Human Molecular Genetics, 2001, Vol. 10, No. 8 807-814
© 2001 Oxford University Press
Deletion of a nuclease-sensitive region between the Igf2 and H19 genes leads to Igf2 misregulation and increased adiposity
Howard Hughes Medical Institute and Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
Received 7 December 2000; Revised and Accepted 9 February 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The insulin-like growth factor 2 gene (Igf2) is imprinted in most somatic tissues of the mouse with the exception of the choroid plexus and leptomeninges of the brain, where it is expressed from both alleles. The imprinting of Igf2 is dependent upon an imprinting control region (ICR) that lies 90 kb 3' of the gene and acts as a chromatin insulator to block enhancers that lie further 3' on the chromosome. Based on this model we would expect that enhancers of brain-specific expression of Igf2 would lie 5' of the ICR, and thus be insensitive to its action. Here we describe a 12 kb deletion of a region 5' of the ICR that is hypersensitive to nuclease digestion in chromatin. Its deletion results in a biallelic decrease in expression of Igf2, but not H19, in the brain, consistent with the proposal that it encodes a positive regulatory element. In addition, the deletion results in a minor relaxation of Igf2 imprinting in skeletal muscle and tongue. Lastly, the reduction in IGFII expression in the adult is accompanied by increased fat deposition and occasional obesity. Overweight animals are hypophagic, suggesting that IGFII affects fat metabolism rather than feeding behavior in adult mice.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The distal region of mouse chromosome 7 harbors one of the most intensively studied imprinted regions in the mammalian genome. Three of the genes within the cluster, the maternally expressed H19 gene and the paternally expressed growth factors, Igf2 and Ins2, are controlled by a 2 kb imprinting control region (ICR) that is located just 5' of the H19 gene (1–3). The ICR exists in two epigenetic states in chromatin: it is methylated and resistant to nuclease digestion on the paternal chromosome and unmethylated and hypersensitive to digestion on the maternal chromosome (4–10). Various deletions of the ICR in mice have been instrumental in demonstrating that the region is required on the paternal chromosome to establish, but not to maintain the DNA methylation that silences the H19 gene. On the maternal chromosome the ICR is required both to establish and maintain the silence of the Igf2 and Ins2 genes (1–3). It is thought to be the site of assembly of a chromatin insulator that blocks the interaction of Igf2 with enhancers that lie 3' of the ICR (11–14). The insulator activity of the ICR in vitro requires the binding of CTCF, a zinc finger protein that has been implicated in the function of multiple vertebrate insulators (15).
Insulators are regulatory elements that lie between a gene and its enhancers and interfere with enhancer–promoter interaction in a position-dependent manner (16–18). One prediction of the insulator model for ICR function is that all the enhancers required for the imprinted expression of Igf2 will lie 3' of the ICR. Consistent with this expectation, both endodermal and mesodermal enhancers have been identified 3' of the H19 gene (13,19–21). However, Igf2 is not imprinted in two cell types in the brain, the choroid plexus and leptomeninges, tissues in which H19 maintains its imprint (22,23). Furthermore, the developmental regulation of the genes is dissociated, as the biallelic expression of Igf2 persists in the adult whereas H19 is down-regulated after birth. Regulatory elements required for Igf2 expression in the brain would be expected to lie 5' of the ICR, and escape its influence.
An effective strategy for identifying enhancers is to search for sequences that are hypersensitive to nucleases in chromatin. Nuclease hypersensitivity has been associated with a variety of cis-acting regulatory elements, including promoters, enhancers and chromatin insulators. Koide et al. (24) identified a region upstream of the ICR that contained three biallelic DNaseI hypersensitive sites (Fig. 1A). The hypersensitive region was unmethylated but was embedded in a region of DNA that was methylated on both chromosomes. When a 2 kb fragment within this region was tested in a transgenic assay, it exhibited enhancer activity in the brain (25). On the other hand, a small 1 kb deletion of the region within the context of a large transgene had no apparent effect on Igf2 expression in the brain (26). To resolve the function of the hypersensitive sites in the biallelic expression of Igf2, we generated a 12 kb deletion of the hypersensitive sites that included the methylated sequences on either side. We report that in the choroid plexus, Igf2 mRNA was reduced upon both maternal and paternal transmission of the mutation, consistent with the presence of a positive regulatory element for Igf2 at the site. In addition the deletion led to a modest relaxation of Igf2 imprinting exclusively in muscle. Finally, the deletion of the hypersensitive sites was accompanied by a consistent increase in adult body weight in both heterozygous and homozygous animals. The weight gain was attributed to increased fat deposition, and occasionally resulted in obesity. Surprisingly, animals were hypophagic, indicating that fat accumulation results from changes in caloric utilization, and/or partitioning, rather than increased food intake.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation of the HS
mutation in miceWe designed a targeting vector to delete a 12 kb region that encompassed the hypersensitive sites and 8.2 and 1.8 kb of heavily methylated DNA 5' and 3' of the hypersensitive sites, respectively (–44 to –32 kb relative to the start of H19 transcription) (Fig. 1A). Following electroporation and neomycin selection of ES cells, two out of 522 clones were correctly targeted, as shown by digestion of genomic DNA with BamHI, EcoRI and HindIII, followed by Southern blot analysis using the probes shown in Figure 1B. From these a mutant line of mice, designated HS
, was established by standard methods. Crosses of heterozygous HS males and females generated viable homozygotes at the expected Mendelian ratio.
Equivalent hypersensitivity of the intergenic region on the two parental chromosomes
The report that the intergenic hypersensitive (HS) region was equivalently hypersensitive on the two parental chromosomes rested on a comparison between normal and MatDi7 embryonic tissue and not a direct comparison of the maternal and paternal chromosomes (24). Furthermore it was possible that the sites were embryo-specific and not relevant to expression in the adult brain. To resolve these issues, we digested nuclei isolated from maternal and paternal HS heterozygous adult brains with DNase I, and used a probe within the deletion to test for digestion. As shown in Figure 2, these sites were detected in brain, with both chromosomes displaying equal sensitivity to DNase I. Similar results were obtained with adult liver nuclei (data not shown). Thus the sites show no parental bias or tissue or temporal specificity.
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HS
reduces Igf2 expression in the choroid plexusTo test the effect of the deletion on Igf2 and H19 expression, we crossed HS
heterozygotes to CAST/Ei mice so that expression on the two parental chromosomes could be independently assessed using allele-specific assays. Maternal transmission of the HS chromosome resulted in a 70% decrease in total brain Igf2 mRNA, whereas paternal transmission resulted in a 45% reduction (Fig. 3A). This finding was confirmed using RNase protection analysis of RNA prepared from choroid plexus tissue, one of the two major sites of Igf2 expression in the adult brain (data not shown). When total Igf2 mRNA levels were measured by RNase protection in brain RNAs from offspring of HS heterozygotes crossed to either C57Bl/6 or 129/Sv mice, a consistent reduction of 70% was detected from the mutant allele irrespective of parental inheritance. Thus the HS region is required on both chromosomes for full expression of Igf2 in the brain.
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The reduction in Igf2 expression in the choroid plexus could result from the deletion of an essential transcriptional regulatory element or from transcriptional interference from the PGK promoter that is directing expression of the neomycin (neo) cassette. To assess the latter, we examined the expression of the neo gene and found no neo transcripts emanating from the HS
in any neonatal or adult organs examined, including the brain (data not shown). This finding is in contrast to the levels of neo transcripts detected in two other mutations at the locus, an intragenic Igf2 deletion generated by De Chiara et al. (27) and H1913, in which the entire H19 gene and its flank is replaced with neo (1) (data not shown). While transcriptional interference by the PGK promoter does not appear to underlie the reduction in Igf2 expression, it remains a formal possibility that retention of the neo gene has resulted in changes in chromatin structure that affect the expression of Igf2.
Brain H19 RNA, which is imprinted in both the choroid plexus and leptomeninges, was generally unaffected by maternal inheritance of HS. However, we occasionally observed elevated levels that were 5- to 15-fold higher than those in wild-type littermates (Fig. 3B), yet all transcripts were maternal in origin (data not shown). Upon paternal inheritance of the HS deletion, the linked H19 gene was silent, indicating that its imprinting was not affected by the mutation.
The effect of inheritance of HS on imprinted Igf2 and H19 expression
We also assessed the consequences of maternal inheritance of HS in the tissues that normally display imprinting of Igf2 and H19. Upon maternal inheritance we observed a partial relaxation of Igf2 imprinting in skeletal muscle and tongue, but not in other organs with significant mesodermal contribution such as the heart and kidney (Fig. 4A). No change in Igf2 silencing was detected in predominantly endodermal tissues such as the liver and gut. A similar finding was recently reported by Ainscough et al. (26) using a 130 kb YAC transgene containing the locus. Quantitation by both allele-specific RNase protection and northern analysis indicated that the level of maternal Igf2 mRNA in HS skeletal muscle and tongue was 
20% of the level of normal paternal expression. This relaxation is significantly less than the complete relaxation of Igf2 silencing observed in H1913 mice in skeletal muscle (Fig. 4A, lane H1913). Post-natal silencing of Igf2 appeared normal as Igf2 mRNA was undetectable in skeletal muscle, tongue, liver, heart, kidney and gut in both wild-type and mutant animals by 4 weeks post-partum (data not shown).
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To investigate the possibility that the relaxation of Igf2 imprinting in muscle was caused by changes at the H19 locus, we analyzed H19 RNA in animals that had inherited HS
maternally. No change was observed in either liver or muscle (Fig. 4B). Similarly, no change was detected in the unmethylated status of the maternal H19 promoter or the ICR in its 5' flank (data not shown). These data suggest that Igf2 mRNA expression in muscle has partially escaped imprinting by an H19-independent mechanism.
Lastly, we tested whether the HS mutation affected the ability of the chromosome to reset in the female germline from a male ‘epigenotype’ to a female epigenotype by comparing the progeny of HS females who had inherited the mutant chromosome from either mothers or fathers. No differences in the expression or imprinting relaxation of Igf2 and H19 were detected (data not shown).
No change in the expression of H19 and Igf2 was detected upon paternal inheritance of HS in any neonatal tissues examined other than brain. This finding was independent of the grandparental origin of the mutant allele, demonstrating that the ability to properly reset the female-specific imprint through the male germline was also unaffected by the HS mutation.
Obesity in HS adults
HS heterozygotes were normal-sized at birth until 6 weeks of age (heterozygote/wild-type weight = 0.99, n = 38 heterozygotes and 37 wild-type animals in 15 litters; P = 0.25). However, a small subset of adult HS heterozygotes became obese with age in initial crosses of F1 and F2 offspring of the original 129/Ola chimeric mice that had been crossed with C57BL/6. The increase in weight was dramatic in the affected animals, with males and females weighing between 50 and 60 g compared with the normal adult body weight of 25–30 g. The onset of the increase in body mass was detected as early as 6.5 weeks and as late as 6 months after birth. Obese mice contained large deposits of fat in the abdominal cavity as well as enlargement of the fat pads surrounding the kidneys in both sexes, the ovarian fat pads in females and the epididymal fat pads in males.
Upon crossing the mutation onto a 129/Sv or C57Bl/6 background, the sporadic obesity decreased from ~5 to 10% of progeny to <1%, suggesting that the 129/Ola background may enhance the penetrance of this phenotype. Nevertheless, among adults derived from crosses with either C57Bl/6 or 129/Sv mates, HS heterozygous males and females were 4–6% heavier on average than their control littermates (Table 1), a difference that was only apparent after 6 weeks post-natal. Homozygous progeny were 7.3% heavier on average than wild-type littermates (Table 1).
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An increase in body weight due to proportional somatic overgrowth has been observed in H19
13 maternal heterozygotes that exhibited deregulation of Igf2 imprinting in all fetal tissues (1). To determine whether the weight gain in HS heterozygotes was proportional or the result of a specific increase in adipose tissue, we weighed the perirenal and perigonadal fat pads of wild-type and homozygous mutants. The perigonadal fat pads were 41% larger in homozygous males and females relative to wild-type littermates and the mesenteric fat pads were 2-fold heavier. Furthermore, when the weights of adipose tissue were normalized to the total body weight of the animal, the increase was still evident in both males and females (Table 1). This increase in adiposity is the most likely explanation for the weight gain, as proportional somatic overgrowth is not detected. To determine whether the increased adiposity was due to increased food intake, the amount of food consumed over a 10 day period was measured in wild-type, heterozygous and homozygous sex-matched littermates. Heterozygotes consumed 7 ± 2.8% less food (measured as mg/gm body weight/day; n = 5 wild-type and 13 heterozygotes; P = 0.015) whereas homozygotes consumed 17 ± 5.4% less food than wild-type controls (n = 3 wild-type and four homozygotes; P = 0.026). These results suggest that the obesity resulted from a change in energy homeostasis rather than over-eating.
Expression of Igf2 and Ins2 in adult mice
In adult mice, Igf2 mRNA is repressed in all tissues except the brain (27). The increased fat deposition in adult HS mice could be explained by a reduced level of IGFII, which is produced in the brain, as IGFII has been shown to cross the blood–brain barrier (28). However, to test the possibility that the phenotype could be due to misregulation of Igf2 in adipose tissue, the expression of Igf2 mRNA was quantitated in white adipose tissue of obese and non-obese HS maternal heterozygotes as well as wild-type controls by RNase protection. Equivalent trace levels of Igf2 mRNA were detected in all samples (data not shown). Thus the only organ that we have examined that displays a reduction in Igf2 mRNA production is the brain.
An alternative candidate for a gene whose misregulation could lead to obesity is the Insulin-2 (Ins2) gene, located 15 kb 5' of Igf2 (Fig. 1A). Insulin plays an important role in regulating body composition and is known to respond to metabolic dysregulation of body fat composition. It is imprinted in the visceral endoderm of the yolk sac during development, but not in the adult pancreas (29). We examined pancreatic Ins2 mRNA levels in HS homozygotes and wild-type littermates. To discriminate between an indirect change in Ins2 expression in response to the obese state versus a change elicited by the HS mutation, Ins2 mRNA levels in pancreatic RNA were measured relative to Ins1 mRNA. Ins1 maps to a different chromosome and should therefore be insensitive to the HS mutation. No change in the levels of Ins2 mRNA was detected (Fig. 5), ruling out a major change in insulin expression as the explanation for the increased body weight or sporadic obesity in HS.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The intergenic region affects Igf2 expression in brain and muscle
Our functional analysis of the hypersensitive sites in the intergenic region between the H19 and Igf2 genes was stimulated by a desire to reconcile the biallelic expression of Igf2 in the brain with the insulator activity of the ICR. The consequences of deleting this region in the genome for the Igf2 gene were restricted to two tissues: the choroid plexus where expression was reduced, and muscle where maternal imprinting was partially relaxed.
Our findings are consistent with the proposal of Ward et al. (25) that the intergenic hypersensitive region contains at least one enhancer required for Igf2 expression in choroid plexus. Furthermore, the enhancer shows little, if any, allele specificity, as its removal affects expression of both alleles. The precise position of the enhancer is unknown, but it presumably lies outside the 1 kb region deleted by Ainscough et al. (26), as they reported no effect of that deletion on expression of Igf2–lacZ in the meninges, as assessed by lacZ staining. The deletion does not completely eliminate expression of Igf2 in the brain, suggesting that there are probably additional regulatory elements required for full Igf2 expression in that organ.
As expected, H19 expression is largely unaffected in the brain by the HS deletion, presumably because it is insulated from the element by the ICR on the maternal chromosome, and its methylation on the paternal chromosome inhibits its expression. We occasionally detected an increased level of H19 RNA in neonatal brain exclusively in HS heterozygotes, which may reflect variable regulation of its repression after birth as an epigenetic response to the mutation. In any case, no decrease in expression was observed, implying that the brain-specific expression of H19 relies on separate enhancers. Indeed, an H19 transgene spanning sequences from –4 to +12 kb relative to the start of transcription is expressed in the brain, where its expression was confined to the choroid plexus (30).
We propose that the biallelic expression of Igf2 in the brain can be partly explained by the location of a critical enhancer 5' of the insulator at the ICR. Webber et al. (31), who integrated the 3' H19 endodermal enhancers 5' of the region deleted in HS, were the first to demonstrate that the position of an enhancer relative to the ICR is a critical determinant for imprinting. With the endodermal enhancers located 5' of the ICR, Igf2 was expressed from the maternal chromosome in liver. More recently, Kaffer et al. (13) moved the ICR to a position between the endodermal and mesodermal enhancer domains 3' of the H19 gene and showed that upon maternal inheritance H19 was biallelic in liver, an endodermal tissue, but silent in muscle. Thus the ICR can function in an ectopic site to insulate enhancers in a position-dependent manner.
The second effect of the HS mutation was a skeletal muscle-specific relaxation of Igf2 imprinting on the maternal chromosome. Tissue-specific relaxation of maternal Igf2 imprinting in muscle has now been observed in four mutations generated within the H19–Igf2 locus. Two deletions of the H19 gene and its promoter displayed modest muscle-specific relaxation of Igf2 imprinting on the maternal chromosome, although imprinting in endodermal tissues such as liver and gut was unaffected (32,33). More recently, a deletion of a differentially methylated region (DMR1) at the 5' end of the Igf2 gene had the same consequence; i.e. relaxation of Igf2 imprinting exclusively in tissues containing mesodermal cells (34). Interestingly, in contrast to the two H19 deletions, in which there was a 5–8% increase in birth weight in response to the increased production of maternal Igf2 mRNA, no increase in birth weight was observed in HS or DMR1 maternal heterozygotes. The explanation for the phenotypic differences between these mutants is unknown, but it is likely to reflect differences in the spatial extent and/or temporal expression of the misregulated maternal Igf2.
The detection of tissue-specific loss of Igf2 imprinting in mesoderm in four separate mutations at the locus is striking. It is conceivable that imprinting in some mesodermal tissues is regulated by a more complex set of cis-acting elements than in endodermal H1913 tissues, where imprinting is sustained in all four mutants. Alternatively, it may be that none of the regions that have been deleted has a direct role in the regulation of imprinting. Rather, these tissues may be particularly sensitive to changes in this region, possibly through effects at the level of chromatin structure. Thus, it may be that the large distances over which the interactions between the mesoderm-specific enhancers and the Igf2 promoter must work place structural constraints on the insulator that are not present in endoderm because of the closer proximity of the enhancers. This issue will require more precise identification of all the enhancers that regulate Igf2 for its resolution.
The finding that the DNase I hypersensitive sites exist in both brain and liver raises the possibility that this region, which is conserved in mice and humans (26), might participate in maintaining biallelic IGF2 expression throughout adulthood in the human brain and liver. Biallelic expression of IGF2 in adult human liver is driven by the P1 promoter, which is absent in rodents (35). Additional enhancers responsible for expression of IGF2 in adult liver have not been defined but would be predicted to lie 5' of the ICR.
Decreased Igf2 mRNA in adult brain leads to increased fat deposition
The most dramatic effect of the HS deletion was adult-onset obesity. As the DNase I hypersensitve sites are not brain-specific and the deletion spanned 12 kb, we considered the possibility that the obesity might have arisen from the deletion of an unidentified gene, or regulatory sequences for such a transcript, that was involved in fat metabolism. However, extensive northern blot analysis using sequences between H19 and Igf2 as probes has yet to uncover additional transcripts (data not shown), consistent with an earlier study (24). Finally, no ESTs have been identified in this region in either human or mouse, leaving the misregulation of Igf2 as the most likely explanation for the mild obesity observed in the HS mice.
The peptide hormone IGFII has been primarily implicated in the regulation of prenatal growth. Mice that are lacking the Igf2 gene as the consequence of a targeted mutation are 60% smaller than wild-type, and mice that over-produce IGFII during development as the result of a variable loss of imprinting in all expressing tissues except the brain show proportional somatic overgrowth (1,27). However Da Costa et al. (36) reported that mice carrying an Igf2 transgene that was expressed in adult skin exhibited a reduced amount of body fat, implying that IGFII expression in the adult might affect energy metabolism. Similarly, a polymorphic variant of IGFII in humans that decreases circulating levels by ~10% is associated with increased adiposity (37). In pigs an imprinted quantitative trait locus with major effect on muscle mass and fat deposition maps to the IGF2 locus (38). Consistent with these findings, our results imply that the increase in adiposity in HS mice is brought about by a decrease in transcription of Igf2 in brain, the major source of circulating IGFII in adult mice. In contrast, it is difficult to conceive that the gain of Igf2 expression in mesoderm could explain the increased fat deposition, as the three other mutations with Igf2 misregulation in mesoderm do not show this effect (32–34).
It has been suggested that the link between IGFII levels and body composition might act through regulation of feeding behavior (39,40). In humans and rats, IGF2 expression has been observed in the hypothalamus in addition to the choroid plexus, and it has been proposed that IGFII acts either in a manner analogous to, or downstream of, insulin in mediating feeding behavior by decreasing levels of neuropeptide Y (41–43). A brain-specific deletion of the insulin receptor, which can bind IGFII, results in mild obesity similar to that observed in HS homozygotes, and was accompanied by hyperphagia (44). In contrast, both heterozygous and homozygous HS mice exhibited hypophagia, leading us to conclude that IGFII may have a unique role in the development of obesity, for example, by affecting the efficiency of caloric utilization and/or partitioning into adipose tissue.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Construction of targeting vector and generation of targeted mice
The 5' arm of the targeting vector was a 3.8 kb SalI fragment obtained from a 129/Sv library (Stratagene), encompassing sequences from –47.8 to –44 kb relative to the transcriptional start of H19. The 3' arm was a 3.5 kb BamHI–EcoRI fragment spanning sequences from –32 to –28.5 kb. These fragments were cloned on opposite sides of the selectable marker cassette containing the neo resistance and thymidine kinase genes (45). The negative selectable marker, diphtheria toxin A chain from pDT101, was cloned immediately 5' to the 3.8 kb arm (46). The targeting vector was linearized with NotI and 20 µg was electroporated into E14.1 cells. After 7 days of selection with G418 (200 µg/ml active), colonies were isolated and expanded in 96-well tissue culture dishes.
Southern blots were used to identify correctly targeted clones. DNA (5–10 µg) was digested with BamHI and fractionated on 0.8 or 1.0% agarose gels in 1x TAE. The DNA was transferred to either Hybond N+ (Amersham) or Zetabind (Cuno) in 0.4 M NaOH. Hybridizations using an external 1.8 kb EcoRI fragment as hybridization probe were carried out in Church buffer at 65°C overnight and washed at 65°C in 0.1x SSC, 0.1% SDS for 20–40 min. Correctly targeted clones were used to produce chimeric mice by standard methods. Chimeric founders were mated to C57Bl/6 mates to establish the line of HS mice.
Genotyping
HS heterozygotes were genotyped by PCR using primers specific for the lox site (5'-GAGGAGTTTACGTCCAGCC-3'), and the PGK promoter of the neo gene (5'-CTTGTGTTAGCGCCAAGTGC-3'). Reactions (20 µl) contained 20 ng of each primer, 100 ng of DNA, 1x PCR buffer (PEC), 0.25 mM dNTPs, 1.5 mM MgCl2, 12% sucrose and 0.02 mM cresol red, and were amplified using 35 cycles of 94°C for 30 s, 55°C for 60 s and 72°C for 60 s in a PEC 9600 thermocycler. Homozygotes were genotyped by Southern blot analysis using 10 µg of EcoRI or EcoRI–HindIII digested DNA and hybridization probes 3 and 4 shown in Figure 1. Probe 3 is a 680 bp fragment containing a portion of the coding region of the neo gene and probe 4 is a 1.9 kb fragment containing the hypersensitive region.
RNA analysis
RNAs were prepared using Trizol (Sigma). All RNase protection assays were carried out using the RPAII kit from Ambion. The assays used to determine Igf2 allele-specific transcripts, H19, rpL32 and Ins levels were performed as described previously (1,29,45,47). For northern analysis, RNAs were fractionated on 1% agarose gels containing 1x MOPS buffer and 0.52% formaldehyde, transferred to Hybond N+ membranes in 10x SSC, followed by hybridization in Church buffer at 65°C overnight and washed at 65°C in 0.1x SSC, 0.1% SDS for 20–40 min. Blots were stripped at 100°C using 0.1x SSC, 0.1% SDS for 10 min.
DNase I hypersensitivity analysis
Nuclei were isolated from adult tissues as described by Bartolomei et al. (4) and washed once in 5 ml RSB (50 mM Tris pH 8.0, 100 mM NaCl, 3 mM MgCl2, 0.1 mM PMSF, 5 mM sodium butyrate), resuspended in RSB containing 0.1 mM CaCl2 and 1 µg/ml DNase I (Worthington) and incubated at 37°C for the times indicated.
Analysis of body weight, adiposity and food intake
Mice were housed in standard conditions with a 14 h light/10 h dark cycle. Water and standard mouse chow were available ad libitum. For comparisons of body weight, size of fat pads and food intake, all mutant animals were compared to sex-matched littermates. Measurements of body length were done by measuring the distance the from the midline point between the ears to the tail. Fat pads were excised and weighed. The food intake assay utilized Precision dustless food pellets (BioServ). Food was weighed and placed in cages containing single mice. Both the mice and the amount of food consumed were weighed four times over the course of 10 days. The weight of each mouse and the amount of food consumed per 24 h was averaged and used to calculate the amount of food consumed per gram of body weight per 24 h interval. All statistical analyses were done using the Student’s t-test to compare the body weights, fat pad weights, body lengths and food intake of wild-type and mutants.
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ACKNOWLEDGEMENTS |
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The authors thank Robert S. Ingram for providing the cloned intergenic DNA. The work was supported by a grant from the NIGMS. B.K.J. was supported by an NRSA postdoctoral fellowship from the NIH and S.M.T. is an Investigator of the Howard Hughes Medical Institute.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 609 259 2900; Fax: +1 609 258 3345; Email: stilghman@molbio.princeton.edu
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REFERENCES |
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