Genomic imprinting. Silence across the border.
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Animals↗
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Evidence for de novo imprinted X-chromosome inactivation independent of meiotic inactivation in mice.
In mammals, one of the two X chromosomes is inactivated in females to enable dosage compensation for X-linked gene products. In rodents and marsupials, only the X chromosome of paternal origin (Xp) is silenced during early embryogenesis. This could be due to a carry-over effect of the X chromosome's passage through the male germ line, where it becomes transiently silenced together with the Y chromosome, during meiotic sex chromosome inactivation (MSCI). Here we show that Xist (X inactive specific transcript) transgenes, located on autosomes, do not undergo MSCI in the male germ line of mice and yet can induce imprinted cis-inactivation when paternally inherited, with identical kinetics to the Xp chromosome. This suggests that MSCI is not necessary for imprinted X-chromosome inactivation in mice. We also show that the Xp is transcribed, like autosomes, at zygotic gene activation rather than being 'pre-inactivated'. We propose that expression of the paternal Xist gene at zygotic gene activation is sufficient to trigger cis-inactivation of the X chromosome, or of an autosome carrying a Xist transgene.
Animals↗
Gamete-specific methylation correlates with imprinting of the murine Xist gene.
We have investigated the potential role of DNA methylation as a regulator of imprinted Xist expression in mouse preimplantation embryos. The active paternal allele was found to be unmodified in sperm at CpG loci near the 5' end of the gene transcription unit. In contrast, on the inactive maternal allele, these same sites are initially methylated in the oocyte and then remain modified in the early embryo. In the male germ line, these methyl moieties are removed during spermatogenesis, and this occurs before the programmed reactivation of Xist in the testis. This represents a clear-cut example of a potential methylation imprinting signal that is reprogrammable and gamete derived.
Alleles↗
Microsatellite variation within and among North American lineages of Phragmites australis.
Over the past century, the spread of the common reed (Phragmites australis) has had a dramatic impact on wetland communities across North America. Although native populations of Phragmites persist, introduced invasive populations have dominated many sites and it is not clear if the two types can interbreed. This study compares patterns of differentiation in 10 microsatellite loci among North American and European Phragmites individuals with results obtained from sequencing of noncoding chloroplast DNA. Three population lineages (native, introduced and Gulf Coast) were previously identified in North America from chloroplast DNA and similar structuring was found in the nuclear genome. Each lineage was distinguished by unique alleles and allele combinations and the introduced lineage was closely related to its hypothesized source population in Europe. Size homoplasy and diagnostic base substitutions distinguishing lineages were evident at several loci, further emphasizing that native, introduced and Gulf Coast North American Phragmites lineages are genetically distinct. Gene flow between lineages was low and invasive introduced populations do not represent a hybrid population type.
Alleles↗
The DELAYED ABAXIAL TRICHOMES Helitron has dual functions in vegetative and pollen development in Arabidopsis thaliana.
Transposons drive genetic diversity and evolution by altering the genomic landscape over time. Here, we describe DELAYED ABAXIAL TRICHOMES (DAB), a Helitron/RC transposable element in Arabidopsis thaliana that has a role in vegetative phase change and gametogenesis. A genome-wide association study (GWAS) for the timing of abaxial trichome development (an adult leaf trait) in A. thaliana revealed a conserved haplotype of polymorphisms within DAB that delays abaxial trichome production. CRISPR-Cas9-induced deletions of DAB are gametophytic pollen-lethal, indicating that this locus is also required for pollen production. DAB produces 24-nucleotide siRNAs with sequence complementarity to genes involved in embryogenesis, gametogenesis, and seed development. DAB also impacts the expression of ARGONAUTE genes, genes involved in RNA-directed DNA methylation (RdDM), as well as genes in several key genetic pathways. This global effect on gene expression suggests that DAB may have functions beyond those identified in this study.
Arabidopsis↗
Three novel families of miniature inverted-repeat transposable elements are associated with genes of the yellow fever mosquito, Aedes aegypti.
Three novel families of transposable elements, Wukong, Wujin, and Wuneng, are described in the yellow fever mosquito, Aedes aegypti. Their copy numbers range from 2,100 to 3,000 per haploid genome. There are high degrees of sequence similarity within each family, and many structural but not sequence similarities between families. The common structural characteristics include small size, no coding potential, terminal inverted repeats, potential to form a stable secondary structure, A+T richness, and putative 2- to 4-bp A+T-biased specific target sites. Evidence of previous mobility is presented for the Wukong elements. Elements of these three families are associated with 7 of 16 fully or partially sequenced Ae. aegypti genes. Characteristics of these mosquito elements indicate strong similarities to the miniature inverted-repeat transposable elements (MITEs) recently found to be associated with plant genes. MITE-like elements have also been reported in two species of Xenopus and in Homo sapiens. This characterization of multiple families of highly repetitive MITE-like elements in an invertebrate extends the range of these elements in eukaryotic genomes. A hypothesis is presented relating genome size and organization to the presence of highly reiterated MITE families. The association of MITE-like elements with Ae. aegypti genes shows the same bias toward noncoding regions as in plants. This association has potentially important implications for the evolution of gene regulation.
Aedes↗
A silencer element identified in Drosophila is required for imprinting of H19 reporter transgenes in mice.
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.
Alkaline Phosphatase↗
Widespread transcription of a Qa region gene in adult mice.
The mouse MHC class I family includes genes encoded in four regions: H-2K, H-2D, Qa and Tla. While K/D genes are well characterized, relatively little is known about Qa or Tla genes. We have studied the transcription of a B10.P Qa region gene. DNA sequence comparisons of the transmembrane region, supported by Southern blot analysis of cosmid and genomic DNAs from BALB/c and C57BL/10, demonstrate the lambda 3a gene corresponds to Q4p. In both Northern blots and RNA protection experiments using probes derived from the 3' noncoding region, we found that Q4, like the H-2K and H-2D genes, is widely transcribed in B10.P tissues. These data demonstrate for the first time widespread transcription of a Qa gene.
Animals↗
Structure and expression of the mouse gene encoding the orphan nuclear receptor TEC.
Translocated in extraskeletal chondrosarcoma (TEC) is an orphan nuclear receptor involved in the control of cell proliferation and apoptosis and is expressed mainly in the mammalian central nervous system. To help understand the regulation of its expression, we have characterized the mouse genomic locus encoding TEC and analyzed its expression pattern in various tissues. The gene spans approximately 40 kb and contains 8 exons, of which the first two are noncoding. The promoter region does not contain any identifiable TATA box or CCAAT box elements; however, several binding sites for the transcription factors cyclic AMP-responsive element binding (CREB) protein and Spl are present. Two types of transcripts generated by alternative splicing were characterized by RT-PCR: one encodes the full-length receptor of 627 amino acids; the other encodes a truncated receptor of 429 amino acids lacking the entire carboxyl-terminal domain. Northern blots and RT-PCR analyses showed that mRNAs encoding both isoforms are expressed in all mouse tissues examined, with the highest levels being found in the brain. This expression pattern suggests that TEC may perform some basic housekeeping cellular function in addition to its role in cell proliferation.
Amino Acid Sequence↗
Turnover of primary transcripts is a major step in the regulation of mouse H19 gene expression.
In the gene expression pathway, RNA biogenesis is a central multi-step process where both message fidelity and steady-state levels of the mature RNA have to be ascertained. An emerging question is whether RNA levels could be regulated at the precursor stage. Until recently, because it was technically very difficult to determine the level of a pre-mRNA, discrimination between changes in transcriptional activity and in pre-mRNA metabolism was extremely difficult. H19 RNA, the untranslated product of an imprinted gene, undergoes post-transcriptional regulation. Here, using a quantitative real-time RT-PCR approach, we accurately quantify its precursor RNA levels and compare these with the transcriptional activity of the gene, assessed by run-on assays. We find that the levels of H19 precursor RNA are regulated during physiological processes and this regulation appears to be related to RNA polymerase II transcription termination. Our results provide direct evidence that turnover of polymerase II primary transcripts can regulate gene expression in mammals.
Animals↗
Paternal imprints can be established on the maternal Igf2-H19 locus without altering replication timing of DNA.
Genomic imprinting in mammals marks the parental alleles in gametes, resulting in differential gene expression in offspring. A number of epigenetic features are associated with imprinted genes. These include differential DNA methylation, histone acetylation and methylation, subnuclear localization and DNA replication timing. While DNA methylation has been shown to be necessary both for establishment and maintenance of imprinting, the connections with the other types of epigenetic marking systems are not clear. Specifically, it is not known whether the other marking systems, either on their own or in conjunction with DNA methylation, are required for imprinting. Here we show that in the mouse mutant Minute (Mnt) the Igf2-H19 locus acquires a paternal methylation imprint in the maternal germline. DNA methylation of the H19 DMR is established in oogenesis, maintained during postzygotic development on the maternal allele, and erased in primordial germ cells. The fact that a paternal type methylation imprint can also be established in the maternal germline indicates that trans-acting factors that target methylation to this imprinted region are likely to be the same in both germlines. Surprisingly, however, asynchrony of DNA replication of the locus is maintained despite the altered expression and methylation imprint of Igf2 and H19. These results show clearly that replication asynchrony of this region is neither the determinant factor for, nor a consequence of, epigenetic modifications that are critical for genomic imprinting. Replication asynchrony may thus be regulated differently from methylation imprints and have a separate function.
Alleles↗
Large-scale comparative sequence analysis of the human and murine Bruton's tyrosine kinase loci reveals conserved regulatory domains.
Large-scale genomic DNA sequencing of orthologous and paralogous loci in different species should contribute to a basic understanding of the evolution of both the protein-coding regions and noncoding regulatory elements. We compared 93 kb of human sequence to 89 kb of mouse sequence in the Bruton's tyrosine kinase (BTK) region. In addition to showing the conservation of both position and orientation of the five functionally unrelated genes in the region (BTK, alpha-D-galactosidase A, L44L, FTP-3, and FCI-12), the comparison revealed conservation of clusters of noncoding sequence flanking the first exon of each gene. Furthermore, in the sequence comparison at the BTK locus, the conservation of clusters of noncoding sequence extends throughout the locus; the noncoding sequence is more highly conserved in the BTK locus in comparison to the flanking loci. This suggests a correlation with the complex developmental regulation of expression of btk. To determine whether a highly conserved 3.5-kb segment flanking the first exon of BTK contains transcriptional regulatory signals, we tested various portions of the segment for promoter and expression activity in several appropriate cell lines. The results demonstrate the contribution of the conserved region flanking the first exon to the cell lineage-specific expression pattern of btk. These data show the usefulness of large scale sequence comparisons to focus investigation on regions of noncoding sequence that play essential roles in complex gene regulation.
Agammaglobulinaemia Tyrosine Kinase↗
Loss of imprinting in human cancer.
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Alleles↗
Transgenic RNAi reveals essential function for CTCF in H19 gene imprinting.
The imprinted regulation of H19 and Insulin-like growth factor 2 expression involves binding of the vertebrate insulator protein, CCCTC binding factor (CTCF), to the maternally hypomethylated differentially methylated domain (DMD). How this hypomethylated state is maintained during oogenesis and the role of CTCF, if any, in this process are not understood. With the use of a transgenic RNA interference (RNAi)-based approach to generate oocytes with reduced amounts of CTCF protein, we found increased methylation of the H19 DMD and decreased developmental competence of CTCF-deficient oocytes. Our results suggest that CTCF protects the H19 DMD from de novo methylation during oocyte growth and is required for normal preimplantation development.
Actins↗
Role of CTCF binding sites in the Igf2/H19 imprinting control region.
A approximately 2.4-kb imprinting control region (ICR) regulates somatic monoallelic expression of the Igf2 and H19 genes. This is achieved through DNA methylation-dependent chromatin insulator and promoter silencing activities on the maternal and paternal chromosomes, respectively. In somatic cells, the hypomethylated maternally inherited ICR binds the insulator protein CTCF at four sites and blocks activity of the proximal Igf2 promoter by insulating it from its distal enhancers. CTCF binding is thought to play a direct role in inhibiting methylation of the ICR in female germ cells and in somatic cells and, therefore, in establishing and maintaining imprinting of the Igf2/H19 region. Here, we report on the effects of eliminating ICR CTCF binding by severely mutating all four sites in mice. We found that in the female and male germ lines, the mutant ICR remained hypomethylated and hypermethylated, respectively, showing that the CTCF binding sites are dispensable for imprinting establishment. Postfertilization, the maternal mutant ICR acquired methylation, which could be explained by loss of methylation inhibition, which is normally provided by CTCF binding. Adjacent regions in cis-the H19 promoter and gene-also acquired methylation, accompanied by downregulation of H19. This could be the result of a silencing effect of the methylated maternal ICR.
Animals↗
Tandem repeat hypothesis in imprinting: deletion of a conserved direct repeat element upstream of H19 has no effect on imprinting in the Igf2-H19 region.
Igf2 and H19 are reciprocally imprinted genes on mouse distal chromosome 7. They share several regulatory elements, including a differentially methylated region (DMR) upstream of H19 that is paternally methylated throughout development. The cis-acting sequence requirements for targeting DNA methylation to the DMR remain unknown; however, it has been suggested that direct tandem repeats near DMRs could be involved. Previous studies of the imprinted Rasgrf1 locus demonstrate indeed that a direct repeat element adjacent to a DMR is responsible for establishing paternal allele-specific methylation at the DMR and therefore allelic expression of the Rasgrf1 transcript. We identified a prominent and conserved direct tandem repeat 1 kb upstream of the H19 DMR and proposed that it played a similar role in imprinted regulation of H19. To test our hypothesis, we generated mice harboring a 1.7-kb targeted deletion of the direct repeat element and analyzed fetal growth, allelic expression, and methylation within the Igf2-H19 region. Surprisingly the deletion had no effect on imprinting. These results together with deletions of other repeats close to imprinted genes suggest that direct repeats may not be important for the targeting of methylation at the majority of imprinted loci and that the Rasgrf1 locus may be an exception to this rule.
Animals↗
Silencing of CDKN1C (p57KIP2) is associated with hypomethylation at KvDMR1 in Beckwith-Wiedemann syndrome.
CONTEXT: Beckwith-Wiedemann syndrome (BWS) arises by several genetic and epigenetic mechanisms affecting the balance of imprinted gene expression in chromosome 11p15.5. The most frequent alteration associated with BWS is the absence of methylation at the maternal allele of KvDMR1, an intronic CpG island within the KCNQ1 gene. Targeted deletion of KvDMR1 suggests that this locus is an imprinting control region (ICR) that regulates multiple genes in 11p15.5. Cell culture based enhancer blocking assays indicate that KvDMR1 may function as a methylation modulated chromatin insulator and/or silencer. OBJECTIVE: To determine the potential consequence of loss of methylation (LOM) at KvDMR1 in the development of BWS. METHODS: The steady state levels of CDKN1C gene expression in fibroblast cells from normal individuals, and from persons with BWS who have LOM at KvDMR1, was determined by both real time quantitative polymerase chain reaction (qPCR) and ribonuclease protection assay (RPA). Methylation of the CDKN1C promoter region was assessed by Southern hybridisation using a methylation sensitive restriction endonuclease. RESULTS: Both qPCR and RPA clearly demonstrated a marked decrease (86-93%) in the expression level of the CDKN1C gene in cells derived from patients with BWS, who had LOM at KvDMR1. Southern analysis indicated that downregulation of CDKN1C in these patients was not associated with hypermethylation at the presumptive CDKN1C promoter. CONCLUSIONS: An epimutation at KvDMR1, the absence of maternal methylation, causes the aberrant silencing of CDKN1C, some 180 kb away on the maternal chromosome. Similar to mutations at this locus, this silencing may give rise to BWS.
Beckwith-Wiedemann Syndrome↗