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Novel conserved elements upstream of the H19 gene are transcribed and act as mesodermal enhancers

Robert A. Drewell^*, Katharine L. Arney^*, Takahiro Arima, Sheila C. Barton, James D. Brenton and M. Azim Surani

Wellcome/CRC Institute of Cancer and Developmental Biology and Physiological Laboratory, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
^* These authors contributed equally to this work
Present address: Department of Reproductive Physiology and Endocrinology, Medical Institute of Bioregulation, Kyushu University, 4546, Tsurumihara, Beppu, Oita 874-0838, Japan
Present address: Cancer Genomics Program, Department of Oncology, University of Cambridge, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 2XZ, UK

View larger version (34K): [in a new window]   Fig. 1. Genomic organisation and conservation of HUC1 and HUC2 sequences in mouse and human. (A) Mouse H19/Igf2 imprinted locus. H19 is expressed only from the maternal allele, Igf2 only from the paternal allele and Rpl23 is bi-allelically expressed. Characterised enhancers downstream of H19 (green circles) mediate expression in both endodermal and some mesodermal tissues (Leighton et al., 1995; Brenton et al., 1999; Ishihara et al., 2000). The ICR at H19 (red ellipse) and the silencer located within DMR1 upstream of Igf2 (blue diamond) (Constancia et al., 2000) are also shown. (B) The conserved structural organisation of sequences at the H19 gene. The extended sequenced region upstream of H19 (AF327412) is indicated. The top line represents mouse DNA sequence, aligned with human sequence on the bottom line (PAC clone pDJ998n23, EM:AC004556) using BlastN software. The numbers above and below the lines relate to the 5' boundary of various regions relative to the H19 gene transcriptional start site. Percentages shown in between the two sequences are the degree of homology at the nucleotide level and the approximate length in kb of the conserved region is shown in brackets. The H19 transcribed region (white box) shows 92% homology between the two species. This degree of conservation is almost matched by HUC1 and HUC2 (yellow boxes), which have a greater level of sequence homology than the characterised downstream enhancer elements (green circles) (Leighton et al., 1995). Conversely, sequence analysis does not reveal a human homologue of the ICR, which comprises both silencer and boundary functions (red ellipse), located upstream of mouse H19. (C) Dotplot sequence comparison of human and mouse H19 upstream region. GCG dotplot software only identified the H19 transcribed sequence and HUC1 and HUC2 regions, as conserved between mouse and human (default parameters) in the upstream region.

View larger version (93K): [in a new window]   Fig. 2. Expression of HUC1 and HUC2 in the mouse. PCR primers and probes at mouse HUC1 and mouse HUC2. (A) PCR primers internal to both conserved regions were used to amplify regions specific to either mouse HUC1 (194 bp), mouse HUC2 (299 bp) or both regions and the intervening sequence (1856 bp, probe A). The PCR primers are described in the Materials and Methods. (B) RT-PCR detection of RNA transcripts at the HUC sequences. PCR amplifications were performed on either genomic DNA (gDNA) or reverse transcribed total RNA (RT cDNA) from 13.5 dpc embryos and the products hybridised to probe A. Mouse HUC1 and mouse HUC2 produced a product when amplified using primers internal to the conserved region (1-1 or 2-2). However, no product was detected from the RT cDNA sample when amplified across the two conserved regions (1-2), suggesting mouse HUC1 and mouse HUC2 are not part of the same transcription unit. Control lanes are water for gDNA (–) and RT- for RT cDNA (–). (C,D) HUC expression is detected from an antisense (HUC AS) probe spanning HUC1 and HUC2 and is restricted to specific tissues in the embryo. (E,F) H19 expression from an antisense (H19 AS) probe (E) detects widespread expression in endodermal and mesodermal tissues. No expression was detected from a HUC sense (HUC S) probe (F), indicating that the HUC sequences are only transcribed in the same orientation as H19. (G-I) Detailed expression pattern detected from the HUC antisense probe in the brain (G), tongue and ear (H) and genital tubercle (I). E, ear; FB, forebrain; G, genital eminence; L, limb; Li, liver; Lu, lung; MB, midbrain. Scale bars: in C, 1 mm for C-F; 0.5 mm in G-I.

View larger version (27K): [in a new window]   Fig. 3. Bi-allelic expression of HUC2 in mouse and human. (A) Tru9I polymorphism in the mouse HUC2 sequence between 129/Sv and M. spretus strains was used to analyse allele-specific expression by RT-PCR in 13.5dpc embryos, as described in Fig. 2. (B) In three separate embryos (1-3), transcription from mouse HUC2 was detected from both parental alleles. The pattern was the same as that from genomic DNA of the F1 hybrid (129/Sv x M. spretus). (C) DdeI polymorphism in the human HUC2 sequence was identified in the genomic DNA of parents in two independent families. (D) RT-PCR analysis was performed on total RNA isolated from post-partum placentas from these families. This demonstrated bi-allelic expression of human HUC2 in placenta (RT+). The pattern was the same as that from genomic DNA of the placental tissue (gDNA).

View larger version (15K): [in a new window]   Fig. 4. Enhancer activity of HUC1 and HUC2 in vitro. (A) Map indicating the regions tested in the cell transfection assay. Regions were cloned upstream of an SV40 promoter driving a firefly luciferase reporter gene. (B) Transcriptional activity of transfected constructs. The light emission obtained from the SV40 promoter alone was normalised to a value of 1. DMD, H19 differentially methylated domain; DMDm, methylated DMD. HUC 1, HUC2 and both regions including the intervening sequence (HUC1+2) were tested. Maximum enhancer activity was detected from HUC1+2 (HUC1+2 with intervening sequence removed). Error bars show calculated standard error of the mean (s.e.m.) values.

View larger version (52K): [in a new window]   Fig. 5. Enhancer activity of the HUC sequences in vivo. (A) The endogenous locus and HUC-PLAP transgene. HUC 1+2 are indicated by the white boxes, the H19 promoter region by the grey box (H) and the PLAP reporter by the black arrow. S (SpeI) and X (XbaI) are restriction sites used for construction. PCR primers used for genotyping the embryos are indicated. Embryos were confirmed transgenic by PCR genotyping (data not shown). (B-D) Three embryos showing typical PLAP staining pattern obtained. This expression pattern was highly consistent between all transgenic embryos. Non-transgenic embryos, as identified by PCR, failed to show any PLAP expression. D, diaphragm; H, heart; K, kidney; M; Meckel’s cartilage; SC, spinal cartilage; T, tongue. Scale bar in B: 1 mm.

View larger version (14K): [in a new window]   Fig. 6. (A,B) Model of proposed functional interaction of the HUC sequences with Igf2 and H19. Activation of gene expression is represented by (+), repression is represented by (–) and inhibition of enhancer function is indicated by a vertical bar. H19 and Igf2 expression in some tissues is controlled by enhancers located downstream of H19 (green circles) (Leighton et al., 1995; Ishihara et al., 2000). On the maternal allele (MAT), the enhancers have access to the H19 promoter but are prevented from activating Igf2 by the boundary function present in the unmethylated ICR (open red ellipse), mediated by CTCF. On the paternal allele (PAT), the ICR is methylated (closed red ellipse), acting to silence H19 (Drewell et al., 2000). The absence of the boundary means the downstream enhancers are free to activate Igf2. In a subset of mesodermal tissues, expression is controlled by the HUC enhancers located upstream of H19 (yellow circles). On the maternal allele, interaction of the HUCs with the H19 promoter may be mediated by a tethering activity within the unmethylated ICR (open red ellipse). Expression of Igf2 by the HUC enhancers in mesodermal tissues is prevented by the unmethylated tissue-specific silencer element located at the Igf2 DMR1 (open blue diamond) (Constancia et al., 2000). On the paternal allele, the HUCs are free to activate expression of Igf2 while H19 is silenced as before.