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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Genomics. Author manuscript; available in PMC Feb 21, 2007.
Published in final edited form as:
PMCID: PMC1802099
NIHMSID: NIHMS16518

Allele-specific histone modifications regulate expression of the Dlk1-Gtl2 imprinted domain

Abstract

Dlk1 and Gtl2 are reciprocally expressed imprinted genes located on mouse chromosome 12. The Dlk1-Gtl2 locus carries three differentially methylated regions (DMRs), which are methylated only on the paternal allele. Of these, the intergenic (IG) DMR, located 12 kb upstream of Gtl2 is required for proper imprinting of linked genes on the maternal chromosome, while the Gtl2 DMR, located across the promoter of the Gtl2 gene, is implicated in imprinting on both parental chromosomes. In addition to DNA methylation, modification of histone proteins is also an important regulator of imprinted gene expression. Chromatin immunoprecipitation was therefore used to examine the pattern of histone modifications across the IG and Gtl2 DMRs. The data show maternal specific histone acetylation at the Gtl2 DMR, but not at the IG DMR. In contrast, only low levels of histone methylation were observed throughout the region, and there was no difference between the two parental alleles. An existing mouse line mouse line carrying a deletion/insertion upstream of Gtl2 is unable to properly imprint the Dlk1-Gtl2 locus, and demonstrates loss of allele-specific methylation at the Gtl2 DMR. Further analysis of these animals now shows that the loss of allele-specific methylation is accompanied by increased paternal histone acetylation at the Gtl2 DMR, with the activated paternal allele adopting a maternal acetylation pattern. These data indicate that interactions between DNA methylation and histone acetylation are involved in regulating the imprinting of the Dlk1-Gtl2 locus.

Keywords: Dlk1, Gtl2, genomic imprinting, mouse, methylation, acetylation, histone

Introduction

Genomic imprinting is the differential expression of a gene based upon parental inheritance. Epigenetic regulation of the ~80 known imprinted genes leads to silencing of either the maternal or paternal copy, but the molecular mechanisms regulating imprinting are understood for only a few genes. For those imprinted genes that have been studied, regulatory mechanisms vary widely. One common regulator of imprinted genes is DNA (CpG) methylation, and many imprinted genes show differentially methylated regions (DMRs) which carry methylation on only one parental allele [1]. In addition to DNA methylation, modification of histone proteins is emerging as an important regulator of genomic imprinting [1]. It has been proposed that the N terminal tails of histone proteins store epigenetic information in specific covalent modifications, including acetylation, phosphorylation, methylation, and ubiquitination [2]. This histone code marks individual nucleosomes and regulates their interaction with other factors; the sum of these marks defines the transcriptional status of a gene. Acetylation of histone proteins is nearly always associated with transcriptional activation, for example, while methylation of histones may be associated with active or inactive genes depending on the specific residue methylated. Methylation of Lys9 and Lys27 of histone H3 (H3K9 and H3K27) are linked to heterochromatin and gene silencing, while methylation of Lys4 (H3K4) is linked to transcriptional activity [3].

Differential histone modification between the active and silent alleles has been reported for several mouse imprinted genes [4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14]. The silent allele of an imprinted gene is generally characterized by compact chromatin, high levels of histone methylation and hypoacetylation, while the expressed allele is characterized by a more open chromatin state and acetylation of histone tails. That histone acetylation is involved in the imprinting mechanism is illustrated by the ability of histone deacetylase (HDAC) inhibitors to alter the expression of some, but not all, imprinted genes [6; 8; 9; 15; 16]. Recent studies suggest that DNA methylation is able to direct histone modifications through the recruitment of HDACs and histone methyltransferases (HMTs) by methyl-binding proteins [17; 18; 19; 20; 21]. This interaction is not essential for histone regulation of imprinting however; at the imprinted human SNRPN locus, for example, histone acetylation is present even in the absence of differential DNA methylation [22]. The particular histone modifications that are needed to confer imprinting are likely to be specific to individual imprinted loci.

Two reciprocally expressed genes, Dlk1 and Gtl2, lie in an imprinted domain located on distal mouse chromosome 12 and human chromosome 14q32 [23; 24; 25]. Dlk1, a gene expressed only from the paternal allele, encodes a transmembrane protein related to Delta/Notch that appears to function as a growth factor, maintaining the proliferation of undifferentiated cells [26; 27; 28; 29; 30]. The Gtl2 gene produces a maternally expressed noncoding RNA transcript whose function is unknown [31]. Several additional genes have been identified within this imprinted domain, although with the exception of Dlk1 and Dio3, most produce noncoding RNAs [32; 33; 34]. Three DMRs are found at the Dlk1-Gtl2 locus, the Dlk1 DMR in the 3′ region of the Dlk1 gene, the intergenic (IG) DMR located 12 kb upstream of Gtl2 and the Gtl2 DMR across the promoter and first exon of the Gtl2 gene [23; 24]. The IG DMR is required for proper imprinting of all genes in the region on the maternal chromosome. Maternal deletion of this region causes the maternal chromosome to adopt a paternal imprinting pattern, with expression of Dlk1 and silencing of Gtl2 [35]. The Gtl2 DMR is also required for imprinting of Dlk1-Gtl2 [36]. Mice carrying a deletion/insertion upstream of the Gtl2 gene (Gtl2Δ5′Neo) show activation of the normally silent gene on the mutant chromosome-Dlk1 upon maternal inheritance and Gtl2 upon paternal inheritance [36]. Additional evidence to inform the imprinting mechanism of Dlk1-Gtl2 comes from mice lacking the Eed gene. EED is a member of the polycomb family of proteins, which function to effect local chromatin structure through interactions with HDACs and HMTs [37; 38; 39; 40]. Eed null animals show biallelic expression of Gtl2, but maintain imprinting of Dlk1 [41]. This loss of Gtl2 imprinting suggests a role for polycomb proteins in the regulation of paternal Gtl2 silencing.

While there is ample evidence indicating a role for DNA methylation in Dlk1-Gtl2 regulation, no data exists on the role of histone modifications in controlling the expression and imprinting of these genes. In this study, the pattern of histone modifications at the IG and Gtl2 DMRs were investigated in an allele-specific manner using chromatin immunoprecipitation (ChIP). The data show that in midgestation mouse embryos there is differential histone acetylation between the maternal and paternal alleles of the Gtl2 DMR, but not the IG DMR. The active maternal allele carries an open chromatin conformation with hyperacetylation of histones H3 and H4, while the silent paternal allele has hypoacetylated histones. Analysis of histone modifications in the Gtl2Δ5′Neo mouse line, which carries a Neo insertion upstream of Gtl2, emphasizes their regulatory importance. In these mice the mutant paternal Gtl2 allele, which shows loss of DNA methylation and inappropriate activation, adopts a maternal pattern of histone hyperacetylation. Removal of the Neo cassette from these animals restores Gtl2 imprinting and paternal DMR methylation, and the wild type histone acetylation pattern is also recovered. Surprisingly, given the apparent role of EED in Gtl2 imprinting, no evidence was found for histone methylation in this region. These data indicate that both DNA methylation and histone acetylation are involved in maintaining the imprinting of the Dlk1-Gtl2 genes.

Results

Methylation analysis of the Gtl2 upstream region

DNA methylation is a known regulator of genomic imprinting, and many imprinted genes are associated with DMRs that play a role in regulating their allele-specific expression. Three DMRs have been identified at the Dlk1-Gtl2 locus, but only the IG DMR located 12 kb upstream of Gtl2 acquires its methylation in the germline, suggesting that this region represents the gametic mark for these genes [24]. The Gtl2 DMR begins approximately 1.5 kb upstream of Gtl2, and continues through the first exon of the Gtl2 gene, but acquires its methylation post-fertilization. This is different from the structurally similar Igf2-H19 region, where the germline DMR is located at -2 to -4 kb upstream of the H19 transcriptional initiation site. Regions further upstream of Gtl2 that correspond to the H19 DMR had not been characterized for their methylation status, so it was possible additional Dlk1-Gtl2 DMRs existed. Allele-specific bisulfite mutagenesis analysis was therefore used to analyze the region upstream of the known Gtl2 DMR. Allele-specific analysis requires an assay that differentiates between the maternal and paternal alleles of a given gene. Towards this end, sequence analysis was used to identify polymorphisms in the Gtl2 upstream region between the Mus musculus domesticus strain C57BL/6 (B6), and the Mus musculus castaneus strain Cast/Ei.

Five sets of nested PCR primer pairs were designed to specifically amplify the mutagenized version of the Gtl2 upstream region, and together span the interval from -1279 through -3887, relative to Gtl2 (Fig. 1). This region contains a total of 32 CpG dinucleotides that could be assayed for their methylation status. Genomic DNAs from e12.5 embryos of crosses between B6 and Cast/Ei animals were subjected to bisulfite mutagenesis, followed by PCR amplification, cloning and sequencing. Bisulfite mutagenesis changes unmethylated cytosines to thymines, but methylated cytosines are protected from modification. Sequencing of multiple individual clones from the resulting PCR product therefore identifies remaining cytosines within CpG dinucleotides as having been methylated in the original DNA. DNA sequence polymorphisms within the analyzed region can be used to assign parental identity to any methylated cytosines identified, and complete mutagenesis is verified by the lack of unmodified cytosines outside of CpG dinucleotides. Bisulfite analysis of the more distal Gtl2 upstream region showed that the region is relatively highly methylated, but there is no allele-specific pattern to this methylation (Fig. 1). The more proximal region showed a paternal methylation bias, but significant methylation was found on the maternal allele as well. As the more distal portion of the region analyzed is technically within the Gtl2 DMR, it suggests that the paternal-specific methylation is relaxed near the end of the DMR. Overall, the maternal allele was 60% methylated (128 of 214 CpG dinucleotides) while the paternal allele was 67% methylated (137 of 205 CpG dinucleotides). Calculating the statistical significance of this data, using a single factor ANOVA to compare the ratio of methylated to unmethylated CpGs on each DNA strand, yields a p-value of 0.87. The difference in methylation between the maternal and paternal alleles is therefore not significant.

Figure 1
Bisulfite analysis of DNA methylation within the Gtl2 upstream region. Schematic of the Gtl2 upstream region and bisulfite sequence analysis. The vertical black line represents the first exon of Gtl2, the horizontal gray bar represents the Gtl2 DMR, and ...

Analysis of allele-specific histone acetylation

Acetylated histones are associated with actively transcribed chromosomal regions [42; 43]. Allele-specific acetylation of lysine residues in the tails of histones H3 and H4 has been identified within the active alleles of imprinted genes, while deacetylation of H3 and H4 is associated with inactive alleles. The regulation of the imprinted Dlk1 and Gtl2 genes is beginning to be elucidated, yet the role of histone modifications in this regulation has not been explored. Chromatin immunoprecipitation (ChIP) was therefore used to investigate the presence of allele specific histone modifications at the Dlk1-Gtl2 locus.

Five individual genomic regions upstream of Gtl2 were analyzed for the presence of allele-specific histone modifications in midgestation B6 x Cg12 embryos (Fig. 2A). The Cg12 mouse line is a congenic strain that carries a distal chromosome 12 derived from M. m. castaneus on a M. m. domesticus background [34]. As the specific elements within the Gtl2 upstream region that regulate imprinting are uncertain, this analysis encompassed the IG and Gtl2 DMRs, as well as several non-differentially methylated regions of interest. The Gtl2 upstream regions analyzed have been designated 1 through 5, moving from proximal to distal, and numbered relative to the Gtl2 transcriptional initiation site. Region 1 is located from -12224 to -11224 bp, within the IG DMR, Region 2 is located at -5145 to -5043 bp, Region 3 is located at -4079 to - 3973, Region 4 is located at -3125 to -2969 bp, within the region analyzed by bisulfite sequencing in this work, and Region 5 is located at -411 to -273, within the Gtl2 DMR. To analyze histone acetylation, antibodies that specifically recognize the acetylated forms of histone H3 and H4 were used. Following immunoprecipitation, a radiolabeled PCR assay was performed with primers that amplify each region from both the input (“In”, sonicated DNA prior to immunoprecipitation) and immunoprecipitated (“IP”) sample. The PCR products were digested with enzymes that distinguish between the B6 and Cg12 alleles, separated by gel electrophoresis, and the M/P ratio calculated. The M/P ratio is the ratio of percent precipitation of the maternal and paternal alleles. When each allele from the IP samples is standardized against the corresponding input sample, there was a >10-fold enrichment of histone H3 acetylation on the maternal chromosome, relative to the paternal, within Region 5 (Fig. 2B). The IG DMR (Region 1) and the intervening Regions 2, 3 and 4 have lower levels of acetylation and no asymmetry between the maternal and paternal alleles (Fig. 2B). Acetylation of histone H4 showed a pattern very similar to that seen for H3, with Region 5 exhibiting a >10-fold enrichment for H4 acetylation on the maternal allele in comparison to the paternal allele (Fig. 2C). The histone acetylation data is shown graphically in Figure 4A. The maternal Gtl2 DMR is therefore enriched for acetylated histones, while the paternal Gtl2 DMR is hypoacetylated. The paternally hypoacetylated Region 5 corresponds to the sequences that carry paternal allele-specific DNA methylation. Note that Region 4 falls within the sequences analyzed in this work by bisulfite mutagenesis. The lack of allele-specific histone acetylation in this region, along with the lack of allele-specific methylation, indicates that it is unlikely to play a role in Dlk1-Gtl2 regulation. Despite its role in regulating Dlk1-Gtl2 imprinting, the IG DMR carries no allele-specific histone acetylation.

Figure 2
Analysis of histone acetylation within the Gtl2 upstream region. (A) Schematic representation of the genomic region upstream of the Gtl2 gene. The vertical black lines indicate the exons of Gtl2, the horizontal gray boxes indicate the differentially methylated ...
Figure 4
Graphical summary of the histone acetylation and methylation data. (A) Summary charts showing the quantitative data from ChIP analysis using antibodies that recognize acetylated histone H3 and H4. In each panel, the black bars represent levels of acetylation ...

Analysis of allele-specific histone methylation

Earlier studies reported loss of imprinting of Gtl2 in the mouse Eed knockout [41]. EED, the mouse homologue of ESC, is a member of the Polycomb group (PcG) protein family. PcG proteins form complexes involved in long term gene silencing during development [44]. PcG complexes containing EED have histone methyltransferase activity, suggesting that PcG-mediated silencing functions through histone methylation [37; 38; 39]. The repressed state of a gene appears to correlate with methylation of histone H3K9 (H3meK9) and H3K27 (H3meK27), which may be mono-, di-, or trimethylated. In imprinted X-chromosome inactivation, for example, an EED complex methylates H3triMeK27 on the inactive X [45]. At the imprinted Kcnq1ot1 locus, repressed genes contain histone dimethylation of H3K9 (H3diMeK9) and histone H3 trimethylation of Lys27 (H3triMeK27) [46].

To determine if the paternal silencing of Gtl2 is due to the methylation of particular residues on histone H3, the ChIP assay was performed using antibodies that individually detect mono-, di- and tri-methylated forms of histone H3 at Lys9 and Lys27. Antibodies specific for dimethylation of H3K9 (H3diMeK9) and H3K27 (H3diMeK27) showed only low levels of histone methylation, and no paternal-specific enrichment of these modifications (Fig. 3A, B). To ensure the results were not due to inefficient precipitation, primers were used that amplify the H19 DMR, a region reported to contain H3diMeK9 [9]. The assay was able to efficiently detect this particular modification at H19 (Fig. 3A). Antibodies specific for the mono- and tri-methylated forms of histone H3 also detected no significant levels of these modifications (data not shown). The histone methylation data is shown graphically in Figure Figure4B4B and and4C.4C. These data suggest that there is little histone methylation at the Dlk1-Gtl2 region, and no allele-specific pattern to the methylation that does exist.

Figure 3
Analysis of histone methylation within the Gtl2 upstream region. (A) Allele-specific ChIP analysis of histone H3 methylation using an antibody against histone H3 di-methyl K9 (H3diMeK9). In both panels, “In” indicates the sonicated input ...

Analysis of CTCF binding to a Gtl2 intronic site

The boundary protein CTCF is required for regulating the imprinting of the Igf2-H19 and Rasgrf1 loci, and is involved in the process of X-chromosome inactivation [47; 48; 49; 50]. At Igf2-H19, CTCF binds to the unmethylated maternal H19 DMR and prevents Igf2 from accessing downstream enhancers, thus keeping the maternal Igf2 allele silent. Paternally, the CTCF binding site is inactivated by DNA methylation, preventing boundary formation. Based upon sequence analysis across the Gtl2 region, a single potential CTCF binding site was identified within the first intron of Gtl2 [51]. Bisulfite analysis of this site revealed differential methylation between the maternal and paternal alleles, although the paternal bias was less absolute than at other DMRs [52]. This is unlike the CTCF binding sites within the H19 DMR, which contain a fully unmethylated maternal and a fully methylated paternal allele [53]. The Gtl2 intronic element had not been analyzed for actual binding of CTCF, so the ChIP assay was performed using a CTCF antibody (Fig. 5A). The results of this analysis indicate that CTCF does not bind to this element in midgestation embryos. A CTCF binding site located in the H19 DMR was used as a positive control, and showed the expected presence of CTCF (Fig. 5A) [9]. No other CTCF sites are found in this region that conform to the consensus typically seen at imprinted loci, so CTCF is unlikely to play a role in Dlk1-Gtl2 regulation.

Figure 5
Analysis of DNA binding protein interactions within the Gtl2 upstream region. (A) ChIP analysis using an antibody that recognizes the CTCF protein. The genomic region analyzed spans a potential CTCF binding site within the first intron of Gtl2. The “ ...

Analysis of YY1 binding to the Gtl2 upstream region

YY1 is a Gli-type zinc finger protein that can function as either a transcriptional activator or a silencer depending on the promoter context [54]. In addition, YY1 interacts with a complex containing EED and the histone methyltransferase EZH2, resulting in the methylation of H3K27 and gene silencing [55; 56; 57]. Similarly to CTCF, YY1 has been shown to have chromatin boundary activity; YY1 binds to sequences in the first intron of the imprinted gene Peg3, where it functions as a methylation-sensitive insulator [58]. Insulators are DNA binding proteins that, when positioned between promoters and enhancers, silence genes by preventing access to the enhancers. Since CTCF does not appear to regulate the Gtl2 gene, it was possible that a YY1-mediated boundary was involved in imprinting regulation at this locus. The lack of H3K27 methylation at the Gtl2 locus does not preclude a role for YY1 if it is acting as a boundary. For example, YY1 regulation of the Peg3 locus is independent of H3K27 methylation. A candidate YY1 site was identified 5072 bp upstream of Gtl2 (within Region 2) that had a single nucleotide mismatch to the YY1 consensus (Fig. 5B). The demonstrated YY1 consensus sequence contains a CpG dinucleotide, and YY1 binding is methylation sensitive, such that methylation of this CpG prevents binding. The sequence found upstream of Gtl2 does not contain the CpG dinucleotide, indicating that it would not be sensitive to DNA methylation. ChIP analysis using an antibody against YY1 did not show binding to the Gtl2 upstream site (Fig. 5B). A genomic region located within the c-myc promoter that has been reported to bind YY1 was used as a positive control [59].

Analysis of methyl-binding protein interaction with the Gtl2 upstream region

One mechanism by which DNA methylation leads to transcriptional silencing is through the actions of methyl-binding proteins. The methyl-binding proteins MeCP2 and MBD2 interact preferentially with methylated DNA and recruit HDACs, leading to transcriptional repression [17; 18; 19]. In addition, MeCP2-bound promoter regions often show methylation of H3K9 [21]. The paternal Gtl2 DMR is methylated and hypoacetylated, suggesting that methyl-binding proteins might be acting to recruit HDACs to the region. The association of methyl-binding proteins with the methylated allele of other imprinted loci has been reported [7; 15]. MBD2 and MeCP2 binding was analyzed by ChIP at the two Gtl2 upstream regions that display DNA methylation, the IG and Gtl2 DMRs. Antibodies against MeCP2 and MBD2 did not show binding to either region (Fig. 5C, D). A direct assay specific for Histone deacetylase 1 (HDAC1) using ChIP with an anti-HDAC1 antibody also did not show interaction with the IG or Gtl2 DMRs (data not shown). MeCP2 has been shown to bind within the 5′ UTR of the imprinted U2af1-rs1 gene, and this region was used as a positive control for MeCP2 immunoprecipitation (Fig. 5C) [7].

Analysis of histone acetylation in a Dlk1-Gtl2 loss of imprinting mouse model

To study the role of the Gtl2 upstream region in regulating the imprinting of Dlk1-Gtl2, a targeted deletion was previously generated in which 2.8 kb of this region is replaced with the Neo selectable marker (Gtl2Δ5′Neo allele) (Fig. 6A) [36]. The deleted region lies immediately adjacent to the Gtl2 DMR, but leaves the DMR itself intact. The Gtl2Δ5′Neo allele causes loss of imprinting at the Dlk1-Gtl2 locus after both paternal and maternal inheritance. Relevant to this work, after paternal inheritance Gtl2Δ5′Neo embryos show activation of the normally silent paternal Gtl2 allele, accompanied by the loss of paternal-specific Gtl2 DMR methylation. When the Neo cassette is excised (Gtl2Δ5′ allele), however, the expression and methylation patterns are wild type (Fig. 6A). These results indicate that it is not the deleted region itself, but rather Neo-induced loss of epigenetic modifications at the adjacent Gtl2 DMR, that is responsible for the Gtl2Δ5′Neo loss of imprinting.

Figure 6
Analysis of histone acetylation patterns in Gtl2Δ5′Neo and Gtl2Δ5′ mice. (A) Schematic showing the Gtl2Δ5′Neo and Gtl2Δ5′ alleles. The vertical black lines represent the Gtl2 exons, the horizontal ...

There is significant evidence for interaction between the DNA methylation and histone modification machineries. For example, the DNA methyltransferases DNMT1 and DNMT3a associate directly with the histone deacetylase HDAC1 [20; 60; 61; 62]. It was important to ask, therefore, whether the loss of paternal-specific methylation at the Gtl2 DMR in Gtl2Δ5′Neo mice was associated with changes in the normal pattern of histone modifications. To assay the Gtl2Δ5′Neo and Gtl2Δ5′ mice in an allele-specific manner, paternal mutants were crossed to Cg12 females and midgestation embryos analyzed. In the Gtl2Δ5′Neo mice, Region 5 on the paternal allele was acetylated on histones H3 and H4 at levels comparable to the wild type maternal allele (Fig. 6B, C, middle). No changes in acetylation were observed at either Region 1 or Region 2 (Regions 3 and 4 are deleted in this mutant). The Gtl2Δ5′ mice in which Neo is excised display wild type patterns of both DNA methylation and histone acetylation (Fig. 6B, C, bottom), which correlates with the recovery of proper Dlk1-Gtl2 imprinting in these animals.

Analysis of enhancer-blocking activity within the Gtl2 upstream region

It was shown previously that the imprinted expression of the Igf2 and H19 genes depends on a CTCF-mediated boundary element located in the H19 DMR [47; 48]. To ask if the Dlk1-Gtl2 locus may also be regulated by a boundary (potentially a non-CTCF boundary given the lack of CTCF interaction by ChIP), a previously reported assay was used to examine the Gtl2 upstream region for enhancer-blocking activity [63]. Enhancer-blocking, the ability of a DNA element to block promoter-enhancer interactions, is one property displayed by chromatin boundary elements. In this assay, DNA fragments to be tested are cloned between an enhancer and a promoter driving expression of the Neomycin resistance gene as a reporter. The constructs are stably transfected into human leukemia K562 cells, and enhancer activity is reflected in the number of Neomycin-resistant colonies formed. A functional boundary cloned between the enhancer and Neo produces a reduction in colony number. Five fragments spanning the Gtl2 upstream region were analyzed for enhancer-blocking (Fig. 7A). None of the Gtl2 upstream fragments displayed any enhancer-blocking activity, as determined by the relative number of colonies generated compared to a negative control (no insert between the enhancer and the Neo cassette) (Fig. 7B). The Igf2-H19 boundary was used as a positive control, and reduced the number of Neo-resistant colonies by 5-10 fold, in line with previous data [48].

Figure 7
Enhancer blocking assay for the Gtl2 upstream region. (A) Schematic of the Gtl2 upstream region and the fragments analyzed for enhancer blocking activity. The vertical black line represents the first exon of Gtl2, and the horizontal gray bars represent ...

Discussion

Differential histone acetylation marks the Gtl2 DMR

The data presented here demonstrate differences in chromatin conformation between the parental alleles of the Gtl2 DMR, as evidenced by the asymmetrical acetylation of histones H3 and H4. The Gtl2 DMR of the silent paternal allele is hypoacetylated on H3 and H4, while the active maternal allele carries high levels of acetylation on both histones. While it is possible this differential acetylation is merely reflective of the transcriptional state of the genes, existing data suggests it is more likely that it is actually required for the imprinted regulation of Gtl2. The promoters of the human housekeeping genes β-ACTIN and GAPDH, for example, are highly transcribed yet do not exhibit increased levels of acetylation [14]. Rather, it may be that the high levels of maternal acetylation are required to keep this allele active and resistant to the silencing that occurs on the paternal allele. It is important to remember that, unlike the IG DMR, methylation of the paternal Gtl2 DMR occurs not in the germline, but in the embryo following fertilization. Silencing is thus occurring in the context of both parental alleles, yet must be restricted to the paternal allele alone.

Histone methylation is not involved in Gtl2 imprinting

Data has shown that the EED protein is required for the silencing of the paternal Gtl2 allele [41]. EED-dependent silencing is typically mediated through histone methylation, however, a modification that was not found at the Gtl2 locus. Additionally, the EED complex would need to be targeted to the Gtl2 region by a specific DNA binding protein. An ideal candidate for this role is YY1, a protein that is known to interact with EED, and has a potential binding site upstream of Gtl2. There is a precedent for YY1 in imprinting regulation, since it functions as an insulator at the Peg3 locus [58]. Chromatin immunoprecipitation assays did not detect YY1 binding at Gtl2, however, suggesting that if a protein does localize EED to the Gtl2 region it is not YY1. The histone deacetylase HDAC1, which is part of the EED complex, was also not detected in this region. Taken together, these data suggest that EED is not a regulator of Gtl2 imprinting, yet the Eed null mice argue for its involvement. How to reconcile these seemingly disparate data? The EED complex binds to the inactive X chromosome early in development, but later becomes undetectable [64; 65]. It may be that the EED repressive complex is necessary for establishing the silent chromatin state through histone methylation, but another mechanism is responsible for its subsequent maintenance. Likely candidates for this additional mechanism are DNA methylation and histone hypoacetylation. Arguing against this model acting at Gtl2, however, is the fact that even in the absence of the EED complex, the histone H3K9 and H3K27 marks persist on the inactive X chromosome [66; 67]. EED may therefore regulate Gtl2 by a previously undescribed mechanism. Further experiments at earlier developmental stages will be required to clarify its role. To examine any link between DNA methylation and histone hypoacetylation, MeCP2 and MBD2 binding to the IG and Gtl2 DMRs was examined. Unlike many other imprinted regions, the data suggest that these methyl-binding proteins do not bind upstream of Gtl2.

Loss of Gtl2 imprinting in Gtl2Δ5′Neo mice correlates with changes in chromatin state

Gtl2Δ5′Neo mice that show loss of Gtl2 imprinting after paternal inheritance also display a gain of acetylation at the now active paternal allele. In Gtl2Δ5′ mice, where the Neo gene is excised, however, imprinting, DNA methylation and histone acetylation are wild type. These data indicate that the deleted region is not causative for the loss of imprinting of Gtl2Δ5′Neo mice, but rather that the presence of the Neo gene prevents silencing from being established or maintained. Although an artificial system, the integration-mediated changes in paternal DNA methylation and histone acetylation, and concomitant activation of Gtl2, indicate a role for these epigenetic marks in silencing the paternal Gtl2 allele. The Neo gene is transcribed towards the promoter of Gtl2, and the failure to silence may be a result of transcription from Neo into the Gtl2 DMR. If this is the case, this phenotype may not be specific to this Neo cassette, but rather result from the insertion of any active transcriptional unit in the same position. This hypothesis is supported by the very similar phenotype of another mutant mouse line, Gtl2lacZ, which carries a lacZ/Neo insertion upstream of Gtl2 and also results in loss of Dlk1-Gtl2 imprinting [31; 36; 68]. This transgene is transcribed away from the Gtl2 promoter, yet still results in paternal expression of Gtl2. Alternatively, the Neo gene may physically disrupt an element or group of elements required for paternal Gtl2 silencing. The Gtl2Δ5′Neo mice demonstrate that loss of DNA methylation at the paternal Gtl2 DMR correlates with gain of histone acetylation within this region. These data suggest a mechanism in which directed histone acetylation is the default state for the Gtl2 allele; this is accomplished on the maternal allele, but prevented on the paternal allele by specific silencing mechanisms. In this model, silencing of the Gtl2 paternal allele is accomplished by EED, through HMTs or another mechanism, and then maintained by DNA methylation and the ability of DNMTs to effect histone hypoacetylation.

Materials and Methods

Mouse maintenance and breeding

Wild type embryos analyzed for the ChIP experiments were the F1 offspring from a cross between the Mus musculus domesticus strain, C57BL/6 (B6), and the congenic mouse strain Cg12, and were analyzed at midgestation (e12-e14) [34]. For analysis of histone modifications in Gtl2Δ5′Neo and Gtl2Δ5′ mice, F1 embryos from crosses to Cg12 were isolated. For the bisulfite mutagenesis assays, genomic DNAs were isolated from F1 offspring of crosses between B6 and pure Mus musculus castaneus (Cast/Ei) animals. Animals were maintained in microisolator cages, on a standard diet with a 14:10 light:dark cycle. All animals used in these experiments were maintained in compliance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and The University of Illinois at Chicago Animal Care Committee guidelines.

Bisulfite mutagenesis

Bisulfite mutagenesis was performed as described [53], with slight modifications. Genomic DNA from e12.5 embryos was digested to completion with EcoRI, and subjected to bisulfite mutagenesis for 16 hours at 55°C. The DNA was purified with the QiaexII kit (Qiagen) according to the manufacturer’s protocol. PCR amplification was accomplished using 5 μl of mutagenized DNA, with two sets of nested primers for each region. PCR primers were generated against the top strand of the regions under analysis, and were designed to recognize only the mutagenized sequence. Primer sets used, and the regions amplified relative to the Gtl2 transcriptional initiation site, are: OL688, 5′-GTTAGGAAATATGTGGTTTAGAG-3′, and OL686, 5′-AAAAAAAACAAAATACCCCAAACC-3′, followed by OL689, 5′-AGATGTTGATATTTGGAGGATAG-3′ and OL687, 5′-AAAACAAAATACCCCAAACCAAC-3′ (-3887 bp to -3049 bp); OL690, 5′-TAGGTTGTTATATTTAGGTTATATAG-3′, and OL684, 5′-CAAATATTAACCTAAAAACTATCACC-3′, followed by OL691, 5′-ATTGTAAGATTGAGGTTAGTTTGG-3′ and OL678, 5′-AACAACATATTTTACCTTCTAACTTC-3′ (-3112 bp to -2457 bp); OL692, 5′-TAGGTTTTGAGTTTTAGAGAAGTTG-3′, and OL679, 5′-AACCCTCAAACACCCAACAAC-3′, followed by OL693, 5′-GAAGTTGATAAATATATTTAAGTATATGG-3′ and OL680, 5′-ACCATTCTCTAAACTCCAAACC-3′ (-2616 bp to -1954 bp); OL694, 5′-TTGTTGTTAGGAATAGGTTTAGG-3′, and OL1088, 5′-AACCCCTAACAAACTAAAAAAACC-3′, followed by OL695, 5′-TTTAGGAGTAAGAGGTTTAGG-3′ and OL1089, 5′-TACAACAAAAAACATAACTCCCAAC-3′ (-2059 bp to -1575 bp) and OL922, 5′-TTTTGTTGATGATTGGTTTTAGTTAG-3′ and OL681, 5′-ACCCCCTATAACCAACAAACC-3′, followed by OL923, 5′-AGAAGGTTTTTTTAGTTTGTTAGG-3′ and OL682, 5′-AACCAACAAACCTAAAATACCAC-3′ (-1279 bp to -1555 bp).

Chromatin immunoprecipitation

Chromatin immunoprecipitation assays were performed using the Upstate Biotechnology ChIP assay kit according to the manufacturer’s protocol with the following changes. Midgestation embryos (0.1 g) were minced with a razor blade and crosslinked with 1% formaldehyde in DMEM for 15 minutes with rocking. The crosslinking reaction was stopped by the addition of 0.125 M glycine for 5 minutes. The tissue was spun at 2000 rpm for 10 minutes, then washed once in 1X PBS containing 1 mM PMSF, 1 μg/μl of pepstatin, and 1 μg/μl of aprotinin, and spun at 2000 rpm for 5 minutes. The tissue was homogenized in a Dounce homogenizer in 1X PBS containing protease inhibitors, spun at 2000 rpm for 5 minutes, and the cells frozen at -80°C. The cells were sonicated in 250 μl of lysis buffer using a Branson model 450 sonicator with a double step tip for 45 seconds (3 cycles of 15 seconds with 2 minutes between each cycle) at 30% power. The chromatin was precleared with 80 μl of protein A or protein G agarose for 1 hour at 4°C with rocking. For immunoprecipitation, 5 μg of anti-acetyl-histone H3 (Upstate Biotechnology, #06-599), 5 μg of anti-acetyl-histone H4 (Upstate Biotechnology, #06-866), 8 μg of anti-monomethyl-histone H3K9 (Upstate Biotechnology, #07-450), 8 μg anti-dimethyl-histone H3K9 (Upstate Biotechnology, #07-441), 8 μg anti-trimethyl-histone H3K9 (Upstate Biotechnology, #07-442), 8 μg anti-monomethyl-histone H3K27 (Upstate Biotechnology, #07-448), 8 μg anti-dimethyl-histone H3K27 (Upstate Biotechnology, #07-452), 8 μg anti-trimethyl-histone H3K27 (Upstate Biotechnology, #07-449), 8 μg anti-MeCP2 (Abcam, #ab3752), 8 μg anti-MBD2 (Upstate Biotechnology, #07-198), 8 μg of anti-YY1 (Santa Cruz Biotechnology, #sc-281, #sc-1703), or 8 μg anti-CTCF (Santa Cruz Biotechnology, #sc-5916, #sc-15914) were added to chromatin overnight at 4°C with rocking. Protein-DNA crosslinks were removed by adding NaCl to a concentration of 250 mM and incubating at 65°C for 4 hours. After proteinase K treatment the DNA was purified using the MiniElute PCR purification kit (Qiagen), and eluted in 50 μl.

PCR amplification

PCR reactions were performed using 5 μl of either the input or immunoprecipitated DNA, with αP-dCTP (0.3 μCi) added for the last four cycles of the 28-cycle reaction. The primers used to analyze the Gtl2 upstream region were (all positions given relative to the Gtl2 transcriptional initiation site): Region 1, OL1085, 5′-ACTCATCAAGGGTAGTTGGGTGGA-3′, and OL1083, 5′-AGCTTTCTGCCCTGGTTTAGGGAA-3′ (-11224 bp to -11124 bp); Region 2 OL982, 5′-CGTGTGTTGTACATGTGCATGAGT-3′, and OL975, 5′-GGGCTTAGGACTTTATTCAAGATGGC-3′ (-5145 bp to -5043 bp); Region 3, OL973, 5′-TGCCTTCTAGAGGAGAGGTCCGTA-3′, and OL974, 5′-CTGTTTCTCCTGCTGTGCTAGGTA-3′ (-4079 bp to -3973 bp); Region 4, OL1086, 5′-TGTAAGACTGAGGTTAGCCTGGAC-3′, and OL1087, 5′-CCCTGGAGATCTAACTCATGGTAT-3′ (-3125 bp to -2969 bp); and Region 5, OL1078 5′-AGCCCCTGACTGATGTTCTG-3′ and OL1079, 5′-TGGAAGGGCGATTGGTAGAC-3′ (-411 bp to -273 bp). The primers used to analyze CTCF binding within Gtl2 intron 1 were OL1082, 5′-AGGTGGTTGGGCTATTGGAGTCTT-3′, and OL1080, 5′-AGGTCACAAGTGTTAGCTGTGTGC-3′ (+1993 bp to +2125 bp). The primers used for the CTCF and H3K9 methylation (H3meK9) controls correspond to the third CTCF site of the H19 DMR as described previously [9]. The primers used for the MeCP2 control amplify a region of the U2af1-rs1 DMR as described previously [7]. The primers for the YY1 control amplify a YY1 binding site in the c-myc promoter [59].

The PCR products were digested with enzymes that distinguish the maternal and paternal alleles as follows: MboI cuts the B6 allele in Region 1; EcoRI cuts the Cg12 allele in Region 2; SspI cuts the Cg12 allele in Region 3; AccI cuts the Cg12 allele in Region 4; and AvaI cuts the B6 allele in Region 5. Input DNA controls (sonicated DNA prior to immunoprecipitation) were included for all regions. The digested fragments were resolved on a 5% polyacrylamide gel and quantified by Phosphorimaging. The percent precipitation was calculated by dividing the immunoprecipitated DNA signal by the input DNA signal. For each region, the percent precipitation given is the average of at least five independent immunoprecipitation experiments.

Enhancer blocking assay

Enhancer blocking activity in the Gtl2 upstream region was analyzed using five fragments that were blunt-end cloned into the KpnI restriction site of the pNI plasmid. The pNI plasmid contains the mouse γ-globin promoter driving expression of the Neomycin resistance gene, the mouse β-globin HS2 LCR and the chicken β-globin insulator [48; 63]. The constructs tested, and their positions relative to the Gtl2 transcriptional initiation site are: Construct 1, the pNI vector with no insert; Construct 2, a 3.9-kb XbaI/DraIII fragment (-4156 bp to -226 bp); Construct 3, a 2.4-kb XbaI/BglI fragment (-4156 bp to -1732 bp); Construct 4, a 1.1-kb XbaI/AflI fragment (-4156 bp to -3031 bp); Construct 5, a 1.3-kb AflI/BglI fragment (-3031 bp to -1732 bp); Construct 6, a 3.9-kb XbaI/DraIII fragment in the reverse orientation (-4156 bp to -226 bp); and Construct 7, a 3.9-kb XbaI/XbaI fragment (-15366 bp to -11450 bp). Construct 8 was a positive control containing a 1.6-kb region of the H19 DMR with demonstrated enhancer-blocking activity [48].

Exponentially growing K562 cells were harvested by centrifugation, washed in PBS and resuspended at 2 x 10 cells/ml. Cells were aliquotted in 0.5 ml portions into pre-chilled 0.4 cm electroporation cuvettes containing 1 μg of linearized DNA. The cuvettes were incubated on ice for 5-10 min, then electroporated at 200V, 500 μF in a Bio-Rad electroporator. Samples were left on ice for another 5 min, and transferred into 20 ml of IMEM media in 75 cm flasks. After 48 hrs incubation, 5 ml of cells were mixed with 27 ml of prewarmed (37°C) media containing 750 μg/ml G418 (active) and 3.5 ml of 3% agar. Each sample was mixed, poured into 150 mm plates, allowed to solidify for 30 min at room temperature and placed at 37°C. The number of colonies surviving G418 selection was counted at 3-4 weeks. At least 3 independent transfections were performed for each construct.

Acknowledgements

The authors thank Gary Felsenfeld for the pNI plasmid. This work was supported by a Kimmel Scholar Award from the Sidney Kimmel Foundation for Cancer Research, and by grant HD042013 from the National Institutes of Health, both to JVS.

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