The Tnfrh1 (Tnfrsf23) gene is weakly imprinted in several organs and expressed at the trophoblast-decidua interface
© Clark et al 2002
Received: 16 May 2002
Accepted: 27 June 2002
Published: 27 June 2002
The Tnfrh1 gene (gene symbol Tnfrsf23) is located near one end of a megabase-scale imprinted region on mouse distal chromosome 7, about 350 kb distant from the nearest known imprinting control element. Within 20 kb of Tnfrh1 is a related gene called Tnfrh2 (Tnfrsf22) These duplicated genes encode putative decoy receptors in the tumor necrosis factor (TNF) receptor family. Although other genes in this chromosomal region show conserved synteny with genes on human Chr11p15.5, there are no obvious human orthologues of Tnfrh1 or Tnfrh2.
We analyzed Tnfrh1 for evidence of parental imprinting, and characterized its tissue-specific expression. Tnfrh1 mRNA is detectable in multiple adult and fetal tissues, with highest expression in placenta, where in situ hybridization reveals a distinctive population of Tnfrh1-positive cells in maternal decidua, directly beneath the trophoblast giant cells. In offspring of interspecific mouse crosses, Tnfrh1 shows a consistent parent-of-origin-dependent allelic expression bias, with relative repression, but not silencing, of the paternal allele in several organs including fetal liver and adult spleen.
Genes preferentially expressed in the placenta are predicted to evolve rapidly, and Tnfrh1 appears to be an example of this phenomenon. In view of its strong expression in cells at the fetal-maternal boundary, Tnfrh1 warrants further study as a gene that might modulate immune or trophic interactions between the invasive placental trophoblast and the maternal decidua. The preferential expression of Tnfrh1 from the maternal allele indicates weak functional imprinting of this locus in some tissues.
A well-studied megabase-scale region of DNA on distal mouse chromosome 7 (Chr7) contains multiple genes subject to parental imprinting. Nearly all genes in this region show conserved synteny with genes on human Chr11p15.5. The extended imprinted region appears to have a bipartite structure in that it contains at least two separate imprinted sub-domains. Each of these subdomains is regulated by a distinct imprinting control element. These correspond to short differentially methylated DNA sequences (DMRs) – one immediately upstream of the H19 gene and another within an intron of the Kcnq1 gene. These two elements control, respectively, the allele-specific expression of the H19/Igf2/Ins2 gene cluster, and the second gene cluster containing Kcnq1, the antisense transcriptKcnq1ot1, p57 Kip2/Cdkn1c, Slc22a1l, Ipl/Tssc3 and possibly additional genes [1, 2]. There is good evidence assigning one border of the overall imprinted region to the DNA immediately downstream of H19 . However, the other border, which must lie upstream of Ipl is less well-defined . Here we show evidence for weak imprinting of a gene, Tnfrh1, which is located upstream of Ipl in mice, and which encodes a putative decoy receptor in the TNF receptor family. We also map the tissue distribution of Tnfrh1 mRNA and discuss the significance of our findings in terms of possible functions of this gene in placentation.
Results and Discussion
The expression of Tnfrh1 only at the fetal-maternal boundary obviously suggests that this gene may play a role in modulating either immune or trophic interactions between the invasive placental trophoblast and the uterine host tissue. Since Tnfrh1 encodes a receptor in the TNFR family lacking a cytoplasmic domain (i.e., a decoy receptor), this gene might function to block the action of TNF-related ligands. A similar scenario has been proposed by Phillips et al. [8, 9] for another TNF ligand-receptor system, namely TRAIL and TRAIL receptors, which are expressed in human placentas. However, in their studies the major decoy receptor for TRAIL, DcR1, was found expressed in trophoblast, not in decidua, while another decoy receptor, DcR2, was expressed by placental macrophages. Fas ligand is also highly expressed by human and murine trophoblast, although its functional role in the placenta is unknown [10, 11].
Other fetal and adult tissues were also assessed in these crosses, and showed a maternal expression bias, but weaker than that observed in fetal liver (data not shown). The parent-of-origin dependent allelic bias in Tnfrh1 mRNA was superimposed on a 'baseline' bias towards hyper-expression of the BL/6 allele (Fig.7). This most likely reflects the effects of polymorphisms in regulatory promoter and/or enhancer sequences. Indeed, sequencing of the BL/6 and CAST Tnfrh1 promoter sequences revealed multiple SNPs in the region -200 to +100 relative to the transcriptional start site (data not shown). The alternative trivial explanation for the baseline bias, polymorphisms within primer binding sites, was excluded by direct sequencing of genomic DNAs.
Sequence variants in Tnfrh1 exons
The findings described here include 'leaky' but consistent imprinting of Tnfrh1 in several organs, and high-level expression of this gene in a distinctive population of cells restricted to the interface between the placental trophoblast and the uterine lining. The absence of identifiable orthologues of Tnfrh1 and Tnfrh2 in the human genome suggests that both of these genes arose subsequent to the divergence of placental mammals. This fact, together with our observation of preferential expression of Tnfrh1 mRNA in cells at the fetal-maternal boundary, highlights Tnfrh1 as a potential example of the rapid evolution of genes with functions specific to the placenta, a process postulated to be driven by conflict between fetal and maternal alleles. Anecdotal examples, including pregnancy-associated glycoproteins, trophoblast interferons, the Pem and Psx homeobox genes, and the placental lactogen genes support the notion that 'placental genes' evolve rapidly [12–17], but counter-examples can also be adduced. Two imprinted genes with placenta-specific expression and function, namely Ipl and Mash2 [6, 7, 18, 19] are highly conserved. This contrasts with the lack of conservation of Tnfrh1, and the species-specific imprinting of at least one placental lactogen gene . Of more immediate interest is the biological function of Tnfrh1, which will need to be determined by knockout experiments. Since the sequence of this gene predicts that it encodes a TNF decoy receptor, a likely possibility is that it acts to dampen immune responses to the fetal semi-allograft.
Imprinting of genes on mouse distal Chr7 is controlled by two DMRs, which act as 'imprinting centers'. Of these two control elements, the closest to Tnfrh1 is KvDMR1, located in an intron of the Kcnq1 gene and giving rise to the Kcnq1ot1 non-translated RNA. This element, which is ~350 kb distant from Tnfrh1, is essential in cis for the imprinting of at least 4 genes, Kcnq1, Cdkn1c, Slc22a1l and Ipl/Tssc3 ([21–23] and M. Higgins, personal communication). All of these genes are relatively repressed on the paternal allele and active on the maternal allele. The simplest explanation for our finding of weak but consistent functional imprinting of Tnfrh1, with relative hyper-expression from the maternal allele, is that the KvDMR1 control element exerts distance-dependent effects. However, studies using KvDMR1-mutant mice will be necessary to confirm this.
Materials and Methods
Genomic and cDNA PCR
Tnfrh1, exon 1
Tnfrh1, exons 5/6
Tnfrh1, exon 6
Tnfrh1, exon 6
Tnfrh2, exon 1
Tnfrh1, exon 3
SSCP and RFLP analysis
PCR labeling was by incorporation of alpha32P-dCTP, using 10 ng of gel isolated PCR product as a template. Cycling employed an initial denaturation at 94°C for 4 min, followed by 6 cycles of denaturation at 94°C for 1 min, annealing at 59°C for 15 sec and extension at 72°C for 1 min with a final extension at 72°C for 7 min. The radiolabeled PCR products were digested with AluI or HpaII, denatured and electrophoresed for 10–16 h at 500 V on non-denaturing 6% acrylamide gels at room temperature . For RFLP analysis, PCR products were digested with MboII and analyzed by electrophoresis on non-denaturing 6% acrylamide gels at 500 V for 3–4 hours.
Fractionation of the placenta by dissection into maternal and fetal portions was done as described previously . RNA was extracted using Trizol, and 6–10 micrograms of total RNA was electrophoresed and transferred to nylon membranes. Northern blots containing total RNA from panels of mouse tissues were purchased from SeeGene (Seoul, Korea). The Tnfrh1 cDNA probes (the longer probe made using primers 1 and 2, and matching the first 506 nt of Genbank Acc. AY046550 plus an additional 11 nt at the 5' end; and a smaller probe made with primers 1 and 6 matching the first 297 nt) were labeled with α-32P (Random primers DNA labeling system, Life Technologies). Blots were prehybridized and hybridized at 42°C in ExpressHyb (Clontech, Palo Alto, CA) and washed in 0.1× SSC/1% SDS for one hour at 65°C.
RNA in situ hybridization
Placentas were fixed in 4% paraformaldehyde overnight at 4 C, transferred to 30% sucrose in 0.1 MPB, equilibrated overnight, and then embedded and snap frozen in standard glycerol-based medium (TBS, Durham, NC). After cryo-sectioning, the sections were post-fixed in paraformaldehyde and then subjected to in situ hybridization (ISH) with digoxigenin-labeled probes (Dig RNA Labeling Kit, Roche), followed by alkaline phosphatase-mediated detection. Anti-sense and sense probes for Tnfrh1 were synthesized from cDNA clone Image 1479846 (Genbank AI156311). The sense probe was used as a control and did not produce a signal.
This work was supported by grants to B.T. from the N.I.H and the Human Frontiers Science Project.
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