Genome demethylation and imprinting in the endosperm

Imprinting occurs in the endosperm of flowering plants. The endosperm, a product of central cell fertilization, is critical for embryo and seed development. Imprinting in the endosperm is mainly due to the inherited differences in gamete epigenetic composition. Studies have also shown that there are differences in genomic DNA methylation patterns between embryo and endosperm. Examining those differences, along with mutations in the DNA demethylase gene DEMETER, gives insight into the number of imprinted genes and how an antagonistic relationship between TE defense and gene regulation could evolutionarily affect imprinting establishment. Finally, studies demonstrate that DEMETER demethylase activity influences endosperm chromatin composition, and could possibly enhance DNA de novo methylation activity.     

Methylation of type II and type I collagen genes in differentiated and dedifferentiated chondrocytes

The methyl-sensitive restriction endonucleases HpaII and HhaI as well as the methyl-insensitive enzyme MspI were used to examine the methylation status of the pro-alpha 1(II) collagen gene of cartilage. Five different cell types with varying abilities to express type II collagen were studied. Chick embryo chondrocytes express type II collagen, while 5-bromodeoxyuridine-treated chondrocytes, retinoic acid-treated chondrocytes, chick embryo fibroblasts, and erythrocytes do not synthesize type II collagen. Both cDNA and genomic probes for the pro-alpha 1(II) collagen gene were used, covering the complete 3' end of the gene and its flanking sequences. The pro-alpha 1(II) collagen DNA was undermethylated in chondrocytes, compared to either fibroblasts or erythrocytes. However, the methylation of the 5-bromodeoxyuridine-treated and retinoic acid-treated chondrocytes was identical to that of control chondrocytes. The methylation pattern of two regions of the gene of the pro-alpha 2(I) collagen chain was identical in all cell types tested, whether or not the gene was expressed. Our results indicate that genes for these collagen chains differ in their methylation pattern. The type II collagen gene shows reduced methylation in expressing cartilage, but does not acquire an increase in methylation in "dedifferentiated" chondrocytes. The changes in DNA methylation that occur during cell differentiation do not appear to be sufficient to explain gene activation and deactivation.     

橡胶树体细胞胚发生过程中DNA甲基化分析

为探讨巴西橡胶树(Hevea brasiliensis)自根幼态无性系与供体间差异产生的原因,应用甲基化敏感扩增多态性扩增技术,对巴西橡胶树体细胞胚发生过程中基因组DNA 胞嘧啶甲基化程度和模式进行了分析。结果表明,在巴西橡胶树体细胞胚发生过程中不同阶段的DNA 甲基化程度不同,以花药的DNA 甲基化程度最高,体细胞胚的DNA 甲基化水平最低。在体细胞胚发生过程中出现了I、Ⅱ和Ⅲ 3 种类型的甲基化多态性带型的改变,包括他们的出现与消失。因此,橡胶树体细胞胚发生过程中可能通过DNA 甲基化甲基化和去甲基化来调控基因的表达。     

Allele-Specific Non-CpG Methylation of the Nf1 Gene during Early Mouse Development

Recent reports of cytosine methylation occurring at CpA and CpT dinucleotides in murine ES cells as well as in Drosophila have renewed interest in methylation at sites other than CpGs. Our examination of the murine neurofibromatosis type 1 gene by sodium bisulfite genomic sequencing has revealed non-CpG methylation primarily in the oocyte and the maternally derived allele of the 2-cell embryo, with markedly lower levels found in sperm. Non-CpG methylation was not found in later stages of embryo development or in adult tissue. Our results suggest that maternal-specific de novo non-CpG methylation has occurred sometime between ovulation and formation of the 2-cell embryo, while during the same period the paternally derived allele has undergone site-specific active demethylation. Our data demonstrate both stage and parent-of-origin specific changes in methylation patterns within the neurofibromatosis type 1 coding region-involving cytosines located at both CpG and non-CpG dinucleotides.     

Methylation patterns of the human apoA-I/C-III/A-IV gene cluster in adult and embryonic tissues suggest dynamic changes in methylation during development.

We describe here a detailed analysis of the methylation patterns of the apoC-III and apoA-IV genes in adult and embryonic tissues. Together with previously reported data on the human apoA-I gene (4), the results presented here constitute a comprehensive study on the methylation pattern of the apoA-I/C-III/A-IV gene cluster. The two genes (apoC-III and apoA-IV) display tissue-specific methylation patterns that correlate with their activity. This gene-specific methylation pattern indicates that the apoA-I/C-III/A-IV gene cluster is not one entity with respect to methylation. The cluster is almost entirely methylated in tissues that do not express any of the genes; however, individual gene regions are unmethylated in the tissue of expression. A comparison of the observed methylation patterns in adult tissues with those in embryonic tissues suggests that the mature tissue-specific methylation patterns are a result of an interplay between demethylation and de novo methylation events in the embryo. These changes in DNA methylation include demethylation in the early embryo followed by de novo methylation at later stages. A second round of tissue-specific demethylation and methylation de novo occurs in the late embryo as well. Evidence presented here supports the idea that CpG islands are protected in general from methylation de novo by a built-in signal and not by CpG density per se.     

Subcellular localization of glutelin-2 in maize (Zea mays L.) endosperm

Accumulation of the 28 KD protein of the glutelin-(G2) fraction was followed in developing maize endosperm, using sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and peak integration of scanned gels. 28 KD glutelin-2 could already be observed from 15 days after pollination and its accumulates reached a plateau during the second half of the development period. The process of biosynthesis of 28 KD glutelin-2 and zeins occurs in a parallel way. Subcellular fractions obtained from linear sucrose gradient centrifugation of developing maize endosperms were analyzed by SDS-PAGE and immunoblotting using a serum reacting against glutelin-2 and 14 KD Z2. Glutelin-2 was found to be present in the protein bodies when subcellular fractionation was carried out without dithiothreitol (DTT). The presence of a reducing agent causes the elution of glutelin-2 from protein bodies. Immunocytochemical labelling using the protein A-colloidal gold technique in protein bodies incubated with anti-G2 IgG revealed that G2 is located mainly in the periphery of protein bodies. These results are interpreted as indicating a structural role for glutelins in protein bodies.     

Nucleic acid (cDNA) and amino acid sequences of the maize endosperm protein glutelin-2.

The cDNA coding for a glutelin-2 protein from maize endosperm has been cloned and the complete amino acid sequence of the protein derived for the first time. An immature maize endosperm cDNA bank was screened for the expression of a beta-lactamase:glutelin-2 (G2) fusion polypeptide by using antibodies against the purified 28 kd G2 protein. A clone corresponding to the 28 kd G2 protein was sequenced and the primary structure of this protein was derived. Five regions can be defined in the protein sequence: an 11 residue N-terminal part, a repeated region formed by eight units of the sequence Pro-Pro-Pro-Val-His-Leu, an alternating Pro-X stretch 21 residues long, a Cys rich domain and a C-terminal part rich in Gln. The protein sequence is preceded by 19 residues which have the characteristics of the signal peptide found in secreted proteins. Unlike zeins, the main maize storage proteins, 28 kd glutelin-2 has several homologous sequences in common with other cereal storage proteins.     

Tissue-specific differences in cytosine methylation and their association with differential gene expression in sorghum

It has been well established that DNA cytosine methylation plays essential regulatory roles in imprinting gene expression in endosperm, and hence normal embryonic development, in the model plant Arabidopsis (Arabidopsis thaliana). Nonetheless, the developmental role of this epigenetic marker in cereal crops remains largely unexplored. Here, we report for sorghum (Sorghum bicolor) differences in relative cytosine methylation levels and patterns at 5'-CCGG sites in seven tissues (endosperm, embryo, leaf, root, young inflorescence, anther, and ovary), and characterize a set of tissue-specific differentially methylated regions (TDMRs). We found that the most enriched TDMRs in sorghum are specific for the endosperm and are generated concomitantly but imbalanced by decrease versus increase in cytosine methylation at multiple 5'-CCGG sites across the genome. This leads to more extensive demethylation in the endosperm than in other tissues, where TDMRs are mainly tissue nonspecific rather than specific to a particular tissue. Accordingly, relative to endosperm, the other six tissues showed grossly similar levels though distinct patterns of cytosine methylation, presumably as a result of a similar extent of concomitant decrease versus increase in cytosine methylation that occurred at variable genomic loci. All four tested TDMRs were validated by bisulfite genomic sequencing. Diverse sequences were found to underlie the TDMRs, including those encoding various known-function or predicted proteins, transposable elements, and those bearing homology to putative imprinted genes in maize (Zea mays). We further found that the expression pattern of at least some genic TDMRs was correlated with its tissue-specific methylation state, implicating a developmental role of DNA methylation in regulating tissue-specific or -preferential gene expression in sorghum.     

Inherited type of allelic methylation variations in a mouse chromosome region where an integrated transgene shows methylation imprinting.

It is still unclear whether or not parent-of-origin-dependent differential methylation observed in some transgenes reflects genomic imprinting of endogenous genes. We have characterized a transgene locus showing such methylation imprinting together with the corresponding native chromosome region. We show that only part of the transgene is affected by methylation imprinting and the methylation pattern is established before early prophase I during spermatogenesis. Interestingly, the native genomic region, which is mapped to the proximal chromosome 11, shows no evidence of methylation imprinting but displays heritable, strain-specific type of allelic methylation differences. The results demonstrate that transgenes do not necessarily reflect the methylation status of either the surrounding or corresponding chromosome region. In addition, inherited type of allelic methylation variations previously described in human may be widespread in mammals.     

Comparison of gene expression and genome-wide DNA methylation profiling between phenotypically normal cloned pigs and conventionally bred controls

Animal breeding via Somatic Cell Nuclear Transfer (SCNT) has enormous potential in agriculture and biomedicine. However, concerns about whether SCNT animals are as healthy or epigenetically normal as conventionally bred ones are raised as the efficiency of cloning by SCNT is much lower than natural breeding or In-vitro fertilization (IVF). Thus, we have conducted a genome-wide gene expression and DNA methylation profiling between phenotypically normal cloned pigs and control pigs in two tissues (muscle and liver), using Affymetrix Porcine expression array as well as modified methylation-specific digital karyotyping (MMSDK) and Solexa sequencing technology. Typical tissue-specific differences with respect to both gene expression and DNA methylation were observed in muscle and liver from cloned as well as control pigs. Gene expression profiles were highly similar between cloned pigs and controls, though a small set of genes showed altered expression. Cloned pigs presented a more different pattern of DNA methylation in unique sequences in both tissues. Especially a small set of genomic sites had different DNA methylation status with a trend towards slightly increased methylation levels in cloned pigs. Molecular network analysis of the genes that contained such differential methylation loci revealed a significant network related to tissue development. In conclusion, our study showed that phenotypically normal cloned pigs were highly similar with normal breeding pigs in their gene expression, but moderate alteration in DNA methylation aspects still exists, especially in certain unique genomic regions.     

Dynamics of DNA Methylation in Recent Human and Great Ape Evolution

DNA methylation is an epigenetic modification involved in regulatory processes such as cell differentiation during development, X-chromosome inactivation, genomic imprinting and susceptibility to complex disease. However, the dynamics of DNA methylation changes between humans and their closest relatives are still poorly understood. We performed a comparative analysis of CpG methylation patterns between 9 humans and 23 primate samples including all species of great apes (chimpanzee, bonobo, gorilla and orangutan) using Illumina Methylation450 bead arrays. Our analysis identified ∼800 genes with significantly altered methylation patterns among the great apes, including ∼170 genes with a methylation pattern unique to human. Some of these are known to be involved in developmental and neurological features, suggesting that epigenetic changes have been frequent during recent human and primate evolution. We identified a significant positive relationship between the rate of coding variation and alterations of methylation at the promoter level, indicative of co-occurrence between evolution of protein sequence and gene regulation. In contrast, and supporting the idea that many phenotypic differences between humans and great apes are not due to amino acid differences, our analysis also identified 184 genes that are perfectly conserved at protein level between human and chimpanzee, yet show significant epigenetic differences between these two species. We conclude that epigenetic alterations are an important force during primate evolution and have been under-explored in evolutionary comparative genomics.     

In vitro analysis of integrated global high-resolution DNA methylation profiling with genomic imbalance and gene expression in osteosarcoma

Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks.     

The Effects of Hormone Treatment on Epigenetic Marks During Organogenesis in <Emphasis Type="Italic">Pinus radiata</Emphasis> D. Don Embryos

Adventitious organogenesis from zygotic embryos is one of the most reliable techniques to propagate clonal Pinus radiata individuals. Regulation of gene expression has an important role to generate a correct caulogenic pattern, and genomic DNA methylation is amongst the main studied regulatory mechanisms in animals and plants. The aim of this work is to evaluate the role of DNA methylation and histone H4 acetylation during different cell reprogramming processes. Embryo macromorphological responses to each growth regulator were correlated to DNA methylation and acetylated H4 histone content over time. Immunodetection of 5-mdC and AcH4 in whole embryos revealed differences in the tissue depending on the growth regulator. Adventitious shoot induction by benzyladenine is associated with a DNA hypomethylation. The distribution along the tissue of the methylation signal suggests a possible inhibition of growth and elongation in the embryos. However, a hypomethylation in the cotyledons during adventitious shoot induction could indicate an active gene expression during the organogenic process. On the other hand, alteration of embryo growth induced by indole butyric acid and 2,4-dichlorophenoxyacetic acid occurs along a wide change in DNA methylation and H4 histone acetylation content and distribution. These results show that the action of the different growth regulators is epigenetically regulated in both apical meristems.     

Tissue-specific methylation patterns and expression of the human apolipoprotein AI gene.

To better understand the tissue-specific expression of the human apolipoprotein (apo)AI gene, we performed a detailed analysis of the pattern of methylation of the gene in various human adult and embryonic tissues and in tissues of transgenic mice harboring the human apo-AI gene. In addition, the gene was analyzed also in liver and intestine-derived human cell lines (HepG2 and Caco2, respectively). Using methyl-sensitive restriction enzymes (HpaII, HhaI, and SmaI) and the appropriate radioactive probes, we were able to determine separately the status of methylation of the 5'-end, the body of the gene, and 3'-end flanking sequences. The apo-AI gene in tissues that express the gene was undermethylated at the 5'-end. However, the 5'-end of the gene in sperm and in all adult tissues that do not express the gene was heavily methylated. The body of the gene which contains a CpG island and the 3'-end flanking sequences were, in general, hypomethylated except for specific sites that showed partial methylation. In contrast, while the gene showed tissue-specific expression already in a 12-week-old embryo, the 5'-end was invariably hypomethylated in all tissues of the embryo. A human apo-AI transgene has recently been shown to be active exclusively in the liver, while the endogenous gene is expressed in both liver and intestine (6). We show here that the 5'-end of the apo-AI transgene was methylated in all tissues of the mouse (including intestine) except liver. The results presented here demonstrate a clear correlation between hypomethylation of the 5'-end and activity of the apo-AI gene. However, the observed methylation pattern of the gene in embryonic tissues suggests that tissue-specific expression precedes formation of the tissue-specific methylation pattern.     

DNA methylation at mammalian replication origins.

In Escherichia coli, DNA methylation regulates both origin usage and the time required to reassemble prereplication complexes at replication origins. In mammals, at least three replication origins are associated with a high density cluster of methylated CpG dinucleotides, and others whose methylation status has not yet been characterized have the potential to exhibit a similar DNA methylation pattern. One of these origins is found within the approximately 2-kilobase pair region upstream of the human c-myc gene that contains 86 CpGs. Application of the bisulfite method for detecting 5-methylcytosines at specific DNA sequences revealed that this region was not methylated in either total genomic DNA or newly synthesized DNA. Therefore, DNA methylation is not a universal component of mammalian replication origins. To determine whether or not DNA methylation plays a role in regulating the activity of origins that are methylated, the rate of remethylation and the effect of hypomethylation were determined at origin beta (ori-beta), downstream of the hamster DHFR gene. Remethylation at ori-beta did not begin until approximately 500 base pairs of DNA was synthesized, but it was then completed by the time that 4 kilobase pairs of DNA was synthesized (<3 min after release into S phase). Thus, DNA methylation cannot play a significant role in regulating reassembly of prereplication complexes in mammalian cells, as it does in E. coli. To determine whether or not DNA methylation plays any role in origin activity, hypomethylated hamster cells were examined for ori-beta activity. Cells that were >50% reduced in methylation at ori-beta no longer selectively activated ori-beta. Therefore, at some loci, DNA methylation either directly or indirectly determines where replication begins.