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Human Molecular Genetics Pages 1979-1985  

Chromatin conformation of the H19 epigenetic mark
Introduction
Results
   Parental-specific nuclease hypersensitivity in the H19 5[prime] flank
   Identification of hypersensitive restriction enzyme sites
   Tissue distribution of hypersensitivity
Discussion
Materials And Methods
   Mice
   Isolation of nuclei
   DNase I digestion of nuclei
   Restriction enzyme digestion of nuclei
   DNA analysis
Acknowledgements
References

Chromatin conformation of the H19 epigenetic mark

Chromatin conformation of the H19 epigenetic mark

Amy T. Hark and Shirley M. Tilghman*

Howard Hughes Medical Institute and Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA

Received July 10, 1998; Revised and Accepted August 28, 1998

Genomic imprinting in mammals is an epigenetic process that results in differential expression of the two parental alleles. The tightly linked murine H19 and Igf2 genes are reciprocally imprinted: H19 is expressed from the maternal chromosome while Igf2 is expressed from the paternal chromosome. A single regulatory region in the 5[prime] flank of the H19 gene has been implicated in silencing both genes. On the paternal chromosome, this region is heavily methylated at CpG residues, leading to repression of the H19 gene. The mechanism by which the same region in an unmethylated state on the maternal chromosome silences Igf2 is less well understood. We have probed the chromatin structure of the region by assessing its sensitivity to nuclease digestion. Two regions of nuclease hypersensitivity that are specific to the maternal chromosome were identified. These coincide with the region that is most heavily methylated on the paternal chromosome. As is the case with paternal methylation, hypersensitivity is present in all tissues surveyed, irrespective of H19 expression. We suggest that the chromatin structure of the maternal 5[prime] flank of the H19 gene may represent an epigenetic mark involved in the silencing of Igf2.

INTRODUCTION

Genomic imprinting is an epigenetic form of gene regulation in which the two parental alleles of a gene are differentially expressed (1,2). This differential expression requires a mechanism by which the transcriptional machinery can distinguish between the parental chromosomes. For the mouse H19 gene, which encodes a maternally expressed RNA of unknown function, that mechanism is thought to involve paternal-specific methylation of the 5[prime] flank of the gene (3-5). As required for a gametic imprinting mark, methylation is found in sperm but not oocytes and is maintained throughout embryogenesis in all tissues (6,7). Furthermore, loss of this methylation in DNA methyltransferase mutant embryos results in expression of the normally silent paternal H19 gene (8). Thus, the function of the methylated epigenetic mark on the paternal chromosome is to silence the H19 gene.

Is there an epigenetic mark on the maternal chromosome? On that chromosome H19 is transcribed, but Igf2, an imprinted gene 90 kb upstream of the H19 gene, is silent (9). No methylation has been detected on the maternal Igf2 gene that could account for its silencing (10,11). Consequently, it has been proposed that imprinting of Igf2 is linked in some way to that of H19 (12). Expression of both genes has been shown to depend upon two endodermal enhancers that lie 3[prime] of the H19 gene (13). Furthermore, when the H19 gene along with its 5[prime] flank was deleted on the maternal chromosome (H19D13), the Igf2 gene was activated (14). A smaller deletion that did not remove the flanking sequence (H19D3) resulted in less pronounced activation of the Igf2 gene, arguing that the H19 5[prime] flank was playing some role in Igf2 silencing (15).

Could the unmethylated maternal H19 5[prime] flank serve as an epigenetic mark involved in regulation of Igf2 imprinting? Although CpG methylation is clearly the leading candidate for an imprinting mark, the potential for differential chromatin structure to also serve as a mark that allows the cell to distinguish between the two parental chromosomes and permit differential gene expression has not been ruled out (16).

To identify a region of the unmethylated flank that might serve as an epigenetic mark for this chromosome, we have examined in detail the chromatin state of the H19 5[prime] flanking sequence in somatic tissues of the mouse. We report the identification of two regions of nuclease hypersensitivity that are specific to the maternal chromosome. The sites coincide precisely with the most heavily methylated domains of the epigenetic mark on the paternal chromosome (6,7,17). Furthermore, the hypersensitivity is present in both expressing and non-expressing tissues. These data imply that there is a somatic function for the epigenetic region on the maternal chromosome that is independent of the transcriptional status of the H19 gene.

RESULTS

Parental-specific nuclease hypersensitivity in the H19 5[prime] flank

To examine the chromatin status of the H19 gene on the two parental chromosomes independently of one another, we used animals heterozygous for H19D13, the targeted deletion of the H19 gene and 10 kb of its 5[prime] flank (14). Nuclei from +/H19D13 and H19D13/+ mice were incubated with DNase I for increasing lengths of time. Following purification, the DNA was digested to completion with SacI and the sites of cleavage were detected using a hybridization probe at the 5[prime] end of a 3.8 kb SacI fragment upstream of the H19 gene (Fig. 1). This analysis revealed the presence of a strong hypersensitive region on the maternal chromosome at approximately -2.4 kb with respect to the H19 promoter (HS2 in Fig. 1). A less hypersensitive region, centered at approximately -3.8 kb, was also detected (HS1). Both HS1 and HS2 appeared to consist of two sites of DNase I cleavage, ~300 bp apart. Both hypersensitive regions were also detected with a probe derived from the 3[prime] end of the SacI fragment, with HS2 exhibiting the greatest degree of hypersensitivity (data not shown). In contrast to the maternal chromosome, no DNase I hypersensitivity was detected on the paternal chromosome (Fig. 1).


Figure 1. Allele-specific DNase I hypersensitivity of the H19 epigenetic region. Liver nuclei from both +/H19D13 (maternal allele) and H19D13/+ (paternal allele) mice were digested for increasing times (min) with DNase I. Following purification, the DNA was digested with SacI and subjected to Southern blot analysis. The blots were probed with a 750 bp SacI-EcoRI fragment, which detects a 3.8 kb SacI fragment in the flank of the H19 gene. The SacI, SacI+EcoRI and SacI+PvuI lanes contain liver DNA digested with the enzymes indicated. The diagram depicts the 5[prime]-end of the H19 gene, with the start of transcription represented by the horizontal arrow, while vertical arrows represent nuclease hypersensitivity detected on the maternal chromosome, designated HS1 and HS2. S, SacI; R, EcoRI; P, PvuI.

To reinforce the conclusion that the maternal chromosome is in a different chromatin conformation than the paternal chromosome, we exploited the methylation-insensitive enzyme HindIII, which cleaves at HS2. Nuclei from both +/H19D13 and H19D13/+ mice were treated with HindIII for increasing lengths of time and the resulting DNAs were digested to completion with EcoRI. On the maternal chromosome, a 3.8 kb EcoRI fragment immediately upstream of the H19 promoter was digested into two fragments by cleavage at the single HindIII site (Fig. 2A). However, no digestion was apparent on the paternal chromosome, even after long exposures of the blot. To confirm that these two experiments were measuring HindIII hypersensitivity in an equivalent manner, the blots were stripped and hybridized with a probe that is specific for a HindIII-hypersensitive site within the [alpha]-fetoprotein (AFP) enhancer region (18). As shown in Figure 2B, the hypersensitivity detected in the AFP gene was identical in the two sets of DNA samples. This observation confirms the findings with DNase I, which suggests that the hypersensitive regions are specific to the maternal chromosome.


Figure 2. Allele-specific hypersensitivity to HindIII. Liver nuclei from +/H19D13 (maternal allele) and H19D13/+ (paternal allele) mice were digested with HindIII for increasing periods of time, indicated in minutes. Following purification, the DNA was digested with EcoRI and subjected to Southern blot analysis. (A) Blots were hybridized to a 3.8 kb EcoRI fragment. The lane designations are similar to those in Figure 1. The diagram depicts the 5[prime] flank of the H19 gene, with the start of transcription indicated by the horizontal arrow. The position of HS2 is indicated by the vertical arrow. H, HindIII; B, BglII; P, PvuI; R, EcoRI. (B) The blots in (A) were stripped and hybridized to a 2.1 kb fragment downstream of the AFP promoter that detects a 10 kb EcoRI fragment. The diagram depicts the structure of the AFP locus, with the start of transcription indicated by the horizontal arrow and the three enhancers by closed ovals. The vertical arrow indicates the position of the hypersensitive HindIII site in enhancer I.

Identification of hypersensitive restriction enzyme sites

We performed more extensive mapping of HS1 and HS2 using 4 and 5 bp recognition restriction enzymes that cleave multiple times within the 5[prime] flank. HhaI allowed us to survey the region on the unmethylated maternal chromosome. However, because the enzyme does not cleave its recognition site (GCGC) when the internal C residue is methylated, it cannot be used to examine the chromatin structure on the paternal chromosome.

Nuclei from +/H19D13 heterozygous mice, in which the intact chromosome is inherited maternally, were treated with HhaI for increasing lengths of time and, following purification, the DNA was digested to completion with EcoRI. With two independent probes that detect the same 3.8 kb EcoRI fragment upstream of the H19 promoter, a strong hypersensitive site that mapped 2.2 kb upstream of the promoter was detected (HS2, Fig. 3A and data not shown). There were several minor cleavage sites surrounding this site, suggesting that the region of hypersensitivity extended beyond the single site. This region coincides with the HS2 region identified in the DNase I experiment. As expected from earlier studies (3,4), cleavage was also observed at the H19 promoter (HS3 in Fig. 3A). These two sets of hypersensitive sites within the 3.8 EcoRI fragment are more accessible than other HhaI sites within the region, which remained uncleaved over the time course of digestion in chromatin (see Fig. 3 for positions of HhaI sites). No preferential digestion of these hypersensitive sites was observed in naked DNA (data not shown). The hypersensitivity at HS2 was confirmed using another enzyme, Sau3AI, which allowed us to survey a different set of potential cleavage sites (data summarized in Fig. 5).


Figure 3. HhaI hypersensitivity in the 5[prime] flanking region of the H19 gene. (A) Liver nuclei from +/H19D13 mice were digested with HhaI for the minutes indicated, followed by DNA purification and complete digestion with EcoRI. The DNA was subjected to Southern blot analysis and the blot was hybridized to a 660 bp XbaI-EcoRI fragment that detects a 3.8 kb EcoRI fragment immediately upstream of the H19 promoter. EcoRI, EcoRI-digested C57BL/6 liver DNA. (B) Brain nuclei from +/H19D13 mice were treated as described in (A). EcoRI+PvuI, C57BL/6 liver DNA digested with EcoRI and PvuI to provide size markers for mapping hypersensitive digestion products. The diagram represents the 5[prime] flank of the H19 gene, with the H19 promoter indicated by a horizontal arrow. The hatch marks above the line represent the positions of HhaI sites within the 3.8 kb EcoRI fragment. Hypersensitive digestion at both the promoter (HS3) and at -2.2 kb (HS2) are indicated. The probe is indicated by the shaded box. R, EcoRI; P, PvuI.

To fine map the cleavage at HS1, we used the restriction enzyme HinfI, which cleaves multiple times in the SacI fragment in naked DNA. As shown in Figure 4, hypersensitive cleavage sites coincided with HS1, as well as HS2 in chromatin. This experiment confirmed the DNase I results at both hypersensitive regions (Fig. 5 and data not shown).


Figure 4. HinfI hypersensitivity in the 5[prime] flanking region of the H19 gene. Liver nuclei from +/H19D13 mice were digested with HinfI for the minutes indicated, followed by DNA purification and complete digestion with SacI. The DNA was subjected to Southern blot analysis and the blot was hybridized to a 0.75 kb SacI-EcoRI fragment. The SacI and SacI+EcoRI lanes contain genomic DNA digested with the enzymes indicated. The diagram depicts the 5[prime] flank of the H19 gene with the start of transcription indicated by the horizontal arrow. The 3.8 kb SacI fragment and the two HS sites are indicated. S, SacI; R, EcoRI.


Figure 5. Epigenetic modifications at the H19 gene. The Igf2/H19 locus is drawn to scale on the first line, with the genes indicated by black rectangles and the 3[prime] endoderm enhancers as closed red circles. The epigenetic region is expanded to illustrate the positions of CpG dinucleotides (CpG), the domains of exclusive paternal methylation (blue rectangles), the position of the element that functions as a silencer in Drosophila (red rectangle) and a G-rich repeat (orange rectangle). The next line represents the potential cleavage sites for the restriction enzymes listed below, color coded by enzyme. The vertical arrows below indicate the individual hypersensitive sites within HS1 and HS2. The hypersensitive HhaI site at the promoter (HS3) is not depicted.

Tissue distribution of hypersensitivity

Nuclease hypersensitivity is associated with several types of cis-acting transcriptional regulatory elements, including promoters and enhancers, as well as chromatin boundary and silencer elements. One strategy for assessing whether the hypersensitivity in the flank of the H19 gene reflects the presence of transcriptional control elements is to investigate whether the hypersensitive sites correlate with transcription of the gene. Using HhaI, we examined the status of H19 chromatin in brain and spleen, two tissues in which H19 RNA is not detectable. In both tissues, HS2 was readily detected (Fig. 3B and data not shown). Thus, the non-nucleosomal structure of the H19 epigenetic mark that is revealed by the presence of nuclease-hypersensitive sites does not require transcription of the gene. This experiment also confirmed a previous finding that the H19 promoter is hypersensitive in non-expressing tissues (Fig. 3B; 3).

DISCUSSION

This report describes the identification of two regions of differential nuclease hypersensitivity in the 5[prime] flank of the H19 gene. A summary of the findings is presented in Figure 5. The promoter-proximal region, HS2, extends from approximately -2.0 to -2.7 kb with respect to the H19 promoter, whereas the more distal hypersensitive region, HS1, is smaller, <500 bp in length, and is centered at -3.8 kb. It is striking that the two hypersensitive regions correspond precisely to two areas of high CpG density that have been shown to be completely methylated at all CpG residues on the paternal chromosome in somatic tissues as well as in sperm. Furthermore, the same regions are completely unmethylated on the maternal chromosome (7,17). These regions are included within 2 kb of the H19 gene 5[prime] flank that Tremblay et al. (7) have proposed as the epigenetic imprinting mark.

The H19 epigenetic mark was initially conceived as the site of allele-specific methylation on the paternal chromosome. It was possible, therefore, that this region had no function on the unmethylated, maternal chromosome. Our findings argue to the contrary, as hypersensitivity to nuclease digestion in chromatin is thought to arise from specific protein-DNA interactions that disrupt the normal nucleosomal array. A function for this region on the maternal chromosome is also suggested by the finding that reactivation of Igf2 expression in H19D13 mice is more extensive than in H19D3 mice that retain the epigenetic region. This implies that the epigenetic region plays a role in silencing the neighboring Igf2 gene.

What are potential functions for the hypersensitive sites? It could be that the function of the HS sites is to bind proteins that prevent DNA methylation on the maternal chromosome. For example, it has been shown that binding of the transcription factor Sp1 to CpG islands in promoters prevents their methylation (19,20). Tucker et al. (21) have shown that in ES cells that are homozygous for a null mutation of Dnmt, the gene encoding DNA methyltransferase, both alleles of the H19 gene are unmethylated. When a DNA methyltransferase cDNA is introduced into these cells, the genome becomes methylated, but the 5[prime] flank of the H19 gene does not, suggesting that once unmethylated this region is resistant to remethylation.

Another possibility is that the hypersensitive sites regulate DNA replication through the region. It has been shown that imprinted gene regions, including the region containing H19 and Igf2, replicate asynchronously, with the paternal chromosome replicating earlier than the maternal (22-24). Differential chromatin conformation may be required for this phenomenon, as is seen with asynchronous replication of the differentially compacted inactive and active X chromosomes (25). In fact, maternal inheritance, but not paternal inheritance, of the H19D13 deletion results in loss of asynchrony (26), implying a specific function for the region on the maternal chromosome.

The hypersensitive region could reflect the presence of positive regulatory elements, acting to enhance H19 transcription. Such a function would be consistent with a long-standing model to explain the reciprocal imprinting of H19 and Igf2 that proposes that the genes compete for shared enhancers (12). According to this model, there is an inverse relationship between the levels of H19 and Igf2 expression. Thus, an element that enhanced H19 expression would have the indirect effect of reducing Igf2 expression. However, in both transgenic mice and transient transfection assays, the upstream region is not required for efficient H19 expression (27-29). In addition, the presence of the HS sites in all tissues surveyed, irrespective of the transcriptional status of the H19 gene, tends to rule out a role for the region in the positive regulation of H19 expression. For example, the 3[prime] H19 endodermal enhancers display hypersensitivity to nucleases in endoderm but are resistant in non-expressing tissues (3).

We favor a fourth option: that the hypersensitive sites reflect the presence of a chromatin boundary element that acts to limit access of the Igf2 gene to the 3[prime] enhancers on the maternal chromosome (Fig. 5). The first boundary elements to be described are those at the Hsp70 locus in Drosophila and were originally identified by their hypersensitivity to nuclease digestion both before and during heat-shock activation of the gene (30-32). Since then, several other boundaries have been functionally identified and, in each instance, the hypersensitivity that helped to define the element was shown to be constitutive in all tissues (33-37). We have shown that hypersensitivity of the maternal epigenetic region of the H19 gene is present in all tissues surveyed, as is its methylation on the paternal chromosome. The coincidence of the maternal hypersensitive sites with the most intense DNA methylation on the paternal chromosome suggests that one function of the methylation may be to block the imposition of a chromatin boundary, allowing Igf2 to be transcribed paternally.

A boundary would help to explain the findings from recent studies that cannot be readily reconciled with a strict enhancer competition model, in which activation of the H19 promoter is sufficient to silence Igf2. First, Webber et al. (38) showed that when the endodermal enhancers are moved from their normal position 3[prime] of the H19 gene to a position midway between the genes, Igf2 is transcribed to the exclusion of H19 on the maternal chromosome. This result can be explained if the enhancers now lie on the other side of a boundary element. Second, Jones et al. (39) have recently shown that an H19 mutation on the paternal chromosome in which the normally silent paternal H19 promoter is partially activated does not affect Igf2 expression. A model of strict promoter competition predicts that transcription at H19 on the paternal chromosome would lead to a decrease in Igf2 expression. Finally, a deletion of a promoter-proximal segment of the epigenetic mark that includes HS2 is sufficient to activate the maternal Igf2 gene (M. Bartolomei, personal communication). This argues that the epigenetic region, independent of the H19 promoter, contributes to Igf2 silencing.

It is intriguing that sequences from this same region have been shown to function as a silencing element in Drosophila (40), although the individual hypersensitive sites do not appear to display either boundary or silencing function (A.T. Hark, unpublished data). It is not clear whether silencing is mediated through binding of proteins that are conserved in evolution between flies and mice or whether it reflects some structural property of the CpG-rich DNA itself. Whatever the case, a similar silencing function has been attributed to the epigenetic mark of the SNRPN gene, which is imprinted in both humans and mice (41).

Epigenetic marks such as paternal-specific methylation of the H19 gene and maternal methylation of the SNRPN gene are established in the germline and sustained throughout development in the offspring (5-7,42). As such, they fulfil the key criterion of an imprinting mark: they can establish and sustain a difference between parental chromosomes. The hypersensitive regions within the H19 flank on the maternal chromosome may represent a different type of epigenetic mark that functions in the soma and is mediated by protein-DNA interaction rather than DNA methylation. To function as a gametic mark, however, it must be shown that this interaction is inherited from the gametes. The ability of protein-DNA interactions to serve as a gametic mark requires a templating mechanism that functions during replication, as has been proposed for the protection of CpG islands from methylation (19,20), mating type silencing in Schizosaccharomyces pombe (43) or position effect variegation in Drosophila (44). Further studies will be required to test these ideas.

MATERIALS AND METHODS

Mice

C57BL/6 mice were purchased from the Jackson Laboratory. The H19D13 strain has been described previously (14).

Isolation of nuclei

Nuclei were isolated from neonatal tissues of approximately seven mice at postnatal days 8-11 as described previously (3) with the following modifications. For the restriction enzyme experiments, pelleted nuclei were resuspended in a total volume of 2 ml digestion buffer. For the DNase I experiments, nuclei were resuspended in 2 ml DNase I digestion buffer (0.3 M sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 15 mM Tris-HCl, pH 7.5, 0.5 mM DTT, 0.1 mM PMSF, 5% glycerol).

DNase I digestion of nuclei

From the resuspended nuclei, one 200 µl aliquot was removed and combined with stop buffer (25 mM EDTA, pH 8.0, 1% SDS, 100 µg/ml proteinase K). Eight microliters of DNase I (1 µg/µl) was added to the remaining nuclei to a final concentration of 4.4 µg/ml and digestion was carried out at room temperature. Aliquots were removed and combined with an equal volume of stop buffer following different digestion times.

Restriction enzyme digestion of nuclei

The resuspended nuclei were divided into 10 200-µl reactions. Restriction enzymes were added to nine of the reactions, to give a final concentration of 0.5 U/µl (HhaI and Sau3AI) or 0.25 U/µl (HinfI and HindIII). Digestion was carried out at 37°C for increasing lengths of time and terminated by addition of an equal volume of stop buffer. A control reaction to which no enzyme was added was kept on ice for 60 min and then quenched with stop buffer. In a second control reaction, digestion was carried out for 9-12 h before the reaction was stopped.

DNA analysis

For all reactions, proteinase K treatment was continued overnight at 37°C. DNA was purified by multiple phenol-chloroform extractions and precipitated with 0.15 mM NaCl and 2 vol ethanol. DNA pellets were washed in 70% ethanol, dried and resuspended in 50-100 µl of 10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA. Fifteen micrograms of DNA were digested with the appropriate restriction enzyme (or enzymes) and separated by gel electrophoresis on 1% agarose gels. The DNA was transferred to nitrocellulose (45) and the filters hybridized to radiolabeled probes prepared by random hexamer labeling (46). The filters were washed twice at room temperature and twice at 55-65°C in 0.1× SSC, 0.1% SDS and exposed to X-ray film with intensifying screens at -70°C. Blots were stripped by incubation in wash solution heated to 95°C and slow shaking until the solution cooled to room temperature.

ACKNOWLEDGEMENTS

We would like to thank members of our laboratory for helpful discussions and especially Dr Paul Schedl for excellent advice throughout the conduct of this work. We would also like to thank Dr Marisa Bartolomei for communicating results prior to publication. This work was supported by a grant from the National Institute for General Medical Sciences. S.M.T. is an Investigator of the Howard Hughes Medical Institute.

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*To whom correspondence should be addressed. Tel: +1 609 258 2900; Fax: +1 609 258 3345; Email: stilghman@molbio.princeton.edu

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