鲫Kaiso基因cDNA的克隆和表达分析

在爪蟾和斑马鱼中, Kaiso是一种在整个基因组范围内与甲基化CpG序列特异性结合的转录抑制因子, 在调控被甲基化基因表达的时间模式中起重要的作用。为深入研究DNA甲基化对我国重要养殖鱼类生殖和发育的影响, 我们克隆了鲫Kaiso基因的cDNA序列, 并对其时空表达模式进行了分析。该cDNA全长3145 bp, 5′-非翻译区132 bp, 3′-非翻译区1117 bp, 开放阅读框1896 bp, 编码631个氨基酸。鲫Kaiso蛋白与其他物种Kaiso蛋白的同源性分析表明, 与其他物种一样, 其 N端和C端分别有高保守性的BTB/POZ结构域和锌指结构域。整胚原位杂交结果显示, Kaiso mRNA在早期胚胎发育的各个时期均广泛表达, 信号均一, 但从尾芽期开始出现组织特异性表达差异。对不同发育阶段胚胎的实时定量PCR检测结果表明: 卵子中有高丰度的母源Kaiso mRNA存在; 在卵裂期至囊胚中期胚胎中Kaiso mRNA的丰度逐渐降低; 从囊胚中期至原肠早期都维持在最低水平状态; 原肠后期其表达水平又逐渐升高, 至尾芽期达到与未受精卵中相当的高水平后在器官发生期的整体水平又稍有下降。Kaiso mRNA丰度在胚胎发育早期的这种变化过程提示在卵裂期检测到的mRNA可能都是母源mRNA, 合子核Kaiso基因可能是在囊胚晚期后才开始转录。对成体不同组织的实时定量PCR检测结果表明Kaiso的表达存在明显的组织特异性差异, 在鲫肌肉、视网膜、心脏和脑中表达水平较高, 而在肾、胰、肝等器官中表达水平很低。Kaiso表达的时间和组织特异性提示其作为甲基化基因的转录抑制因子参与了胚胎和成体基因表达时空模式的调控。这些结果为进一步研究Kaiso和DNA甲基化修饰在鲫发育调控和遗传育种中的作用提供了基础资料。     

The oocyte-specific methylated region of the U2afbp-rs/U2af1-rs1 gene is dispensable for its imprinted methylation

Imprinted genes harbor discrete regions which are differentially methylated in gametes; usually the final differential methylation patterns in adults are established during embryogenesis through modifications of the initial methylation patterns in gametes. Previous reports have shown that a 200-bp region termed region II within the CpG island of the mouse imprinted U2afbp-rs gene is methylated in oocytes but not in sperm, suggesting that this region is a center for the propagation of methylated CpGs on the maternal allele and is also a candidate for an imprinting control element. To determine whether region II is required for the imprinted methylation of this gene at the endogenous locus, we generated mice carrying a deletion of this region. We herein show that parental methylation differences still exist in the CpG island on the region II-deleted allele. These findings suggest that region II is dispensable for the imprinted methylation of the U2afbp-rs gene.     

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.     

Genomic imprinting in mammals

A review of the data on the mechanisms and effects of genomic imprinting, an epigenetic phenomenon regulating the development in placentate mammals, is presented. In contrast to the majority of gene loci with biallelic expression, the expression of imprinted loci is monoallelic. In humans and mice, more than 300 imprinted loci have been identified, in which maternal or paternal alleles may either be expressed or be found in a repressed state during ontogeny. Imprinting is established during gametogenesis, and the repression of an allele of the imprinted locus is determined by methylation of the key regulatory element of this allele. Both the maternal and paternal chromosome sets are required for normal development in mammals. This is why parthenogenesis and androgenesis in these animals are impossible in nature. As a result of differential gene expression of many imprinted loci, the balance of gene activity is established, which is necessary for normal proliferation and differentiation of various cell clones in embryogenesis. Many human developmental abnormalities and syndromes are determined by defective genomic imprinting. In particular, the loss of imprints, which is followed by the occurrence of biallelic expression of some imprinted loci, may cause malignant tumors.     

Immunological detection of changes in genomic DNA methylation during early zebrafish development.

DNA methylation reprogramming, the erasure of DNA methylation patterns shortly after fertilization and their reestablishment during subsequent early development, is essential for proper mammalian embryogenesis. In contrast, the importance of this process in the development of non-mammalian vertebrates such as fish is less clear. Indeed, whether or not any widespread changes in DNA methylation occur at all during cleavage and blastula stages of fish in a fashion similar to that shown in mammals has remained controversial. Here we have addressed this issue by applying the techniques of Southwestern immunoblotting and immunohistochemistry with an anti-5-methylcytosine antibody to the examination of DNA methylation in early zebrafish embryos. These techniques have recently been utilized to demonstrate that development-specific changes in genomic DNA methylation also occur in Drosophila melanogaster and Dictyostelium discoideum, both organisms for which DNA methylation was previously not thought to occur. Our data demonstrate that genome-wide changes in DNA methylation occur during early zebrafish development. Although zebrafish sperm DNA is strongly methylated, the zebrafish genome is not detectably methylated through cleavage and early blastula stages but is heavily remethylated in blastula and early gastrula stages.     

Histone H1 affects gene imprinting and DNA methylation in Arabidopsis

Imprinting, i.e. parent-of-origin expression of alleles, plays an important role in regulating development in mammals and plants. DNA methylation catalyzed by DNA methyltransferases plays a pivotal role in regulating imprinting by silencing parental alleles. DEMETER (DME), a DNA glycosylase functioning in the base-excision DNA repair pathway, can excise 5-methylcytosine from DNA and regulate genomic imprinting in Arabidopsis. DME demethylates the maternal MEDEA (MEA) promoter in endosperm, resulting in expression of the maternal MEA allele. However, it is not known whether DME interacts with other proteins in regulating gene imprinting. Here we report the identification of histone H1.2 as a DME-interacting protein in a yeast two-hybrid screen, and confirmation of their interaction by the in vitro pull-down assay. Genetic analysis of the loss-of-function histone h1 mutant showed that the maternal histone H1 allele is required for DME regulation of MEA, FWA and FIS2 imprinting in Arabidopsis endosperm but the paternal allele is dispensable. Furthermore, we show that mutations in histone H1 result in an increase of DNA methylation in the maternal MEA and FWA promoter in endosperm. Our results suggest that histone H1 is involved in DME-mediated DNA methylation and gene regulation at imprinted loci.     

Parental-specific methylation of an imprinted transgene is established during gametogenesis and progressively changes during embryogenesis.

Genomic imprinting is a regulatory process that requires a cell to recognize the parental origin of alleles. To understand how these alleles are distinguished, we have assessed changes in the DNA methylation of an imprinted transgene as it switches from one inheritance pattern to another while moving through gametogenesis and embryogenesis. We find that both maternally and paternally inherited methylation patterns are erased in primordial germ cells and that distinctive patterns emerge during germ cell maturation. In the case of the maternal allele, the methylation pattern is fully acquired during oogenesis. In the case of the paternal allele, the methylation pattern found in sperm undergoes further modification during embryogenesis. Thus, the distinction between "erased" maternal and paternal alleles is first established during their residence in different germ cells and then may be maintained by the recognition of the distinctive patterns that each allele displays in the zygote.     

Genomic Imprinting in Mammals

A review of the data on the mechanisms and effects of genomic imprinting, an epigenetic phenomenon regulating the development in placentate mammals, is presented. In contrast to the majority of gene loci with biallelic expression, the expression of imprinted loci is monoallelic. In humans and mice, more than 30 imprinted loci have been identified, in which maternal or paternal alleles may either be expressed or be found in a repressed state during ontogeny. Imprinting is established during gametogenesis, and the repression of an allele of the imprinted locus is determined by methylation of the key regulatory element of this allele. Both the maternal and paternal chromosome sets are required for normal development in mammals. This is why parthenogenesis and androgenesis in these animals are impossible in nature. As a result of differential gene expression of many imprinted loci, the balance of gene activity is established, which is necessary for normal proliferation and differentiation of various cell clones in embryogenesis. Many human developmental abnormalities and syndromes are determined by defective genomic imprinting. In particular, the loss of imprints, which is followed by the occurrence of biallelic expression of some imprinted loci, may cause malignant tumors.     

Potential for parthenogenesis of virgin females in a bisexual population of the geographically parthenogenetic mayfly Ephoron shigae (Insecta: Ephemeroptera,Polymitarcyidae)

The burrowing polymitarcyid mayfly Ephoron shigae is a geographically parthenogenetic species. Interestingly, the distributions of the bisexual and unisexual populations overlap broadly in their respective geographic ranges. In this mayfly, obligatory diploid thelytoky appears within unisexual populations. In the present study, we examined the potential for parthenogenesis or the parthenogenetic ability of females in a bisexual population aiming to understand the emergence of unisexual populations. The results obtained revealed that females in the examined bisexual populations showed a potential for diploid thelytoky as also seen in the unisexual populations, although, in females from bisexual populations, the development success rates of their unfertilized eggs were considerably lower than those of virgin females from unisexual populations. In the three bisexual reproducing species (Ephemera japonica, Ephemera strigata, and Ephemera orientalis) in the closely‐related family Ephemeridae, diploid thelytoky (i.e. tychoparthenogenesis; < 3%) was also observed. However, in this case, the parthenogenetic development success rates of unfertilized eggs were significantly lower than those of virgin females in the bisexual (Hino‐yosui Irrigation Canal) population of E. shigae. Accordingly, we suggest that parthenogenetic ability (i.e. tychoparthenogenesis or facultative parthenogenesis) in bisexual populations of E. shigae may facilitate the evolutionary transition to unisexual populations with fully obligatory parthenogenesis. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 326–334.     

MIRA-SNuPE,a quantitative,multiplex method for measuring allele-specific DNA methylation

5-methyl-C (5mC) and 5-hydroxymethyl-C (5hmC) are epigenetic marks with well-known and putative roles in gene regulation, respectively. These two DNA covalent modifications cannot be distinguished by bisulfite sequencing or restriction digestion, the standard methods of 5mC detection. The methylated CpG island recovery assay (MIRA), however, specifically detects 5mC but not 5hmC. We further developed MIRA for the analysis of allele-specific CpG methylation at differentially methylated regions (DMRs) of imprinted genes. MIRA specifically distinguished between the parental alleles by capturing the paternally methylated H19/Igf2 DMR and maternally methylated KvDMR1 in mouse embryo fibroblasts (MEFs) carrying paternal and maternal duplication of mouse distal Chr7, respectively. MIRA in combination with multiplex single nucleotide primer extension (SNuPE) assays specifically captured the methylated parental allele from normal cells at a set of maternally and paternally methylated DMRs. The assay correctly recognized aberrant biallelic methylation in a case of loss of imprinting. The MIRA-SNuPE assays revealed that placenta exhibited less DNA methylation bias at DMRs compared to yolk sac, amnion, brain, heart, kidney, liver and muscle. This method should be useful for the analysis of allele-specific methylation events related to genomic imprinting, X chromosome inactivation and for verifying and screening haplotype-associated methylation differences in the human population.Key words: epigenetics, imprinting, DMR, MIRA, MBD, DNA methylation, SNuPE     

Rasgrf1 imprinting is regulated by a CTCF-dependent methylation-sensitive enhancer blocker

Imprinted methylation of the paternal Rasgrf1 allele in mice occurs at a differentially methylated domain (DMD) 30 kbp 5' of the promoter. A repeated sequence 3' of the DMD regulates imprinted methylation, which is required for imprinted expression. Here we identify the mechanism by which methylation controls imprinting. The DMD is an enhancer blocker that binds CTCF in a methylation-sensitive manner. CTCF bound to the unmethylated maternal allele silences expression. CTCF binding to the paternal allele is prevented by repeat-mediated methylation, allowing expression. Optimal in vitro enhancer-blocking activity requires CTCF binding sites. The enhancer blocker can be bypassed in vivo and imprinting abolished by placing an extra enhancer proximal to the promoter. Together, the repeats and the DMD constitute a binary switch that regulates Rasgrf1 imprinting.     

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.     

A novel imprinted transgene located near a repetitive element that exhibits allelic imbalance in DNA methylation during early development

A mouse line carrying a lacZ transgene driven by the human EEF1A1/EF1alpha promoter was established. Although the promoter is known to show ubiquitous activity, only paternal transgene alleles were expressed, resulting in a transgene imprinting. At mid‐gestation, the promoter sequence was differentially methylated, hypomethylated for paternal and hypermethylated for maternal alleles. In germline, the promoter was a typical differentially methylated region. After fertilization, however, both alleles were hypermethylated. Thus, the differential methylation of the promoter required for transgene imprinting was re‐established during later embryonic development independently of the germline differential methylation. Furthermore, also a retroelement promoter closely‐flanking imprinted transgene and its wild type counterpart displayed similar differential methylation during early development. The retroelement promoter was methylated differentially also in germline, but in an opposite pattern to the embryonic differential methylation. These results suggest that there might be an unknown epigenetic regulation inducing transgene imprinting independently of DNA methylation in the transgene insertion site. Then, besides CpG dinucleotides, non‐CpG cytosines of the retroelement promoter were highly methylated especially in the transgene‐active mid‐gestational embryos, suggesting that an unusual epigenetic regulation might protect the active transgene against de novo methylation occurring generally in mid‐gestational embryo.     

Identification of a methylation imprint mark within the mouse Gnas locus

The imprinted mouse gene Gnas produces the G protein alpha-subunit G(S)alpha and several other gene products by using alternative promoters and first exons. G(S)alpha is maternally expressed in some tissues and biallelically expressed in most other tissues, while the gene products NESP55 and XLalphas are maternally and paternally expressed, respectively. We investigated the mechanisms of Gnas imprinting. The G(S)alpha promoter and first exon are not methylated on either allele. A further upstream region (approximately from positions -3400 to -939 relative to the G(S)alpha translational start site) is methylated only on the maternal allele in all adult somatic tissues and in early postimplantation development. Within this region lies a fourth promoter and first exon (exon 1A) that generates paternal-specific mRNAs of unknown function. Exon 1A and G(S)alpha mRNAs have similar expression patterns, making competition between their promoters unlikely. Differential methylation in this region is established during gametogenesis, being present in oocytes and absent in spermatozoa, and is maintained in preimplantation E3. 5d blastocysts. Therefore, this region is a methylation imprint mark. In contrast, differential methylation of the NESP55 and XLalphas promoter regions (Nesp and Gnasxl) is not established during gametogenesis. The methylation imprint mark that we identified may be important for the tissue-specific imprinting of G(S)alpha.     

Diploid parthenogenetic embryos adopt a maternal-type methylation pattern on both sets of maternal chromosomes

Epigenetic modifications are closely associated with embryo developmental potential. One of the epigenetic modifications thought to be involved in genomic imprinting is DNA methylation. Here we show that the maternally imprinted genes Snrpn and Peg1/Mest were nearly unmethylated or heavily methylated, respectively, in their differentially methylated regions (DMRs) at the two-cell stage in parthenogenetic embryos. However, both genes were gradually de novo methylated, with almost complete methylation of all CpG sites by the morula stage in parthenogenetic embryos. Unexpectedly, another maternally imprinted gene, Peg3, showed distinct dynamics of methylation during preimplantation development of diploid parthenogenetic embryos. Peg3 showed seemingly normal methylation patterns at the two-cell and morula stages, but was also strongly de novo methylated in parthenogenetic blastocysts. In contrast, the paternally imprinted genes H19 and Rasgrf1 showed complete unmethylation of their DMRs at the morula stage in parthenogenetic embryos. These results indicate that diploid parthenogenetic embryos adopt a maternal-type methylation pattern on both sets of maternal chromosomes and that the aberrantly homogeneous status of methylation imprints may partially account for developmental failure.     

Methylation status of differentially methylated regions at Igf2/H19 locus in porcine gametes and preimplantation embryos

The aim of this study was to demonstrate how differential methylation imprints are established during porcine preimplantation embryo development. For the methylation analysis, the primers for the three Igf2/H19 DMRs were designed and based upon previously published sequences. The methylation marks of Igf2/H19 DMRs were analysed in sperm and MII oocytes with our results showing that these regions are fully methylated in sperm but remain unmethylated in MII oocytes. In order to identify the methylation pattern at the pronuclear stage, we indirectly compared the methylation profile of Igf2/H19 DMR3 in each zygote derived by in vitro fertilization, parthenogenesis, and androgenesis. Interestingly, this region was found to be differently methylated according to parental origins; DMR3 was hemimethylated in in vitro fertilized zygotes, fully methylated in parthenogenetic zygotes, and demethylated in androgenetic zygotes. These results indicate that the methylation mark of the paternal allele is erased by active demethylation, and that of the maternal one is de novo methylated. We further examined the methylation imprints of Igf2/H19 DMR3 during early embryonic development. The hemimethylated pattern as seen in zygotes fertilized in vitro was observed up to the 4-cell embryo stage. However, this mark was exclusively demethylated at the 8-cell stage and then restored at the morula stage. These results suggest that methylation imprints are established via dynamic changes during early embryonic development in porcine embryos.