Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on August 4, 2004
Human Molecular Genetics 2004 13(19):2233-2245; doi:10.1093/hmg/ddh244

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Human Molecular Genetics, Vol. 13, No. 19 © Oxford University Press 2004; all rights reserved

Promoter-restricted histone code, not the differentially methylated DNA regions or antisense transcripts, marks the imprinting status of IGF2R in human and mouse

Thanh H. Vu^*,, Tao Li and Andrew R. Hoffman

Medical Service, Veteran Affairs Palo Alto Health Care System and Department of Medicine, Stanford University, Palo Alto, CA 94304, USA

Received May 7, 2004; Revised July 8, 2004; Accepted July 20, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Imprinting of the mouse Igf2r depends upon an intronic differentially methylated DNA region (DMR) and the presence of the Air antisense transcript. However, biallelic expression of mouse Igf2r in brain occurs despite the presence of Air, and biallelic expression of human IGF2R in peripheral tissues occurs despite the presence of an intronic DMR. We examined histone modifications throughout the mouse and human Igf2r/IGF2R using chromatin immuno-precipitation (ChIP) assays in combination with quantitative real time PCR. Methylation of Lys4 and Lys9 of histone H3 in the promoter regions marks the active and silenced alleles, respectively. We measured di- and tri-methyl Lys4 and Lys9 across the Igf2r and Air promoters. While both di- and tri-methyl Lys4 marked the active Igf2r and the active Air allele, tri-methyl Lys9, but not di-methyl Lys9, marked the suppressed Air allele. We show here that enrichment of parental allele-specific histone modifications in the promoter region, rather than the presence of DNA methylation or antisense transcription, correctly identifies the tissue- and species- specific imprinting status of Igf2r/IGF2R. We discuss these findings in light of recent progress in identifying specific components of the epigenetic marks in imprinted genes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Genomic imprinting is a parent-of-origin epigenetic mechanism whereby one of the two parental alleles is preferentially suppressed, while the other parental allele is normally transcribed. In the mouse genome, 38 maternally imprinted (paternally expressed genes, PEG) and 35 paternally imprinted (maternally expressed genes, MEG) genes have been identified to date (http://www.mgu.har.mrc.ac.uk/imprinting/imprinting.html). In imprinted genes, the epigenetic information that is transmitted independently of DNA sequence is conveyed through alterations in nucleosome structure, resulting from covalent modifications of DNA (methylation) and of histones (e.g. acetylation and methylation). Recent studies of the epigenetic marks associated with imprinted genes have revealed that these epigenetic modifications occur with a differential preponderance on the expressed and silenced alleles (1–12).

Imprinted genes often cluster on large chromosome regions, forming imprinted domains that are regulated by cis-acting imprinting control regions (reviewed in 13,14). One of the well-characterized imprinted domains contains the gene that encodes the insulin-like growth factor-II (IGF-II) receptor/mannose-6-phosphate receptor (IGF2R). The gene is imprinted (maternally expressed) in rodents, marsupials and artiodactyls, but it is biallelically expressed in primates, including humans (15–17). The IGF-II receptor regulates IGF-II, a potent mitogen, by binding it, internalizing it and then transporting it to lysosome for degradation. Loss of function of the IGF-II receptor by mutation in the coding regions and by loss of heterozygosity of IGF2R has been observed in numerous malignancies (18,19). It has been suggested therefore that IGF2R is a tumor suppressor gene (20).

The mouse Igf2r gene encodes two reciprocally imprinted transcripts, each of which is associated with a differentially methylated DNA region (DMR) (21). The first DMR (DMR1) includes the promoter for the sense Igf2r transcript, whereas DMR2, which is located within the second intron of the gene, includes the promoter for an antisense transcript, Air. The paternally expressed Air RNA suppresses the expression of the sense Igf2r as well as Slc22a2 and Slc22a3 on the paternal chromosome (22). Deletion or premature termination of Air leads to loss of Igf2r locus imprinting (22,23). However, Igf2r expression in the CNS does not appear to be regulated by Air. Although Air is paternally expressed in the CNS as well as in peripheral tissues, Igf2r sense transcripts are biallelically expressed in brain (24,25).

The human IGF2R gene contains a single DMR in intron 2 (26), but no antisense transcripts have been detected and the gene is biallelically expressed in all tissues including Wilms' tumors (27). Thus, it appears that although the DNA is ‘marked’ for imprinting, the putative imprint is never read. To understand this lack of epigenetic readout, we used a chromatin immuno-precipitation (ChIP) assay in combination with quantitative real time (Q)-PCR (Q-PCR) to scan for enrichment of various histone modifications throughout the mouse and human Igf2r/IGF2R. We have found that the human DMR lacks enrichment of acetylated and methylated histones. The absence of differentially modified histones (DMH) in the human DMR may account for the epigenetic readout failure. We show here that enrichment of parental allele-specific histone modifications in the promoter region, rather than the presence of DNA methylation or antisense transcription, correctly identifies the tissue- and species-specific imprinting status of Igf2r/IGF2R.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Histone acetylation and Lys4 methylation are enriched in the human IGF2R promoter exon and are absent in the DMR
We ran triplicate Q-PCR assays (PCR primers shown in Table 1) on ChIP preparations of human embryonic fibroblast cells that maintain the normal imprinting of all tested imprinted genes (28 and unpublished data). We used a panel of antibodies against acetyl lysines (H3 and H4) and methyl lysines (Lys4 and Lys9), as reported previously (11,12). Relative enrichment compared to input chromatin DNA was calibrated with GAPD (measured 0.8 kb downstream of the GAPD transcription site; Fig. 1C, right panel), calculated as previously described (11) and plotted on the same graph (under appropriate scales for comparison) in Figure 1A. We observed a specific and symmetric distribution of acetylated histones (H4-Ac and H3-Ac) across the 3 kb region of the IGF2R promoter exon (Fig. 1A, green lines). The enrichment of acetylated histones near the IGF2R transcription site was often >10-fold when compared with the enrichment of acetylated histones at 1.5 kb upstream or at 2 kb downstream of the transcription site. Low levels of acetylated histones were found in the intronic DMR or in other exons. Methyl Lys4 of histone 3 (H3 K4-Me) was also enriched in the promoter exon and was essentially absent in the DMR (Fig. 1A, blue line), whereas methyl Lys9 (H3 K9-Me) was depleted near the IGF2R transcription site (Fig. 1A, red line). Histone acetylation and H3-Lys4 methylation have been found in transcriptionally active genes (29,30); the absence of these activating histone modifications in the human DMR correlates with the absence of a potential human ‘AIR’ antisense transcript (27).

View this table: [in this window] [in a new window]   Table 1. PCR primers for ChIP–PCR of human IGF2R, GAPD and ß-ACTIN

 

View larger version (37K): [in this window] [in a new window]   Figure 1. Distribution of modified histones across the promoter regions of human and mouse IGF2R/Igf2r. Top panel shows the IGF2R map and details of the promoter and the DMR. IGF2R is transcribed from both parental alleles with no detectable antisense transcript from DMR. (A) ChIP–Q-PCR assay across human IGF2R showing enrichment of activating histone modifications (acetylation and H3-Lys4 methylation) near the IGF2R transcription site and low levels of modifications in the intronic DMR, exon 10 and exon 34 regions. H3-Lys9 methylation was absent near the transcription site. (B) ChIP–Q-PCR assay across mouse Igf2r showing enrichment of H3-Lys4 methylation in both promoter regions of Igf2r and Air (DMR1 and DMR2). (C) ChIP–Q-PCR analysis of the promoter, upstream and downstream regions of human GAPD and ß-ACTIN showing a broad distribution of histone modifications in the housekeeping genes. The promoter regions were examined by two sets of adjacent PCR primers a and b that were separated by less than 50 b. The histone enrichment in the GAPD (+0.8 kb downstream) served as a calibration control.

 
Histone H3 Lys4 and Lys9 methylation are enriched in mouse Igf2r DMR1 and DMR2
We scanned the distribution of methyl Lys4 and methyl Lys9 of histone H3 across the mouse Igf2r in cultured fetal skin cells where Air antisense is actively transcribed from the paternal allele (23). Allele-specific histone modifications in the promoter region of Igf2r and Air have been reported previously (10,11) but the distribution of modified histones across the Igf2r promoter region was not clarified. As shown in Figure 1B (blue line), tri-methyl Lys4 demonstrated peak enrichments within 3 kb region of both DMRs, confirming the promoter-specific enrichment of activating modified histone in transcriptionally active regions. The peak enrichments occurred near the transcription site of Igf2r and Air despite the fact that our Igf2r/Air scanning results reflect the sum from both transcriptionally active and inactive parental alleles in each region. Examining the enrichment of di- and tri-methyl Lys4 on each parental allele in the Igf2r and Air promoter regions suggests a similar pattern of di-methylation and tri-methylation of H3-Lys4 in various tissues and in fetal cells (Fig. 3). This result suggests that H3-Lys4 methylation marks the active promoter region of mouse and human Igf2r/IGF2R gene and that the absence of histone acetylation and Lys4 methylation marks in the human DMR is associated with lack of active gene transcription. Tri-methyl Lys9 of histone 3 was more abundant than di-methyl Lys9 in fetal skin cells (Fig. 3C), and it was more enriched in the Igf2r DMR2 than in DMR1 (Fig. 1B, red line).

View larger version (164K): [in this window] [in a new window]   Figure 3. Allele-specific histones modifications accompany imprinting status of Igf2r and Air. Top panel shows the Igf2r map, promoter, DMRs and imprinted Igf2r and Air transcripts. ChIP–Q-PCR was performed on tissues from newborn C57BL/6J xM. spretus F1 mice (A and B) and from primary culture cells derived from skin (C) and brain (D) of the newborn F1 mice. ChIP–Q-PCR using allele-specific primers (Materials and Methods and Table 2) was performed to quantify the enrichment of the modified histones (acetylated histone H4 and H3, di-methylated H3-Lys4 and-Lys9, and tri-methylated H3-Lys4 and Lys9) on each maternal and paternal chromosomes at three locations (–0.2 kb in DMR1, and +1 and +0.01 kb in DMR2). Doublet columns at the three polymorphic locations depict the data of maternal versus paternal chromosomes as lighter versus darker colored columns. Each column represents the mean values of three PCR reactions. On top of each panel, the predominant allele—an enrichment of a modified histone in a particular parental allele—was identified as ‘M’ (maternal), ‘P’ (paternal), ‘Bi’ or ‘(Bi)’ (biallelic) by criteria described in the text. Low levels of modified histones were marked as ‘x’. Comparable data were obtained at nearby locations (0 and +0.1 kb, Fig. 3A and B), or by using allele-specific primers versus allele-common primers (–0.2 kb, Fig. 3C and D, compare left two panels).

 
Absence of promoter-restricted enrichment of histone modifications in human GAPD and ß-ACTIN
We examined various histone modifications across two housekeeping, autosomal non-imprinted genes, GAPD and ß-ACTIN (Fig. 1C). GAPD and ß-ACTIN are relatively small genes, and they are embedded in gene-rich regions on chromosomes 12p13 and 7p22, respectively. GAPD spans a short DNA region of 3.85 kb located 2.87 kb downstream of CNAP1 whereas ß-ACTIN spans 3.44 kb, 1.92 kb upstream of the putative transcript (LOC 402247). Because of the presence of other nearby transcripts, we examined two locations upstream of the GAPD and ß-ACTIN promoters (1 kb), two locations downstream (ß-ACTIN, 1 kb) and two adjacent locations near the GAPD and ß-ACTIN transcription sites (promoter a and b, Fig. 1C; for PCR primer, see Table 1). The patterns of various histone modifications observed at the two adjacent locations near the transcription site were similar, confirming our reliable ChIP–Q-PCR assays and revealing low levels of histone modifications in the promoter region, when compared with those in the upstream or downstream regions. This result is in sharp contrast to the enrichment of acetylated histones and H3-methyl Lys4 near the human and mouse IGF2R/Igf2r transcription site in Figure 1A and B.

Absence of allele-specific histone modifications in human IGF2R
To evaluate histone modifications on each of the two parental alleles of IGF2R, we genotyped five fetal subjects for potential SNPs across the promoter and the DMR (we selected SNPs of >10% heterozygous frequency from the NCBI database). We identified two subjects (HFB #1 and HFB #5) who were informative for allelic analysis in the DMR and the IGF2R promoter. We performed ChIP assays using a panel of six antibodies, and we ran triplicate RFLP–PCR (with duplicate samples) on the two informative fetal skin cell lines. To enhance detection sensitivity we used [³²P] dCTP radioisotopes, and we quantified by PhosphoImager the relative enrichment in each parental allele with reference to the parental allele ratio observed in ‘input DNA’. Figure 2 shows representative RFLP–PCR gels with relative allelic ratios from duplicate samples. Although the parental origins are unknown, it is clear that all histone modifications including acetylation (H3 and H4) and methylation (di- and tri-methylation of both H3-Lys4 and H3-Lys9) were enriched equally in both parental chromosomes at the locations near the promoter region (+1.9 kb) and near the intronic DMR (0.3 kb upstream of exon 3). In contrast to the allele-specific histone modifications in the DMRs of the mouse Igf2r/Air (10,11), the absence of allele-specific histone modifications in the human IGF2R promoter is consistent with the biallelic expression of the human IGF2R.

View larger version (36K): [in this window] [in a new window]   Figure 2. Allelic distribution of histone modifications at sites near the IGF2R promoter and the DMR by ChIP RFLP–PCR. ChIP DNAs from human fetal skin HFB # 5 were PCR amplified across a polymorphic +1884 AclI site (SNP no. 1015155). Digestion with AclI revealed an undigested 170C allele and a digested 134T allele. ChIP samples from HFB #1 were amplified and digested with MscI (SNP no. 8191728) to reveal an undigested 112G and a digested 81A alleles. PCR with (plus) and without (minus) input DNA served as positive and negative controls, respectively. An internal restriction site was integrated to the PCR primers (asterisk), which served as an internal digestion control. Traces of incomplete digestion products were marked (minor bands) along with the major digested products (major bands). Results of quantification by PhosphoImager from duplicate samples, after calibration with control input DNA (equal parental alleles), are shown at the bottom of each panel.

 
Surprisingly, despite the presence of allele-specific DNA methylation in the intronic DMR (26), both parental chromosomes harbor equally low levels of histone modifications. The absence of these DMHs near the intronic DMR highlights the discordance between histone modifications (both acetylation and methylation) and DNA methylation in the ‘non-functional’ DMR. Furthermore, the observation of very low levels of histone modifications in the intronic DMR in both parental chromosomes correctly identifies the absence of antisense ‘AIR’ transcription from both parental alleles.

Allele-specific histone acetylation and Lys4 methylation in the mouse DMR2
To examine histone modifications on each of the two parental chromosomes across DMR1 and DMR2 of Igf2r, we performed ChIP–Q-PCR from various tissues (kidney, liver and CNS) and embryonic cells (skin and CNS) from interspecific F1 mice (C57/BL/6J femalexMus spretus male). To this end, we designed PCR primers to amplify both parental alleles and three sets of parental allele-specific primers encompassing the three polymorphic sites (11) to amplify each parental allele (Table 2). In Figure 3, activating modifications are shown as green (acetyl lysines of H3 and H4) and blue columns (H3-methyl Lys4), whereas the silencing modification (H3-methyl Lys9) is shown in red color. The dark, light and medium intensities of the three colors (green, blue and red) represent paternal, maternal and (pat+mat) alleles, respectively (see color code in Fig. 3B and D).

View this table: [in this window] [in a new window]   Table 2. Allele-specific primers and biallelic PCR primers for ChIP–PCR assay of mouse Igf2r

 
To simplify the allelic analysis of various histone modifications across the three polymorphic sites, we have marked on top of each panel in Figure 3 the predominant allele, ‘M’ (maternal) or ‘P’ (paternal) when the ratio of the predominant versus the less abundant allele was greater than 3.0. When both alleles were enriched (enrichment >1.0-fold), the panel was marked as ‘Bi’ for ‘bi-allelic enrichment’ if the allelic ratio was within 1.0–2.0; or marked as ‘(Bi)’—in parentheses—if the allelic ratio was 2.0–3.0. Low levels of modified histones on both parental alleles (enrichment <1.0-fold) were marked as ‘x’.

In kidney, liver and fetal skin cells, activating modifications (green and blue columns) were found exclusively in the maternal allele in the Igf2r promoter (Fig. 3A and C, –0.2 kb panel). These activating modifications switched exclusively to the paternal allele in the Air promoter region (Fig. 3A and C, +1 and +0.1 kb panels). In contrast, in CNS and in cultured brain cells (predominantly fetal astrocytes), despite the paternal allele-specific modifications (H3-acetylation, H3-Lys4 di- and tri-methylation) observed at Air (Fig. 3B and D, +1 and +0.1 kb panels), the activating modifications marked both paternal and maternal chromosomes in the Igf2r promoter (–0.2 kb panel). Although there was likely a case of maternal bias in the tri-methyl Lys4 in brain cells (Fig. 3D, –0.2 kb panel), this case might be considered as a marginal ‘(Bi)’ since both parental alleles were enriched (enrichment >1.0-fold).

Tri-methyl Lys9 methylation marks the suppressed allele in the mouse Igf2r DMR2
The silencing modification, methyl Lys9 of H3, occurred primarily in DMR2 of the maternal chromosome (Air promoter) but was much lower in DMR1 (Igf2r promoter) (Fig. 3, red columns). This was more obvious in fetal skin and brain cells (Fig. 3C and D) than in kidney and liver (Fig. 3A and B), and was observed exclusively in tri-methyl Lys9 but not in di-methyl Lys9 (Fig. 3C and D, panels +1 kb and +0.1 kb, compare d-Me and t-Me). As DMR2 is a DNA methylation imprint that is inherited from the female gamete, whereas DMR1 CpG methylation is not completed until post-natal day 4 (31), DMR2 has been referred to as the primary DNA-gametic imprint. Our ChIP–Q-PCR results indicate that tri-methylation of Lys9 of histone H3 accompanies the primary gametic imprint of the Igf2r. In the human IGF2R gene, the absence of such a marker in the intronic DMR (Fig. 1A) corresponds to the absence of a potential imprinted antisense ‘AIR’ and the presence of biallelic expression of IGF2R in all tissues.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
The epigenetic code of imprinted genes is likely to consist of both DNA methylation (DMR) and histone modifications. The DNA methylation code appears to be clear: CpG methylation near the promoter region correlates with suppression of transcription. In contrast, a variety of histone modifications including acetylation and methylation (mono-, di- and tri-) at various lysine residues and other modifications constitutes a complex histone code (29,32). In this report, we examined histone acetylation and di- and tri-methylation throughout the mouse and human Igf2r/IGF2R by ChIP–PCR in order to clarify the epigenetic marks governing tissue- and species-specific imprinting of the Igf2r/IGF2R.

We have shown that histone modifications were enriched in the promoter region of Igf2r/IGF2R and that the promoter-specific enrichment was independent of the tissue- or species-specific imprinting status of the gene. The enrichment of histone acetylation and Lys4 methylation near the transcription site within a 3 kb promoter region of human IGF2R was striking. We confirmed this observation by ChIP assays in fetal skin cells from three independent subjects. These activating modifications were normally found in active chromosome domains encompassing the entire gene or cluster of active genes. However, to our knowledge, the sharp increase with a ‘Gaussian distribution pattern’ across a gene promoter has not been demonstrated before, although sharp increases of histone acetylation were noted at a silencing boundary in the chicken ß-globin (33) and in the mouse Gnas gene (12). The sharp increase of activating histones does not simply reflect the transcriptional activity at the promoter region since other non-imprinted, autosomal housekeeping genes such as the human GAPD or ß-ACTIN demonstrated no such sharp enrichment near their promoters. Scanning of histone modifications across the promoters of other imprinted genes may shed light on the role of this promoter-specific epigenetic mark in imprinted genes.

Methyl Lys9 of histone H3, which is considered to be a silencing modification (reviewed in 29,30) was enriched in a pattern reciprocal to that of the methyl Lys4 modification. This inverted pattern was more obvious in the human IGF2R promoter region, which showed a profound dip near the transcription site (Fig. 1A, red line). Although higher levels of methyl Lys9 were present outside of the promoter region, the human DMR and exon 10 showed low levels of methyl Lys9 modification.

The pattern of methyl Lys9 distribution in the mouse Igf2r appeared to be more complicated, because we presented data that resulted from the sum of opposing modifications in the active and inactive parental alleles that were measured together (Fig. 1B). Nonetheless, we observed enrichment of methyl Lys9 in DMR2 that regulates the Air transcript (Fig. 1B). Differentiating the two parental alleles at three polymorphic sites using allele-specific PCR primers and antibodies against the three levels (mono-, di- and tri-) of methylation at lysine residues also confirms high levels of tri-methyl Lys9 in DMR2 versus DMR1. The allele-specific PCR demonstrated that the tri-methyl Lys9 modification was exclusively from the suppressed maternal allele. ChIP assays using antibodies against mono-methyl Lys9 (and mono-methyl Lys4) yielded weak signals (data not shown), whereas di-methyl Lys9, in contrast to di-methyl Lys4, did not mark the suppressed allele in the Igf2r/Air promoters (Fig. 3, red columns).

Promoter-restricted enrichment of DMH, rather than CpG methylation in the DMR or the presence of an antisense transcript, correctly identifies the tissue- and species-specific imprinting status of Igf2r/IGF2R. Figure 4 summarizes the tissue- and species-specific epigenetic marks, including DNA methylation (DMR) and histone modifications (DMH), in the Igf2r/IGF2R region and in the human intronic DMR or mouse DMR2. In the mouse, the presence of a DMR and of a DMH correlates with the imprinting status of Igf2r/Air in peripheral tissues. However, there is a dissociation of Air imprinting versus Igf2r non-imprinting in brain. Absence of Air imprinting and dissociation of Air/Igf2r imprinted expression also have been observed in mouse uniparental, androgenetic and parthenogenetic fetuses even in the presence of a complete DMR2 (34). The histone epigenetic marks in these uniparental fetuses, have not been investigated. In human, we have now shown that despite the presence of a DMR, the absence of activating histone modifications corresponds to the absence of a human ‘AIR’ antisense, whereas biallelic presence of activating histones (i.e. absence of a DMH) in the sense promoter accompanies biallelic expression of IGF2R.

View larger version (25K): [in this window] [in a new window]   Figure 4. Schematic representing the association of epigenetic marks with tissue-specific and species-specific imprinting of Igf2r/IGF2R. In mouse liver, both Igf2r and Air are marked by DNA methylation DMR and histone DMH, and are expressed exclusively from the maternal and the patenal allele, respectively. In mouse brain, Air is marked by the presence of a DMR and a DMH, and is imprinted, whereas the absence of DMR and DMH correlates with biallelic expression of Igf2r. In human IGF2R, we show here that the absence of DMH, even in the presence of DMR, correctly identifies the biallelic expression of IGF2R and the absence of ‘AIR’ antisense.

 
Rougeulle et al. (35) have recently suggested that the enrichment of H3-Lys4 di-methylation in the promoter versus exon regions is an epigenetic mark for monoallelic expression of X-inactivated genes and of three imprinted autosomal genes, Igf2, Ube3A and Peg3. They did not discuss mono-methyl or tri-methyl H3-Lys 4, however. Histone scanning of the mouse Igf2r/Air indicates that the predominance of Lys4 methylation at the promoter (versus exons 3, 20 and 30) of Igf2r/Air is not restricted to di-methylation: both di-methyl Lys4 and tri-methyl Lys4 demonstrated a similar ‘epigenetic mark’ pattern. Interestingly, the species-specific non-imprinted human IGF2R also exhibits the enrichment of Lys4 di-methylation mark in the promoter region, whereas other autosomal housekeeping genes failed to exhibit the Lys4 methylation mark. We suggest that, at least in the case of the imprinted Igf2r and Air, promoter-restricted enrichment of both di- and tri-methyl Lys4 (in the active allele) is the governing epigenetic mark. Using mouse embryonic stem cells (129 ES), we have verified the presence of the di- and tri-methyl Lys4 marks in the promoter region of X-inactivated genes (Cdx and G6pd) and in the promoter of a number of imprinted genes, including MEGs (Cdkn1, Ascl2, Grb10 and Meg3) and PEGs (Dlk, Nnat, Snrpn and Peg3) (Vu et al., unpublished data). Histone scanning of imprinted genes using a panel of specific antibodies against modified histones, including tri-methyl Lys9 and phosphorylated Ser10, may further define the specific components of ‘epigenetic marks’ in imprinted genes.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Human tissues, interspecific mice and primary culture cells
Normal human fetal skin tissues of 6–10 weeks of gestation were obtained from the Central Laboratory for Human Embryology Tissue, University of Washington, Seattle, WA, USA. To generate F1 interspecific mice, M. spretus male mice were mated with M. musculus female mice. Housing and all procedures were performed according to protocols approved by the Institutional Care and Use Committee at the Veterans Affairs Palo Alto Health Care System. Liver, kidney and CNS tissues from newborn F1 mice were dissected, snap-frozen in liquid nitrogen and stored at –70°C. Human fetal skin cells, skin cells and brain cells from newborn F1 mice were removed, minced and cultured as described previously (11). Skin fibroblast cells at passage 3–6 were used in this study.

Antiserum against modified histones
The panel of antisera used in this report has been used in numerous studies (data from the suppliers) and in our previous studies (11,12). Antiserum specific for histone H4 acetylation (acetyl Lys5, 8, 12 and 16; cat. no. 06-866), antiserum for H3 acetylation (di-acetyl Lys9 and Lys14; cat. no. 06-599), antiserum for H3 methylation (di-methyl Lys4, cat. no. 07-030 and di-methyl Lys9, cat. no. 07-212) were obtained from Upstate Biotechnology (Waltham, MA, USA). Antiserum specific for H3-Lys9 acetylation (cat. no. 617) was from Cell Signaling Technology (Beverly, MA, USA). Antiserum specific for H3 tri-methyl Lys4 (cat. no. ab8580) and H3 tri-methyl Lys9 (cat.no. ab8898) were obtained from Abcam (Cambridge, UK).

Human IGF2R polymorphisms and RFLP–PCR analysis
We searched the SNPs database (GenBank, National Center for Biotechnology Information) and used RFLP–PCR to genotype five human fetal subjects for potential SNPs across 4 kb of the IGF2R promoter and 4 kb of the DMR. Only SNPs creating a restriction site and having high frequency of heterozygosity (>10%) were tested. We identified two informative subjects. The HFB #1 was a heterozygote having A/G alleles at 305 bases upstream of exon 3 (–305A/G); the two A and G parental alleles could be identified by the MscI restriction enzyme that recognizes the TGGCCA sequence of the A-allele. Input and ChIP DNA samples were amplified using primer p 2257 (5'-TTTGA TGGCC ACACT GGTGC AAGAT GGATG AG-3') and p 2237 (5'-CGCTA AAACC ACTAC CTGCG CT-3') to yield a 120 bp PCR product that was then digested by MscI to produce 112 bp G-allele and 81 bp A-allele. The HFB #5 was a +1884C/T heterozygote having an AclI polymorphic site AACGTT at 1884 bases downstream of IGF2R transcription (bold letters represent the digested allele). The DNA samples were amplified using primer p 2256 (5'-TTCAA CAACG TTAGG CCAGC TGGGT TAATT TC-3') and p 2253 (5'-GTCCT CCCAG TTAAG GGAGG CTGA-3') to yield a 178 bp PCR product that was then digested by AclI to produce 170 bp C-allele and 134 bp T-allele. To create an internal control for the restriction digestion, which is critical in RFLP–PCR analysis, we added an internal restriction site (underlined sequence, bold and italic nucleotides) 8 bases from the 5' end of the forward primers. Remaining fragments of any undigested products were detected as minor bands 8 bp longer than the major bands on a polyacrylamide gel (Fig. 2).

Chromatin immuno-precipitation
ChIP assay was performed as previously described (11,12). Briefly, about 5 million cells were fixed with 1% formaldehyde, and were then sonicated for 180 s (10 s on and 5 s off) on ice by a Branson sonicator with a 2 mm microtip and setting of 40% for output control and 90% for duty cycle. The sonicated chromatin (0.6 ml) was clarified by centrifugation, aliquoted and snap-frozen in liquid nitrogen. To perform ChIP, sonicated chromatin (20 µl) was diluted 10-fold, cleared with salmon sperm DNA/protein A-agarose (80 µl) and purified with specific antiserum (2–5 µl) and protein A-agarose (60 µl). The DNA from the bound chromatin after cross-linking reversal and proteinase K treatment was further purified by MiniElute PCR purification kit (Quiagen, Valencia, CA, USA) and finally eluted in 100 µl of low-TE buffer (1 mM Tris, 0.1 mM EDTA).

ChIP–PCR and restriction enzyme digestion
Duplicate PCR reactions (5 µl under liquid wax) contained 2 µl ChIP (or input) DNA, 0.1 µM appropriate primer pairs (Table 1), 50 µM deoxynucleotide triphosphate and 0.2 units KlenTaq I (Ab Peptides, St Louis, MO, USA). Standard PCR conditions were 95°C for 60 s, followed by 30 cycles of 95°C for 10 s and 65°C annealing (and extension) temperature for 90 s and finally 72°C for 10 min. All primer sets were tested for the absence of primer–dimer products. The PCR products were digested with appropriate enzymes (New England Biolabs, MA, USA; 1 unit) in a total volume of 10 µl for 6–12 h under liquid wax. The digested products were separated on a 5% polyacrylamide-urea gel and quantified by a PhosphoImager (Molecular Dynamics, Sunnyvale, CA, USA).

(Q)-PCR
Enrichment of modified histones in DNA obtained from the ChIP assays was determined by Q-PCR using an ABI Prism 7900HT sequence detector following the ABI protocol. The Q-PCR assays were run in triplicate on 384-well plates. We designed allele-specific oligonucleotide primers to amplify specifically each parental allele (Table 2). All primer sets for Q-PCR were free of primer–dimer products. We used SYBR Green in our Q-PCR assays. At the end of the Q-PCR amplification we run a ‘melting curve analysis’ to confirm the homogeneity of all Q-PCR products. Relative enrichment of a given target sequence by a specific antibody is determined by a ‘delta Ct and delta–delta Ct’ calculation by an ABI protocol with reference to human GAPD or mouse ribosomal L7 protein gene control.

	ACKNOWLEDGEMENTS

 
We thank J.F. Hu and Y. Yang for pioneering the ChIP protocols in the laboratory, and G. Ulaner, E. Littman, X.M. Yao, Z. Zeng, M. Daniels, Q. Wang and H. Chen for technical assistance. We acknowledge the Central Laboratory for Human Embryology Tissue, University of Washington, Seattle, for human fetal tissues. This work was supported by NIH Grant DK36054 and the Research Service of the Department of Veterans Affairs.

	FOOTNOTES

 
^* To whom correspondence should be addressed at: Stanford Medical School, 3801 Miranda Avenue, Palo Alto, CA 94304, USA. Tel: +1 6504935000 ext. 63185; Fax: +1 6508568024; Email: thanhvu{at}stanford.edu

The authors wish it be known that, in their opinion, the first two authors should be regarded as joint First Authors.

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