Allele-specific histone modifications regulate expression of the Dlk1-Gtl2 imprinted domain

Dlk1 and Gtl2 are reciprocally expressed imprinted genes located on mouse chromosome 12. The Dlk1-Gtl2 locus carries three differentially methylated regions (DMRs), which are methylated only on the paternal allele. Of these, the intergenic (IG) DMR, located 12 kb upstream of Gtl2, is required for proper imprinting of linked genes on the maternal chromosome, while the Gtl2 DMR, located across the promoter of the Gtl2 gene, is implicated in imprinting on both parental chromosomes. In addition to DNA methylation, modification of histone proteins is also an important regulator of imprinted gene expression. Chromatin immunoprecipitation was therefore used to examine the pattern of histone modifications across the IG and Gtl2 DMRs. The data show maternal-specific histone acetylation at the Gtl2 DMR, but not at the IG DMR. In contrast, only low levels of histone methylation were observed throughout the region, and there was no difference between the two parental alleles. An existing mouse line carrying a deletion/insertion upstream of Gtl2 is unable to imprint the Dlk1-Gtl2 locus properly and demonstrates loss of allele-specific methylation at the Gtl2 DMR. Further analysis of these animals now shows that the loss of allele-specific methylation is accompanied by increased paternal histone acetylation at the Gtl2 DMR, with the activated paternal allele adopting a maternal acetylation pattern. These data indicate that interactions between DNA methylation and histone acetylation are involved in regulating the imprinting of the Dlk1-Gtl2 locus.     

Imprinting disruption of the CDKN1C/KCNQ1OT1 domain: the molecular mechanisms causing Beckwith-Wiedemann syndrome and cancer

Human chromosomal region 11p15.5, which is homologous to mouse chromosome region 7F5, is a well-known imprinted region. The CDKN1C/KCNQ1OT1 imprinted domain, which is one of two imprinted domains at 11p15.5, includes nine imprinted genes regulated by an imprinting center (IC). The CDKN1C/KCNQ1OT1 IC is a differentially methylated region of KCNQ1OT1(KCNQ1OT-DMR) with DNA methylation on the maternal allele and no methylation on the paternal allele. CDKN1C (alias p57KIP2), an imprinted gene with maternal expression, encoding a cyclin-dependent kinase inhibitor, is a critical gene within the CDKN1C/KCNQ1OT1 domain. In Beckwith-Wiedemann syndrome (BWS), approximately 50% of patients show loss of DNA methylation accompanied by loss of histone H3 Lys9 dimethylation on maternal KCNQ1OT-DMR, namely an imprinting disruption, leading to diminished expression of CDKN1C. In cancer, at least three molecular mechanisms--imprinting disruption, aberrant DNA methylations at the CDKN1C promoter, and loss of heterozygosity (LOH) of the maternal allele--are seen and all three result in diminished expression of CDKN1C. Imprinting disruption of the CDKN1C/KCNQ1OT1 domain is involved in the development of both BWS and cancer and it changes the maternal epigenotype to the paternal type, leading to diminished CDKN1C expression. In this review, we describe recent advances in epigenetic control of the CDKN1C/KCNQ1OT1 imprinted domain in both humans and mice.     

Towards unravelling the Igf2/H19 imprinted domain

Genomic imprinting is an epigenetic marking process that confers parent-of-origin-dependent expression on certain genes. These imprinted genes are sometimes found in clusters, suggesting a possible involvement of higher order regulatory elements controlling expression and imprinting of genes organised in such clusters. In the distal chromosome 7 there are at least four imprinted genes: Mash2, Ins2, Igf2 and H19. Recent evidence⁽¹⁾ suggests that imprinting and expression of at least Igf2 and H19 may be mechanistically linked.     

Recent assembly of an imprinted domain from non-imprinted components

Genomic imprinting, representing parent-specific expression of alleles at a locus, raises many questions about how—and especially why—epigenetic silencing of mammalian genes evolved. We present the first in-depth study of how a human imprinted domain evolved, analyzing a domain containing several imprinted genes that are involved in human disease. Using comparisons of orthologous genes in humans, marsupials, and the platypus, we discovered that the Prader-Willi/Angelman syndrome region on human Chromosome 15q was assembled only recently (105–180 million years ago). This imprinted domain arose after a region bearing UBE3A (Angelman syndrome) fused with an unlinked region bearing SNRPN (Prader-Willi syndrome), which had duplicated from the non-imprinted SNRPB/B′. This region independently acquired several retroposed gene copies and arrays of small nucleolar RNAs from different parts of the genome. In their original configurations, SNRPN and UBE3A are expressed from both alleles, implying that acquisition of imprinting occurred after their rearrangement and required the evolution of a control locus. Thus, the evolution of imprinting in viviparous mammals is ongoing.     

A tripartite paternally methylated region within the Gpr1-Zdbf2 imprinted domain on mouse chromosome 1 identified by meDIP-on-chip

The parent-of-origin specific expression of imprinted genes relies on DNA methylation of CpG-dinucleotides at differentially methylated regions (DMRs) during gametogenesis. To date, four paternally methylated DMRs have been identified in screens based on conventional approaches. These DMRs are linked to the imprinted genes H19, Gtl2 (IG-DMR), Rasgrf1 and, most recently, Zdbf2 which encodes zinc finger, DBF-type containing 2. In this study, we applied a novel methylated-DNA immunoprecipitation-on-chip (meDIP-on-chip) method to genomic DNA from mouse parthenogenetic- and androgenetic-derived stem cells and sperm and identified 458 putative DMRs. This included the majority of known DMRs. We further characterized the paternally methylated Zdbf2/ZDBF2 DMR. In mice, this extensive germ line DMR spanned 16 kb and possessed an unusual tripartite structure. Methylation was dependent on DNA methyltransferase 3a (Dnmt3a), similar to H19 DMR and IG-DMR. In both humans and mice, the adjacent gene, Gpr1/GPR1, which encodes a G-protein-coupled receptor 1 protein with transmembrane domain, was also imprinted and paternally expressed. The Gpr1-Zdbf2 domain was most similar to the Rasgrf1 domain as both DNA methylation and the actively expressed allele were in cis on the paternal chromosome. This work demonstrates the effectiveness of meDIP-on-chip as a technique for identifying DMRs.     

Evolution and control of imprinted FWA genes in the genus Arabidopsis

A central question in genomic imprinting is how a specific sequence is recognized as the target for epigenetic marking. In both mammals and plants, imprinted genes are often associated with tandem repeats and transposon-related sequences, but the role of these elements in epigenetic gene silencing remains elusive. FWA is an imprinted gene in Arabidopsis thaliana expressed specifically in the female gametophyte and endosperm. Tissue-specific and imprinted expression of FWA depends on DNA methylation in the FWA promoter, which is comprised of two direct repeats containing a sequence related to a SINE retroelement. Methylation of this element causes epigenetic silencing, but it is not known whether the methylation is targeted to the SINE-related sequence itself or the direct repeat structure is also necessary. Here we show that the repeat structure in the FWA promoter is highly diverse in species within the genus Arabidopsis. Four independent tandem repeat formation events were found in three closely related species. Another related species, A. halleri, did not have a tandem repeat in the FWA promoter. Unexpectedly, even in this species, FWA expression was imprinted and the FWA promoter was methylated. In addition, our expression analysis of FWA gene in vegetative tissues revealed high frequency of intra-specific variation in the expression level. In conclusion, we show that the tandem repeat structure is dispensable for the epigenetic silencing of the FWA gene. Rather, SINE-related sequence is sufficient for imprinting, vegetative silencing, and targeting of DNA methylation. Frequent independent tandem repeat formation events in the FWA promoter led us to propose that they may be a consequence, rather than cause, of the epigenetic control. The possible significance of epigenetic variation in reproductive strategies during evolution is also discussed.     

MiR-1188 at the imprinted Dlk1-Dio3 domain acts as a tumor suppressor in hepatoma cells

The aberrant expression of microRNAs (miRNAs) has frequently been reported in cancer studies; miRNAs play roles in development, progression, metastasis, and prognosis. Recent studies indicate that the miRNAs within the Dlk1-Dio3 genomic region are involved in the development of liver cancer, but the role of miR-1188 in hepatocellular carcinoma (HCC) and the pathway by which it exerts its function remain largely unknown. Here we demonstrate that miR-1188 is significantly down-regulated in mouse hepatoma cells compared with normal liver tissues. Enhanced miR-1188 suppresses cell proliferation, migration, and invasion in vitro and inhibits the tumor growth of HCC cells in vivo. Moreover, overexpressed miR-1188 promotes apoptosis, enhances caspase-3 activity, and also up-regulates the expression of Bax and p53. MiR-1188 directly targets and negatively regulates Bcl-2 and Sp1. Silencing of Bcl-2 and Sp1 exactly copies the proapoptotic and anti-invasive effects of miR-1188, respectively. The expression of apoptosis- and invasion-related genes, such as Vegfa, Fgfr1, and Rprd1b, decreases after enhancement of miR-1188, as determined by gene expression profiling analysis. Taken together, our results highlight an important role for miR-1188 as a tumor suppressor in hepatoma cells and imply its potential role in cancer therapy.     

Role of the CDKN1A/p21, CDKN1C/p57, and CDKN2A/p16 Genes in the Risk of Atherosclerosis and Myocardial Infarction

Atherosclerotic is characterised by excessive proliferation of neointimal leukocytes and vascular smooth muscle cells (VSMCs). In mice, the manipulation of cell cycle inhibitors such as CDKN1B (p27) and CDKN1A (p21) modifies the risk of developing atherosclerosis. In humans, CDKN1A, CDKN1B, and CDKN1C (p57) are differentially expressed in normal versus atherosclerotic vessels. A DNA-polymorphism within the CDKN1B promoter has been associated with myocardial infarction (MI). In the present study, we analysed the effect of CDKN1A, CDKN1C, and CDKN2A (p16) polymorphisms on MI-risk. A total of 316 patients (all male,     

Specific differentially methylated domain sequences direct the maintenance of methylation at imprinted genes

Landmark features of imprinted genes are differentially methylated domains (DMDs), in which one parental allele is methylated on CpG dinucleotides and the opposite allele is unmethylated. Genetic experiments in the mouse have shown that DMDs are required for the parent-specific expression of linked clusters of imprinted genes. To understand the mechanism whereby the differential methylation is established and maintained, we analyzed a series of transgenes containing DMD sequences and showed that imperfect tandem repeats from DMDs associated with the Snurf/Snrpn, Kcnq1, and Igf2r gene clusters govern transgene imprinting. For the Igf2r DMD the minimal imprinting signal is two unit copies of the tandem repeat. This imprinted transgene behaves identically to endogenous imprinted genes in Dnmt1o and Dnmt3L mutant mouse backgrounds. The primary function of the imprinting signal within the transgene DMD is to maintain, during embryogenesis and a critical period of genomic reprogramming, parent-specific DNA methylation states established in the germ line. This work advances our understanding of the imprinting mechanism by defining a genomic signal that dependably perpetuates an epigenetic state during postzygotic development.     

Gene expression pattern of IGF2, PHLDA2, PEG10 and CDKN1C imprinted genes in spontaneous miscarriages or fetal deaths

Genomic imprinting is defined as an epigenetic modification that leads to parent-of-origin specific monoallelic expression. Some current research on the fetal control growth has been focused on the study of genes that display imprinted expression in utero. Four imprinted genes, two paternally expressed (IGF2 and PEG10) and two maternally expressed (PHLDA2 and CDKN1C), are well known to play a role in fetal growth and placental development. Pregnancy loss in the general reproductive population is a very common occurrence and other genetic causes beyond chromosomal abnormalities could be involved in spontaneous miscarriages or fetal deaths, such as alteration of expression in imprinted genes particularly those related to fetal or placental growth. Quantitative Real Time PCR was performed to evaluate gene expressions patterns of the four mentioned genes in spontaneous miscarriages or fetal deaths from 38 women. Expression levels of PHLDA2 gene were upregulated in the first trimester pregnancy cases and all four imprinted genes studied were upregulated in the second trimester of pregnancy cases comparing with controls. In third trimester PEG10 was downregulated in fetal samples group. This is the first study presenting data from human imprinted genes expression in spontaneous miscarriages or fetal deaths cases from the three trimesters of pregnancy.     

The Prader-Willi/Angelman imprinted domain and its control center

The present review focuses on the recent advances towards understanding the mode of operation of the imprinting center (IC) within the Prader-Willi/Angelman syndromes (PWS/AS) domain. Special emphasis is put on the elucidation of the functional interaction between the two parts of the center, AS-IC and PWS-IC. The recent studies, on which the review is based, reveal cis-acting elements and trans-acting proteins that constitute the two parts of the IC and presumably provide the molecular mechanism for this interaction. AS-IC acquires the primary imprint during gametogenesis by establishing the maternal epigenotype. The unmethylated maternal allele of the AS-IC binds, very likely, a trans-acting factor that confers methylation on the PWS-IC maternal allele after fertilization. It is assumed that the PWS-IC paternal epigenotype, once established, spreads across the entire PWS/AS domain in the soma.     

Embryonic inheritance of the chromatin organisation of the imprinted H19 domain in mouse spermatozoa

Insulin-like growth factor 2 (Igf 2) and H19 genes are oppositely imprinted and as such have been most extensively studied imprinted genes both genetically and at the molecular level. Imprints of the H19 gene, being established during spermatogenesis, are epigenetically transmitted to the somatic cells of the embryo. Current hypotheses attempting to explain the allele-specific silence of the H19 gene include DNA methylation and chromatin condensation. In order to understand the molecular basis of H19 epigenesis, it is crucial to identify the markings in the chromatin organising the imprinted domain in spermatozoa. Using Micrococcal nuclease (MNase), DNase I and Methidiumpropyl-EDTA. iron II (MPE·Fe(II)) as chromatin probes, we demonstrate that in mouse epididymal spermatozoa, at least 4 kb DNA upstream of the H19 ‘cap’ site, containing the imprinted and differentially methylated domain (DMD), is heterochromatic. The cleavage sites in this domain (−2 to −4 kb) exhibit ~425 bp periodicity. This structure is maintained in the paternal allele of normal embryos and is disrupted at −2.2, −2.65 and at −3.5 kb in embryos maternally disomic for the distal end of chromosome 7 (MatDp 7). The hypersensitive sites in chromatin precisely register the MPE·Fe(II) cleavage sites in chromosomal DNA. Therefore, the DNA sequences in the imprinted domain constrain the chromatin structure in a way similar to that of 1.688 g/cm³ Drosophila satellite chromatin. In addition, we find that condensation of the paternal allele correlates with methylation-dependent alteration in the structure of DNA sequences in DMD. These results suggest that CpG-methylation induces localised changes in DNA conformation and these facilitate consequent remodelling of chromatin thereby allowing the paternal and maternal H19 alleles to be distinguished.     

Evolution of domain promiscuity in eukaryotic genomes--a perspective from the inferred ancestral domain architectures

Most eukaryotic proteins are composed of two or more domains. These assemble in a modular manner to create new proteins usually by the acquisition of one or more domains to an existing protein. Promiscuous domains which are found embedded in a variety of proteins and co-exist with many other domains are of particular interest and were shown to have roles in signaling pathways and mediating network communication. The evolution of domain promiscuity is still an open problem, mostly due to the lack of sequenced ancestral genomes. Here we use inferred domain architectures of ancestral genomes to trace the evolution of domain promiscuity in eukaryotic genomes. We find an increase in average promiscuity along many branches of the eukaryotic tree. Moreover, domain promiscuity can proceed at almost a steady rate over long evolutionary time or exhibit lineage-specific acceleration. We also observe that many signaling and regulatory domains gained domain promiscuity around the Bilateria divergence. In addition we show that those domains that played a role in the creation of two body axes and existed before the divergence of the bilaterians from fungi/metazoan achieve a boost in their promiscuities during the bilaterian evolution.