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Human Molecular Genetics Advance Access originally published online on July 21, 2005
Human Molecular Genetics 2005 14(17):2511-2520; doi:10.1093/hmg/ddi255
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

Neuron-specific relaxation of Igf2r imprinting is associated with neuron-specific histone modifications and lack of its antisense transcript Air

Yoko Yamasaki1,2,6,{dagger}, Tomohiko Kayashima1,{dagger}, Hidenobu Soejima4, Akira Kinoshita1,6, Ko-ichiro Yoshiura1,6, Naomichi Matsumoto1,6, Tohru Ohta3,6, Takeshi Urano5, Hideaki Masuzaki2, Tadayuki Ishimaru2, Tsunehiro Mukai4, Norio Niikawa1,6 and Tatsuya Kishino3,6,*

1Department of Human Genetics, 2Department of Obstetrics and Gynecology, Graduate School of Biomedical Sciences, 3Division of Functional Genomics, Center for Frontier Life Sciences, Nagasaki University, Nagasaki 852-8523, Japan, 4Department of Biomolecular Sciences, Saga University School of Medicine, Saga 849-8501, Japan, 5Department of Biochemistry II, Graduate School of Medicine, Nagoya University, Nagoya 466-8550, Japan and 6CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan

* To whom correspondence should be addressed at: Division of Functional Genomics, Center for Frontier Life Sciences, Nagasaki University, Sakamoto 1-12-4, Nagasaki 852-8523, Japan. Tel: +81 958497120; Fax: +81 958497178; Email: kishino{at}net.nagasaki-u.ac.jp

Received May 11, 2005; Accepted July 8, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The mouse insulin-like growth factor II receptor (Igf2r) gene and its antisense transcript Air are reciprocally imprinted in most tissues, but in the brain, Igf2r is biallelically expressed despite the imprinted Air expression. To investigate the molecular mechanisms of such brain-specific relaxation of Igf2r imprinting, we analyzed its expression and epigenetic modifications in neurons, glial cells and fibroblasts by the use of primary cortical cell cultures. In glial cells and fibroblasts, Igf2r was maternally expressed and Air was paternally expressed, whereas in the primary cultured neurons, Igf2r was biallelically expressed and Air was not expressed. In the differentially methylated region 2 (DMR2), which includes the Air promoter, allele-specific DNA methylation, differential H3 and H4 acetylation and H3K4 and K9 di-methylation were maintained in each cultured cell type. In DMR1, which includes the Igf2r promoter, maternal-allele-specific DNA hypomethylation, histones H3 and H4 acetylation and H3K4 di-methylation were apparent in glial cells and fibroblasts. However, in neurons, biallelic DNA hypomethylation and biallelic histones H3 and H4 acetylation and H3K4 di-methylation were detected. These data indicate that lack of reciprocal imprinting of Igf2r and Air in the brain results from neuron-specific relaxation of Igf2r imprinting associated with neuron-specific histone modifications in DMR1 and lack of Air expression. Our observation of biallelic Igf2r expression with no Air expression in neurons sheds light on the function of Air as a critical effector in Igf2r silencing and suggests that neuron-specific epigenetic modifications related to the lineage determination of neural stem cells play a critical role in controlling imprinting by antisense transcripts.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Genomic imprinting is a mode of gene regulation in which two parental alleles are differentially expressed. Most genes are expressed equally from both parental alleles, whereas imprinted genes are expressed exclusively or preferentially from either the paternal or maternal allele. The allelic expression of imprinted genes is dependent on whether the allele is inherited from the egg or sperm, because differential epigenetic marking occurs during gametogenesis (1Go–3Go).

The mouse insulin-like growth factor II receptor (Igf2r) gene is one of the most intensively analyzed genes showing tissue-specific imprinting (4Go). The gene encodes two reciprocally imprinted transcripts, sense Igf2r RNA and the non-coding Air RNA that overlaps the Igf2r promoter in antisense orientation and extends 79 kb upstream (5Go). In somatic tissues other than brain, the sense Igf2r is exclusively expressed from the maternal allele, whereas the antisense Air RNA is expressed from the paternal allele. However, in neonatal and adult brains, Igf2r escapes imprinting; expression of Igf2r is detected from both parental alleles and expression of Air is detected only from the paternal allele (6Go). The expression of Igf2r in somatic tissues is associated with differentially methylated regions (DMRs) on the two parental chromosomes. The first region (DMR1) includes the Igf2r promoter, and the second (DMR2) is located in intron 2 of Igf2r, encompassing the Air promoter. DMR2 acquires its maternal methylation during oogenesis and the differential methylation persists in all somatic tissues, whereas DMR1 acquires paternal methylation after fertilization (7Go,8Go). DMR1 is paternally methylated in most somatic tissues, but is biallelically hypomethylated in neonatal and adult brains, where Igf2r is biallelically expressed despite the imprinted Air expression. Thus, parental expression of Igf2r correlates with DMR1 CpG methylation rather than with Air transcript in the brain (4Go,6Go), suggesting that DNA methylation of the Igf2r promoter rather than the antisense transcript Air may be a direct effector of Igf2r imprinting (9Go).

Molecular mechanisms of Igf2r imprinting associated with antisense Air expression have been investigated using knockout mice with deletion of DMR2 (10Go) and with a short truncated Air transcript (11Go). Evidence from these knockout mice has indicated that the intact antisense Air transcript from the paternal allele was required for the repression of the overlapped Igf2r expression as well as the non-overlapped Slc22a2 and Slc22a3 expression from the paternal allele (11Go). A recent report of knockout mice with the exogenous promoter that fully replaces the Igf2r promoter and with complete deletion of the Igf2r promoter showed that the Igf2r promoter had no role in a silent or an active form in imprinted genes including Igf2r (12Go). The Air transcript is now thought to be essential for establishment of the Igf2r imprint, however, the previous observations in the brain (4Go,6Go) allow the possibility that Air might not be enough to maintain Igf2r imprinting during the brain development or to re-establish Igf2r imprinting if DNA methylation silencing of the Igf2r promoter is released in the brain (9Go).

Recently, mouse genes with brain-specific imprinting patterns have been reported. They are Ube3a, Grb10 and Murr1 with neuron-specific, brain-promoter-specific and brain-developmental-stage-specific expressions, respectively. Ube3a is biallelically expressed in most tissues, but expressed exclusively from the maternal allele only in neurons, leading to apparent partial imprinting with predominant maternal Ube3a expression in the whole brain (13Go). Grb10 is maternally expressed in most tissues but predominantly paternally expressed only in the brain. Such reciprocally imprinted Grb10 expression in the brain and other tissues is thought to depend on the alternative usage of promoters (14Go,15Go) and insulator activity in DMR (14Go). Murr1 is imprinted in the adult brain, especially in mature neurons, but not in embryonic and neonatal brains (16Go). These lines of evidence suggest that brain-specific imprinting may be regulated partly by epigenetic modifications, depending on specification and maturation of cell lineages in the developing brain.

Here, we hypothesize that brain-specific relaxation of Igf2r imprinting may also depend on brain cell lineage-specific epigenetic modifications. We thought that there might be two cell types with imprinted or non-imprinted Igf2r expression in the brain, depending on brain cell lineage. To examine our hypothesis, we performed epigenetic analysis in brain cells with the aid of primary cortical cell cultures, where neurons or glial cells were cultured separately from products of crosses between the C57BL/6 and PWK strains (divergent strains of Mus musculus). In each type of culture, Igf2r and Air expression and epigenetic factors such as DNA methylation and histone modifications were analyzed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Imprinting analysis of Igf2r in the tissues and primary cultured cells
Imprinting of Igf2r is known to be established during the early post-implantation stage (17Go–19Go), whereas biallelic Igf2r expression is demonstrated in the brain at embryonic day (E) 15 (4Go). To examine whether relaxation of Igf2r imprinting seen in the brain is developmental-stage-specific, imprinting status in the fetal and neonatal tissues of F1 mice, crosses between the BL6 and PWK strains were analyzed by RT–PCR using a BalI polymorphism in exon 48 of the gene (Fig. 1). Because neurogenesis in the mouse cerebral cortex commences around E10, peaks around E15 and finishes around birth (20Go,21Go), we examined the telencephalon to see the imprinting status in the brain before neurogenesis. In the E10 telencephalon examined, the brain precursor cell marker, Nestin, was strongly expressed and the earliest neuronal marker, Tuj1, expressed in neurons at an early stage of development just after the last mitosis (22Go) was detected by RT–PCR, while Map2, expressed in neurons at a later stage of development was not detected (data not shown). Imprinting analysis in the E10 telencephalon showed predominant maternal expression with extremely faint paternal expression (Fig. 2A), suggesting that imprinting of Igf2r is maintained before neurogenesis. We then performed imprinting analysis in primary cultures of neurons and glial cells to examine whether neurons, but not glial cells, escape Igf2r imprinting. Prior to the analysis, we confirmed that over 95% of the two cultured cell types were post-mitotic neurons and astrocytes, by immunostaining and RT–PCR (13Go) with brain precursor, neuronal and glial markers (data not shown). Imprinting analysis clearly showed that imprinting of Igf2r was lost in the cultured neurons, but not in glial cells and embryonic fibroblasts (Fig. 2B). Because quantitative RT–PCR revealed that Igf2r was expressed almost equally in the cultured neurons and glial cells, biallelic expression of Igf2r in the cultured neurons was not related to the abundance of the gene expression (Fig. 2C). The existence of novel neuron-specific promoters to initiate biallelic Igf2r expression was not supported by 5'-RACE of neuron-derived cDNA (data not shown). Each experiment was repeated twice and the results were also confirmed in genetically reciprocal F1 mice.



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  		Figure 1. Map of mouse Igf2r and Air. Location of primers used was shown: for expression analysis: 1F/1R and AS1F/AS1R for Igf2r and Air expressions, respectively; for methylation analysis: DMR1F/DMR1R and DMR2F/DMR2R; for ChIP assay: Chip1F/Chip1R and Chip2F/Chip2R. Imprinting analysis of Air expression was performed using primer Chip1F/Chip1R. Primers for quantitative analysis of immunoprecipitated DNA were Chip1F-2/Chip1R-2 and Chip2F-2/Chip2R-2 for DMR1 and DMR2, respectively. White and gray boxes indicate coding and untranslated regions, respectively. Asterisks indicate polymorphic sites used for allelic difference. One asterisk denotes polymorphic DdeI site, two asterisks denote single nucleotide (G/A) polymorphism and three asterisks denote polymorphic BalI site.

 


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  		Figure 2. Imprinting analysis of Igf2r in tissues and primary cultured cells. (A) Imprinting analysis in embryonic (E10), neonatal (P1) tissues from F1 hybrids. RT–PCR products were digested with BalI, which digests only the product from the C57BL/6 allele. Digested products are, 126 and 136 bp fragments in size, seen as two adjacent bands. (B) Imprinting analysis in cultured cells derived from F1 hybrids (PWKxC57BL/6). (C) Quantitative analysis of Igf2r expression in cultured cells by real-time PCR. Standard errors of mean values are indicated by bars. Ce, cerebrum; L, liver; Te, telencephalon; M, maternal-allele origin; P, paternal-allele origin.

 
Expression analysis of Air in the brain tissues and primary cultured cells
It is thought that the non-coding Air transcript, which overlaps the Igf2r promoter in the antisense orientation, is required for the silencing of sense Igf2r, Slc22a2 and Slc22a3 from the same allele (11Go). Previous reports have demonstrated Air expression in the whole embryo at E9.5 (23Go) and in the neonatal brain (6Go). To see whether Igf2r and Air imprinting can be coupled in brain cells, we first examined Air expression in the embryonic brains by primer-specific RT–PCR. RT–PCR products were detected as Air expression in the E10 telencephalon-derived and E15 cerebrum-derived cDNA primed only by reverse primer AS1R (Fig. 3A). Then, Air expression in embryonic tissues and cultured cells was evaluated by quantitative RT–PCR using cDNA primed by random-hexamers. High-level Air expression was found in embryonic brain tissues between E10 and E15 (Fig. 3B), however, expression in neurons was almost undetected (Fig. 3D). Paternal allele-specific expression of Air was confirmed in identical cDNA by RT–PCR using a DdeI polymorphism in the upstream region of Igf2r exon 1 (Figs 1 and 3C).



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  		Figure 3. Expression analysis of Air. (A) Primer-specific RT–PCR in embryonic brain (E10 and E15). F, R and minus sign indicate cDNA samples by forward priming (AS1F), reverse priming (AS1R) and without priming in the presence of RT, respectively. (B and D) Quantitative analysis of Air expression in embryonic tissues and cultured cells. cDNA primed by random hexamers was used for the analysis. Standard errors of mean values are indicated by bars. (C) Imprinting analysis of Air expression in cultured cells from F1 hybrids (C57BL/6xPWK). Plus and minus signs indicate with and without DdeI digestion, respectively.

 
DNA methylation analysis of Igf2r
DNA methylation in DMR2, the Air promoter region located in Igf2r intron 2, is a primary gametic imprint, whereas that in DMR1, the Igf2r promoter region, is a secondary imprint, acquired during early post-implantation development (8Go,24Go). We analyzed DNA methylation status in DMR1 and DMR2 by bisulfite method. The allele-specific methylation of 34 CpGs in DMR2 was examined using a G/A polymorphism in PCR products of bisulfite-modified DNA. Sequencing of the PCR products and clones with the PCR products revealed maternal allele-specific methylation in the adult tissues (data not shown) and in the cultured cells (Fig. 4A and B, right columns). In contrast, the analysis of the methylation status of 12 CpGs spanning an A/G polymorphism in DMR1 showed that in the adult liver, 12 CpGs were methylated almost exclusively on the paternal allele, whereas in the adult cerebrum, they were methylated only in half of the products originated from the paternal allele (Fig. 4C, right column). In cultured glial cells, methylated CpGs were found exclusively on the paternal allele, whereas in cultured neurons, both alleles were hypomethylated (Fig. 4A and B, left columns). Because adult brain tissue mainly consists of neurons and glial cells, our results using the in vitro culture system can explain those using the adult cerebrum tissue in vivo. To see when such neuron-specific relaxation of paternal methylation in DMR1 occurs during development, methylation status in each developmental stage was analyzed (Fig. 4C). In the telencephalon and whole embryo at E10, the paternal allele was preferentially methylated, but less in the telencephalon than in the whole embryo; 25 and 60% of total CpGs in the paternal clones were methylated, respectively. At E15 and P1, allele-specific methylation was completely established in the liver, whereas in the cerebrum, paternal allele became hypomethylated; 15% of total CpGs in the paternal clones were methylated.



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  		Figure 4. Methylation analysis of DMRs. (A) Chromatograms of direct sequence of PCR products. Original sequences without bisulfite treatment are shown in the lowest row. Sequences of bisulfite-modified DNA are shown in upper three rows. Unmethylated cytosines are converted to thymines by bisulfite treatment. (B) DNA methylation status in DMR1 and DMR2 in cultured neurons and glial cells. (C) DNA methylation status in DMR1 at each developmental stage. Twenty to thirty clones from the maternal (M) and paternal (P) alleles were sequenced. Each row corresponds to an individual strand of DNA and each circle represents a CpG on that strand. Parental alleles are distinguished by DNA polymorphism between C57BL/6 and PWK. Filled and open circles indicate methylated and unmethylated sites, respectively. Numbers on the both side of the top strands represent nucleotide positions, given according to GenBank accession nos L06445 for DMR1 and L06446 for DMR2. Green and red lines under the lowest row of the chromatograms (A) denote CpGs in DMR1 and DMR2, respectively, corresponding to the same positions of CpGs in DNA strands (B and C).

 
Histone modifications of DMRs
To determine whether neuron-specific relaxation of Igf2r imprinting is associated with histone modifications, chromatin immunoprecipitation (ChIP) assay was performed in cultured cells and E10 embryonic tissues. The level of histone modifications in DMR1 and DMR2 was quantitatively analyzed by real-time PCR (Fig. 5). In DMR1, enrichment for acetyl histone H3 (H3Ac), acetyl histone H4 (H4Ac) and di-methyl Lys4 histone H3 (H3mK4) was detected similar to that in Gapdh promoter region, which was used as a positive control region for H3Ac, H4Ac and H3mK4. In DMR2, enrichment for H3mK4 was similar to that in Gapdh, whereas the lower level of H3Ac and H4Ac than that in Gapdh was detected. We could not find any significant differences in the histone modifications between neurons and glial cells. Both in DMR1 and DMR2, the level of di-methyl Lys9 histone H3 (H3mK9) was similar to that in D13Mit55, which is a locus near the centromere of mouse chromosome 13 and used as a positive control region for H3mK9 (25Go,26Go).



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  		Figure 5. Quantitative histone modification analysis in DMRs. Percentage of IP by antibodies for H3Ac, H4Ac, H3mK4 and H3mK9. Immunoprecipitated DNA in each cultured cells was quantitatively analyzed at Igf2r DMR1, DMR2, Gapdh and D13Mit55 by real-time PCR. Standard errors of mean values are indicated by bars.

 
After evaluation of ChIP DNA by allele-specific histone modifications in the Lit1 promoter region as a control (25Go), we analyzed allele-specific histone modifications in DMR1 and DMR2. Single strand conformation polymorphisms (SSCPs) analysis of PCR products was performed. In DMR2, histones H3 and H4 were hyperacetylated, and H3K4 was hypermethylated on the paternal chromosome, whereas H3K9 was hypermethylated predominantly on the maternal chromosome in each cultured cell (Fig. 6A). Allele-specific histone modifications in DMR1 were analyzed by hot-stop PCR (25Go,27Go) (Fig. 6B). The ratio (M/P) of the maternal to the paternal band intensities was corrected for the M/P in the input sample. In fibroblasts, M/P values of H3Ac, H4Ac and H3mK4 were over 10, whereas in glial cells, they were between 3.6 and 5. This result indicates that in glial cells and fibroblasts, histones H3 and H4 were hyperacetylated and H3K4 was hypermethylated on the maternal chromosome, although less pronounced allele-specific histone modifications in glial cells. In contrast, in neurons, M/P value of each histone modification was approximately 1, indicating no allelic differences in histone acetylation and di-methylation. Histone modifications in E10 telencephalon tissues and whole embryos revealed the similar but less pronounced allelic histone modification pattern than that in glial cells (Fig. 6B). In DMR1, we could not detect a clear allelic difference in H3mK9 in cultured cells and E10 tissues (Fig. 6B).



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  		Figure 6. Alellic histone modification analysis in DMRs. The chromatin of the primary cultured cells and E10 tissues derived from F1 hybrids (C57BL/6xPWK) were analyzed. (A) Histone modifications in DMR2 by SSCP analysis. PCR products of chromatin DNA immunoprecipitated by H3Ac, H4Ac, H3mK4 and H3mK9 antibodies were electrophoresed in MDE gel. PCR products of input DNA (Inp) and precipitated DNA without rabbit (no r {alpha})/mouse antibodies (no m {alpha}) by protein A/protein G agarose were also electrophoresed as positive and negative controls, respectively. (B) Histone modifications in DMR1 in cultured cells and E10 tissues by hot-stop PCR. Ratios of maternal intensity to paternal intensity (M/P), corrected by M/P in input chromatin (Inp), are indicated below each lane. PCR products of C57BL/6 and PWK genomic DNAs were shown as homozygous controls in first two lanes of the left panels.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Recently, primary cortical cell cultures have been used for neural epigenetic analysis (28Go–30Go) and provided useful models for determining the contribution of epigenetic mechanisms (31Go). As far as neurons in our culture system are concerned, cells harvested are thought to be adjusted at ages equivalent for neurons at P0–2. It is conceivable that epigenetic status of these cells might not be reflective of that of matured or aged neurons and that the epigenetic controls which preserve stable patterns of gene expression in vivo might be lost even in the primary culturing. Considering these limitations, the results from the experiments using primary brain cell culture system were compared with the results in the whole cortices from embryos and adult mice and interpreted consistently. In this study, we demonstrated that Igf2r imprinting and its relaxation of imprinting were associated with epigenetic modifications, depending on the brain cell types: neurons, glial cells and fibroblasts. We showed that neurons, but not glial cells, escape Igf2r imprinting (Fig. 2B). The neuron-specific relaxation of Igf2r imprinting in vitro is supported by the in vivo results that Igf2r was imprinted in the telencephalon before neurogenesis, but biallelically expressed in the neonatal brain after neurogenesis (Fig. 2). This indicates that biallelic Igf2r expression previously reported in the brain (4Go,6Go) results from neuron-specific relaxation of Igf2r imprinting and that neuron-specific epigenetic modifications alter allele-specific Igf2r expression.

As imprinting mechanisms of Igf2r, some epigenetic modifications have been reported. Non-coding Air transcript that overlaps the Igf2r promoter in antisense orientation is one of the epigenetic factors, which can affect the imprinted expression of Igf2r. The expression study in several knockout mice suggested that Air might have intrinsic silencing properties with direct action on Igf2r expression (12Go). However, the previous observations in the brain that Igf2r was biallelically expressed despite the imprinted Air expression and that the Igf2r promoter was biallelically hypomethylated (4Go,6Go) have supported that Igf2r promoter methylation might be the more direct effector of Igf2r silencing than Air expression (9Go). Here, we showed that Air was not expressed in cultured neurons, where Igf2r was biallelically expressed, whereas Igf2r was imprinted with Air expression in glial cells. In addition, reciprocal imprinting of Igf2r and Air was coupled in E10 telencephalon, where DNA methylation at the Igf2r promoter was not yet completely established. These results support arguments that Air transcript is involved in the initial establishment of Igf2r imprint during the early post-implantation development (9Go) directly or indirectly through other epigenetic modifications before establishment of DNA methylation at the Igf2r promoter.

DNA methylation at CpG dinucleotides is one of the cis-acting epigenetic modifications associated with genomic imprinting. In the imprinted Igf2r gene, DNA methylation in DMR2 is a primary imprint to differentiate parental alleles for Igf2r expression, whereas that in DMR1 is thought to be a secondary imprint that affects imprinted expression of Igf2r (4Go,32Go). Our methylation analysis by bisulfite method revealed significant allelic difference of DNA methylation in DMR1 in the adult cerebrum; the maternal allele is not methylated but half of paternal alleles are hypermethylated (Fig 4B). This result contrasts with previous reports of the DMR1 methylation status by Southern blot analysis (4Go) and by modified combined bisulfite restriction analysis (32Go), indicating that both parental alleles were hypomethylated in the adult brain. However, our in vitro result that DMR1 is biallelically hypomethylated in neurons but paternally methylated in glial cells supports the hypothesis that only the paternal alleles from glial cells are methylated in the whole cerebrum tissue in vivo. The discrepancy in the methylation status in the brain may be explained by the ratio of neurons to other cells including glial cells in the brain.

Igf2r imprinting is reported to be present in the early post-implantation stage at E6.5–7.5 (18Go,19Go), however, it remains unknown whether paternal methylation in DMR1 is acquired parallel to imprinted Igf2r expression. Our methylation analysis in tissues at each developmental stage revealed that the paternal DMR1 became methylated during embryogenesis in the whole embryo except in the brain, where it became hypomethylated during neurogenesis (Fig. 4C). Interestingly, in the E10 embryo including the telencephalon, Igf2r showed imprinted expression but allele-specific DNA methylation was not completely established. Recently, domain-wide silencing of placentally imprinted genes on mouse distal chromosome 7 was reported to be based on histone modifications in trophoblasts, independent of DNA methylation (33Go,34Go). The discrepancy between imprinted Igf2r expression and DNA methylation in DMR1 may also be explained by histone modifications. Even in the embryonic tissues, tissue-specific imprinting or relaxation of imprinting may be controlled by histone modifications, which are acquired prior to DNA methylation as secondary imprints during differentiation.

Parental-origin-specific histone modifications are recently reported to represent the determinant of epigenetic features as well as DNA methylation. Recently, Fournier et al. (8Go) and Yang et al. (32Go) reported allele-specific histone modifications along the Igf2r gene; chromosome in both DMR1 and DMR 2 showed allele-specific H3 and H4 acetylation and H3 di-methylation in the liver, whereas in the brain, allelic differences in histone acetylation and di-methylation were detected in DMR2, but not in DMR1. In our in vitro culture system, two interesting results about the relationship between histone modifications and gene expressions were obtained. One is the histone modification in DMR1 associated with the Igf2r expression and the other is that in DMR2, which is independent of the Air expression.

In DMR1, there were no allelic difference in histone acetylation and di-methylation on both active parental chromosomes in neurons, whereas histone acetylation and H3K4 di-methylation were present on the expressed maternal chromosome in glial cells and fibroblasts (Fig. 6). This indicates that such histone modifications in each cell correlate well with Igf2r expression and may explain the previous reports about histone modifications in the whole adult brain (8Go,32Go). We could not detect pronounced allelic difference of H3K9 di-methylation in cultured cells, which is previously reported in tissues by Fournier et al. (8Go). In contrast, despite <1% of chromatin precipitated by the H3mK9 antibody, the precipitated samples showed allelic difference of H3mK9 at germ line DMRs (8Go,25Go,32Go): DMR2 of Igf2r (Fig. 6A) and the promoter region of Lit1 (data not shown). One plausible explanation is that H3K9 di-methylation in germ cells is maintained as a stable and heritable marker for a primary imprint, but may not be established as a secondary imprint during development. Recently, di- and tri-methyl H3K4 and tri-methyl H3K9 rather than di-methyl H3K9 were reported in DMR2 to be primary epigenetic marks of Igf2r imprinting (35Go). Fournier et al. (8Go) also described paternal dominant H3K9 methylation using {alpha}-branched antibody, which recognizes both di- and tri-methyl H3K9 (8Go). As our H3mK9 antibody is the antiserum to a linear peptide with K9 di-methylation, which recognizes di-methyl H3K9 only (36Go), further analysis concerning the allelic occurrence of other histone modifications is needed, especially both di- and tri-methylation as primary and/or secondary imprint marks.

In DMR2, histones H3 and H4 were paternally acetylated in neurons, where Air expression was not detected (Fig. 6). We first expected that histones might be biallelically hypoacetylated in neurons and paternally hyperacetylated in glial cells, leading to the paternal-allele-specific histone acetylation in the whole brain (8Go,32Go), because Air was not detected in neurons but paternally expressed in glial cells. However, the total level of histone acetylation in DMR2 was almost equal in neurons and glial cells (Fig. 5). Such discrepancy between histone acetylation and gene silencing is seen in other genes. In the Ube3a promoter region, histones H3 and H4 were biallelically hyperacetylated in neurons (unpublished data), where Ube3a was maternally expressed and antisense Ube3a was paternally expressed (13Go). In the mouse ß-globin locus, the transcriptionally inactive ß-minor promoter was hyperacetylated in yolk sac (37Go). Our data support that there is no strict correlation between acetylation and tissue-specific active transcription and that hyperacetylation near promoters establishes transcriptional competence, but additional factors would be necessary for tissue-specific transcriptional activation (37Go).

In summary, the results presented here show that DNA methylation, histone acetylation and H3K4 di-methylation in DMR1 and Air expression are dynamically changed during neurogenesis and correlate well with Igf2r expression (Fig. 7). It should be noted that Air was expressed in brain precursor cells and glial cells but not in neurons, where Igf2r was biallelically expressed. In addition, before establishment of DNA methylation in DMR1, allele-specific histone modifications were detected with imprinted Igf2r expression in brain precursor cells. These indicate that imprinted Igf2r expression is associated with Air expression and that histone modifications in DMR1 might be earlier epigenetic marks than DNA methylation as secondary imprints. There is increasing evidence that neuronal differentiation responding the extrinsic signals is regulated, at least partly, by epigenetic mechanism at the level of histone modifications and/or DNA methylation (28Go–30Go). Further experiments to examine how alteration of imprinting during neurogenesis are controlled by the factors intrinsic to brain precursor cells will provide new clues to the molecular mechanism of neuron-specific imprinting and relaxation of imprinting.



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  		Figure 7. Summary of expression of sense and antisense transcripts of Igf2r and epigenetic modifications in neural lineage. In brain precursor cells and glial cells, sense transcript (Igf2r) and antisense transcript (Air) are expressed exclusively from the maternal (M) and paternal alleles (P), respectively, whereas in neurons, sense transcript (Igf2r) is biallelically expressed with no expression of antisense transcript (Air). Parentheses indicate the promoter regions of Igf2r (DMR1) and Air (DMR2) and circles indicate histone modifications: open circles indicate histone hyperacetylation and H3K4 hypermethylation and closed circles indicate histone hypoacetylation and H3K4 hypomethylation. Hypermethylated DNA regions are indicated as ‘m’ in DMRs.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Tissues
All procedures were performed with approval from the Nagasaki University Institutional Animal Care and Use Committee. Cerebral cortices and other tissues were prepared from E10, E15, P1 and adult (P42) products of crosses between the C57BL/6 and PWK strains, divergent strains of M. musculus. Cerebral cortices were freed from meninges. Tissues were used for RNA and DNA extraction and for primary cultures.

Primary culture
Primary cultures of cortical neurons, glial cells and embryonic fibroblasts were described elsewhere (13Go). In brief, E15 cerebral cortices without meninges were trypsinized to dissociate brain cells. For neuron culture, dissociated cells were cultured in NeurobasalTM (Gibco BRL) with B27 supplement (Gibco BRL). Cells were plated on polyethyleneimine-coated 3.5 cm plastic dishes at a concentration of 1x106 cells/ml. Cultures were maintained in 5% CO2 at 37°C for 5 days. For glial cells culture, dissociated brain cells were cultured overnight in Dulbecco's modified Eagle's medium (DMEM) (Sigma) supplemented with 10% FCS on the same dishes and at the same concentrationmentioned earlier, and then medium was changed to Neurobasal (Gibco BRL) with G5 supplement (Gibco BRL). After culture for 5–7 days, glial components were subcultured on new dishes and maintained in 5% CO2 at 37°C for a total of 14 days. For fibroblasts, embryonic skin tissues derived from E15 embryos were cultured in DMEM supplemented with 10% FCS on plastic dishes and maintained in a 5% CO2 at 37°C.

cDNA synthesis
Total RNA was isolated from cultured cells and tissues with RNAeasy kit (Qiagen) according to the manufacturer's protocol. The first-strand cDNA primed by oligo (dT)12–18 was synthesized at 42°C and that by specific primers was synthesized at 65°C for 50 min by SUPERSCRIPT II RNase H-reverse transcriptase (Gibco BRL). The first-strand cDNA primed by random hexamers was synthesized at 25°C for 5 min, followed by 50 min at 50°C by SUPERSCRIPT III (Gibco BRL). Then, mRNA–cDNA chains were denatured and the reverse transcriptase activity was arrested by heating at 70°C for 15 min. As a control for genomic DNA contamination, an identical reaction was carried out without the reverse transcriptase. Sequences of primers used for specific primings for Air expression as follows are the same (nos 8-04 and 6458) as designed by Hu et al. (6Go): AS1F (forward), 5'-GCACGAGCGCCAGGTACCTACTCGA-3' and AS1R (reverse): 5'-GGTGCTGGA CGGGGAAACTGAGGT-3'.

Imprinting analysis
cDNA primed by oligo (dT)12–18 and by random hexamer from cultured cells and tissues was used to perform PCR for Igf2r and Air amplification, respectively. Following primers were used for allele-specific expression of Igf2r and Air: 1F: 5'-CAGAAGAAGCTCGGGCGTGTCCTAC-3'; 1R: 5'-CTCCGCTCCTCGGCCTAGT GAACT-3';, Chip1F: 5'-CATGAGTCGGAAACTCCCAC-3'; and Chip1R: 5'-CTCTCTCTTCTTCAACCGAG-3'. PCR for Igf2r was performed through 35 cycles at 94°C for 30 s, 65°C for 30 s and 72°C for 30 s. PCR product of Igf2r was digested by restriction enzyme BalI and electrophoresed in 4% polyacrylamide gel or 2% agarose gel. PCR for Air was performed through 35 cycles at 94°C for 30 s, 60°C for 30 s and 72°C for 30 s. PCR product of Air was digested by restriction enzyme DdeI and electrophoresed in 5% agarose gel.

Bisulfite analysis
Isolated DNA was treated with sodium bisulfite using CpGenome DNA Modification Kit (Chemicon International, Inc.). The bisulfite-modified DNA was amplified by PCR using following primers. DMR1F: 5'-GGGATTTTAGAAAGATTGATTTT-3'; DMR1R: 5'-AAACCTAACAACCCCAAAATTACTCAC-3'; DMR2F: 5'-TTAAGGGT GAAAAGTTGTATAAGGAG-3'; and DMR2R: 5'-CTAATAAAACACCTTCATTTACATAACCAA-3'. PCR was performed under the following conditions: 40–45 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 30 s. PCR products were directly sequenced on ABI PRISM Model3100 (Applied Biosystems) or ligated into PCR2.1 vector by TOPO TA Cloning Kit (Invitrogen) before sequencing.

ChIP assay
ChIP assay was performed by ChIP assay kit (Upstate Biotechnology), according to the manufacturer's protocol. In brief, the chromatin of cultured cells was prepared from ~1.0x106 cells and treated with formaldehyde to cross-link DNA to protein in situ, sonicated to an average size of 0.5 kb and immunoprecipitated with antibodies. Antibodies against H3Ac (cat no. 06-599), H4Ac (cat no. 09-866) and H3mK4 (cat no. 07-030) were obtained from Upstate Biotechnology and the monoclonal antibody against H3mK9 was originally developed (36Go). The chromatin of E10 tissues was prepared as described elsewhere (8Go) and immunoprecipitated. DNA recovered from immunoprecipitated complex was subjected to PCR. ChIP was performed independently twice. Following primers were used for the analysis of allele-specific histone acetylation and methylation: Chip1F, Chip1R, Chip2F: 5'-TCATGCATAGCCAGGATAGC-3' and Chip2R: 5'-TATCCTGAGGGTG CAAACTG-3'.

SSCP and hot-stop PCR
Immunoprecipitated DNA associated with acetylated or methylated histones in DMR2 was analyzed by SSCP. PCR was performed in the presence of [{gamma}-32P] ATP-labeled primers under the following conditions: 35 cycle of 94°C for 30 s, 55°C for 30 s and 72°C for 30 s. PCR products were resolved by electrophoresis in MDE gel, non-denaturing acrylamide gel (FMC Bioproduct). Hot-stop PCR for analysis of allele-specific histone modifications in DMR1 was performed as follows: after a number of PCR cycles sufficient to detect a product, a primer labeled by [{gamma}-32P]ATP was added to the mixture and then one cycle of PCR was performed. The PCR product was digested with DdeI and was electrophoresed in 4% polyacrylamide gel. The intensity of the PCR products was measured with a BAS 5000 Bioimaging Analyzer (Fujifilm).

Quantitative PCR
cDNA primed by oligo (dT)12–18 and by random hexamers was applied to real-time PCR for quantitative expression analysis of Igf2r and Air, respectively, using SYBR Green and an ABI Prism 7900 (Applied Biosystems). Each PCR was run at least triplicate to control for PCR variation. The relative amounts of Igf2r and Air RNAs were calculated by normalizing their values with the housekeeping gene Gapdh. Each experiment was repeated on independent RNAs two to three times. Primers used for PCR were 1F and 1R for Igf2r, AS1F and AF1R for Air and Gapdh forward and Gapdh reverse for Gapdh (13Go).

Immunoprecipitated DNA by antibodies and input DNA were also analyzed by real-time PCR using the same protocol. The results were represented at each region of Igf2r DMR1, DMR2 and Gapdh or D13Mit55 as a percentage of IP, calculated by dividing the average value of the immunoprecipitated DNA by the average value of the corresponding input DNA. Primers used for PCR were as follows: Chip1F-2: 5'-GACTGACCTCTTAACCCTGC-3'; Chip1R-2: 5'-TTCAACCGAGACCAGTACG-3'; Chip2F-2: 5'-ATGCATAGCCAGGATAGCGC-3'; Chip2R-2: 5'-GCTCAAAAGTGCCATGTTACAG-3'; and primers for Gapdh and D13Mit55 (25Go).


   ACKNOWLEDGEMENTS
 
We are grateful to Dr Joseph Wagstaff for critical reading of the manuscript. N.N. and T.K. were supported partly by a Grant-in-Aid for Scientific Research on Priority Areas (no. 12204010 for N.N and T.K and no. 17024046 for T.K.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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