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Human Molecular Genetics, 2004, Vol. 13, No. 8 819-828
DOI: 10.1093/hmg/ddh098

Tissue-specific imprinting of the G protein Gs{alpha} is associated with tissue-specific differences in histone methylation

Akio Sakamoto, Jie Liu, Andrew Greene{dagger}, Min Chen and Lee S. Weinstein*

Metabolic Diseases Branch, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA

Received December 9, 2003; Accepted February 10, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The G protein Gs{alpha} is imprinted in a tissue-specific manner, being primarily expressed from the maternal allele in some tissues, such as renal proximal tubules. The Gs{alpha} promoter is unmethylated, but is downstream of a differentially methylated region [the exon 1A differentially methylated region (DMR)] that is methylated on the maternal allele. Maternal Gs{alpha} null mutations or loss of maternal-specific exon 1A methylation leads to pseudohypoparathyroidism types 1A or 1B, respectively. We now have examined the chromatin state of each parental allele within the exon 1A-Gs{alpha} promoter region by chromatin immunoprecipitation of samples derived from mice with heterozygous deletions within the region using antibodies to covalently modified histones. The exon 1A DMR had allele-specific differences in histone acetylation and methylation, with histone acetylation and H3 lysine 4 (H3K4) methylation of the paternal allele, and H3 lysine 9 (H3K9) methylation of the maternal allele. Both parental alleles had similar levels of histone acetylation and H3K4 methylation within the Gs{alpha} promoter and first exon, with no H3K9 methylation. In liver, where Gs{alpha} is biallelically expressed, both parental alleles had similar levels of tri- and dimethylated H3K4 within the Gs{alpha} first exon. In contrast, in renal proximal tubules there was a greater ratio of tri- to dimethylated H3K4 of Gs{alpha} exon 1 in the more transcriptionally active maternal as compared with the paternal allele. These results show that allele-specific differences in Gs{alpha} expression correlate in a tissue-specific manner with allele-specific differences in the extent of H3K4 methylation, and are the first demonstration that chronic transcriptional activation in mammals is correlated with trimethylation of H3K4.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Genomic imprinting is a process by which specific genes undergo allele-specific epigenetic changes that lead to allele-specific differences in gene expression. One or more of these epigenetic changes is established in the male or female germline during gametogenesis. In many cases imprinting is associated with allele-specific differences in DNA methylation at CpG dinucleotides. Allele-specific DNA methylation of some regions is established during gametogenesis and maintained throughout development (referred to as gametic imprint marks). In many cases silencing of one parental allele is complete and associated with CpG methylation of the gene promoter on the same allele. These regions often have allele-specific differences in covalent modifications of histones, particularly acetylation and methylation of histones H3 and H4 (1–12). In other cases imprinting may be incomplete, with only partial silencing of one allele, or may be tissue-specific. How tissue-specific imprinting is established and whether there are concordant tissue-specific differences in the epigenetic state of the promoter regions in these genes is not well studied.

One example of incomplete and tissue-specific imprinting is provided by the heterotrimeric G protein {alpha}-subunit Gs{alpha} that couples seven transmembrane receptors to adenylyl cyclase and is required for receptor-stimulated intracellular cAMP production (13). Gs{alpha} is biallelically expressed in most tissues, but in a few tissues, such as renal proximal tubules and some endocrine organs, is expressed primarily from the maternal allele (14–18). As a result of this tissue-specific imprinting, patients with Albright hereditary osteodystrophy who inherit Gs{alpha} null mutations from their mother develop multihormone resistance (a condition known as pseudohypoparathyroidism type 1A, PHP1A), while those who inherit the same mutations from their father fail to develop hormone resistance (19).

Gs{alpha} is one product of the GNAS locus at 20q13 (Gnas on distal chromosome 2 in mouse), a complex imprinted locus that generates multiple gene products through the use of multiple promoters and first exons that splice onto a common set of downstream exons (13) (Fig. 1). Far upstream of the Gs{alpha} promoter are two oppositely imprinted promoters that generate transcripts for the maternally expressed gene product NESP55 and paternally expressed gene product XL{alpha}s, respectively, as well as a promoter for paternally expressed antisense transcripts. For all of these promoters one allele is methylated and transcriptionally inactive. Recent studies show that a gametic imprint mark may be located within the antisense/XL{alpha}s promoter region that may be required to establish the imprinting patterns of these upstream promoter regions (20).



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  		Figure 1. Overview of the Gnas locus. The maternal (Mat, above) and paternal (Pat, below) alleles are represented with regions of differential methylation (METH) shown above and splicing patterns shown below each allele. Transcriptionally active promoters are indicated with horizontal arrows. Four alternative promoters and first exons for NESP55 (NESP), XL{alpha}s, exon 1A, and Gs{alpha} (1) are shown individually and common downstream exons 2–13 are represented by a single box. The promoter and first exon of a paternally expressed antisense transcript are also shown (NESPAS). Exon 1A, XL{alpha}s and NESP55 are 2.5, 30 and 45 kB upstream of Gs{alpha} exon 1 respectively (21,38). The dashed arrow for the Gs{alpha} promoter on the paternal allele indicates that this promoter is relatively inactive in some, but not all tissues.

 
The Gs{alpha} promoter, which is ~35 kB downstream of XL{alpha}s, is within a CpG island that is unmethylated on both alleles in all tissues, despite the fact that in some of these tissues there are allele-specific differences in promoter activity (13,21). Just upstream of this CpG island is another ~2 kb GC-rich region that appears to carry a maternal gametic imprint mark in that it is densely methylated on the maternal allele in all tissues and this methylation is established during oogenesis and maintained throughout development (21) (Figs 1 and 2). Within this differentially methylated region (DMR) is an alternative first exon (exon 1A) that generates untranslated transcripts from only the paternal allele, and we therefore refer to this region as the exon 1A DMR (21). The exon 1A DMR and unmethylated CpG island are separated by a ~250 bp AT-rich region located 638–889 bp upstream of the Gs{alpha} translational start site. In patients with PHP1B (pseudohypoparathyroidism type 1B), an isolated form of renal parathyroid hormone resistance, methylation of the exon 1A on the maternal allele is absent, and based upon this we have proposed that the exon 1A DMR has one or more cis-acting elements, such as a silencer or insulator, that negatively regulates Gs{alpha} expression on the same allele in a methylation-sensitive and tissue-specific manner (22).



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  		Figure 2. H3K4 and H3K4 methylation of the exon 1A DMR-Gs{alpha} promoter region. The exon 1A DMR-Gs{alpha} promoter region is depicted with the methylation status of each HpaII site within the maternal (Mat) and paternal (Pat) alleles, respectively, above and a plot of the observed/expected frequency of CpG dinucleotides below (21). Exon 1A is shown as a grey box and Gs{alpha} exon 1 is shown as a white box with the position of the Gs{alpha} translational start site (+1) indicated as a vertical line. Methylated HpaII sites are indicated with solid boxes and unmethylated sites with open boxes. At the top are the positions of the segments analyzed by ChIP assay. Results of amplification of DNA segments A–I after ChIP of liver nuclei from wild type (WT), +/{Delta}, or {Delta}/+ mice with anti-dimethyl H3K4 (left) or anti-dimethyl (right) H3K9 antibodies are shown below. Each panel shows the results of ethidium bromide staining after PCR of DNA from ChIP (C), DNA from ChIP with no antibody added (N), and input DNA (I). The positions of the 5' and 3' boundaries of each DNA segment is indicated on the left. For A and B, mutant mice had the {Delta}–6267/-1601 allele while for the remainder, mutant mice had the {Delta}–1601/+419 allele.

 
In this study we made use of mice with maternal or paternal deletions within the exon 1A DMR-Gs{alpha} promoter region to perform an initial characterization of allele-specific patterns of histone methylation and acetylation within this region. Our results show reciprocal patterns of histone modification within the exon 1A DMR. The paternal allele was associated with H3 and H4 acetylation and methylation of H3 lysine 4 (H3K4) but not H3 lysine 9 (H3K9), histone modifications that are generally associated with euchromatin, while the maternal allele was associated with H3K9 methylation and absence of H3K4 methylation or histone acetylation, a pattern typical for heterochromatin. In contrast, the unmethylated CpG island including the Gs{alpha} promoter had a euchromatic pattern on both alleles, with histone acetylation and methylation of H3K4 but not H3K9. While there were no allele-specific differences in the relative amount of tri- to dimethyled H3K4 in liver, a tissue in which Gs{alpha} is not imprinted, in renal proximal tubules higher Gs{alpha} transcriptional activity in the maternal allele is associated with a higher ratio of tri- to dimethylated H3K4 within Gs{alpha} exon 1. This is the first demonstration of the association of transcriptional activity with a specific increase in trimethylation of H3K4 in mammals and shows that tissue-specific imprinting can lead to tissue-specific epigenetic changes in the absence of DNA methylation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
H3K4 and H3K9 methylation patterns in the exon 1A-Gs{alpha} region
The Gs{alpha} promoter and first exon are located within a CpG island that is unmethylated on both parental alleles (Fig. 2) (21). Located just upstream of this CpG island is a GC-rich DMR that includes exon 1A and that is a gametic imprint mark which is methylated on the maternal allele. These two regions are separated by a ~250 bp AT-rich region. In this study we examined the chromatin states of both the exon 1A DMR and the unmethylated CpG island containing the Gs{alpha} promoter region by chromatin immunoprecipitation (ChIP) to determine the covalent modifications of their associated histones. In order to examine the histone modifications on each parental allele individually, we performed ChIP assays in tissues obtained from mice with heterozygous deletions of this region that were generated in our laboratory. One mutant allele ({Delta}–6267/–1601) had a deletion from position –6267 to –1601 relative to the translational start site of Gs{alpha}, while the other mutant ({Delta}–1601/+419) had a deletion from –1601 to +419. In mice with paternal deletion (+/{Delta}), only the maternal allele of the region of interest is present while in the maternal deletion ({Delta}/+) only the paternal allele is present. Neither deletion had any effect on DNA methylation within the Gnas locus (unpublished data).

After crosslinking of chromatin in liver nuclei isolated from wild type (WT), +/{Delta}, and {Delta}/+ mice, ChIP assays were performed using anti-dimethyl H3K4 and -dimethyl H3K9 antibodies, respectively (Fig. 2). After ChIP and reverse crosslinking, PCR reactions were performed on immunoprecipitated DNA (C), negative controls in which ChIP was performed with no antibody (N), and input DNA (I; 1% of the DNA subjected to ChIP). For primer pairs A and B, DNA from {Delta}–6267/–1601 heterozygotes were used, while {Delta}–1601/+419 heterozygotes were used for the primer pairs C-I which amplify segments downstream of position –1601.

ChIP of WT chromatin with anti-dimethyl H3K4 showed relatively weak amplification of the most upstream segments and stronger amplification of segments within the transition from DMR to unmethylated CpG island and within the unmethylated CpG island itself. For segments within the DMR (A–E) the ChIP assay was positive in chromatin derived from {Delta}/+, but not +/{Delta} mice, consistent with H3K4 methylation only associated with the paternal allele. Sequencing of ChIP–PCR products from WT CD1x129Sv mice using primer pair B, which contains a polymorphic HpaII site (21), confirmed that only the paternal allele was immunoprecipitated with the anti-dimethyl H3K4 antibody (data not shown). In contrast H3K4 was strongly methylated on both parental alleles within the unmethylated CpG island based upon the fact that products F–I amplified to a similar extent from both +/{Delta} and {Delta}/– samples after ChIP with the anti-dimethyl H3K4 antibody. Results in experiments examining renal proximal tubule samples were similar to the results with liver samples (data not shown).

Similar analysis of WT samples using the anti-dimethyl H3K9 antibody showed strong amplification of segments within the exon 1A DMR (A–C; Fig. 2). Segments A and B were amplified after ChIP in the +/{Delta}–6267/–1601, but not the {Delta}–6267/–1601/+ samples, indicating that H3K9 methylation within the DMR was only associated with the maternal allele. Segments G–I failed to amplify after ChIP of the WT sample, indicating that H3K9 methylation is absent from both alleles downstream of the DMR within the unmethyated CpG island. Results in experiments examining renal proximal tubule samples were similar to the results with liver samples (data not shown). In summary, the exon 1A DMR had reciprocal methylation of H3K4 and H3K9 on the paternal and maternal alleles, respectively, while the unmethylated CpG island associated with the Gs{alpha} promoter had H3K4 methylation, but no H3K9 methylation, on both parental alleles.

H3 and H4 acetylation patterns in the exon 1A DMR-Gs{alpha} promoter region
Similar experiments were performed using antibodies to acetylated histones H3 and H4, respectively (Fig. 3). In these experiments duplex PCR was performed using primers for {alpha}-tubulin and primers for each respective Gnas segment. Negative controls, which are not shown, generated no bands. Both H3 and H4 acetylation was detected in WT samples in all segments examined throughout the exon 1A DMR-Gs{alpha} promoter region. Within the DMR (segments A–C), H3 and H4 acetylation was easily detected in the {Delta}–6267/–1601/+ samples and minimally detected in the +/{Delta}–6267/–1601 samples, indicating that H3 and H4 acetylation is almost exclusively associated with the paternal allele within the DMR. Towards the downstream end of the DMR (segment E) H4 acetylation appears to be similar within the two parental alleles, but H3 acetylation is still present to a greater extent in the paternal allele. Within the unmethylated CpG island including the Gs{alpha} promoter and first exon (segments G, H and J), histones H3 and H4 are acetylated to similar extents on both parental alleles. Similar results were obtained in renal proximal tubules, despite the fact that Gs{alpha} is preferentially expressed from the maternal allele in this tissue (14) (data not shown).



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  		Figure 3. H3 and H4 acetylation of the exon 1A DMR-Gs{alpha} promoter region. The exon 1A DMR-Gs{alpha} promoter region and position of the DNA segments that were amplified are depicted at the top as in Figure 1. Below are the results of duplex PCR of each respective Gnas segment and {alpha}-tubulin after ChIP with anti-acetylated H3 (H3) or anti-acetylated H4 (H4) antibodies, as well as input DNA (I). DNA after ChIP without added antibody failed to amplify any bands (not shown). For A and B, mutant mice had the {Delta}–6267/-1601 allele while for the remainder, mutant mice had the {Delta}–1601/+419 allele.

 
Quantitation of di- and trimethylated H3K4 within the exon 1A DMR-Gs{alpha} promoter region
Gs{alpha} is imprinted in a tissue-specific manner with predominant expression from the maternal allele in a small number of hormone-responsive tissues. However no corresponding allele-specific differences in the chromatin state of the Gs{alpha} promoter region have been identified, as both parental alleles lack DNA methylation in somatic tissues (21,22) and there are no gross allele-specific differences in histone acetylation or methylation even in renal proximal tubules where Gs{alpha} is known to be expressed preferentially from the maternal allele. Recent studies in yeast suggest that transcriptional activation is correlated with the extent of H3K4 methylation and that in activated genes there is a greater conversion of dimethyated H3K4 to trimethylated H3K4 in the initial portion of the coding region (23,24). We therefore examined whether there were allele-specific differences in the ratio of tri- to dimethylated H3K4 that correlated with allele-specific differences in Gs{alpha} expression.

ChIP was performed using anti-dimethyl and -trimethyl H3K4 antibodies, respectively, on samples derived from +/{Delta}–1601/+419 and {Delta}–1601/+419/+ mice. The relative quantity of each Gnas segment that was immunoprecipitated was determined by performing duplex PCR with Gnas and {alpha}-tubulin primers and determining the ratio of Gnas segment/{alpha}-tubulin in the ChIP sample versus the same ratio in the input DNA sample. The number of PCR cycles was optimized to be sure that the reactions were not saturated and control experiments were performed to show that the readout of relative quantity was not significantly affected by cycle number. Using this approach we determined the relative amounts of tri- and dimethylated H3K4 in liver, a tissue where there are no allele-specific differences in Gs{alpha} expression, and in renal proximal tubules where Gs{alpha} is expressed primarily from the maternal allele. Because the dimethyl H3K4 antibody used has some cross-reactivity with monomethyl H3K4 (Technical Services, Upstate Biotechnology), the quantitative results using the dimethyl H3K4 antibody may to some extent also reflect the level of monomethyl H3K4.

Quantitative results from one representative experiment are shown in Figure 4. Within the DMR (segment C) levels of both di- and trimethyl H3K4 are relatively low with a higher level of methylation in the {Delta}–1601/+419/+ samples, consistent with the results in Figure 2 and the fact that the paternal allele is not CpG methylated in this region. While the levels of H3K4 methylation are somewhat higher within the AT-rich region that is located between the exon 1A DMR and the unmethylated CpG island (segment G), the highest levels of di- and trimethylation are located within the most upstream portion of the CpG island (segment K). This high level of H3K4 methylation may serve as a barrier to prevent the spread of heterchromatization towards the Gs{alpha} promoter (25). In liver the levels of di- and trimethylated H3K4 and the tri/dimethylation ratio is similar in both {Delta}–1601/+419/+ and +/{Delta}–1601/+419 samples, indicative of the fact that the two parental alleles have similar epigenetic characteristics. In contrast, in proximal tubules there are differences in di- and trimethylated H3K4 within the Gs{alpha} exon 1 coding region (segment J) between the {Delta}–1601/+419/+ and +/{Delta}–1601/+419 samples, indicative of the two parental alleles having epigenetic differences with exon 1. Note that in the initial coding region (segment J) the ratios of tri- to dimethylation of H3K4 in the liver samples and +/{Delta}–1601/+419 proximal tubule samples are similar whereas the ratio in the {Delta}–1601/+419/+ proximal tubule sample is much lower. This is consistent with suppressed transcriptional activity of the Gs{alpha} promoter on the paternal allele in renal proximal tubules. The higher levels of trimethylated H3K4 of segment H in liver compared to proximal tubule possibly reflects the fact that Gs{alpha} expression levels are much higher in this tissue.



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  		Figure 4. Quantitation of dimethyl H3K4, trimethyl H3K4, and tri/dimethyl H3K4 within the exon 1A DMR-Gs{alpha} promoter region. The location of the amplified segments are shown at the top. ChIP was performed on liver samples (left) or renal proximal tubules (right) from +/{Delta}–1601/+419 (+/{Delta}, open bars) and {Delta}–1601/+419/+ mice ({Delta}/+, black bars), respectively. Duplex PCR was performed for each segment and {alpha}-tubulin and the relative amount of di- and trimethylated H3K4 for each segment was calculated as described in the text. The results for dimethyl H3K4, trimethyl H3K4, and tri-/dimethyl H3K4 ratio from one representative experiment are shown.

 
Table 1 shows the combined results from two independent experiments with independently derived tissue samples which both gave similar results. Each parameter is expressed as the ratio of the quantity immunoprecipitated from the +/{Delta}–1601/+419 versus {Delta}–1601/+419/+ samples, which reflects the relative proportion of each parameter in the maternal versus paternal allele. There were no allele-specific differences in the amount of di- or trimethyl H3K4 or the ratio of tri- to dimethyl H3K4 within the AT-rich region (segment G) or the upstream portions of the unmethylated CpG island in either tissue. In liver there were no obviously allele-specific differences in the ratio of tri- to dimethylated H3K4 throughout the exon 1A DMR-Gs{alpha} promoter region including Gs{alpha} exon 1 (segment J), consistent with similar transcriptional activity of the Gs{alpha} promoter on both parental alleles. In contrast, the ratio of tri-to dimethyl H3K4 in the coding region of Gs{alpha} exon 1 (segment J) was 5-fold greater in the maternal allele as compared with the paternal allele in renal proximal tubules. These changes were due to a relatively lower level of dimethyl H3K4 and higher level of trimethyl H3K4 in the maternal allele relative to the paternal allele. The allele-specific differences in the extent of H3K4 methylation in proximal tubules were present only in the nucleosomes of Gs{alpha} exon 1, as there were no allele-specific differences observed in the other more upstream segments.


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  		Table 1. Allele-specific ratios (maternal/paternal) of tri-, di- and tri/dimethyl H3K4 within the exon 1A DMR-Gs{alpha} promoter regiona
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study we show that the region involved in the tissue-specific imprinting of Gs{alpha} has a complex pattern of histone modifications that correlate with its pattern of DNA methylation. Within the exon 1A DMR the maternal allele has H3 and H4 hypoacetylation and H3K9 methylation without H3K4 methylation, characteristics of heterochromatin that are often associated with DNA methylation (25,26) (Fig. 5). In contrast, the paternal allele in this region has histone acetylation and H3K4 methylation without H3K9 methylation, characteristics of euchromatin and active transcription. These epigenetic differences are consistent with the paternal-specific expression from the exon 1A promoter (21,22). Similar allele-specific differences in histone modifications have been identified in DMRs associated with other imprinted genes (1–12). Our results provide further evidence that imprinted genes are associated with allele-specific differences in histone modifications, and that these differences in the ‘histone code’ may be important in the imprinting mechanism.



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  		Figure 5. Summary of allele-specific epigenetic differences within the exon 1A DMR-Gs{alpha} promoter region. The exon 1A DMR-Gs{alpha} promoter region is depicted as in Figure 2 with the epigenetic status of the maternal allele (Mat) shown above and the paternal allele (Pat) shown below. Methylated and unmethylated CpG dinucleotides are indicated with solid and open boxes, respectively. Relative levels of H3K4 methylation (H3K4 Me), histone acetylation (H Acet) and H3K9 methylation (H3K9 Me) throughout the region are depicted with plus and minus signs. Above are shown the positions of the exon 1A DMR, the unmethylated CpG island including the Gs{alpha} promoter, and a highly AT-rich region that lies between them. The vertical hash marks indicate 1000 bp intervals beginning with the Gs{alpha} translational start site. Tissue-specific differences in tri- versus dimethylation of H3K4 within Gs{alpha} exon 1 are not shown.

 
Although DNA methylation is thought to be the primary epigenetic mark of imprinting, recent studies suggest that histone modifications may also be primary imprinting marks for some genes. Evidence in Neurospora crassa (27) and Arabidopsis (28) demonstrate that in these organisms DNA methylation is dependent upon H3K9 methylation, and the presence of such histone modifications could explain why some methylation imprints are lost during the blastocyst stage and reaquired during later development. In mouse embryonic stem (ES) cells deficient in G9a, a methylase for H3K9 and H3K27, DNA methylation and imprinting of the Prader–Willi imprinting control region is lost, while ES cells deficient in the DNA methyltransferase Dnmt1 lose DNA methylation without affecting histone methylation or imprinting of the gene (5). Another study provided evidence that imprinting of the Prader–Willi imprinting center is dependent upon an upstream region (the Angelman syndrome imprinting center) with allele-specific differences in histone modifications but not in DNA methylation (4). We have shown that methylation of the exon 1A DMR is established during oogenesis in mice (21). A recent study has identified a region 220 kb upstream of the exon 1A DMR that is deleted in familial PHPIB and is associated with loss of methylation within the DMR when present on the maternal allele (29). Further studies will be required to determine how this putative upstream cis-acting element leads to imprinting of the exon 1A DMR and whether histone modification or DNA methylation is the primary epigenetic event.

Spread of the heterochromatic state is believed to be mediated by binding of the chromodomain protein HP-1 to methylated H3K9, which then can recruit H3K9 methylases and possibly DNA methyltransferases (30). Downstream spread of heterochromatization from the exon 1A DMR on the maternal allele appears to be inhibited ~1000 bp upstream of the Gs{alpha} translational start site. The short AT-rich region that separates the DMR and unmethylated CpG island may be a boundary that prevents the spread of heterochromatization into the CpG island (Fig. 5). Alternatively there may be a barrier to heterochromatin spreading within the most upstream portion of the CpG island, as we showed this region to have very high levels of H3K4 methylation, a characteristic present within a similar barrier region of the chicken ß-globin locus (25). In either case the CpG island containing the Gs{alpha} promoter region remains unmethylated and has histone modifications characteristic of euchromatin (histone hyperacetylation, H3K4 methylation, no H3K9 methylation), which is consistent with Gs{alpha} being ubiquitously and biallelically expressed in most tissues.

Gs{alpha} is imprinted in a tissue-specific manner, being primarily expressed from the maternal allele in some tissues (e.g. renal proximal tubules) despite an absence of DNA (21,22) or H3K9 methylation within its promoter. In yeast a complex containing the H3K4 methylase Set1 (the yeast analog of MLL) binds to RNA polymerase II during the initial phase of elongation, leading to trimethylation of H3K4 within the initial portion of the coding region (23,24,31), and similar patterns have also been observed in chicken genes (32). While both di- and trimethylation have been associated with inducible transcription in higher organisms (33–35), there is no evidence that the extent of H3K4 methylation (tri- versus dimethylation) directly correlates with the transcriptional activity of mammalian genes in their native state. In liver, a tissue in which Gs{alpha} is not imprinted, we found no allele-specific differences in the extent of di- or trimethylation of H3K4 within the Gs{alpha} upstream region. In contrast, in renal proximal tubules lower Gs{alpha} expression from the paternal allele is associated with a decrease in the ratio of trimethyl versus dimethyl H3K4 within the Gs{alpha} exon 1 coding region, which is due to both a decrease in trimethyl H3K4 and an increase in dimethyl H3K4. The increased levels of dimethyl H3K4 in the paternal allele may be due to accumulation resulting from decreased conversion to trimethyl H3K4, or may be a reflection of the fact that the overall levels of bound histones in this region is greater due to lower rates of transcription and chromatin remodeling. This is the first demonstration that trimethylation of H3K4 in the initial coding region correlates with chronic expression levels of a mammalian gene and shows that genes that undergo tissue-specific imprinting can have corresponding allele-specific differences in histone methylation even in the absence of DNA methylation. Although it has been suggested that trimethylation of H3K4 in the initial coding region may act as a molecular memory for gene transcriptional activity (23,36), it has not been definitively established whether this histone modification leads to increased transcription or is a result of increased transcription.

Although our results define an association between tissue-specific imprinting and epigenetic differences within the Gs{alpha} promoter region, they do not answer the question of how these tissue-specific differences are established or maintained. We had previously proposed that tissue-specific imprinting is dependent upon a cis-acting negative regulatory element within the exon 1A DMR that is both methylation-sensitive and tissue-specific (13,22). This model predicts that a tissue-specific factor such as a repressor can bind to the unmethylated paternal allele and inhibit the Gs{alpha} promoter. Evidence for this model is provided by the fact that loss of methylation on the maternal allele in the DMR leads to PTH resistance in PHP1B patients, presumably due to loss of expression of the Gs{alpha} signaling protein from both parental alleles (22). More direct evidence for this model is provided by preliminary results suggesting that paternal deletion of the exon 1A DMR results in increased Gs{alpha} expression in the proximal tubules of mice (J.L. and L.S.W., unpublished data). Although there appears to be a barrier to the spread of heterochromatization between the exon 1A DMR and Gs{alpha} exon 1, results showing that the exon 1A DMR impacts on Gs{alpha} promoter activity suggests that there is no functional insulator between these two regions. Experiments examining the chicken ß-globin locus recently demonstrated that barrier and insulator functions are not dependent on identical sequence elements (37). Future studies to identify of the binding proteins and cis-acting elements that mediate the Gs{alpha} imprinting process will provide important insights into the mechanisms involved in tissue-specific imprinting.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Mice
Mice heterozygous for the {Delta}–6267/–1601 and {Delta}–1601/+419 alleles were generated by the insertion of loxP sites at each respective position using targeted mutagenesis in embryonic stem cells and injection of mutant cells into blastocysts, followed by mating of offspring with EIIa-cre mice. The numbers indicate extent of the deletion based upon the position in bp relative to the Gs{alpha} translational start site. Details of how these mutant mice were generated will be provided elsewhere. All mice were mated to WT CD1 mice to generate mice with paternal (+/{Delta}) or maternal ({Delta}/+) deletions of the indicated regions, respectively. WT mice were CD1 background (Charles River Laboratories, Wilmington, MA, USA) unless indicated.

Tissue isolation and chromatin immunoprecipitation
Renal cortices were dissected and proximal tubule suspensions were prepared by collagenase digestion and isolation on a Percoll gradient as previously described (14). Proximal tubule fragments from five mice of the same genotype were combined and immediately suspended in formaldehyde for cross linking. Liver samples (300–500 mg wet weight) were snap frozen and homogenized at 4°C in 10 ml Dulbecco's phosphate-buffered saline (DPBS without Ca and Mg), 5 mM EDTA and 40 µl proteinase inhibitor cocktail (P8340, Sigma, St Louis, MI, USA) using a Teflon/glass homogenizer (size AA). The homogenate was poured through cheese cloth and then centrifuged at 2000 g at 4°C for 5 min. The pellet was resuspended in 7 ml of 4% formaldehyde in DPBS, 5 mM EDTA, 10 µl proteinase inhibitor using a size A teflon/glass homogenizer (one stroke) and cross-linked on ice for 25 min and recentrifuged as before. Pellets were resuspended in 7 ml of homogenizing buffer (0.15 mM spermine, 0.5 mM spermidine, 60 mM KCl, 15 mM NaCl, 15 mM Tris–HCl, pH 7.6, 2 mM EDTA, 0.5 mM EGTA, 0.5% NP-40, with 20 µl proteinase inhibitor), homogenized (three to five strokes), incubated for 10 min on ice, rehomogenized and recentrifuged. The pellets were resuspended in homogenizing buffer without NP-40 and separated into three microcentrifuge tubes, centrifuged at 3000 rpm for 5 min at 4°C, and rewashed with the same buffer. Pellets were resuspended in SDS lysis buffer (Upstate Biotechnology, Lake Placid, NY, USA) using the manufacturer's instructions and sonicated four times for 10 s at 30% maximal power to generate fragments <500 bp in length with peak length of 2–300 bp, as determined by agarose gel electrophoresis. Sonicated samples were centrifuged (13 000 rpm for 10 min at 4°C) and supernatants were stored at –80°C in 200 µl aliquots. Chromatin precipitation and reverse crosslinking were performed using the protocol and reagents provided by Upstate Biotechnology. After dilution to 2 ml with ChIP dilution buffer and two precleanings with protein A-agarose beads, half the sample (1 ml) was incubated with antibody to modified histone, and the other half was treated identically but without addition of antibody (negative control). Prior to the second precleaning 10 µl of sample was removed and saved as the input DNA sample. This sample was diluted to 500 µl with water prior to reverse crosslinking. The antibodies used for ChIP assay (with volume used in parentheses) were anti-acetyl-H3 (10 µl, Upstate, 06-599), anti-acetyl-H4 (20 µl, Upstate, 06-866), anti-dimethyl-H3K4 (5 µl, Upstate, 07-030), anti-trimethyl-H3K4 (3 µl, Abcam, Cambridge, MA, USA, ab8898) and anti-dimethyl-H3K9 (70 µl, United States Biological, Swampscott, MA, H5110-14E). ChIP performed with normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 2.5 µg/ml final concentration) consistently gave negative results (data not shown).

PCR
DNA derived after reverse crosslinking (1 µl) was amplified by PCR in 25 µl reactions consisting of 1xPCR buffer (Invitrogen, Carlsbad, CA, USA), 0.3 µl Platinum Taq polymerase (Invitrogen), 2 mM MgCl2, oligonucleotide primers (0.4 µM each), and dNTPs (400 µM each). The sequences of oligonucleotides used are listed in Table 2. In some reactions 6 µl betaine (Sigma) was included to allow amplification of GC-rich regions. The PCR cycling profile included an initial denaturation at 94°C for 5 min, followed by 30–35 cycles of annealing (62°C, 30 s), extension (72°C, 1 min), and denaturation (94°C, 30 s) and a final cycle with a 7 min extension. Cycle number was adjusted to be two cycles beyond the cycle in which a band was first seen for the input DNA, and ranged from 30 to 32 cycles for liver tissue and 32–34 cycles for proximal tubules. Products were assessed by acrylamide gel electrophoresis (6% TBE gels, Invitrogen) and ethidium bromide staining. Duplex PCR was performed using the same procedure as for monoplex PCR except for the annealing temperature, which was 58°C, and cycle numbers, which ranged from 30 to 35 and were adjusted to avoid saturation of either product. Gnas and {alpha}-tubulin primer concentrations were 0.2 and 0.4 µM, respectively, for the {Delta}–1601/+419 samples and 0.4 and 0.2 µM, respectively, for the {Delta}–6267/–1601 samples. Ethidium bromide stained bands were quantified using Alpha Ease FC software (Alpha Innotech, San Leandro, CA, USA). To determine the relative amount of histone modification in a given Gnas segment, the ratio of intensities of the Gnas-specific to {alpha}-tubulin-specific band in the ChIP sample was divided by the Gnas/{alpha}-tubulin ratio in the input DNA sample (26). We confirmed that the quantitative analysis was not affected by the PCR cycle number, the amount of PCR product that was loaded or the exposure time.


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  		Table 2. PCR oligonucleotide primer sequences
 

   ACKNOWLEDGEMENTS
 
This work was supported by the Intramural Research Program of the National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, US Department of Health and Human Services.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +1 3014022923; Fax: +1 3014020374; Email: leew{at}amb.niddk.nih.gov

{dagger} Present address: Columbia University College of Physicians and Surgeons, New York, USA. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Fournier, C., Goto, Y., Ballestar, E., Delaval, K., Hever, A.M., Esteller, M. and Feil, R. (2002) Allele-specific histone lysine methylation marks regulatory regions at imprinted mouse genes. EMBO J., 21, 6560–6570.[CrossRef][Web of Science][Medline]

  2. Fulmer-Smentek, S.B. and Francke, U. (2001) Association of acetylated histones with paternally expressed genes in the Prader-Willi deletion region. Hum. Mol. Genet., 10, 645–652.[Abstract/Free Full Text]

  3. Pedone, P.V., Pikaart, M.J., Cerrato, F., Vernucci, M., Ungaro, P., Bruni, C.B. and Riccio, A. (1999) Role of histone acetylation and DNA methylation in the maintenance of the imprinted expression of the H19 and Igf2 genes. FEBS Lett., 458, 45–50.[CrossRef][Web of Science][Medline]

  4. Perk, J., Makedonski, K., Lande, L., Cedar, H., Razin, A. and Shemer, R. (2002) The imprinting mechanism of the Prader–Willi/Angelman regional control center. EMBO J., 21, 5807–5814.[CrossRef][Web of Science][Medline]

  5. Xin, Z., Tachibana, M., Guggiari, M., Heard, E., Shinkai, Y. and Wagstaff, J. (2003) Role of histone methyltransferase G9a in CpG methylation of the Prader–Willi syndrome imprinting center. J. Biol. Chem., 278, 14 996–15 000.

  6. Grandjean, V., O'Neill, L., Sado, T., Turner, B. and Ferguson-Smith, A. (2001) Relationship between DNA methylation, histone H4 acetylation and gene expression in the mouse imprinted Igf2-H19 domain. FEBS Lett., 488, 165–169.[CrossRef][Web of Science][Medline]

  7. Gregory, R.I., Randall, T.E., Johnson, C.A., Khosla, S., Hatada, I., O'Neill, L.P., Turner, B.M. and Feil, R. (2001) DNA methylation is linked to deacetylation of histone H3, but not H4, on the imprinted genes Snrpn and U2af1-rs1. Mol. Cell. Biol., 21, 5426–5436.[Abstract/Free Full Text]

  8. Yang, Y., Li, T., Vu, T.H., Ulaner, G.A., Hu, J.-F. and Hoffman, A.R. (2003) The histone code regulating expression of the imprinted mouse Igf2r gene. Endocrinology, 144, 5658–5670.[Abstract/Free Full Text]

  9. Xin, Z., Allis, C.D. and Wagstaff, J. (2001) Parent-specific complementary patterns of histone H3 lysine 9 and H3 lysine 4 methylation at the Prader–Willi syndrome imprinting center. Am. J. Hum. Genet., 69, 1389–1394.[CrossRef][Web of Science][Medline]

  10. Saitoh, S. and Wada, T. (2000) Parent-of-origin specific histone acetylation and reactivation of a key imprinted gene locus in Prader–Willi syndrome. Am. J. Hum. Genet., 66, 1958–1962.[CrossRef][Web of Science][Medline]

  11. Higashimoto, K., Urano, T., Sugiura, K., Yatsuki, H., Joh, K., Zhao, W., Iwakawa, M., Ohashi, H., Oshimura, M., Niikawa, N. et al. (2003) Loss of CpG methylation is strongly correlated with loss of histone H3 lysine 9 methylation at DMR-LIT1 in patients with Beckwith-Wiedemann syndrome. Am. J. Hum. Genet., 73, 948–956.[CrossRef][Web of Science][Medline]

  12. Yang, Y., Hu, J., Ulaner, G.A., Li, T., Yao, X., Vu, T.H. and Hoffman, A.R. (2003) Epigenetic regulation of Igf2/H19 imprinting at CTCF insulator binding sites. J. Cell Biochem., 90, 1038–1055.[CrossRef][Web of Science][Medline]

  13. Weinstein, L.S., Yu, S., Warner, D.R. and Liu, J. (2001) Endocrine manifestations of stimulatory G protein {alpha}-subunit mutations and the role of genomic imprinting. Endocr. Rev., 22, 675–705.[Abstract/Free Full Text]

  14. Yu, S., Yu, D., Lee, E., Eckhaus, M., Lee, R., Corria, Z., Accili, D., Westphal, H. and Weinstein, L.S. (1998) Variable and tissue-specific hormone resistance in heterotrimeric Gs protein {alpha}-subunit (Gs{alpha}) knockout mice is due to tissue-specific imprinting of the Gs{alpha} gene. Proc. Natl Acad. Sci. USA, 95, 8715–8720.[Abstract/Free Full Text]

  15. Liu, J., Erlichman, B. and Weinstein, L.S. (2003) The stimulatory G protein {alpha}-subunit Gs{alpha} is imprinted in human thyroid glands: implications for thyroid function in pseudohypoparathyroidism types 1A and 1B. J. Clin. Endocrinol. Metab., 88, 4336–4341.[Abstract/Free Full Text]

  16. Hayward, B.E., Barlier, A., Korbonits, M., Grossman, A.B., Jacquet, P., Enjalbert, A. and Bonthron, D.T. (2001) Imprinting of the Gs{alpha} gene GNAS1 in the pathogenesis of acromegaly. J. Clin. Invest., 107, R31–R36.[CrossRef][Web of Science][Medline]

  17. Mantovani, G., Ballare, E., Giammona, E., Beck-Peccoz, P. and Spada, A. (2002) The Gs{alpha} gene: predominant maternal origin of transcription in human thyroid gland and gonads. J. Clin. Endocrinol. Metab., 87, 4736–4740.[Abstract/Free Full Text]

  18. Germain-Lee, E.L., Ding, C.-L., Deng, Z., Crane, J.L., Saji, M., Ringel, M.D. and Levine, M.A. (2002) Paternal imprinting of G{alpha}s in the human thyroid as the basis of TSH resistance in pseudohypoparathyroidism type 1a. Biochem. Biophys. Res. Commun., 296, 67–72.[CrossRef][Web of Science][Medline]

  19. Davies, S.J. and Hughes, H.E. (1993) Imprinting in Albright's hereditary osteodystrophy. J. Med. Genet., 30, 101–103.[Abstract/Free Full Text]

  20. Coombes, C., Arnaud, P., Gordon, E., Dean, W., Coar, E.A., Williamson, C.M., Feil, R., Peters, J. and Kelsey, G. (2003) Epigenetic properties and identification of an imprint mark in the Nesp–Gnasxl domain of the mouse Gnas imprinted locus. Mol. Cell. Biol., 23, 5475–5488.[Abstract/Free Full Text]

  21. Liu, J., Yu, S., Litman, D., Chen, W. and Weinstein, L.S. (2000) Identification of a methylation imprint mark within the mouse Gnas locus. Mol. Cell. Biol., 20, 5808–5817.[Abstract/Free Full Text]

  22. Liu, J., Litman, D., Rosenberg, M.J., Yu, S., Biesecker, L.G. and Weinstein, L.S. (2000) A GNAS1 imprinting defect in pseudohypoparathyroidism type IB. J. Clin. Invest., 106, 1167–1174.[Web of Science][Medline]

  23. Ng, H.H., Robert, F., Young, R.A. and Struhl, K. (2003) Targeted recruitment of Set1 histone methylase by elongating Pol II provides a localized mark and memory of recent transcriptional activity. Mol. Cell, 11, 709–719.[CrossRef][Web of Science][Medline]

  24. Santos-Rosa, H., Schneider, R., Bannister, A.J., Sherriff, J., Bernstein, B.E., Emre, N.C.T., Schreiber, S.L., Mellor, J. and Kouzarides, T. (2002) Active genes are tri-methylated at K4 of histone H3. Nature, 419, 407–411.[CrossRef][Medline]

  25. Litt, M.D., Simpson, M., Gaszner, M., Allis, C.D. and Felsenfeld, G. (2001) Correlation between histone lysine methylation and developmental changes at the chicken ß-globin locus. Science, 293, 2453–2455.[Abstract/Free Full Text]

  26. Noma, K., Allis, C.D. and Grewal, S.I.S. (2001) Transitions in distinct histone H3 methylation patterns at the heterochromatin domain boundaries. Science, 293, 1150–1155.[Abstract/Free Full Text]

  27. Tamaru, H. and Selker, E.U. (2001) A histone H3 methyltransferase controls DNA methylation in Neurospora crassa. Nature, 414, 277–283.[CrossRef][Medline]

  28. Jackson, J.P., Lindroth, A.M., Cao, X. and Jacobsen, S.E. (2002) Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. Nature, 416, 556–560.[CrossRef][Medline]

  29. Bastepe, M., Frohlich, L.F., Hendy, G.N., Indridason, O.S., Josse, R.G., Koshiyama, H., Korkko, J., Nakamoto, J.M., Rosenbloom, A.L., Slyper, A.H. et al. (2003) Autosomal dominant pseudohypoparathyroidism type Ib is associated with a heterozygous microdeletion that likely disrupts a putative imprinting control element of GNAS. J. Clin. Invest., 112, 1255–1263.[CrossRef][Web of Science][Medline]

  30. Grewal, S.I.S. and Moazed, D. (2003) Heterochromatin and epigenetic control of gene expression. Science, 301, 798–802.[Abstract/Free Full Text]

  31. Santos-Rosa, H., Schneider, R., Bernstein, B.E., Karabetsou, N., Morillon, A., Weise, C., Schreiber, S.L., Mellor, J. and Kouzarides, T. (2003) Methylation of histone H3 K4 mediates association of the Isw1p ATPase with chromatin. Mol. Cell, 12, 1325–1332.[CrossRef][Web of Science][Medline]

  32. Schneider, R., Bannister, A.J., Myers, F.A., Thorne, A.W., Crane-Robinson, C. and Kouzarides, T. (2004) Histone H3 lysine 4 methylation patterns in higher eukaryotic genes. Nat. Cell Biol., 6, 73–77.[CrossRef][Web of Science][Medline]

  33. Martens, J.H.A., Verlaan, M., Kalkhoven, E. and Zantema, A. (2003) Cascade of distinct histone modifications during collagenase gene activation. Mol. Cell. Biol., 23, 1808–1816.[Abstract/Free Full Text]

  34. Kim, J., Jia, L., Tilley, W.D. and Coetzee, G.A. (2003) Dynamic methylation of histone H3 at lysine 4 in transcriptional regulation by the androgen receptor. Nucl. Acids Res., 31, 6741–6747.[Abstract/Free Full Text]

  35. Sedkov, Y., Cho, E., Petruk, S., Cherbas, L., Smith, S.T., Jones, R.S., Cherbas, P., Canaani, E., Jaynes, J.B. and Mazo, A. (2003) Methylation at lysine 4 of histone H3 in ecdysone-dependent development of Drosophila. Nature, 426, 78–83.[CrossRef][Medline]

  36. Turner, B.M. (2003) Memorable transcription. Nat. Cell Biol., 5, 390–393.[CrossRef][Web of Science][Medline]

  37. Recillas-Targa, F., Pikaart, M.J., Burgess-Beusse, B., Bell, A.C., Litt, M.D., West, A.G., Gaszner, M. and Felsenfeld, G. (2002) Position-effect protection and enhancer blocking by the chicken beta-globin insulator are separable activities. Proc. Natl Acad. Sci. USA, 99, 6883–6888.[Abstract/Free Full Text]

  38. Kelsey, G., Bodle, D., Miller, H.J., Beechey, C.V., Coombes, C., Peters, J. and Williamson, C.M. (1999) Identification of imprinted loci by methylation-sensitive representational difference analysis: application to mouse distal chromosome 2. Genomics, 62, 129–138.[CrossRef][Web of Science][Medline]


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