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Mealybugs are aggressive pests with world-wide distribution and are suitable for the study of different phenomena like genomic imprinting and epigenetics. Genomic approaches facilitate these studies in absence of robust genetics in this system. We sequenced, de novo assembled, annotated Maconellicoccus hirsutus genome. We carried out comparative genomics it with four mealybug and eight other insect species, to identify expanded, specific and contracted gene classes that relate to pesticide and desiccation resistance. We identified horizontally transferred genes adding to the mutualism between the mealybug and its endosymbionts. Male and female transcriptome analysis indicates differential expression of metabolic pathway genes correlating with their physiology and the genes for sexual dimorphism. The significantly lower expression of endosymbiont genes in males relates to the depletion of endosymbionts in males during development. • We sequenced the genome and transcriptome of the mealybug Maconellicoccus hirsutus , an aggressive plant pest. • Highly expanded gene classes conferring resistance to pesticides and desiccation were identified by comparative genomics. • Transcriptome analysis led to the identification of differentially expressed genes in adult males and females. • Metabolic genes and genes conferring sexual dimorphism were found to be differentially expressed. • Lower expression of endosymbiont genes in males correlates with the depletion of endosymbionts during development. [ABSTRACT FROM AUTHOR]

Tambaqui (Colossoma macropomum) is the major native species in Brazilian aquaculture, and we have shown that females exhibit a higher growth compared to males, opening up the possibility for the production of all-female population. To date, there is no information on the sex determination and differentiation molecular mechanisms of tambaqui. In the present study, transcriptome sequencing of juvenile trunks was performed to understand the molecular network involved in the gonadal sex differentiation. The results showed that before differentiation, components of the Wnt/β-catenin pathway, fox and fst genes imprint female sex development, whereas antagonistic pathways (gsk3b , wt1 and fgfr2), sox9 and genes for androgen synthesis indicate male differentiation. Hence, in undifferentiated tambaqui, the Wnt/β-catenin exerts a role on sex differentiation, either upregulated in female-like individuals, or antagonized in male-like individuals. • Before differentiation, Wnt/β-catenin pathway genes imprint female sex development. • Antagonistic pathways, sox9 and androgen synthesis genes imprint male development. • Molecular paths towards sex differentiation can be detected in larvae from 2 cm. [ABSTRACT FROM AUTHOR]

Mammalian imprinted domains are regulated through small genomic regions termed Imprinting Control Regions (ICRs). In the current study, the evolution patterns of the ICRs of Peg3 and H19-imprinted domains were analyzed using the genomic sequences derived from a large number of mammals. The results indicated that multiple YY1 and CTCF binding sites are localized within the Peg3 and H19-ICR in all the mammals tested. The numbers of YY1 and CTCF binding sites are variable among individual species, yet positively correlate with the presence of tandem repeats within the Peg3 and H19-ICRs. Thus, multiple YY1 and CTCF binding sites within the respective ICRs may have been maintained through tandem repeats/duplications. The unit lengths of tandem repeats are also non-random and locus-specific, 140 and 400 bp for the Peg3 and H19-ICRs. Overall, both Peg3 and H19-ICRs may have co-evolved with two unique features, multiple transcription factor binding sites and tandem repeats. • Multiple YY1 and CTCF binding sites are localized within the Peg3 and H19-ICR in all the mammals tested. • Multiple YY1 and CTCF binding sites within the Peg3 and H19-ICRs may have been maintained through tandem duplications. • The unit lengths of tandem repeats are non-random and locus-specific, 140 and 400 bp for the Peg3 and H19-ICRs. [ABSTRACT FROM AUTHOR]

DNA methylation has crucial roles in regulating the expression of genes involved in skeletal muscle development. However, the DNA methylation pattern of lncRNA during sheep skeletal muscle development remains unclear. This study investigated previous WGBS and LncRNA data in skeletal muscle of sheep (fetus and adult). We then focused on LncRNA GTL2, which is differentially expressed in skeletal muscle and has multiple DMRs. We found that the expression level of GTL2 decreased with age. GTL2 DMRs methylation levels were significantly higher in adult muscle than in fetal muscle. After 5AZA treatment, GTL2 expression was significantly increased in a dose-dependent manner.The dCas9-DNMT3A-sgRNA significantly reduced the expression level of GTL2 in cells, but increased GTL2 DMR methylation levels. The above studies indicate that dCas9-DNMT3A can effectively increase the methylation level in the DMR region of GTL2, the expression level of GTL2 is regulated by DNA methylation during muscle development., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract: Genomic imprinting at the Delta-like 1 (Dlk1)–Maternally expressed gene 3 (Meg3) locus is regulated by the Meg3 differentially methylated region (DMR), but the mechanism by which this DMR acts is unknown. The goal of this study was to analyze the Meg3 DMR during imprinting establishment and maintenance for the presence of histone modifications and trans-acting DNA binding proteins using chromatin immunoprecipitation. In embryonic stem (ES) cells, where Meg3 is biallelically expressed, the DMR showed variable DNA methylation, with biallelic methylation at one region but paternal allele-specific methylation at another. All histone modifications detected at the Meg3 DMR of ES cells were biallelic. In embryonic day 12.5 (e12.5) embryos, where Meg3 is maternally expressed, the paternal Meg3 DMR was methylated, and activating histone modifications were specific to the maternal DMR. DNA-binding proteins that represent potential regulatory factors were identified in both ES cells and embryos. [Copyright &y& Elsevier]

Abstract: Beckwith–Wiedemann syndrome (BWS) is an overgrowth syndrome associated with genetic or epigenetic alterations in one of two imprinted domains on chromosome 11p15.5. Rarely, chromosomal translocations or inversions of chromosome 11p15.5 are associated with BWS but the molecular pathophysiology in such cases is not understood. In our series of 3 translocation and 2 inversion patients with BWS, the chromosome 11p15.5 breakpoints map within the centromeric imprinted domain, 2. We hypothesized that either microdeletions/microduplications adjacent to the breakpoints could disrupt genomic sequences important for imprinted gene regulation. An alternate hypothesis was that epigenetic alterations of as yet unknown regulatory DNA sequences, result in the BWS phenotype. A high resolution Nimblegen custom microarray was designed representing all non-repetitive sequences in the telomeric 33Mb of the short arm of human chromosome 11. For the BWS-associated chromosome 11p15.5 translocations and inversions, we found no evidence of microdeletions/microduplications. DNA methylation was also tested on this microarray using the HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) assay. This high-resolution DNA methylation microarray analysis revealed a gain of DNA methylation in the translocation/inversion patients affecting the p-ter segment of chromosome 11p15, including both imprinted domains. BWS patients that inherited a maternal translocation or inversion also demonstrated reduced expression of the growth suppressing imprinted gene, CDKN1C in Domain 2. In summary, our data demonstrate that translocations and inversions involving imprinted domain 2 on chromosome 11p15.5, alter regional DNA methylation patterns and imprinted gene expression in cis, suggesting that these epigenetic alterations are generated by an alteration in “chromatin context”. [Copyright &y& Elsevier]

Abstract: A substantial number of transcription factor families have been investigated from all kingdoms of life, but a particular class of plant-specific TIFY transcription factors, characterized by a highly conserved TIFY domain, lacks a systemic analysis of its origin and evolutionary relationships among different plant species. After exhaustive genome-wide searches against 14 genomes, TIFY transcription factors were identified and classified into four subfamilies TIFY, PPD, JAZ and ZML according to their different domain architectures. Results show that the TIFY domain of the ZML subfamily possesses a core “TLS[F/Y]XG” motif rather than the “TIFYXG” motif that is dominant in the other three subfamilies. A comprehensive survey of the TIFY family allowed us to discover a new group within the JAZ subfamily and to identify several novel conserved motifs via phylogenetic analysis. Evolutional analysis indicates that whole genome duplication and tandem duplication contributed to the expansion of the TIFY family in plants. [Copyright &y& Elsevier]

Abstract: The Dlk1-Dio3 imprinted domain on mouse chromosome 12 contains IG-DMR and Gtl2-DMR, whose methylation patterns are established in the germline and after fertilization, respectively. In this study, we determine that acquisition of DNA methylation at the paternal allele of the Gtl2-DMR is initiated after the blastocyst stage and completed by embryonic day 6.5, and that Gtl2 (approved symbol: Meg3) is monoallelically expressed from the maternal allele as early as the blastocyst. Therefore, DNA methylation at the Gtl2-DMR is not a prerequisite for the imprinted expression of Gtl2, which may be involved in the control of proliferation and differentiation of cells during early gestation. We also reveal that a subregion of the IG-DMR exhibits tissue-specific differences in allelic methylation patterns. These results add to the growing body of knowledge elucidating the mechanism whereby parent-of-origin-dependent DNA methylation at the IG-DMR leads to the imprinted expression of the Dlk1-Dio3 cluster. [Copyright &y& Elsevier]

Abstract: The recent identification of an intragenic differentially methylated region (DMR) within the last exon of the bovine Insulin-like growth factor 2 (IGF2) gene provides a diagnostic tool for in-depth investigation of bovine imprinting and regulatory mechanisms which are active during embryo development. Here, we used bisulfite sequencing to compare sex-specific DNA methylation patterns within this DMR in bovine blastocysts produced in vivo, by in vitro fertilization and culture, SCNT, androgenesis or parthenogenesis. In in vivo derived embryos, DNA methylation was removed from this intragenic DMR after fertilization, but partially replaced by the time the embryo reached the blastocyst stage. Among embryos developing in vivo, the level of DNA methylation was significantly lower in female than in male blastocysts. This sexual dimorphism was also found between parthenogenetic and androgenetic embryos, and followed the donor cell sex in SCNT derived blastocysts and is evidence for correct methylation reprogramming in SCNT embryos. [Copyright &y& Elsevier]

Abstract: In mammals, both the maternal and paternal genomes are necessary for normal embryogenesis due to parent-specific epigenetic modification of the genome during gametogenesis, which leads to non-equivalent expression of imprinted genes from the maternal and paternal alleles. In this study, we identified a paternally expressed imprinted gene, Zdbf2, by microarray-based screening using parthenogenetic and normal embryos. Expression analyses showed that Zdbf2 was paternally expressed in various embryonic and adult tissues, except for the placenta and adult testis, which showed biallelic expression of the gene. We also identified a differentially methylated region (DMR) at 10 kb upstream of exon 1 of the Zdbf2 gene and this differential methylation was derived from the germline. Furthermore, we also identified that the human homolog (ZDBF2) of the mouse Zdbf2 gene showed paternal allele-specific expression in human lymphocytes but not in the human placenta. Thus, our findings defined mouse chromosome 1 and human chromosome 2 as the loci for imprinted genes. [Copyright &y& Elsevier]

Abstract: Genomic imprinting is widely conserved amongst placental mammals. Imprinted expression of IGF2R, however, differs between mice and humans. In mice, Igf2r imprinted expression is seen in all fetal and adult tissues. In humans, adult tissues lack IGF2R imprinted expression, but it is found in fetal tissues and Wilms'' tumors where it is polymorphic and only seen in a small proportion of tested samples. Mouse Igf2r imprinted expression is controlled by the Air (Airn) ncRNA whose promoter lies in an intronic maternally-methylated CpG island. The human IGF2R gene carries a homologous intronic maternally-methylated CpG island of unknown function. Here, we use transfection and transgenic studies to show that the human IGF2R intronic CpG island is a ncRNA promoter. We also identify the same ncRNA at the endogenous human locus in 16–40% of Wilms'' tumors. Thus, the human IGF2R gene shows evolutionary conservation of key features that control imprinted expression in the mouse. [Copyright &y& Elsevier]

Abstract: Genetic imprinting may have played a more notable role in shaping embryonic development of plants, animals, and humans than previously appreciated. Quantitative trait loci that are imprinted (iQTL) exert monoallelic effects, depending on the parent of origin, which is an exception to the laws of Mendelian genetics. In this article, we present a modified random effect-based mapping model to use in a genome-wide scan for the distribution of iQTL that contribute to genetic variance for a complex trait in a structured pedigree. This model, implemented with the maximum likelihood method, capitalizes on a network of relatedness for maternally and paternally derived alleles through identical-by-descent sharing, thus allowing for the discrimination of the genetic variances due to alleles derived from maternal and paternal parents. The model was employed to map iQTL responsible for canine hip dysplasia in a multihierarchical canine pedigree, founded with seven greyhounds and six Labrador retrievers. Of eight significant QTL detected, three, located on CFA1, CFA8, and CF28, were found to trigger significant parent-of-origin effects on the age of femoral capital ossification measured at the left and right hips of a canine. The detected iQTL provide important candidate regions for fine-mapping of imprinted genes and for studying their structure and function in the control of complex traits. [Copyright &y& Elsevier]

Abstract: Using multiple mammalian genomic sequences, we have analyzed the evolution and imprinting of several genes located in the Peg3 domain, including Mim1 (approved name, Mimt1), Usp29, Zim3, and Zfp264. A series of comparative analyses shows that the overall genomic structure of this 500-kb imprinted domain has been well maintained throughout mammalian evolution but that several lineage-specific changes have also occurred in each species. In the bovine domain, Usp29 has lost its protein-coding capability, Zim3 has been duplicated, and the expression of Zfp264 has become biallelic in brain and testis, which differs from paternal expression of mouse Zfp264 in brain. In contrast, the two transcript genes of cow, Mim1 and Usp29, both lacking protein-coding capability, are still expressed mainly from the paternal allele, indicating the imprinting of these two genes in cow. The imprinting of Mim1 and Usp29 along with Peg3 is the most evolutionarily selected feature in this imprinted domain, suggesting significant function of these three genes, either as protein-coding or as untranslated transcript genes. [Copyright &y& Elsevier]

Abstract: Beckwith–Wiedemann syndrome (BWS) is a model human imprinting disorder resulting from altered activity of one or more genes in the 11p15.5 imprinted gene cluster. Approximately 20% of BWS cases have uniparental disomy (UPD) of chromosome 11. Such cases appear to result from mitotic recombination occurring in early embryogenesis and offer a rare opportunity to study mitotic recombination in nonneoplastic cells. We analyzed a cohort of 52 children with BWS and UPD using a panel of microsatellite markers for chromosome 11. All cases demonstrated mosaic paternal isodisomy, and IGF2 and H19 were included in the segment of UPD in all cases. However, the extent of segmental disomy was variable, with no evidence of clustering of the proximal UPD breakpoint. In most cases (92% of those informative) UPD did not involve 11q, but 4 patients demonstrated UPD for the whole of chromosome 11. In contrast to meiotic recombination, the mitotic recombination frequency did not decline near the centromere. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Imprinting control regions (ICRs) often harbor tandem arrays of transcription factor binding sites, as demonstrated by the identification of multiple YY1 binding sites within the ICRs of Peg3, Nespas, and Xist/Tsix domains. In the current study, we have sought to characterize possible roles for YY1 in transcriptional control and epigenetic modification of these imprinted domains. RNA interference-based knockdown experiments in Neuro2A cells resulted in overall transcriptional up-regulation of most of the imprinted genes within the Peg3 domain and also, concomitantly, caused significant loss in the DNA methylation of the Peg3 differentially methylated region. A similar overall and coordinated expression change was also observed for the imprinted genes of the Gnas domain: up-regulation of Nespas and down-regulation of Nesp and Gnasxl. YY1 knockdown also resulted in changes in the expression levels of Xist and Snrpn. These results support the idea that YY1 plays a major role, as a trans factor, in the control of these imprinted domains. [Copyright &y& Elsevier]

Abstract: Imprinting centers (IC) can be defined as cis-elements that are recognized in the germ line and are epigenetically modified to bring about the full imprinting program in a somatic cell. Two paternally expressed human genes, HYMAI and PLAGL1 (LOT1/ZAC), are located within human chromosome 6q24. Within this region lies a 1-kb CpG island that is differentially methylated in somatic cells, unmethylated in sperm, and methylated in mature oocytes in mice, characteristic features of an IC. Loss of methylation of the homologous region in humans is observed in patients with transient neonatal diabetes mellitus and hypermethylation is associated with a variety of cancers, suggesting that this region regulates the expression of one or more key genes in this region involved in these diseases. We now report that a transgene carrying the human HYMAI/PLAGL1 DMR was methylated in the correct parent-origin-specific manner in mice and this was sufficient to confer imprinted expression from the transgene. Therefore, we propose that this DMR functions as the IC for the HYMAI/PLAGL1 domain. [Copyright &y& Elsevier]

Abstract: The role of imprinting in shaping development has been ubiquitously observed in plants, animals, and humans. However, a statistical method that can detect and estimate the effects of imprinted quantitative trait loci (iQTL) over the genome has not been extensively developed. In this article, we propose a maximum likelihood approach for testing and estimating the imprinted effects of iQTL that contribute to variation in a quantitative trait. This approach, implemented with the EM algorithm, allows for a genome-wide scan for the existence of iQTL. This approach was used to reanalyze published data in an F2 family derived from the LG/S and SM/S mouse strains. Several iQTL that regulate the growth of body weight by expressing paternally inherited alleles were identified. Our approach provides a standard procedure for testing the statistical significance of iQTL involved in the genetic control of complex traits. [Copyright &y& Elsevier]

Abstract: Imprinted genes are expressed from only one allele in a parent-of-origin-specific manner. We here describe a systematic approach to identify novel imprinted genes using quantification of allele-specific expression by Pyrosequencing, a highly accurate method to detect allele-specific expression differences. Sixty-eight candidate imprinted transcripts mapping to known imprinted chromosomal regions were selected from a recent expression profiling study of uniparental mouse embryos and analyzed. Three novel imprinted transcripts encoding putative non-protein-coding RNAs were identified on the basis of parent-of-origin-specific monoallelic expression in E11.5 (C57BL/6 × Cast/Ei)F1 and informative (C57BL/6 × Cast/Ei) × C57BL/6 backcross embryos. In addition, four transcripts with preferential expression of a strain-specific allele were found. Intriguingly, a vast majority of the analyzed transcripts showed no imprinting-associated expression in F1 embryos. These data strengthen the view that a large fraction of nonimprinted genes is differentially expressed between parthenogenetic and androgenetic embryos and question the efficiency of expression profiling of uniparental embryos to identify novel imprinted genes. [Copyright &y& Elsevier]

Abstract: On pig chromosome 6, the SW71 microsatellite is located in the region corresponding to several quantitative trait loci (QTL), such as those for intramuscular fat content and for body weight at 4 weeks of age. The genomic sequence of approximately 909 kb was obtained from seven BAC clones encompassing the SW71 region corresponding to human 18q11.21–q11.22. By searching the NCBI GenBank using BLASTX and BLASTN, this 909-kb segment was found to contain eight genes, RAB31, TXNDC2, VAPA, APCDD1, NAPG, FAM38B, C18orf30, and C18orf58, and one putative gene (DN119777). The average G + C content in the sequence of this contig was 45.75% and 33 CpG islands were detected. CpG islands were scattered throughout the region in which most of the putative genes were located. Dense CpG islands of approximately 840 bp were observed, including within the 5′ UTR and exon 1 of the orthologs of the RAB31, VAPA, APCDD1, and NAPG genes. Comparative analysis of conserved segments of six species showed that K a/K s ratios of the TXNDC2 gene in collinear and rearranged segments were significantly different at 4.1 and 1.3, respectively. In conclusion, we demonstrated the genomic organization of pig chromosome 6, including the gene order surrounding SW71, which provides important information for comparative mapping. Moreover, the genes revealed in this study may be positional candidate genes associated with QTL on chromosome 6 that affect fat deposition in pigs. [Copyright &y& Elsevier]

Abstract: The paternal repression of several genes in human chromosome 11p15.5 (mouse chromosome 7) is associated with paternal expression of a transcript called KCNQ1OT1 (also known as LIT1). This long transcript originates from a promoter that resides in a CpG island in intron 10 of the KCNQ1 gene and runs in an antisense orientation to the direction of the coding KCNQ1 transcript. The CpG island is maternally methylated but paternally nonmethylated. The CpG island loses its maternal methylation in over 50% of cases of Beckwith–Wiedemann syndrome who lack uniparental disomy. This loss is usually accompanied by biallelic expression of the KCNQ1OT1 transcript. We have examined the methylation status of this CpG island in somatic cell hybrids and diploid lymphoblasts using Southern hybridization and bisulfite sequencing techniques. We find that the maternal copy of the CpG island is methylated at all CpGs examined within the CpG island and uniformly paternally unmethylated. In addition, in BWS patients who have lost methylation of the CpG island, this loss occurs throughout the CpG island. Finally, we find that there is a switch in methylation patterns outside the CpG island from maternal methylation within the island to predominantly paternal methylation at sites flanking the CpG island. [Copyright &y& Elsevier]

Maternal inheritance of targeted loss of function alleles encoding either the cyclin-dependent kinase inhibitor 1C (Cdkn1c) or the insulin-like growth factor 2 receptor (Igf2r) leads to fully penetrant perinatal lethality in C57BL/6J mice due to genomic imprinting. Here, we demonstrate that there is a marked enhancement in postnatal viability of F1 mice carrying either the ablated Igf2r (∼32%) or Cdkn1c (∼83%) when the paternal genome was derived from the inbred Mus musculus musculus CzechII/Ei strain. Genetic and molecular analyses indicated that the increased viability was not caused by relaxation of imprinted gene expression, but is the consequence of unidentified polygenic modifiers that are not imprinted. In the course of this study, restriction-site polymorphisms between 129S1 and CzechII/Ei in 21 imprinted and 14 biallelically expressed genes were identified. These polymorphisms may prove useful in determining the effects of different mutant backgrounds on genomic imprinting. [Copyright &y& Elsevier]

The human 11p15.5 region contains several maternally and paternally imprinted genes. Dysregulation of imprinting of some of these genes occurs in the Beckwith–Wiedemann syndrome and several tumors. Imprinting in this region is controlled by two imprinting control regions (ICR). ICR1 acts as an insulator that regulates the reciprocal imprinting of the IGF2 and H19 genes. A differentially methylated region in ICR2 regulates the expression of a long transcript called KCNQ1OT1. This paternally expressed transcript negatively regulates several paternally imprinted genes around ICR2. Biallelic expression of the KCNQ1OT1 transcript is the primary molecular defect in over 50% of cases of Beckwith–Wiedemann syndrome. To understand the role of KCNQ1OT1 in regulating ICR2 we characterized its promoter. The critical promoter is ∼300 bp and it is surrounded by inhibitory elements within the CpG island. The promoter activity is strongly inhibited by cytosine methylation in keeping with the finding that the inactive maternal promoter is methylated in vivo. We have identified the transcription start sites and four CCAAT boxes upstream of the 5′-most start site. Mutation of the CCAAT boxes produced impairment of promoter activity. Transfection and gel mobility shift experiments suggest that binding of the factor NF-Y to the CCAAT boxes is important for promoter activity. [Copyright &y& Elsevier]

The regulation of genomic imprinting, the allele-specific expression of an autosomal gene, is complex and poorly understood. Imprinted genes are organized in clusters, where cis-acting regulatory elements are believed to interact to control multiple genes. We have used BAC transgenesis in the mouse to begin to delineate the region of DNA required for proper expression and imprinting of the mouse Delta-like1 (Dlk1) and Gene-trap locus2 (Gtl2) imprinted genes. We demonstrate that the Gtl2 gene is expressed from a BAC transgene in mouse embryo and placenta only upon maternal inheritance, as is the endogenous Gtl2 gene. Gtl2 is therefore properly imprinted on the BAC in an ectopic chromosomal location and must carry with it all necessary imprinting regulatory elements. Furthermore, we show that the BAC Gtl2 gene is expressed at levels approaching those of the endogenous gene only in the brain of adult animals, not in other sites of endogenous expression such as the pituitary, adrenal, and skeletal muscle. These data localize the enhancer(s) for brain Gtl2 expression, but not those for other tissues, to the DNA contained within the BAC clone. As the Dlk1 gene is not expressed from the BAC in any tissues, it must require additional elements that are different from those necessary for Gtl2 expression. Our data refine the interval for future investigation of Gtl2 imprinting and provide evidence for distinct regulation of the linked Dlk1 and Gtl2 genes. [Copyright &y& Elsevier]

The human DIO3 gene and its mouse homolog, Dio3, map to chromosomes 14q32 and 12F1, respectively, and code for the type 3 deiodinase, an enzyme that inactivates thyroid hormones and is highly expressed during pregnancy and development. Mouse Dio3 is imprinted and preferentially expressed from the paternal allele in the fetus. We analyzed the human DIO3 genomic region and identified a gene (DIO3OS) that is transcribed in the antisense orientation. Multiple DIO3OS transcripts are expressed in most tissues. The structure of several DIO3OS cDNAs obtained by RT-PCR-based techniques reveals the occurrence of numerous splice variants. The exon–intron structures of DIO3OS are similar in mouse and human, but the homology of the exonic sequence is very low, except for the first exon, and no conserved open reading frame is present. We also detected DIO3 transcripts containing additional 5′ untranslated sequence and a potential alternative upstream promoter for mouse Dio3. Exonic sequence of a Dio3os cDNA overlaps with the Dio3 promoter and strong promoter activity in the antisense orientation is detected in a genomic fragment located 3′ of mouse and human DIO3 but not in the DIO3 promoter region. These results suggest that the DIO3 gene may lie within the structure of the antisense gene, a complex arrangement often observed in imprinted loci. [Copyright &y& Elsevier]

Insulin-like growth factor 2 receptor (Igf2r), a maternally expressed imprinted gene, is triplicated in Ts43H trisomic mice. To verify its expression in a trisomic mode, we examined the allele-specific transcripts and relative levels of Igf2r mRNA in trisomic and control mice. Igf2r was expressed from the maternal allele(s) and silenced on the paternal allele(s) in most tissues of maternally or paternally derived Ts43H mice. The triallelic expression was observed in adult brain and testis and in embryonic head, but the antisense transcript, Air, was strictly paternal, and methylation of region 2 was strictly maternal in all tissues. The Igf2r expression in maternally derived trisomics, with two active copies of the gene, did not exceed the average mRNA levels of euploid controls with monoallelic expression. Thus, an indication is presented for a dosage compensation mechanism of Igf2r in the trisomic context. [Copyright &y& Elsevier]

Human 11p15.5, as well as its orthologous mouse 7F4/F5, is known as the imprinting domain extending from IPL/Ipl to H19. OBPH1 and Obph1 are located beyond the presumed imprinting boundary on the IPL/Ipl side. We determined full-length cDNAs and complete genomic structures of both orthologues. We also investigated their precise imprinting and methylation status. The orthologues resembled each other in genomic structure and in the position of the 5′ CpG island and were expressed ubiquitously. OBPH1 and Obph1 were predominantly expressed from the maternal allele only in placenta, with hypo- and not differentially methylated 5′ CpG islands in both species. These results suggested that the imprinting domain would extend beyond the presumed imprinting boundary and that methylation of the 5′ CpG island was not associated with the imprinting status in either species. It remains to be elucidated whether the gene is under the control of the KIP2/LIT1 subdomain or is regulated by a specific mechanism. Analysis of the precise genomic sequence around the region should help resolve this question. [Copyright &y& Elsevier]

DNA methylation differences between maternal and paternal alleles of many imprinted genes are inherited from the male and female gametes and subsequently maintained during development. However, the stages of gametogenesis during which methylation imprints are established have not been well defined. In this study, we used bisulfite sequencing to determine the methylation dynamics of the imprinted genes small nuclear ribonucleoprotein N (Snrpn), insulin-like growth factor 2 receptor (Igf2r), mesoderm-specific transcript (Mest; formerly Peg1), paternally expressed gene 3 (Peg3), and H19 fetal liver mRNA (H19). We identified regions in the maternally imprinted genes (Snrpn, Mest, and Peg3) that were unmethylated in sperm but 100% methylated in mature oocytes. Igf2r, which is expressed from the maternal allele, was completely methylated within intronic differentially methylated region 2 in oocytes and unmethylated in sperm. The 5′ region of H19, a paternally imprinted gene, was completely methylated in sperm and unmethylated in oocytes. We examined the methylation status of Snrpn during oocyte growth and maturation. Whereas the DNA of non-growing oocytes was mostly unmethylated, mid-size growing oocytes had a mosaic pattern of allelic methylation, and full acquisition of the methylation imprint was complete by metaphase II. We have identified regions within imprinted genes that show gamete-specific methylation patterns in mature germ cells and demonstrated that maternal methylation imprints on at least one imprinted gene, Snrpn, are established during the postnatal growth phase of oogenesis. Thus, whereas paternal imprints seem to be established early (in diploid gonocytes well before the onset of meiosis), maternal imprints are established late (in growing oocytes that are arrested in the diplotene stage of meiosis). These findings raise the possibility that assisted reproductive technologies that involve in vitro maturation of oocytes may result in developmental abnormalities due to incomplete methylation imprints in immature oocytes. [Copyright &y& Elsevier]

We have identified a novel, maternally expressed imprinted gene encoding a C/D-box small nucleolar RNA (snoRNA) called MBII-343, which may regulate RNA editing or alternative splicing of an as yet unknown target gene. This gene is closely linked to an imprinted gene, Meg3, on mouse distal chromosome 12, which is syntenic to human chromosome 14. The paternal duplication of mouse distal chromosome 12 leads to late embryonal/neonatal lethality, growth promotion, and cardiomyopathy, whereas maternal duplication leads to late embryonal lethality and growth retardation. Human paternal uniparental disomy for chromosome 14 leads to musculoskeletal problems and mental retardation, whereas maternal uniparental disomy leads to intrauterine growth retardation, motor developmental delay, premature puberty, hypotonia, joint laxity, macrocephaly, short statue, neonatal poor sucking, skill with jigsaw puzzles, skin picking, obesity, and maturity onset diabetes of the young. [Copyright &y& Elsevier]