The reciprocally imprinted H19 and Igf2 genes form a co-ordinately regulated 130 kb unit in the mouse controlled by widely dispersed enhancers, epigenetically modified silencers and an imprinting control region (ICR). Comparative human and mouse genomic sequencing between H19 and Igf2 revealed two novel regions of strong homology upstream of the ICR termed H19 upstream conserved regions (HUCs). Mouse HUC1 and HUC2 act as potent enhancers capable of driving expression of an H19 reporter gene in a range of mesodermal tissues. Intriguingly, the HUC sequences are also transcribed bi-allelically in mouse and human, but their expression pattern in neural and endodermal tissues in day 13.5 embryos is distinct from their enhancer function. The location of the HUC mesodermal enhancers upstream of the ICR and H19, and their capacity for interaction with both H19 and Igf2 requires critical re-evaluation of the cis-regulation of imprinted gene expression of H19 and Igf2 in a range of mesodermal tissues. We propose that these novel sequences interact with the ICR at H19 and the epigenetically regulated silencer at differentially methylated region 1 (DMR1) of Igf2.

The imprinted H19-Igf2 locus is subject to complex regulation involving a differentially methylated cis-acting silencer, an imprinting control region (ICR), comprising both boundary/insulator and silencer functions, enhancers downstream of H19, and other transcriptional units of unknown function (Fig. 1A) (Arney et al., 2001; Onyango et al., 2000; Surani, 1998). The cis-elements mediate expression of H19 as well as regulating access to shared enhancers by the paternal Igf2 and maternal H19 alleles (Leighton et al., 1995). Indeed, the H19 transcriptional unit is dispensable for correct imprinting at the locus (Ripoche et al., 1997). The differentially methylated ICR proximate to H19 and silencers both upstream of Igf2 and within the intergenic region are the known key regulatory regions at this locus (Ainscough et al., 2000a; Ainscough et al., 2000b; Constancia et al., 2000; Drewell et al., 2000; Srivastava et al., 2000; Thorvaldsen et al., 1998). The ICR upstream of H19 itself is a complex multipartite regulatory region. On the unmethylated maternal chromosome, the region acts as a boundary/insulator, recruiting the CTCF protein to block access to Igf2 by enhancers located downstream of H19 and thus allowing H19 expression (Bell and Felsenfeld, 2000; Hark et al., 2000; Kanduri et al., 2000). On the methylated paternal allele, a silencer element present in the ICR acts to silence H19 in specific tissues (Brenton et al., 1999; Drewell et al., 2000). Methylation of the ICR prevents CTCF binding and formation of the boundary, allowing the downstream enhancers free access to the Igf2 promoter.

Unlike the H19 gene, which is differentially methylated at the promoter, both parental alleles of Igf2 are unmethylated at the promoter and potentially active (Sasaki et al., 1992). To combat this, both the boundary element upstream of H19 and a silencer element located at differentially methylated region 1 (DMR1) upstream of Igf2 acts to prevent transcription of Igf2 from the maternal allele in specific tissues (Constancia et al., 2000). How precisely the complex regulation of the two genes is achieved by distant enhancers and a variety of cis-regulatory elements remains to be fully elucidated. In this study, we have identified two novel regulatory elements upstream of the ICR, which act as strong mesodermal enhancers in specific tissues. They are also bi-allelically transcribed. The location of these elements between H19 and Igf2 is unexpected and they, in concert with other cis-elements at the locus, may drive expression of H19 and Igf2 in a variety of mesodermal tissues.

RNA isolation and RT-PCR

RNA was isolated from mouse embryos and organs using Trizol™ (Life Technologies) according to the manufacturer’s instructions. RT-PCR was performed using the Qiagen One-Step RT-PCR kit, or as previously described (Ainscough et al., 1997). PCR products were electrophoresed, blotted onto Hybond N+ membranes (Amersham Pharmacia Biotech) and hybridised to α32P-dCTP labelled probes, according to standard procedures.

PCR primer sequences were as follows:

mouse HUC1S 5′-ATCCTGCTGGTATCCTGAGG-3′;

mouse HUC1AS 5′-ACTTATGCGTTCAGTCACTTCC-3′;

mouse HUC2S 5′-AAGAGAATGGACAGGACCCAGG-3′;

mouse HUC2AS 5′-CATTCAAAAGGGAACAAGGGC-3′;

human HUC2S 5′-AGGGGAATGGACAGGGCCCAGG-3′; and

human HUC2AS 5′-GATTCAAGAGGGAGCGAGGGC-3′.

To determine allelic expression of HUC 2, RT-PCR products were purified using GeneClean™ (Bio 101) and digested to completion with either Tru9I (mHUC2) or DdeI (hHUC2).

In situ hybridisation analysis

Sense and antisense riboprobes (relative to the direction of H19 transcription) were prepared from a 1865 bp clone containing HUC1, HUC2 and the intervening sequence by in vitro transcription using a DIG RNA labelling kit (Boehringer Mannheim). An antisense probe spanning the H19 transcriptional unit (Drewell et al., 2000) was used as a control. Sagittal sections (8 μm) from 13.5 days post coitum (dpc) mouse embryos were used for in situ hybridisation, essentially as described previously (Wilkinson and Nieto, 1993). Sections were counterstained with Eosin.

Cell transfection assay

Regions were amplified by PCR and cloned into the pGL3-Promoter vector (Promega), upstream of an SV40 promoter driving a firefly luciferase reporter gene, and sequenced to confirm integrity and orientation. PCR primers for HUC1 and HUC2 are described above. Other PCR primers were as follows:

DMDS 5′-TGCCTACAGTTCCCGAATCACC-3′;

DMDAS 5′-CGGCATCGTCTGTCCATTTAGC-3′;

Downstream enhancers S 5′-ATCATTACATCCTGGTGCCTCC-3′; and

Downstream enhancers AS 5′-TAGGCAGTTGGATGATGGCACC-3′.

The HUC1+2 construct was obtained by using the HUC1S and HUC2AS primers. The 1+2Δ construct was obtained by ApaI digestion of the 1+2 construct followed by re-ligation, removing 1.1kb of sequence between HUC1 and HUC2.

DNA constructs were transfected into HeLa cells, cultured for 20 hours, lysed and luciferase readings assayed. Firefly luciferase values were normalised against a co-transfected Renilla luciferase reporter gene driven by a Thymidine Kinase (TK-Renilla) promoter, as described in the DLR assay protocol (Promega). Each construct was tested in triplicate in each experiment, and the experiment was repeated. The H19 differentially methylated domain was tested both unmethylated (DMD), and after in vitro methylation by SssI (DMDm). Cells transfected with TK-Renilla alone demonstrated no firefly luciferase activity (negative control).

Mouse transgenic assay

The region encompassing HUC1 and HUC2 was amplified using the following primers:

tgHUCS 5′- CAGGCAGTCAGTCATCTCAGCC-3′; and

tgHUCAS 5′- GCATTCAAAAGGGAACAAGGGC-3′.

SpeI sites were incorporated into the primers allowing this fragment to be cloned upstream of the H19 promoter (–816 bp to +5 bp relative to the transcriptional start site) and a PLAP reporter gene (see Fig. 5A). This reporter was generated by XbaI excision of the H19-PLAP transgene described by Brenton et al. (Brenton et al., 1999), followed by cloning into pBluescript. Vector sequences were removed and transgene injection, embryo recovery, subsequent fixation and staining of embryos performed as described in Brenton et al. (Brenton et al., 1999). Embryos were removed at 13.5 days following oviduct transfer and bisected to allow penetration of stain. DNA was prepared from yolk sacs using standard procedures and used for PCR genotyping of the embryos.

PCR primers for genotyping were as follows:

PLAPS 5′- TTGGTTGACAGAGTAGGGGC-3′; and

PLAPAS 5′- GAGCAAAGATCAGGTCAGCC-3′

Identification of conserved regions upstream of human and mouse H19.

To identify additional cis-regulatory elements involved in the control of imprinted gene expression of H19 and Igf2, we used genomic DNA sequence upstream of the mouse H19 gene from both published sequence (AF049091) and from sequencing we performed ourselves. This sequence extended from the transcriptional start site of H19 to approximately 11.5 kb upstream (GenBank Accession Number, AF327412) (Fig. 1A). Using Nucleotide Identification X (NIX, HGMP Resource Centre, UK) sequence analysis, we identified two novel sequences upstream of H19, which we have termed HUC1 and HUC2 (H19 Upstream Conserved). They exhibit very strong homology to genomic sequence from a human P1 artificial chromosome (PAC) clone (AC004556) (Fig. 1B). The PAC clone contains the equivalent H19 upstream region and transcription unit.

The conserved sequences revealed similar genomic organisation in the same orientation upstream of the H19 gene. They were approximately 400 bp each in size and were separated by approximately 1.4 kb of unconserved sequence (Fig. 1B,C). These conserved sequences do not contain any recognisable repetitive elements and have no homology to any other sequences in the human or mouse genome databases. HUC1 and HUC2 therefore represent two novel DNA sequence blocks, which are conserved at the H19 upstream region in mice and humans. Cross-species Southern hybridisation analysis also revealed the presence of the HUC sequences in other mammals, including rat, dog and muntjac deer (data not shown). The HUCs are relatively poor in CpG content and are therefore unlikely to be a target for regulation by DNA methylation. We failed to detect any significant level of CpG methylation on either parental chromosome when analysed by methylation-sensitive restriction enzyme digestion (data not shown).

HUC sequences are bi-allelically transcribed

As highly conserved sequences are potentially indicative of transcribed regions, we examined whether the HUC regions are transcribed sequences. We initially examined expression from HUC1 and HUC2 using RT-PCR in 13.5 days post coitum (dpc) whole mouse embryos and detected transcription from these sequences using primers internal to mouse Huc1 and Huc2 (Fig. 2A,B). However, we did not detect a message with primers spanning the two conserved regions, indicating they are not part of the same transcript (Fig. 2B). We also attempted RT-PCR with primers from the Huc sequences and exon 1 of H19 and again failed to detect a linked transcript (data not shown). This observation suggests that mouse Huc1 and Huc2 represented two separate novel RNAs. We also examined expression by Northern hybridisation analysis in 11.5 dpc and 13.5 dpc whole embryos, where we detect relatively weak low molecular weight bands (data not shown), indicating that the mouse Huc transcripts are not part of a larger abundant mRNA.

RNA in situ hybridisation analysis of 13.5 dpc embryos revealed specific expression from the HUC sequences in a number of tissues, including forebrain and midbrain, developing ear, limbs, liver, lungs and the genital eminence (Fig. 2C,D). Comparison with control embryonic sections hybridised to a H19 probe showed that Huc (Fig. 2C,D) and H19 (Fig. 2E) transcription was neither mutually exclusive, nor shared in all tissues. The direction of the probe used indicated that transcription was orientated in the same direction as the H19 transcript. The probe in the opposite direction detected no transcripts from the HUC region (Fig. 2F). Detailed analysis of Huc expression revealed that the pattern was consistently restricted to the telencephalon and choroid plexus in the forebrain and the cerebellar primordium in the midbrain (Fig. 2G), the cochlea, olfactory epithelium, muscles of the tongue (Fig. 2H) and the genital tubercle (Fig. 2I).

As the HUC regions lie within an imprinted domain, we also tested whether the mouse Huc transcripts were generated from only one of the parental chromosomes. Using a restriction polymorphism between 129/Sv and M. spretus mouse strains identified in the most highly conserved region, mouse Huc2, we performed RT-PCR analysis on RNA isolated from 13.5 dpc whole embryos (Fig. 3A). This showed that mouse Huc2 was transcribed from both chromosomes (Fig. 3B). Furthermore, we also found that the human HUC2 sequence was transcribed in human placental tissues. A polymorphic site was identified in the parental genomic DNAs of two families which demonstrated that hHUC2 was also expressed from both alleles (Fig. 3C,D). Therefore, in both mice and humans, Huc2 is bi-allelically expressed, within a chromosomal domain thought previously to contain only mono-allelically expressed genes.

Our analysis suggests that the Huc transcripts are not exons of a larger transcript – indeed no consensus splice acceptor or donor sites can be detected around the HUC sequences using standard analysis software. It is therefore possible that Huc1 and Huc2 represent small non-coding RNAs. We could also detect expression by RT-PCR (data not shown), through the previously characterised silencer element at H19 (Drewell et al., 2000), suggesting that there may be other transcripts of unknown function at the H19/Igf2 locus. Such noncoding transcripts have been characterised at several other imprinted loci (Arima et al., 2000; Moore et al., 1997; Takada et al., 2000; Wutz et al., 1997), although in many cases their function remains enigmatic.

HUC sequences demonstrate enhancer activity in vitro

As demonstrated previously, short regions of very highly conserved sequence between species may also be indicative of cis-regulatory elements. Long range regulatory elements (Loots et al., 2000) and transcriptional enhancers (Aparicio et al., 1995) have been identified in this way. The enhancers identified to date for the H19 and Igf2 genes are located downstream of the H19 gene (Fig. 1B) (Ishihara et al., 2000; Leighton et al., 1995). It is, however, possible that the HUC sequences represent additional enhancer elements. We first investigated the ability of the HUC sequences to act as enhancers in a HeLa cell transfection system. In this assay, HUC1 and HUC2 demonstrated enhancer activity that was approximately fourfold greater than that of the previously characterised endodermal enhancers located downstream of H19 (Fig. 4) (Leighton et al., 1995; Brenton et al., 1999). Interestingly, a drop in enhancer activity was observed when both HUC1 and HUC2 and the intervening sequence was tested. However, when this intervening sequence was removed, the enhancer activity detected was significantly stronger than that of HUC1 or HUC2 alone. In comparison, the H19 differentially methylated domain (DMD) or ICR, which is responsible for silencing the paternal H19 allele when methylated (Drewell et al., 2000), mediates transcriptional repression in this assay (Fig. 4). Therefore, this in vitro assay appears to be a reliable indicator of both enhancer and silencer activity of cis-regulatory elements from imprinted loci, in agreement with our previous studies (Arima et al., 2001).

HUC sequences can act as enhancers in vivo

To test whether the enhancer activity we detected in vitro represents a genuine in vivo function, we used a transgenic approach. A region containing both HUC1 and HUC2, plus the intervening sequence, was placed upstream of a reporter gene comprising a 0.8 kb H19 promoter and the placental alkaline phosphatase (PLAP) gene (Henthorn et al., 1988). It has been previously demonstrated that transgenes containing 3.7 kb of 5′ flank upstream of the H19 gene have no transcriptional activity in the absence of enhancer elements (Elson and Bartolomei, 1997).

Six pre-germline transgenic embryos, carrying varying numbers of copies of the transgene, were recovered at day 13.5 of gestation. The PLAP staining patterns were remarkably consistent between the transgenic embryos, with reporter expression principally in the developing cartilage in the ribs and spinal column, heart and developing kidney (Fig. 5B-D). Expression could also be detected in the diaphragm and tongue, and in the lung and skeletal muscle of the head. This highly tissue specific expression pattern strongly suggests that the HUCs are exclusively mesodermal enhancers, as opposed to harbouring an intrinsic general transcriptional activation activity associated with a promoter sequence. Significantly, PLAP expression was never detected in the liver where both endogenous H19 and Igf2 are highly expressed. Expression of these genes in the liver and other endodermal tissues has previously been shown to be controlled by enhancers located downstream of H19 (Leighton et al., 1995).

It is important to note that the expression pattern observed is appropriate for a subset of tissues when compared with the full expression pattern of the endogenous Igf2 and H19 genes (Fig. 2E) (Leighton et al., 1995). This rules out the possibility that the transgene displays ectopic expression, and supports the notion that the HUCs are major enhancers for mesodermal tissues and can interact with the H19, and probably, Igf2 promoters. Furthermore, we also note that the previously identified enhancers at the H19/Igf2 locus do not account for the full expression patterns of these two genes (Ishihara et al., 2000; Leighton et al., 1995). What is unusual, however, is the location of these HUC enhancers. For the first time, such enhancers have been detected upstream of H19 and the ICR. This is significant because the ICR contains a proposed insulator element that is suggested to play a crucial role in regulating promoter-enhancer communication (Bell and Felsenfeld, 2000; Hark et al., 2000; Kanduri et al., 2000). The location of major mesodermal enhancers upstream of the ICR necessitates re-evaluation of the mechanism of imprinted expression of the H19 and Igf2 genes in the mesodermal tissues described here.

The HUC sequences represent novel DNA elements within the mouse and human H19 and Igf2 imprinted domain. We show that HUC1 and HUC2 are enhancers both in vitro and in vivo. Indeed, in HeLa cells, the HUC sequences have enhancer activity which is significantly greater than that of previously characterised enhancers downstream of H19. This may be a reflection of differences in their activity or, more likely, a demonstration of the underlying differences in their tissue specificity.

HUC enhancer tethering to the H19 promoter

The HUC enhancers are clearly capable of driving expression of the H19-PLAP reporter gene from a minimal H19 promoter in a wide range of mesodermally derived tissues in day 13.5 embryos. This expression is in a subset of tissues in which the endogenous maternal H19 gene is normally expressed (see Fig. 2E), strongly suggesting that they interact with the endogenous H19 promoter. It is interesting to note that a 140 kb H19 BAC transgene extending only –6 kb upstream of the H19 transcriptional start site (and therefore not containing the HUCs) shows significantly reduced expression in the heart and kidney (Kaffer et al., 2000), tissues in which we detect strong transcriptional activation by the HUCs. However, the HUCs and the endogenous H19 gene are located on opposite sides of the ICR, which harbours a proposed insulator on the maternal chromosome. (Fig. 6A) (Bell and Felsenfeld, 2000; Hark et al., 2000; Kanduri et al., 2000). We propose that a key function of the ICR is to tether enhancers that are active in these distinct tissues to the H19 promoter. In this case, the interaction of the HUCs with the maternal H19 gene may be mediated by the insulator itself. The insulator may potentially act as a tethering element by recruiting a protein complex capable of directing these enhancers (and possibly the other downstream mesendodermal enhancers) to the H19 promoter (Fig. 6A). Such a positive regulatory function for the unmethylated ICR was revealed by an extensive deletion of this region at the endogenous locus, which resulted in a decrease in the level of H19 expression (Thorvaldsen et al., 1998).

It is not possible to directly attribute a tethering/boundary function to the ICR in mesodermal tissues, as only a very limited number of tissues (predominantly neonatal liver) were studied in mice carrying a comprehensive deletion of the ICR (Thorvaldsen et al., 1998). The hypersensitive sites that map to the ICR when maternally inherited have been found in all tissues examined to date; including, liver, brain and ES cells, irrespective of the transcriptional status of H19 (Hark and Tilghman, 1998; Khosla et al., 1999). This consistent chromatin organisation suggests that the maternal ICR has the potential to function similarly in all tissues. Taken together with the absence of sufficient data about the role of the ICR in a variety of tissues this by no means rules out a role for the ICR in mesodermal tissues.

There is a precedent for similar promoter-enhancer interactions at complex genetic loci in Drosophila, where facilitator proteins bound to insulator elements are thought to specifically direct enhancers to promoters over long distances (Dorsett, 1999; Sipos et al., 1998), although the mechanisms have yet to be fully elucidated. Indeed, in the case of GAGA-mediated tethering activity at the eve promoter the stimulatory effect is disrupted when GAGA is separated from the promoter. The situation at the H19 ICR appears to be different, in that the tethering activity acts over 2 kb. However, similar to the complex situation at the H19 ICR, Drosophila insulators are also believed to be multipartite with the capacity to interact with enhancers (Geyer, 1997).

The role of the HUCs in regulating Igf2 expression

On the paternal chromosome, the H19 gene is silenced by DNA methylation through the recruitment of a repressive protein complex (R. A. D., unpublished). This would release the enhancers to interact with the closely linked paternal Igf2 gene (Fig. 6B). As noted before, unlike the H19 gene, both parental alleles of Igf2 are potentially active (Sasaki et al., 1992). Therefore, the mechanism by which the maternal Igf2 gene remains repressed is different from that governing H19 silencing by DNA methylation. The regulation of the Igf2 gene is governed by accessibility to the shared enhancers. The enhancers downstream of H19 are apparently prevented from interacting with the maternal Igf2 allele through the presence of an active insulator function within the ICR (Fig. 6A). This mechanism would not apply to the HUCs, which are located upstream of the ICR. However, there is, in addition, an epigenetically regulated silencer element upstream of Igf2 at DMR1 that ensures silencing of the maternal Igf2 gene in specific tissues (Fig. 6A). This was demonstrated by deletion of DMR1, resulting in the activation of the maternal Igf2 gene. However this activation of Igf2 was observed only in certain mesodermal tissues such as the heart and kidneys (Constancia et al., 2000). Expression of H19, and by inference the function of the H19 ICR, was unaffected in these animals. This reactivated expression pattern shows striking resemblance to that conferred by the HUC enhancers described here. This suggests that there is an interaction between the HUC enhancers and the Igf2 DMR1 in these tissues.

The combined data relating to the cis-regulation of the endogenous Igf2 and H19 genes provides a compelling explanation for how access to the HUCs and other enhancers may be regulated by the ICR upstream of H19 and DMR1 upstream of Igf2. However, expression of H19/Igf2 from a 130 kb YAC transgene is relatively low or absent in the heart, kidney and other tissues (Ainscough et al., 2000a; Ainscough et al., 1997). These are tissues in which the HUC enhancers play a significant role in driving expression. The YAC clone does not contain the Igf2 DMR1 silencer element or further sequence upstream of Igf2. This suggests that the complete imprinted expression of Igf2 and H19 is dependent on the interaction of a complex and extensive network of cis-regulatory elements at the locus, some of which may remain to be identified. In addition, the role of the transcripts at the HUC sequences (mouse Huc1 and mouse Huc2) is also unclear. Although the presence of small non-coding RNAs has long been established at imprinted regions (Arima et al., 2000; Moore et al., 1997; Takada et al., 2000) and other complex loci (Ashe et al., 1997; Zhou et al., 1999), their role remains largely enigmatic. We are currently generating a targeted deletion at the endogenous mouse locus to elucidate the role(s) of the HUCs as transcribed sequences, as enhancers or other as yet unknown functions in their normal in vivo context.

The authors thank S. Khosla for assistance with the sequence alignment and J. F.-X. Ainscough for advice. R. A. D. was funded by a Wellcome Trust Prize Fellowship. K. L. A. was supported by an Elmore Research Studentship from Gonville and Caius College, Cambridge. T. A. was funded by a Newton Trust grant. J. D. B. received a Research Fellowship for a Clinician (CRC). This work was supported by a grant from the Wellcome Trust to M. A. S.

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