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Human Molecular Genetics Pages 555-566  

In vivo nuclease hypersensitivity studies reveal multiple sites of parental origin-dependent differential chromatin conformation in the 150 kb SNRPN transcription unit
Introduction
Results
   SNRPN exon 1 is associated with prominent parental allele-specific nuclease-hypersensitive sites
   Hypersensitivity tests using MspI restriction nuclease
   Hypersensitivity experiments using DNase I
   Differential cytosine methylation explains size differences of SmaI and XmaI fragments
   Fine mapping of paternal-allele-specific nuclease-hypersensitive sites flanking SNRPN exon 1
   The SNRPN transcriptional unit contains several sites that are preferentially nuclease hypersensitive on the maternal allele
   A differentially methylated region in intron 7 corresponds to differentially nuclease-hypersensitive sites
   Differential nuclease hypersensitivity associated with the putative imprinting center
Discussion
Materials And Methods
   Isolation and labeling of genomic probes
   Cell lines and cell culture
   Cell permeabilization and nuclease hypersensitivity tests
Acknowledgements
Abbreviations
References

In vivo nuclease hypersensitivity studies reveal multiple sites of parental origin-dependent differential chromatin conformation in the 150 kb SNRPN transcription unit

In vivo nuclease hypersensitivity studies reveal multiple sites of parental origin-dependent differential chromatin conformation in the 150 kb SNRPN transcription unit

Johannes Schweizer1, Debra Zynger2 and Uta Francke1,2,*

1Howard Hughes Medical Institute and 2Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5323, USA

Received October 5, 1998; Revised and Accepted January 8, 1999

Human chromosome region 15q11-q13 contains a cluster of oppositely imprinted genes. Loss of the paternal or the maternal alleles by deletion of the region or by uniparental disomy 15 results in Prader-Willi syndrome (PWS) or Angelman syndrome (AS), respectively. Hence, the two phenotypically distinct neurodevelopmental disorders are caused by the lack of products of imprinted genes. Subsets of PWS and AS patients exhibit ‘imprinting mutations’, such as small microdeletions within the 5[prime] region of the small nuclear ribonucleoprotein polypeptide N (SNRPN) transcription unit which affect the transcriptional activity and methylation status of distant imprinted genes throughout 15q11-q13 incis. To elucidate the mechanism of these long-range effects, we have analyzed the chromatin structure of the 150 kb SNRPN transcription unit for DNase I- and MspI-hypersensitive sites. By using an in vivo approach on lymphoblastoid cell lines from PWS and AS individuals, we discovered that the SNRPN exon 1 is flanked by prominent hypersensitive sites on the paternal allele, but is completely inaccessible to nucleases on the maternal allele. In contrast, we identified several regions of increased nuclease hypersensitivity on the maternal allele, one of which coincides with the AS minimal microdeletion region and another lies in intron 1 immediately downstream of the paternal-specific hypersensitive sites. At several sites, parental origin-specific nuclease hypersensitivity was found to be correlated with hypermethylation on the allele contributed by the other parent. The differential parental origin-dependent chromatin conformations might govern access of regulatory protein complexes and/or RNAs which could mediate interaction of the region with other genes.

INTRODUCTION

In mammals, some genes are expressed from only one allele of specific parental origin. These genes are subject to genomic imprinting, a process which epigenetically marks alleles according to their parental origin during gametogenesis and results in monoallelic transcription in somatic cells (1). The mechanisms regulating genomic imprinting are not yet fully understood. Allele-specific DNA methylation has been shown to play an important role in maintenance of the imprinted status (2,3). Nevertheless, many lines of evidence suggest that regulation of genomic imprinting is quite complex and involves more than a parent-of-origin-specific methylation pattern of an imprinted gene’s promoter (1,4).

Within the genome, most of the imprinted genes are organized in clusters that generally are conserved in human and mouse. Human chromosome region 15q11-q13 and mouse chromosome 7 band D represent such a conserved imprinted domain. Numerous genes in the region are expressed from the paternally inherited allele only, including small nuclear ribonucleoprotein polypeptide N (SNRPN) (5-7), IPW (8), NDN (9), ZNF127 (10), and the PAR-1 and PAR-5 transcripts (11). At least one gene, UBE3A, generates a maternal-specific transcript in brain (Fig. 1A) while, in other tissues, it is expressed from both parental alleles. Mutations in UBE3A are responsible for some cases of familial Angelman syndrome (AS) (12-14). Another conserved cluster of imprinted genes, located on human chromosome band 11p15 and distal mouse chromosome 7 band F3-5, contains the monoallelically expressed loci H19 (15), Igf2 (16), Ins2 (17), Mash2 (18) and p57 kip2 (19,20).


Figure 1. (A)Schematic map of known imprinted transcripts in the human 15q11-q13 region. Arrows indicate allele-specific expression from the paternal (above) or maternal (below) allele, but not necessarily the direction of transcription. IC denotes the location of the putative imprinting center. (B)Known exons of the SNRPN transcriptional unit. Black boxes indicate untranslated exons, and white boxes coding exons. The nuclease-hypersensitive sites/regions (HS) identified in this study are indicated. The size and orientation of the vertical arrows correspond to abundance and allele specificity of nuclease-hypersensitive sites. On the paternal allele, there are two sites flanking exon 1 (pHS1 and pHS2) that are strong and strictly allele specific. The six sites designated mHS1-6 identify regions of weak nuclease hypersensitivity that predominate on the maternal allele. Horizontal bars on arrowheads indicate the regions that contain the mHS sites. Hatched boxes indicate the location of probes in the 5[prime]-upstream SNRPN region that are not shown on the detailed map in Figure 2B. The PWS/AS microdeletions which define the IC critical region map within the SNRPN transcription unit.

AS (21) and Prader-Willi syndrome (PWS) (22) are two distinct neurodevelopmental disorders caused by the lack of products of imprinted genes in the 15q11-q13 region. In ~70% of individuals affected with PWS or AS, the monoallelically expressed genes in the region are lost due to recurrent de novo deletions extending over 4-5 Mb. Deletion of the paternal 15q11-q13 region results in PWS, and deletion of the same region on the maternal chromosome causes AS. Another mechanism that leads to the loss of the expressed allele of an imprinted gene is uniparental disomy (UPD). Approximately 28% of PWS patients have maternal UPD of chromosome 15, and ~2% of all AS cases are due to paternal UPD (23,24).

In rare cases, PWS and AS are caused by ‘imprinting mutations’ such as submicroscopic deletions of variable size (10-1000 kb) within the 5[prime] region of the SNRPN transcription unit (Fig. 1B). These microdeletions affect the expression of distant genes (25). Microdeletions on the paternal allele, which result in PWS, not only suppress SNRPN expression, but also abrogate paternal expression of IPW, and are associated with changes in paternal allele-specific methylation at the ZNF127 locus. ZNF127 is located ~1.5 Mb centromeric and IPW at least 150 kb telomeric to the site of the microdeletion (9). Maternal microdeletions in the 5[prime]-untranslated region of the SNRPN transcription unit result in AS. Thus, the microdeletions define one or more cis-acting regulatory regions upstream of the SNRPN coding exons, referred to as the ‘imprinting center’ (IC). Since all known microdeletions causing PWS include SNRPN exon 1 and all known microdeletions causing AS are located upstream of SNRPN exon 1, a bipartite structure for the IC has been proposed (26,27).

The observation that regulation of imprinted genes in the PWS/AS region not only occurs at the level of individual promoters, but that cis-acting elements may be crucial for their coordinated regulation, is reminiscent of the regulation of other imprinted or non-imprinted gene clusters. In mouse, for example, parent-of-origin-specific expression of the clustered genes H19, Igf2 and Ins2 depends on a cis regulatory element associated with the H19 enhancers (28-30). A classical example of long-range cis regulation is the non-imprinted human [beta]-globin gene cluster, where the globin genes [epsis], [lambda]G, [lambda]A, [delta] and [beta] are arranged in the order of their developmental stage-specific expression. The temporal expression is under control of a locus control region (LCR). The distance between the LCR and the most distal [beta]-globin gene is ~70 kb in humans (31). The mechanism by which such long-range cis-acting regulation is mediated is still unknown. It has been shown, however, that chromatin containing regulatory elements of the Ins2-Igf2-H19 imprinted domain (32-34), the [beta]-globin gene cluster LCR (35) and other regulatory regions is characterized by hypersensitivity to nucleases. The hypersensitivity is thought to reflect relatively decondensed chromatin that would allow interactions of regulatory elements with their target genes via a protein or protein-RNA complex.

In this study, a chromatin region including the 150 kb SNRPN transcription unit on human chromosome 15 was examined for nuclease-hypersensitive sites to identify the locations of candidate regulatory regions and to characterize the chromatin structure of the putative IC previously identified by studies of spontaneously occurring microdeletions.

RESULTS

SNRPN exon 1 is associated with prominent parental allele-specific nuclease-hypersensitive sites

Parent-of-origin-specific expression and methylation of imprinted loci (SNRPN, IPW and ZNF127) in the PWS/AS deletion region previously have been shown to be maintained in human lymphoblastoid cell lines (LCLs) (8). Furthermore, microdeletions in the IC region result in the abrogation of the paternally expressed genes IPW and SNRPN not only in tissues where these genes are predominantly expressed, but also in LCLs (36), suggesting that the mechanism by which parent-of-origin-specific expression of these genes is achieved is maintained in LCLs. Therefore, we used as our experimental system LCLs derived from individuals with PWS and AS. To search for chromatin sites showing hypersensitivity to nucleases, we used a recently described in vivoapproach in which whole cells are permeabilized briefly with a mild detergent prior to incubation with nuclease (37,38). Hypersensitive sites identified by the in vivo technique are more likely to represent loci of chromatin decondensation in the living cell as compared with sites identified by approaches that involve the isolation of nuclei prior to nuclease digestion.

When nuclease hypersensitivity of chromatin is tested by digestion with DNase I, only prominent hypersensitive sites are detected because the DNA is degraded at higher DNase I concentrations. In order to detect less prominent hypersensitive sites and subtle differences in allele-specific hypersensitivity, we used MspI instead of DNase I for certain hypersensitivity experiments. Since MspI cuts only in its sequence-specific recognition site (5[prime]-CCGG-3[prime]), bands indicating hypersensitive sites are stable even at high enzyme concentrations. A drawback of this approach is that only those hypersensitive regions that contain MspI sites are detected.

Hypersensitivity tests using MspI restriction nuclease

Seventeen probes corresponding to different regions of the SNRPN transcription unit were tested using MspI as an in vivo nuclease on LCLs from controls, AS and PWS individuals. Cell lines weretreated with increasing amounts of MspI (10-500 U) or not treated with MspI (negative control). The restriction enzymes XmaI or EcoRI, and in some cases HindIII, were used for subsequent Southern analysis. As shown in Figure 2, a 6.9 kb XmaI fragment revealed by SNT6, a probe from a region just upstream of SNRPN exon 1, disappeared almost completely in the AS-derived samples with increasing amounts of MspI. Three new bands appeared, a faint one of 5.7 kb and two prominent ones of 4.1 and 3.9 kb. These bands were also seen in samples derived from the control cell lines, but here the 6.9 kb fragment was retained. With all samples derived from the PWS patients in whom the paternal allele was missing or not expressed, only the 6.9 kb fragment was detected, even at the highest concentration of endonuclease (500 U MspI/ml). The results identify two paternal allele-specific nuclease-hypersensitive sites. pHS1 is defined by the 4.1 and 3.9 kb fragments. The weak 5.7 kb fragment indicates site pHS2 that is located nearer the telomere. With the probe used (SNT6), this fragment is only seen when no cleavage has occurred at pHS1. Therefore, to evaluate further the strength of the pHS2 site, we needed to generate more downstream probes.


Figure 2. (A) MspI nuclease hypersensitivity experiment with control, AS and PWS lymphoblastoid cell lines (LCL). XmaI was used in Southern analysis. Probe SNT6 reveals strong hypersensitive sites on the paternal allele, indicated by the disappearance of the 6.9 kb band and generation of 5.7, 4.1 and 3.9 kb bands in AS LCLs upon treatment with increasing amounts of MspI, while the 6.9 kb fragment remains undigested in PWS LCLs. (B) The SNRPN genomic region including exons 1-10a. The centromere is to the left and the telomere to the right. Gray boxes represent coding exons. The location of hypersensitive sites on the paternal (pHS) and maternal (mHS) allele are indicated by arrows. The locations of probes used in hypersensitivity experiments are shown as hatched boxes. EcoRI (E), HindIII (H), MspI (M) and SmaI-XmaI-MspI (X) restriction sites are indicated. MspI sites (including SmaI and XmaI sites) are numbered from 1 to 17 centromeric to telomeric. For their precise location within the genomic sequence, see Table 1. CpG denotes the CpG island surrounding exon 1. Horizontal arrows indicate restriction fragments corresponding to bands in (A) and in Figures 3 and 4.

For the MspI hypersensitivity experiments shown in Figure 3, probes SNT7.2 and SNT7.3 were used and Southern analysis was performed with EcoRI. Probes SNT7.1, SNT7.2 and SNT7.3 correspond to the region downstream of SNRPN exon 1 and the CpG island (Fig. 2B). Upon treatment of cells with MspI, SNT7.2 reveals three bands (3.5, 2.3 and 1.1 kb; Fig. 3A) in the control sample, in addition to the undigested 11 kb EcoRI fragment (Fig. 2B). The 1.1 kb fragment was absent in PWS samples, because it derived from a second paternal allele-specific hypersensitive site named pHS2. In contrast, the 3.5 and the 2.3 kb bands appeared only very faintly in AS samples but were much stronger in PWS samples. Therefore, they identify sites that are hypersensitive preferentially on the maternal allele. In MspI hypersensitivity experiments where Southern analysis was performed with EcoRI, probe SNT7.1 (Fig. 2B) revealed the same bands as probe SNT7.2 (data not shown). These results clearly indicate that the paternal allele in the AS cell lines is highly sensitive to in vivo MspI digestion at sites associated with SNRPN exon 1. In contrast, the maternal allele retained in PWS cell lines is completely nuclease resistant at these sites, suggesting a distinctly different chromatin organization. In addition, our results indicate that SNRPN intron 1 contains hypersensitive regions that are substantially more accessible on the maternal allele as compared with the paternal allele.


Figure 3. (A) MspI hypersensitivity experiment with control, AS and PWS LCLs. Southern analysis was performed with EcoRI that generates an 11 kb fragment. After treatment of LCLs with MspI, probe SNT7.2 detects hypersensitive sites specific to either parental allele, as reflected by the appearance of the 1.1 kb band in AS samples and 3.5 and 2.3 kb bands in PWS samples (Fig. 2B). The 1.1 kb paternal allele-specific band in this experiment and the 5.7 kb paternal allele-specific band in Figure 2A identify the same hypersensitive site, pHS2. Formation of the 1.1 kb band results in a 9.9 kb derivative of the 11 kb EcoRI fragment. In this gel system, these two fragments are not separated. The 3.5 and 2.3 kb fragments identify two differentially hypersensitive sites with strong preference for the maternally derived allele (mHS3 and mHS4). (B) MspI hypersensitivity experiment performed as in (A) and using probe SNT7.3 for hybridization. SNT7.3 corresponds to the distal portion of SNT7.2 and, therefore, reveals only the 2.3 and 3.5 kb fragments identifying mHS3 and mHS4, but not the 1.1 kb fragment (Figs 2B and 3A).

Hypersensitivity experiments using DNase I

The existence of a prominent paternal allele-specific nuclease-hypersensitive region detected with probes SNT6, SNT7.1 and SNT7.2was confirmed by nuclease hypersensitivity tests performed with DNase I (Fig. 4). Cells of LCL 1497 (control), LCL 1201 (AS) and LCL 865 (PWS) were treated with 0 (negative control), 2, 5 and 10 U/ml of DNase I. As predicted by the DNA sequence, probe SNT6 revealed a 6.9 kb SmaI fragment in Southern blots in negative controls without DNase I (Fig. 4A). This band was seen together with a band corresponding to an ~10 kb fragment with DNA prepared from a PWS LCL and control LCL, but not with samples prepared from an AS LCL. In addition, probe SNT6 revealed a high molecular weight smear (>15 kb) in the AS LCL and in the control LCL, but not in the PWS LCL. The larger bands were due to differential methylation, as explained below. The two different band patterns each correspond to the parental allele of maternal or paternal origin, respectively, and the combination of both patterns was seen in the control LCL 1497. Upon addition of DNase I, the high molecular weight smear of the AS LCL disappeared and bands smaller than the expected 6.9 kb SmaI fragment were generated (3.8 and 5.5 kb), indicating that the region complementary to probe SNT6 contains DNase I-hypersensitive sites on the paternal allele. No DNase I-induced changes in band size were seen for the PWS LCL, demonstrating that the DNase I-hypersensitive sites revealed by SNT6 are not present on the maternal allele. Slightly faster migration and fading intensity of signals with increasing amounts of DNase I is due to DNA degradation. The same DNase I-generated fragments of 3.8 and 5.5 kb were observed in AS samples but not in PWS LCLs when DNA was cleaved with the less methylation-sensitive isoschizomer XmaI(Fig. 4B). Although in the experiment shown there was a lower extent of overall DNA digestion by DNase I, the results were reproducible. DNase I hypersensitivity specific for the paternal allele was also detected with probe SNT7.2 (data not shown). The ~3.8 and 5.5 kb DNase I-XmaI fragments correspond to the 3.9/4.1 kb and 5.7 kb fragments seen in the MspI-XmaI experiments. The results provide independent confirmation for the existence of the pHS1 and pHS2 sites. As the two bands are of about equal intensity, it is likely that the level of hypersensitivity is similar at both sites.


Figure 4. (A)In vivo digestion with increasing concentration ofDNase I, followed by SmaI Southern analysis and hybridization with probe SNT6. Probe SNT6 reveals two hypersensitive sites on the paternal allele (pHS1 and pHS2, see Fig. 2B) as indicated by the appearance of 5.5 and 3.8 kb fragments in AS and control but not in PWS. The 6.9 kb SmaI fragment in PWS overlaps the SNT6 probe, as shown in Figure 2B, and the 10 kb fragment is generated by incomplete cleavage of the centromeric SmaI-XmaI site and includes the 3.4 kb fragment. This site (site 2 in Fig. 2B) is largely methylated on the maternal allele present in the PWS cells. Neither the 10 nor the 6.9 kb fragment is seen in AS samples because the SmaI-XmaI site at the telomeric end of the 6.9 kb fragment (site 10 in Fig. 2B)is methylated consistently on the paternal allele and the next SmaI-XmaI site lies further 3[prime] between two coding exons. (B) DNase I hypersensitivity experiment. Southern analysis was done with XmaI, an SmaI isoschizomer that is less methylation sensitive than SmaI. In this experiment the DNase I digest was less complete than in the experiment shown in (A), as reflected by the abundance of the 6.9 kb fragment. The 5.5 and the 3.8 kb bands that identify pHS1 and pHS2 appear upon DNase I treatment of AS LCLs but not of PWS LCLs. (C) Probing the filter used for the experiment shown in (A) with SNT3 does not reveal hypersensitive sites, but ensures equal loading and digestion efficiency of the samples. SNT3 maps further upstream of SNRPN exon 1, as shown in Figure 1B.

No distinct DNase I-dependent changes in restriction fragment length were seen with any of the other 12 probes tested that are distributed over the entire SNRPN transcription unit (Figs 1B and 2B). For example, with SNT3, a probe from a region ~15 kb upstream of the SNRPN exon 1, the same band pattern was seen with samples from all cell lines tested (Fig. 4C). Hybridization with SNT3 to the filter corresponding to the experiment shown in Figure 4A ensured equal sample loading and efficiency of the SmaI restriction endonuclease digest.

Differential cytosine methylation explains size differences of SmaI and XmaI fragments

When Southern analysis of DNA derived from PWS or AS LCLs was performed with SmaI, a 10 kb band and a high molecular weight DNA smear were revealed by probe SNT6 in addition to the expected 6.9 kb band, as outlined above. The possibility of SmaI restriction fragment length polymorphism was ruled out by using the SmaI isoschizomer XmaI, which is less sensitive to methylation, and MspI, which cuts within all SmaI-XmaI sites (data not shown). Therefore, the longer fragments are most likely generated by parent-of-origin-specific methylation at SmaI restriction sites which prevent SmaI from cutting. The ~10 kb fragment produced by SmaI digestion of the maternal allele (PWS LCL) consists of the 6.9 kb fragment plus the adjacent centromeric 3.4 kb SmaI fragment, due to an uncut SmaI restriction site (site 2 in Fig. 2B). This finding is not unexpected, since virtually all cytosines of the 5[prime]-CpG-3[prime] dinucleotides in the region surrounding SNRPN exon 1 are methylated on the silent maternal allele (39,40). More surprisingly, we also found sites of paternal hypermethylation, as reflected by the high molecular weight smear seen after SmaI digestion and hybridization with probe SNT6 in control and AS LCLs, but not in PWS LCL (Fig. 4A). Interestingly, the SmaI site (site 10 in Fig. 2B)showing paternal-specific methylation corresponds to a nuclease-hypersensitive site on the maternal allele (mHS3; see below).

Fine mapping of paternal-allele-specific nuclease-hypersensitive sites flanking SNRPN exon 1

The 6.9 kb SmaI (XmaI) fragment revealed by probe SNT6 includes SNRPN exon 1 and contains seven MspI sites. Five sites (sites 5-9) are located downstream, one (site 4) is within and one (site 3) lies upstream of exon 1 (Fig. 2B). Probe SNT6 reveals only this most centromeric (5[prime]) MspI site. The distance between the centromeric end of the 6.9 kb XmaI fragment and MspI site 3 is 3.9 kb, and this distance is 4.1 kb from MspI site 5 that is located in exon 1. The 3.9 and 4.1 kb bands produced in MspI hypersensitivity tests performed with control and AS LCLs correspond to the centromeric part of the 6.9 kb XmaI fragment. Thus, the results obtained in the MspI hypersensitivity tests show that MspI sites 3 and 4, which are located within and immediately 5[prime] of SNRPN exon 1, map within a nuclease-hypersensitive region on the paternal allele (called pHS1).

Another prominent hypersensitive site (pHS2) maps 3[prime] to SNRPN exon 1, as reflected by the appearance of the weak ~5.7 kb fragment seenon XmaI Southern analysis with probe SNT6 (Fig. 2A) and the 1.1 kb fragment seen on EcoRI Southern analysis with probes SNT7.2 (Fig. 3A) and SNT7.1 (data not shown) upon MspI treatment of control and AS cells. These fragments are due to a hypersensitive site that includes MspI site 9, but not MspI sites 7 and 8 that are situated ~200 and 800 bp more centromeric but still downstream of SNRPN exon 1 (Fig. 2B). Probes SNT6 and SNT7.2 were also used in MspI hypersensitivity tests where Southern analysis was performed with BamHI or with MfeI plus XmaI. The results of those experiments (data not shown) are entirely consistent with the localization of pHS1 and pHS2 as described above. Taken together, our MspI and DNase I hypersensitivity experiments have discovered two distinct nuclease-hypersensitive sites associated with SNRPN exon 1 on the paternally derived chromosome. One site (pHS1) is located just upstream of SNRPN exon 1, as indicated by the 3.8 kb band in the DNase I experiment, and the other (pHS2) is ~1.7 kb downstream of exon 1 within SNRPN intron 1 (Fig. 2C).

The SNRPN transcriptional unit contains several sites that are preferentially nuclease hypersensitive on the maternal allele

The first indication for the existence of distinct sites of preferential hypersensitivity on the maternal allele was provided by the MspI hypersensitivity experiments shown in Figure 3. On Southern analysis with EcoRI, all three SNT7 probes revealed 3.5 and 2.3 kb bands in controls and PWS samples that were only very faintly present in AS samples. These results were reproduced in repeated experiments. The 3.5 and 2.3 kb bands are generated by digestion at MspI sites 10 and 11 but not at MspI sites 7-9, the last being part of pHS2 (Fig. 2B). The sites were designated mHS3 and mHS4. As shown in Figure 3B, probe SNT7.3 that corresponds to the distal part of the 2.3 kb EcoRI-XmaI fragment does not detect the paternal allele-specific 1.1 kb EcoRI-MspI fragment in AS LCLs that hybridizes with probes SNT7.2 (Fig. 3A) and SNT7.1 (data not shown), nor does SNT7.3 reveal the 3[prime] adjacent 1.2 kb MspI-XmaI fragment in any of the three LCLs. The 2.3 kb EcoRI-XmaI and the 3.4 kb EcoRI-MspI fragments, however, are maintained in Southern analysis with SNT7.3, thus clearly demonstrating that mHS3 and mHS4 occur preferentially on the maternal allele. Likewise, when probe SNT8 (Fig. 2B) was used for hybridization in an MspI hypersensitivity experiment where Southern analysis was done with XmaI, the 1.2 kb MspI-XmaI fragment generated by cleavage at mHS4 was detected with control LCLs and PWS LCLs, but hardly seen with AS LCLs, thus again confirming that mHS4 is preferentially nuclease sensitive on the maternal allele (data not shown). Since mHS3 and mHS4 are less prominent than the paternal allele-specific sites flanking SNRPN exon 1, they are not detectable in DNase I hypersensitivity tests and, therefore, the distal boundary has not been delineated. Proximally, however, the boundary is defined. The transition from the paternal allele-specific hypersensitive site 3[prime] of SNRPN exon 1 (pHS2) to the maternal allele-specific hypersensitive region that contains the mHS3 and mHS4 sites occurswithin the 1.2 kb between MspI sites 9 and 10 (Fig. 2B).

To search the SNRPN transcriptional unit further downstream of exon 1 for candidate regions with regulatory function, we generated probes SNT9 and SNT10 that correspond to the regions immediately upstream of SNRPN exon 2 and exon 3, respectively (Fig. 2B). In Southern analysis, both probes revealed an XmaI band representing the ~13 kb fragment 3[prime] adjacent to the 6.9 kb XmaI fragment. The prominent paternal allele-specific hypersensitive region pHS2 was not detected with probe SNT9, which confirms its telomeric boundary as being within the 6.9 kb XmaI fragment. The ~13 kb XmaI fragment, however, exhibited slightly increased sensitivity to MspI nuclease on the maternal allele, as reflected by the ~7.5 kb fragment that appeared upon treatment of the cells with MspI in the control and in PWS samples, but to a substantially lower degree in AS samples (Fig. 5A). The 7.5 kb band is due to a hypersensitive site that includes MspI site 12 in intron 2, that is located 3.1 kb downstream of exon 2. This site defines a region predominantly nuclease-hypersensitive on the maternal allele as compared with the paternal allele, called mHS5 (Fig. 2B). Further experiments using probe SNT10 on a HindIII blot indicated that MspI site 13 in intron 4 is not located within a nuclease-hypersensitive region (data not shown).


Figure 5. MspI-hypersensitive sites on the maternal allele detected with four different SNRPN-associated probes. SNT9 (A) corresponds to the region immediately upstream of SNRPN intron 2 and SNT11 (B) to the central portion of SNRPN intron 7. Probe SNT2 (C)correspondsto the minimal AS microdeletion region, and IC1B[prime][prime] (D)corresponds to an SNRPN upstream non-coding exon (Figs 1B and 2B). Southern analysis was performed with EcoRI (probes SNT11 and IC1B[prime][prime]) or XmaI (probes SNT2 and SNT9). All four probes revealed higher sensitivity to the endonuclease of the maternal allele, as indicated by additional bands appearing in PWS and control, but to a lower extent in AS cells upon MspI treatment (see text for details). Probes IC1A (A and C) and SNT1 (B and D) that do not reveal any hypersensitive sites in XmaI or EcoRI Southern analysis, respectively, were used as loading controls.

A differentially methylated region in intron 7 corresponds to differentially nuclease-hypersensitive sites

The SNRPN intron 7 contains several 5[prime]-CpG-3[prime] sites which are methylated on the expressed paternal allele but unmethylated on the transcriptionally silent maternal allele (41). This methylation pattern is reminiscent of the imprinted Igf2r gene in mouse, where specific methylation of an intronic sequence on the expressed maternal allele is required for Igf2r expression (42). To test whether the SNRPN intron 7 differential allelic methylation pattern corresponds to differential allele-specific chromatin conformation, a probe corresponding to a part of intron 7 (SNT11) was generated and used for hypersensitivity tests. Probe SNT11 revealed an ~15 kb EcoRI fragment in Southern analysis. Upon treatment of cells with MspI, seven shorter fragments were produced, with five fragments (11, 5.5, 4.8, 4.3 and 4.0 kb) detected in control and PWS cell lines but not in AS samples (Fig. 5B). Similar results were obtained when HindIII was used instead of EcoRI (data not shown). Thus, the maternal allele of the SNRPN intron 7 contains sites of increased nuclease hypersensitivity as compared with the paternal allele. We conclude from the location of SNT11 and from the length of the fragments generated that this hypersensitive region (mHS6) covers all three MspI sites (sites 14-16) of intron 7 and probably extends downstream through SNRPN exon 9 (MspI site 17), but does not include the 3[prime]-untranslated exons 10a, 11 and 12 (43). This conclusion is consistent with results obtained using probe SNT12 from exon 10a (data not shown).

Differential nuclease hypersensitivity associated with the putative imprinting center

The genomic region covered by the smallest AS microdeletion (Fig. 1B) was suggested to contain a cis-acting regulatory element constituting the centromeric part of a bipartite IC (the ‘imprintor’), with the region around SNRPN exon 1 representing the telomeric portion of the IC [the ‘switch initiation site’ (27)]. To test whether the region upstream of SNRPN exon 1, including the putative IC, contains allele-specific hypersensitive sites, genomic probes were generated by subcloning cosmids and by PCR (Fig. 1). Since these probes originate from regions of previously unknown sequence, double digests and genomic Southern blotting were first carried out to ensure that the fragments revealed by the probes contain MspI sites testable for hypersensitivity (data not shown).

Probes IC2 and SNT3 did not detect any hypersensitive sites, and none of the other probes covering 5[prime]-untranslated exons of the SNRPN transcription unit revealed a paternal allele-specific MspI-hypersensitive site. Two probes mapping within the AS microdeletion regions (SNT1 and SNT2), however, revealed MspI-sensitive sites that are substantially stronger on the maternal allele than on the paternal allele. In hypersensitivity experiments using HindIII for Southern analysis, probe SNT1 revealed three smaller fragments in addition to the >13 kb HindIII fragment predominantly in PWS and control samples that were much weaker in AS samples (data not shown). A similar result was obtained with probe SNT2 when Southern analysis was performed with XmaI. Bands of ~5.2, 4.3, 3.7 and 3.2 kb in length were generated by MspI treatment of PWS and control samples, but in AS samples these bands were seen only at substantially higher nuclease concentrations. The results shown in Figure 5C were reproducible in repeat experiments (data not shown). Probes SNT1 and SNT2 do not overlap with each other, but they both map to the centromeric part of cosmid c106 which overlaps with the telomeric end of clone c119 (Fig. 1B). This overlap of ~20 kb includes the >13 kb HindIII fragment. The proximal end of the overlap region maps ~28 kb centromeric to SNRPN exon 1. Probes SNT1 and SNT2 may detect the same maternal hypersensitive sites (collectively labeled mHS2) within this c119-c106 overlap region.

To test whether the SNRPN untranslated upstream exons IC1A and IC1B[prime][prime] are associated with allele-specific nuclease-hypersensitive sites, the two corresponding probes IC1A and IC1B[prime][prime] were used in MspI hypersensitivity experiments. SNRPN exon IC1A maps ~100 kb centromeric to SNRPN exon 1, close to D15S63, a locus characterized by allele-specific methylation of the maternal allele (44). No hypersensitive sites were detected with probe IC1A (data not shown). SNRPN exon IC1B[prime][prime] maps ~130 kb centromeric to exon 1. Probe IC1B[prime][prime] revealed a strong ~8.5 kb EcoRI fragment and two weak fragments of 4.5 and 2 kb. Since the latter two fragments did not change with increasing MspI concentration, we assume that they are due to cross-hybridization with a related sequence. MspI treatment of the cells produced five additional fragments (8.0, 3.2, 2.4, 1.7 and 0.7 kb; in Fig. 5D, the two smallest fragments are not shown). The 3.2 and 2.4 kb fragments were seen in PWS and control samples but only very weakly in AS cells (Fig. 5D). Hence, this hypersensitive region (mHS1) is specific to the maternal allele. The overall abundance of these sites, however, is relatively low since the 8.5 kb EcoRI band did not decrease substantially in intensity upon appearance of the shorter fragments when the loading control data are taken into consideration (Fig. 5D). To confirm these conclusions independently, we carried out Southern experiments with XmaI-digested DNA probed with IC1B[prime][prime]. With increasing concentrations of MspI in the in vivo hypersensitivity assay, weak fragments of 5.5 and 5 kb were generated in the PWS and control samples, but not in AS cells (data not shown).

DISCUSSION

In many instances, chromatin of cis-acting regulatory elements has been found to be hypersensitive to nucleases. Therefore, we systematically examined the 150 kb SNRPN transcription unit (27,44,45) that encompasses the putative IC for nuclease-hypersensitive sites. We hypothesized that distinct and allele-specific chromatin organization may play a role in the regulation of imprinted genes on chromosome 15 region q11-q13. Table 1 contains a summary of our results. We found regions of preferential or exclusive nuclease hypersensitivity on a parental origin-specific allele, as well as regions without any detectable hypersensitvity.

Table 1. Combinations of probes, MspI sites and restriction enzymes that detect nuclease-hypersensitive sites in the SNRPN transcription unit
Probe name Hypersensitive region mat/pat allelea Hypersensitive MspI siteb Southern analysis restriction enzymec
IC1B[prime][prime] mHS1, mat ND EcoRI
IC1A none detected    
IC2 none detected    
SNT1 mHS2, mat ND HindIII (not EcoRI)
SNT2 mHS2 ND XmaI, BamHI
SNT3 none detected    
SNT4 none detected    
SNT5 none detected    
SNT6 pHS1, pHS2, pat 3, 4, 9 SmaI, XmaI, EcoRI
SNT7.1 pHS2 pat
mHS3, mHS4, mat		 9-11 XmaI, EcoRI
SNT7.2 pHS2 pat
mHS3, mHS4, mat		 9-11 XmaI, EcoRI
SNT7.3 mHS3, mHS4, mat 10, 11 EcoRI
SNT8 mHS4, mat 11 XmaI
SNT9 mHS5, mat 12 XmaI
SNT10 mHS5, mat 12 XmaI
SNT11 mHS6, mat 14-17 EcoRI, HindIII
SNT12 mHS6, mat 14-17 EcoRI, HindIII
aAll mHS sites are preferentially hypersensitive on the maternal (mat) allele; all pHS sites are hypersensitive only on the paternal (pat) allele.
bNumbering of MspI sites is as in Figure 2B. The exact positions of MspI sites in the sequence of cosmid c102, GenBank accession no. U41384, are: site 1 corresponds to N 22806, site 2 to N 19406, site 3 to N 15502, site 4 to N 15354, site 5 to N 15285, site 6 to N 15153, site 7 to N 14469, site 8 to N 13877, site 9 to N 13676, site 10 to N 12492, site 11 to N 11238 and site 12 to N 4966. MspI sites 13-17 refer to our unpublished sequence. ND, MspI sites whose existence within a given restriction endonuclease fragment has been demonstrated, but their exact position is unknown.
cSNT1 reveals a hypersensitive site only in combination with a particular restriction endonuclease.

The ~33 kb extending from SNRPN exon 3 to ~20 kb upstream of exon 1 had been sequenced previously (GenBank accession no. U41384) and could be searched completely for hypersensitive sites. In DNase I and MspI hypersensitivity tests, we detected two prominent nuclease-hypersensitive sites specific to the paternal allele, one immediately upstream (pHS1) and the other downstream of exon 1 (pHS2). This region contains the CpG island from which transcription of SNRPN exons 1-12 initiates (39,43), and overlaps with the PWS microdeletions which define the telomeric part of the putative IC (Fig. 1B). The hypersensitive site just upstream of SNRPN exon 1 (pHS1) may merely reflect the open promoter conformation associated with SNRPN transcription. Site pHS2, however, maps to a defined region ~1.7 kb downstream of exon 1 which is not likely to coincide with the open promoter complex associated with initiation of the SNRPN coding transcript (46). Both hypersensitive sites detected on the paternal allele are extremely prominent, strictly allele-specific, and have precise boundaries. They are the only nuclease hypersensitive sites, identified by DNase I and by MspI hypersensitivity experiments, on the paternally derived allele and, consequently, are likely to represent the regulatory region that controls the paternal-specific epigenotype.

Previously, the chromatin structure of three imprinted genes, H19, Igf2 and U2af-rs1, has been studied by nuclease hypersensitivity tests in the mouse (for review, see ref. 47). Both allele-specific and non-specific sites have been detected. The U2af-rs1 gene demonstrates general DNase I and MspI sensitivity over its entire length and has three hypersensitive sites in the promoter region on the expressed paternal allele (48,49). Igf2 and H19 have hypersensitive sites only in the promoter and enhancer regions. Interestingly, the Igf2 promoter region and the H19 enhancers are nuclease hypersensitive on both parental alleles, whereas the H19 promoter exhibits an open chromatin configuration only on the expressed maternal allele (32-34,50). It has now been proven conclusively that regulatory sequences associated with the H19 gene and their location are key elements controlling the monoallelic expression of the Igf2 and Ins2 genes (28,29,32,51). SNRPN appears to be analogous to the H19 associated regulators in that the 5[prime]-untranslated portion of SNRPN contains the cis-acting IC, the SNRPN exon 1-associated CpG island exhibits prominent maternal allele-specific methylation, and onset of parent-of-origin-specific gene expression occurs very early (27,40,52). We report now that, in addition to these features, the human SNRPN IC is characterized by two prominent paternal allele-specific DNase I-MspI hypersensitive sites that are flanked distally by a sudden transition from paternal allele-specific to maternal allele-specific chromatin conformation.

On the paternal allele, the hypomethylated SNRPN exon 1 CpG island and associated pHS1 and pHS2 sites can formally be considered as an activator of paternally expressed imprinted genes in the PWS/AS region, since its loss by microdeletions in PWS patients (27) results in preventing expression of imprinted genes on 15q11-q13. In gene-targeted mice, a 40 kb deletion including the Snrpn exon 1 site mimicked the PWS phenotype and silenced all known paternally imprinted genes in the region, while the disruption of the Snrpn coding region alone failed to generate similar effects (53). The SNRPN exon 1 IC might physically interact with its target gene via a protein complex, similarly to a mechanism which has been suggested for target gene activation by the [beta]-globin LCR (32). The open chromatin conformation on the active paternal allele could be crucial for this interaction to be established.

The centromeric part of the SNRPN-associated IC which is defined by the AS microdeletions might interact in a similar manner with the AS-associated gene UBE3A or other potential genes involved in AS. In this model, the smallest overlap of AS microdeletions (AS-SRO) would be expected to show nuclease hypersensitivity on the maternal allele. The hypersensitive site mHS2 that we identified co-localizes with the AS-SRO. It is interesting, therefore, to speculate that maternal allele-specific chromatin decondensation at the AS-SRO locus is associated functionally with the regulation of maternally expressed transcripts in the PWS/AS deletion region. Dittrich et al. suggested a different model explaining the functional mechanism of the SNRPN-associated IC (27). According to their model, the SNRPN exon 1 IC contains a ‘switch initiation site’ (SIS) from where, depending on the sex of germline passage, either the paternal or maternal epigenotype would spread over the PWS/AS region, similarly to X chromosome inactivation starting from the X inactivation center. This model further suggests the upstream ‘IC’ exons act as an ‘imprintor’ by triggering the epigenotype switch at the SIS in the female germline (27). Therefore, expression of the IC exons on the paternal allele in the female germline is thought to be in competition with expression of SNRPN, IPW, NDN and perhaps other imprinted genes on the same allele. Recently, a paternally expressed antisense RNA was found that overlaps the maternally expressed UBE3A transcript in brain (54). The idea that competition of transcription from the same allele is a key element in regulation of genomic imprinting underlies many of the current models (for review, see ref. 55).

Our finding of highly nuclease-hypersensitive sites associated with the SNRPN exon 1 IC has important implications with regard to any of the scenarios that have been suggested: if the SNRPN exon 1 IC functions by physically interacting with its target genes, such interactions would be impossible on the maternal allele, where the regulatory sequence is inaccessible due to chromatin condensation characterized by complete nuclease insensitivity. If the SNRPN exon 1 IC functions as a silencer from where heterochromatinization spreads over 15q11-q13, the mechanisms would clearly be different from that of X chromosome inactivation since, according to our findings, the ‘active’ hypersensitive IC and the transcribed imprinted genes are contained on the same allele. Our findings are also incompatible with the idea of a euchromatin-like state spreading continuously from the SNRPN exon 1 region over 15q11-q13, because pHS1 and pHS2 show clear-cut boundaries and are flanked by maternal allele-specific hypersensitive regions. Interestingly, part of the SNRPN exon 1 CpG island as well as an H19 5[prime]-upstream element have been shown to function as gene silencers when tested in Drosophila (56,57). These findings are not directly applicable to the human situation because deletion from the paternal allele of the sequences studied causes the silencing of genes in the entire 15q11-q13 region in cis. It seems more likely that the maternal epigenotype is not dependent on the activity or presence of the silencer sequences identified in the fly studies. Rather, the SNRPN exon 1 regulatory region is required to establish and maintain the paternal epigenotype while the maternal epigenotype may represent the default state. Notably, however, the same ~200 bp element upstream of SNRPN exon 1 that confers silencing in the Drosophila system must be present in a hypomethylated state for SNRPN expression (46), and corresponds to a strong paternal allele-specific hypersensitive site, as reported in this study.

With respect to the question of whether the maternal epigenotype is also actively controlled, we have identified six regions of increased nuclease sensitivity on the maternal allele. Although much less abundant than the hypersensitive sites flanking the SNRPN exon 1 on the paternal allele,several of these differentially hypersensitive sites co-localize with regions which might contain elements potentially involved in gene regulation within the PWS/AS deletion region. The EcoRI fragment revealed by probe SNT2 overlaps with the smallest reported AS microdeletion [AS-H (27)], suggesting that the region including site mHS2 may be transcriptionally active or have an open chromatin configuration on the maternal allele. Probe SNT11 that detects site mHS6 corresponds to a region within SNRPN intron 7 which is hypomethylated on the silent maternal allele but hypermethylated on the expressed paternal allele (27). A maternal allele-specific hypersensitive region that includes sites mHS3 and mHS4 is located in intron 1 directly downstream of the SNRPN exon 1 IC. Interestingly, we also detected specific methylation on the paternal allele at the site corresponding to mHS3. Notably, the human SNRPN intron 1 and the mouse Snrpn intron 1 show conservation of G-rich clustered repeats. These conserved sequence elements have been suggested to play a role in establishing an allele-specific SNRPN methylation pattern(58).Our findings suggest the possibility that transcripts could be initiated from this region or from intron 7 on the maternal allele, while the paternal allele would be protected by hypermethylation. Such transcripts, if synthesized in the antisense direction, might interfere with the assembly of the transcription machinery at the SNRPN promoter on the maternal allele.

Slight allelic differences in nuclease hypersensitivity might reflect features other than allelic differences in chromatin condensation. For example, matrix attachment sites were shown previously to be less sensitive to nucleases (59), and the parental alleles might show a different distribution, number or strength of chromatin attachment sites to the nuclear matrix (60,61). Such allelic differences in chromatin organization within the nuclear structure could directly affect allele-specific long-range regulation of distant targets by the SNRPN IC and phenomena such as asynchronous replication in the PWS/AS region (62-64). In this context, it would be interesting to test the protein-binding properties of the hypersensitive regions to candidate proteins, such as components of the nuclear matrix. The SNRPN exon 1 downstream region representing the locus of transition from paternal allele-specific to maternal allele-specific chromatin hypersensitivity might be of particular interest in this regard. For example, a protein complex that forms at the maternal allele-specific hypersensitive sites adjacent to the SNRPN exon 1-associated IC region could prevent the latter from decondensation on the maternal allele. Alternatively, the absence of decondensed chromatin in the vicinity of the SNRPN exon 1-associated IC might increase chances of proteins binding to the unique ‘open conformation’ site (the SNRPN exon 1-associated IC) embedded within relatively condensed chromatin, thus increasing the specificity of protein complex formation at the IC. To elucidate further the mechanism by which the SNRPN exon 1-surrounding region regulates gene expression in cis, its effect on reporter sequences other than SNRPN itself could be tested by either transfection studies in cultured cells or a transgenic mouse system.

MATERIALS AND METHODS

Isolation and labeling of genomic probes

Fifteen probes mapping within the ~150 kb SNRPN transcription unit were generated by PCR from genomic DNA and two were subcloned from cosmids. Seven PCR-generated probes map within the sequenced cosmid clone c102 (11) (GenBank accession no. U41384), which covers ~33 kb upstream of SNRPN exon 3 and includes the putative IC region upstream of exon 1 (Fig. 1). Another five PCR-generated probes correspond to the 5[prime] SNRPN untranslated IC exons 1A, 1B[prime][prime] and 2 (27), and to exon 10a (43) (GenBank accession no. U81001) and intron 7 (our unpublished data). For PCR, the Perkin Elmer (Foster City, CA) Gene-Amp kit was used according to the supplier’s recommendations. For all PCR-generated probes, 50-100 ng of genomic DNA derived from control LCLs were used as template. Denaturation for 3 min at 94°C was followed by 35 cycles at 94°C for 45 s, 60°C for 45 s and 72°C for 45 s, and one final extension at 72°C for 5 min. The MgCl2 concentration was 1.5 mM.

Sequences of PCR primers used for probes were: probe IC1B[prime][prime], revealed mHS1, a 71 bp fragment, 5[prime]-ACGAAGGTAATTGGGACTCC-3[prime] and 5[prime]-CCTTACCTGGAGAAGTGCCC-3[prime]; probe IC1A, a 171 bp fragment, 5[prime]-AGCTGAGCTCAGAGCCTTCT-3[prime] and 5[prime]-TTACCAGGAAAGCCTGAAGC-3[prime]; probe IC2, a 71 bp fragment, 5[prime]-CCTTACCTGGAGAAGTGCCC-3[prime] and 5[prime]-ACGAAGGTAATTGGGACTCC-3[prime] (27); probe SNT3, a 714 bp fragment of cosmid c102, N 30987-N 31700, 5[prime]-AATGGCCAAGAAGCACATCC-3[prime] and 5[prime]-AGGAGGCAGAAGATAGGAGT-3[prime]; probe SNT4, a 598 bp fragment of cosmid c102, N 24122-24719, 5[prime]-ATTAGGAAACTGATGCCCAG-3[prime] and 5[prime]-GAATATGTGCAAAGGCCATG-3[prime]; probe SNT5, a 548 bp fragment of cosmid c102, N 21351-21898, 5[prime]-GGCAAATCCATATAGACAGA-3[prime] and 5[prime]-ATGTGTCTCATATGTTTCCC-3[prime]; probe SNT6, revealed pHS1 and 2, a 1005 bp fragment of cosmid c102, N 15721-N 16725, 5[prime]-GTCTTCCTATGTGCGGTACA-3[prime] and 5[prime]-ATTGACTCCCGTGATCAGAG-3[prime]; probe SNT7.1, revealed pHS2, mHS3 and mHS4, an 889 bp fragment of cosmid c102, N 13922-N 14810, 5[prime]-AAACTCCGAAACGCAGAAG-3[prime] and 5[prime]-GTAGGTGTATAATAGTGACC-3[prime]; probe SNT7.2, revealed pHS2, mHS3 and mHS4, a 1298 bp fragment of cosmid c102, N 12917-N 14124, 5[prime]-GGGAAAACACGGAGGTT-3[prime] and 5[prime]-TGTAGTAGCTCCAACTTCAA-3[prime]; probe SNT7.3, revealed mHS3 and mHS4, a 499 bp fragment of cosmid c102, N 12917-N 13415, 5[prime]-GGGAAAACACGGAGGTT-3[prime] and 5[prime]-TCAGCAAGCTCTCATTAATG-3[prime]; probe SNT8, revealed mHS4, an 847 bp fragment of cosmid c102, N 11494-12340, 5[prime]-TCAAGCCTGTACTAACAGG-3[prime] and 5[prime]-CCTTATGTTGTTGTCTAAGG-3[prime]; probe SNT9, revealed mHS5, a 1091 bp fragment of cosmid c102, N 8376-N 9466, 5[prime]-GGTAGACTGCATCAATCTATG-3[prime] and 5[prime]-AGTGGCATAAATGTGTCTGTC-3[prime]; probe SNT10, a 961 bp fragment of cosmid c102, N 3023-N 3983, 5[prime]-GTCAGAGGATGACACTATCT-3[prime] and 5[prime]-GCACATTAGCGTATATCACC-3[prime]; probe SNT11, revealed mHS6 in SNRPN intron 7, a 184 bp fragment, 5[prime]-GTCATACAACTGTAAACAGC-3[prime] and 5[prime]-TCAACTGTGGTATATCCATG-3[prime]; probe SNT12, revealed mHS6, a 989 bp fragment, N 27-N 1015 (GenBank accession no. HSU81001), 5[prime]-CTGTTGATTTTGATGAGATC-3[prime] and 5[prime]-ATCAAGTCAGTAACAGGAAC-3[prime].

Cosmid c106 was digested with EcoRI, HindIII or PstI. On a Southern blot hybridized with labeled total genomic DNA, two fragments that failed to hybridize, and presumably lacked repetitive sequences, were subcloned and used as probes: SNT1, a 4 kb EcoRI fragment, and SNT2, a 0.7 kb PstI fragment. For [[alpha]-32P]dCTP labeling of DNA fragments, the ‘Multiprime DNA labelling system RPN 1601Z’ kit (Amersham, Arlington Heights, Il) was used. For labeling of short fragments (<300 bp), a sequence-specific (PCR) primer was added to the reaction at 0.4 µM. Prior to labeling, DNA fragments were purified by agarose gel electrophoresis.

Cell lines and cell culture

Epstein-Barr virus (EBV)-transformed lymphoblasts from control individuals (LCL 1425 and LCL 1497), PWS patients [LCL 863 and LCL 865: del (15) (q11.2q13.1), GM 09133 and GM 10184, obtained from NIGMS (Camden, NJ); LCL 1309: O family microdeletion, obtained from A. Beaudet (Baylor College of Medicine, Houston, TX); LCL 1514: maternal UPD (15), obtained from V. Lindgren (University of Chicago, Chicago, Il)] and AS patients [LCL 1101: del (15) (q11q13) mat transformed in our laboratory; LCL 1201: del (15) (q11q13) mat, GM 11404, obtained from NIGMS] were cultured in RPMI 1640 medium containing 10% fetal bovine serum (Gibco, Gaithersburg, MD), glutamine and antibiotics.

Cell permeabilization and nuclease hypersensitivity tests

DNase I and MspI in vivo hypersensitivity tests were performed according to the protocols published by Ymer and Jans (38) and Eberhart and Warren (37), with minor modifications. Per sample, 1 × 106 lymphoblasts of control, PWS or AS were centrifuged for 5 min at 15°C and 200 g. For DNase I tests, cells were resuspended in 90 µl of nuclear cell permeabilization buffer [NB; 60 mM KCl, 15 mM NaCl, 1 mM MgCl2, 0.1 mM EGTA, 15 mM Tris-HCl, pH 7.4, 0.5 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 0.3 M sucrose and 0.01% lysolecithin (Sigma, St Louis, MO)]. After 15 min incubation on ice, cell suspensions were transferred to a 25°C waterbath and 10 µl of NB containing 0 (negative control), 2, 5 or 10 U of DNase I (Boehringer Mannheim, Indianapolis, IN) were added. The nuclease digest was stopped by addition of 100 µl of stop solution (20 mM EDTA, 1% SDS, 0.5 mg/ml proteinase K) and samples were incubated overnight at 55°C. Subsequently, genomic DNA was extracted, following a phenol, phenol-chloroform and chloroform protocol and was precipitated with ethanol (65). After resuspension, restriction digests were performed with the appropriate restriction endonucleases and the DNA was reprecipitated prior to Southern analysis (65). Hybridization conditions used were as recommended by Amersham for Hybond N membranes.

In MspI nuclease hypersensitivity experiments, aliquots of growing cells were washed in phosphate-buffered saline (pH 7.4), then resuspended in 1 ml of permeabilization buffer [PB; 150 mM sucrose, 80 mM KCl, 35 mM HEPES (pH 7.4), 5 mM KPO4, 1 mM MgCl2, 0.5 mM CaCl2] and chilled on ice. Permeabilization was started by adding lysolecithin at 25 µg/ml. After 2 min, permeabilization was stopped by adding PB without lysolecithin in 3-fold excess and recentrifuging cells at 200 g at room temperature. Cells were resuspended in MspI reaction buffer (New England Biolabs, Beverley, MA) containing 0 (negative control), 10, 100 or 500 U of MspI. Digests were performed for 1 h at 37°C, followed by 10 min incubation at 65°C to inactivate the enzyme. Subsequently, samples were treated as for the DNase I experiments.

ACKNOWLEDGEMENTS

We thank J. Sutcliffe and A. Beaudet for cosmid clones spanning part of the PWS region, A. Beaudet and V. Lindgren for cell lines, E. Valero for transforming and maintaining LCLs, and J. Giacalone for sequencing data. We are grateful to Yvonne Franke for her helpful comments on this manuscript. The work was supported by the Howard Hughes Medical Institute (U.F. and J.S.) and by an NSF predoctoral scholarship to D.Z.

ABBREVIATIONS

AS, Angelman syndrome; IC, imprinting center; Igf2, insulin-like growth factor type 2; Igf2r, insulin-like growth factor type 2 receptor; ins2, insulin 2 gene; LCL, lymphoblastoid cell line; LCR, locus control region; N, nucleotide; NDN, necdin; PWS, Prader-Willi syndrome; SNRPN, small nuclear ribonucleoprotein polypeptide N; UPD, uniparental disomy; ZNF127, zinc finger protein 127.

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*To whom correspondence should be addressed. Tel: +1 650 725 8089; Fax: +1 650 725 8112; Email: francke@cmgm.stanford.edu

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