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. 2000 May 15;14(10):1186-95.

H19 and Igf2 monoallelic expression is regulated in two distinct ways by a shared cis acting regulatory region upstream of H19

M Srivastava  1 , S Hsieh, A Grinberg, L Williams-Simons, S P Huang, K Pfeifer
Affiliations

H19 and Igf2 monoallelic expression is regulated in two distinct ways by a shared cis acting regulatory region upstream of H19

M Srivastava et al. Genes Dev. .

Abstract

H19 and Igf2 are expressed in a monoallelic fashion from the maternal and paternal chromosomes, respectively. A region upstream of H19 has been shown to regulate such imprinted expression of both genes in cis. We have taken advantage of a loxP/cre recombinase-based strategy to delete this region in mice in a conditional manner to determine the temporal requirement of the upstream region in initiating and maintaining the imprinted expression of H19 and Igf2. Analysis of allele-specific expression of H19 and Igf2 and DNA methylation at the H19 promoter demonstrates that this region controls the monoallelic expression of the two genes in different ways, suggesting that it harbors two functionally distinct regulatory elements. Continued presence of the region is required to silence maternal Igf2 in accordance with its proposed role as an insulator. However, it does not have a direct role in keeping the paternal H19 promoter silenced. Instead, on the paternal chromosome, the upstream element mediates epigenetic modifications of the H19 promoter region during development, leading to transcriptional silencing of H19. Thereafter, its presence is redundant for preventing transcription. Presently, this temporal requirement of the silencing element appears to be a unique cis activity in the mammalian system. However, it is likely that other cis-acting elements, positive and negative, have the ability to effect stable changes in the chromatin structure and are not constantly required to give signals to the transcriptional machinery.

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Figures

Figure 1
Figure 1
Strategy for conditional deletion of the −7.0-kb to −0.8-kb region upstream of H19. (a) Targeting vector; (b) wild-type chromosome; (c) targeted chromosome, DMRfloxNeo; (d) targeted chromosome DMRflox after excision of the Neor selection marker; (e) the DMRΔ chromosome after cre recombinase-mediated excision at specific stages. (f,g) Southern hybridization of ES cell DNA to confirm the correctly targeted clones (DMRfloxNeo). Genomic DNA isolated from the ES cells was digested with XbaI (f) and hybridized to an EcoRI/XbaI fragment from the 5′ end of the targeted locus. Clones that have undergone homologous recombination (lane 2) yield a 9.5-kb band in addition to a 8.0-kb band derived from the endogenous non-targeted allele (lane 1). Similarly, BglII digests of the genomic DNA (g) hybridized to an XbaI/BglII fragment from the 3′ end of the targeted locus yield 11.5-kb and 10.2-kb bands from the wild-type (lane 1) and targeted alleles (lane 2), respectively, due to the presence of the additional BglII site at the −0.8-kb position. (h) DNA amplified from ES cell clones (DMRflox) correctly recombined by cre recombinase in vitro. The DMRflox allele was a result of cre recombinase-mediated excision of the Neo resistance gene. This was identified by amplifying the region around the −7.0-kb HindIII site (primers PrA and PrB). The endogenous locus (lane 1) gives a PCR product of 387 bp and the presence of the loxP site as a result of the correct excision event increases the size of this fragment to 520 bp. Presence of the other loxP site at −0.8-kb XbaI was confirmed by amplifying the region around it (Primers PrC and PrD). Wild-type (lane 3) and loxP carrying DNA samples yield PCR products of 177 and 234 bp, respectively. The correct clones carrying the DMRflox allele (lanes 2, 4) carry the loxP sites at both of these positions. (i) DNA amplified from genomic DNA of neonates (DMRΔ), in which DMR has been excised due to in vivo expression of cre recombinase under the control of different promoters (described in text). The excision was verified by PCR amplification (primers PrA and PrD) to give a 340-bp product. The authenticity of all of the PCR products was confirmed by sequencing. (j) Positions of the primers used for the PCR- based amplifications shown in h and i. Enhancers (circles), loxP sites (solid triangles), regions used as flanks (thick lines), transcription start site for H19 (arrow above H19 gene) and fragments used as probes (hatched lines above b) are shown. (DTA) Diptheria toxin A gene; (Bg) BglII; (H) HindIII; (R1) EcoRI; (X) XbaI; (M) DNA size markers.
Figure 2
Figure 2
SNuPE assays for H19 with control DNA templates. (a) DNA from castaneus (cas), domesticus (dom) and from castaneus × domesticus F1 (F1) mice. Castaneous DNA incorporates dGTP and domesticus DNA incorporates dATP. F1 DNA, representing a 1:1 mix of the castaneus and domesticus templates, incorporates both of the nucleotides. dATP incorporation in F1 is higher than dGTP incorporation, although, theoretically, it should be equivalent (see text). The experimentally observed relative incorporation of the two nucleotides by F1 DNA was used to derive a correction factor (see Materials and Methods) for dGTP incorporation. (b) Castaneus and domesticus DNA mixed in known proportions as indicated, amplified, and assayed by SNuPE. The expected values of A/(A+G) on the basis of relative concentrations of domesticus and castaneus DNA used in the mix are compared with those observed.
Figure 3
Figure 3
Effect of the DMR deletion mutations on H19 and Igf2 allele-specific expression. Skeletal muscle RNA samples were DNase treated, amplified for H19 and Igf2 by RT–PCR, and analyzed by SNuPE assays. (a,f) Demonstration of the efficiency of the assay for H19 and Igf2 respectively, by testing pure DNA templates, castaneus (cas), domesticus (dom), and DNA from castaneus × domesticus F1 mouse (F1), which represents a 1:1 mix of the templates. On the basis of a polymorphism between castaneus and domesticus alleles, the relative incorporation of the radiolabeled nucleotides dATP and dGTP during primer extension gives an estimate of relative abundance of the DNA from the two parental alleles. H19 expression was analyzed in neonates carrying a mutated paternal allele (b–e). Igf2 expression was analyzed in neonates carrying mutated maternal allele (g–j). Control littermates are either wild-type or DMRflox as indicated. The experimental chromosome (paternal for H19 and maternal for Igf2) is always domesticus, whereas the other chromosome is wild-type castaneus. For each RNA sample analyzed, RT-minus controls were used. These exhibited no amplification products, demonstrating the absence of any DNA contamination in the DNase treated RNA samples.
Figure 4
Figure 4
CpG methylation in the H19 promoter region as assayed by digestion with methylation-sensitive enzymes. (a) Restriction map of the region. Wild-type and DMRflox alleles are distinguished by the presence of a BglII site at −0.8-kb (top). Cre recombinase mediated excision of the DMR keeps the BglII site and also brings in juxtaposition a HindIII site (bottom). (Thick lines) The probes used for hybridization; (*) BglII site present only on the DMRflox and DMRΔ alleles; (**) HindIII site present only on DMRΔ allele; (arrow) transcription start site for H19. (b) Maternal and paternal inheritance of DMRΔG. DNA prepared from skeletal muscle of neonates inheriting DMRΔG either maternally or paternally was digested with BamHI and BglII. Subsequent hybridization with a 1.4-kb probe (see a) reveals 2.5- and 1.5-kb bands representing the wild-type and mutant chromosomes, respectively. Additional digestion with HpaII and HhaI was performed to monitor methylation. (c) Deletion of the DMR mediated by MCK-cre recombinase. DNA was prepared from neonates in which the DMRΔS mutation was generated on the maternal or paternal chromosome. Control DNA is from +/DMRflox mutants not carrying the MCK-cre transgene. Digestion with BamHI and HindIII and subsequent hybridization as above reveals three bands of 2.6, 2.5, and 1.6 kb representing DMRflox, wild-type, and DMRΔS alleles, respectively. Portions of the enzyme digest were also digested with HpaII and HhaI and methylation of the DMRΔS allele was monitored by the disappearance of the 1.6-kb band. (B) BamHI; (Bg) BglII; (H) HindIII; (Hh) HhaI; (Hp) HpaII, and (X) XbaI.
Figure 5
Figure 5
A model to explain H19 and Igf2 imprinting. Hypermethylation and other associated changes (close vertical bars) at the DMR on the paternal chromosome accomplish two functions. They inactivate the insulator function residing in the DMR to allow Igf2 promoter-enhancer interaction and also direct epigenetic modification (sparse vertical bars) of the H19 promoter region leading to its transcriptional silencing. The shared endodermal enhancers for H19 and Igf2 expression have been mapped from the +7- to +9-kb region downstream of H19 (Leighton et al. 1995). The enhancers for mesodermal expression of H19 are located downstream to +12 kb of H19 and on the basis of identical expression patterns of the two genes, are presumably shared by Igf2. (D) DMR; (E) enhancer.
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