinity of the imprinted gene MEST, at chromosome 7q32, we identified the carboxypeptidase A4 gene ( CPA4) in a gene cluster of the carboxypeptidase family, 200 kb centromeric to MEST. Because CPA4 was originally identified as a protein induced in a prostate cancer cell line (PC-3) by histone deacetylase inhibitors, and was located at the putative prostate cancer-aggressiveness locus at 7q32, we investigated its imprinting status in fetal tissues and in adult benign hypertrophic prostate (BPH). RT-PCR using four intragenic polymorphisms as markers showed that CPA4 was expressed preferentially from the maternal allele in the fetal heart, lung, liver, intestine, kidney, adrenal gland, and spleen, but not in the fetal brain. It was also preferentially expressed in the BPH. These findings support that CPA4 is imprinted and may become a strong candidate gene for prostate cancer-aggressiveness. As a Silver-Russell syndrome (SRS) locus has been proposed to be located to a region near MEST and to be involved in imprinting, CPA4 would have been a candidate gene for SRS. However, analysis of ten SRS patients revealed no mutations in CPA4.     

A narrow segment of maternal uniparental disomy of chromosome 7q31-qter in Silver-Russell syndrome delimits a candidate gene region

Maternal uniparental disomy of chromosome 7 (matUPD7), the inheritance of both chromosomes from only the mother, is observed in approximately 10% of patients with Silver-Russell syndrome (SRS). It has been suggested that at least one imprinted gene that regulates growth and development resides on human chromosome 7. To date, three imprinted genes-PEG1/MEST, gamma2-COP, and GRB10-have been identified on chromosome 7, but their role in the etiology of SRS remains uncertain. In a systematic screening with microsatellite markers, for matUPD7 cases among patients with SRS, we identified a patient who had a small segment of matUPD7 and biparental inheritance of the remainder of chromosome 7. Such a pattern may be explained by somatic recombination in the zygote. The matUPD7 segment at 7q31-qter extends for 35 Mb and includes the imprinted gene cluster of PEG1/MEST and gamma2-COP at 7q32. GRB10 at 7p11.2-p12 is located within a region of biparental inheritance. Although partial UPD has previously been reported for chromosomes 6, 11, 14, and 15, this is the first report of a patient with SRS who has segmental matUPD7. Our findings delimit a candidate imprinted region sufficient to cause SRS.     

Genomic imprinting of IGF2, p57(KIP2) and PEG1/MEST in a marsupial, the tammar wallaby

Genomic imprinting is widespread amongst mammals, but has not yet been found in birds. To gain a broader understanding of the origin and significance of imprinting, we have characterized three genes, from three separate imprinted clusters in eutherian mammals in the developing fetus and placenta of an Australian marsupial, the tammar wallaby Macropus eugenii. Imprinted gene orthologues of human and mouse p57(KIP2), IGF2 and PEG1/MEST genes were isolated. p57(KIP2) did not show stable monoallelic expression suggesting that it is not imprinted in marsupials. In contrast, there was paternal-specific expression of IGF2 in almost all tissues, but the biased paternal expression of IGF2 in the fetal head and placenta, demonstrates the occurrence of tissue-specific imprinting, as occurs in mice and humans. There was also paternal-biased expression of PEG1/MESTalpha. The differentially methylated region (DMR) of the human and mouse PEG1/MEST promoter is absent in the wallaby. These data confirm the existence of common imprinted regions in eutherians and marsupials during development, but suggest that the regulatory mechanisms that control imprinted gene expression differ between these two groups of mammals.     

Differential methylation persists at the mouse Rasgrf1 DMR in tissues displaying monoallelic and biallelic expression

A subset of mammalian genes exhibits genomic imprinting, whereby one parental allele is preferentially expressed. Differential DNA methylation at imprinted loci serves both to mark the parental origin of the alleles and to regulate their expression. In mouse, the imprinted gene Rasgrf1 is associated with a paternally methylated imprinting control region which functions as an enhancer blocker in its unmethylated state. Because Rasgrf1 is imprinted in a tissue-specific manner, we investigated the methylation pattern in monoallelic and biallelic tissues to determine if methylation of this region is required for both imprinted and non-imprinted expression. Our analysis indicates that DNA methylation is restricted to the paternal allele in both monoallelic and biallelic tissues of somatic and extraembryonic lineages. Therefore, methylation serves to mark the paternal Rasgrf1 allele throughout development, but additional factors are required for appropriate tissue-specific regulation of expression at this locus.     

Igf-2r expression regulated by epigenetic modification and the locus of gene imprinting disrupted in cloned cattle

Epigenetic reprogramming has a crucial role in establishing nuclear totipotency in normal development and in cloned animals. Insulin-like growth factor-2 receptor (Igf-2r) is a tissue-specifically and species-dependently imprinted gene, regulated by epigenetic modifications. The diversity of Igf-2r imprinting suggests that the success of animal cloning may be species-dependent. To determine the relation between epigenetic modifications and Igf-2r expression in cattle, and explore whether this gene was correctly imprinted and reprogrammed after nuclear transfer, we quantified Igf-2r mRNA in a cattle cell line after treated with an inhibitor of DNA methylation transferase or an inhibitor of histone deacetylase, and confirmed that DNA methylation and histone acetylation could regulate this gene expression. CpG island searching showed that there is a conservative imprinting control region (ICR) within the second intron of Igf-2r in cattle, analogous to mice and sheep, regulating this gene imprinting. DNA methylation analysis in sperm and blood cells showed that DNA methylation at Igf-2r ICR2 was reprogrammed in normal cattle. The methylation at Igf-2r ICR2 showed significant variation in tissues, such as blood, liver, brain, heart and heart. It suggested that Igf-2r imprinting was tissue-specifically regulated. In cloned cattle, DNA methylation at Igf-2r ICR2 was markedly altered in comparison with normal fetus, while patterns of DNA methylation at Igf-2r 3'-UTR (3-terminal untranslated region) were similar to normal fetus, it indicated that 3'-UTR was not significantly altered by cloning procedures, but DNA methylation at the locus of gene imprinting was disrupted and not completely reprogrammed after nuclear transfer.     

An enhancer element at the Igf2/H19 locus drives gene expression in both imprinted and non-imprinted tissues

The insulin-like growth factor 2 (Igf2) gene encodes a potent growth factor that is expressed in multiple tissues during embryonic development. Expression at this locus is mediated by genomic imprinting. In the developing endodermal tissues, imprinting of Igf2 is mediated by the interaction of a set of enhancers downstream of the linked H19 gene with a differentially methylated domain (DMD) that lies approximately 2-4 kb upstream of H19 that has a boundary or insulator function in the hypomethylated state. In the remainder of tissues that express Igf2 and H19, the cis elements that drive their correct expression and imprinting are not well understood. In addition, enhancers driving expression of Igf2 in the choroid plexus and leptomeninges, tissues where the gene is thought not to be imprinted, have not been isolated. Here we show that biallelic (non-imprinted) expression within the choroid plexus is restricted to the epithelium, and we provide evidence that a conserved intergenic region functions as an enhancer for Igf2 both in tissues where the gene is imprinted, and where Igf2 is biallelically expressed. The presence of an enhancer for imprinted tissues in the intergenic region argues for the existence of imprinting controls distinct from the DMD, which may be provided by differential methylation at sites proximal to Igf2.     

Culture of preimplantation mouse embryos affects fetal development and the expression of imprinted genes

Culture of preimplantation mammalian embryos and cells can influence their subsequent growth and differentiation. Previously, we reported that culture of mouse embryonic stem cells is associated with deregulation of genomic imprinting and affects the potential for these cells to develop into normal fetuses. The purpose of our current study was to determine whether culture of preimplantation mouse embryos in a chemically defined medium (M16) with or without fetal calf serum (FCS) can affect their subsequent development and imprinted gene expression. Only one third of the blastocysts that had been cultured from two-cell embryos in M16 medium complemented with FCS developed into viable Day 14 fetuses after transfer into recipients. These M16 + FCS fetuses were reduced in weight as compared with controls and M16 fetuses and had decreased expression of the imprinted H19 and insulin-like growth factor 2 genes associated with a gain of DNA methylation at an imprinting control region upstream of H19. They also displayed increased expression of the imprinted gene Grb10. The growth factor receptor binding gene Grb7, in contrast, was strongly reduced in its expression in most of the M16 + FCS fetuses. No alterations were detected for the imprinted gene MEST: Preimplantation culture in the presence of serum can influence the regulation of multiple growth-related imprinted genes, thus leading to aberrant fetal growth and development.