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news and views production. It is tempting now to speculate that the haploinsufficiency for ribosomal protein genes in DBA causes failure of red cell production via induction of p53-mediated apoptosis. The work also suggests a mechanism for one of the key puzzling features of DBA: that shortage of a ubiquitously expressed ribosomal protein can have such a cell-specific effect. Indeed, other recent studies also show cell-specific effects of ribosomal defects mediated by p53. For example, in zebrafish, deficiency of Rps19 and other ribosomal proteins has p53-mediated effects on erythropoiesis and development10. Moreover, in a mouse model of Treacher Collins syndrome, in which deficiency of the nucleolar protein Treacle causes impaired ribosome biogenesis and craniofacial defects, inhibition of p53 rescued the phenotype completely11. One question is how much of the cell specificity resides in ribosome biogenesis, for example, if different components are limiting
in different cells, and how much lies in events downstream of p53. It is important now to characterize the precise molecular chain of events whereby defective ribosome biogenesis in the nucleolus leads to p53 stabilization6. We may then have candidate molecules for possible therapeutic intervention in DBA, Treacher Collins and other ribosome-mediated diseases. Not surprisingly, the effects of p53 are species specific as well as cell type specific, and individuals with DBA do not show pigmentation defects. Interestingly, two other bone marrow failure syndromes, Fanconi anemia and dyskeratosis congenita, do have pigmentation abnormalities. In both cases, p53 has been shown to be upregulated as a result of defects in DNA repair12 and telomere maintenance13,14, respectively. Whether the pigmentation phenotype associated with these, and possibly other, conditions is due to the effects of elevated p53 levels
and induction of Kit ligand in keratinocytes remains to be seen. 1. Ellis, S.R. & Lipton, J.M. Curr. Top. Dev. Biol. 82, 217–241 (2008). 2. Gazda, H.T. et al. Br. J. Haematol. 127, 105–113 (2004). 3. Draptchinskaia, N. et al. Nat. Genet. 21, 169–175 (1999). 4. McGowan, K.A. et al. Nat. Genet. 40, 963–970 (2008). 5. Volarevic, S. et al. Science 288, 2045–2047 (2000). 6. Panic, L., Montagne, J., Cokaric, M. & Volarevic, S. Cell Cycle 6, 20–24 (2007). 7. Wehrle-Haller, B. Pigment Cell Res. 16, 287–296 (2003). 8. Perdahl, E.B., Naprstek, B.L., Wallace, W.C. & Lipton, J.M. Blood 83, 645–650 (1994). 9. Miyake, K. et al. Stem Cells 26, 323–329 (2008). 10. Danilova, N., Sakamota, K.M. & Lin, S. Blood advance online publication, doi:10.1182/blood-2008–01– 132290 (30 May 2008). 11. Jones, N.C. et al. Nat. Med. 14, 125–133 (2008). 12. Kennedy, R.D. & D’Andrea, A.D. Genes Dev. 19, 2925– 2940 (2005). 13. Ogden, G.R., Lane, D.P. & Chisholm, D.M. J. Clin. Pathol. 46, 169–170 (1993). 14. Artandi, S.E. & Attardi, L.D. Biochem. Biophys. Res. Commun. 331, 881–890 (2005).
H19 in the pouch Marisa S Bartolomei, Sebastien Vigneau & Michael J O’Neill The linked maternally expressed H19 and paternally expressed Igf2 genes use a CTCF-dependent DNA methylation– sensitive insulator to govern their allele-specific imprinting patterns. Contrary to expectations, a new study shows that the noncoding H19 RNA has a marsupial ortholog and that key features of the locus are similar, indicating that the imprinting regulation of this locus is conserved among therian mammals. In 1991, the first three imprinted genes were described in mice, with H19 and Igf2 as two of the three1. For well over a decade, numerous groups have been frustrated by attempts to identify a marsupial ortholog of the H19 noncoding RNA (ncRNA), suggesting that one may not exist. However, on page 971 of this issue, Smits and colleagues discover that marsupials have an H19 gene and that its most notable features, including gene structure, miRNA, imprinted status and Igf2 proximity, are conserved2. Identification of the H19 ncRNA H19 was initially cloned in a differential cDNA screen designed to identify genes that were Marisa S. Bartolomei and Sebastien Vigneau are at the Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA. Michael J. O’Neill is at the Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269, USA. e-mail:
[email protected]
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controlled similarly to the mouse Afp (alphafetoprotein) gene, which is highly expressed in fetal liver and repressed after birth3. One gene that fulfilled the screen criteria was H19, named to designate its filter position. H19 was subsequently identified in other cDNA screens. Most notable was the appearance of H19 in the screen for myoblast-specific cDNAs (designated myoH), where myoD was identified4. Although H19 was highly expressed and evident in multiple screens, it soon became clear that it did not encode a protein and was rather an unconventional mRNA. The gene is organized into five exons (Fig. 1). The longest ORF within the 2.2-kb mRNA resides in exon 1 and is sufficient to encode a protein of only 14 kDa, but this ORF is not conserved in human5. This apparent absence of a protein product was atypical at the time (before the ncRNA revolution) and led many to believe that H19 did not have a function. Whereas one study suggested that the human H19 gene encoded a tumor suppressor6, more bad news came from H19 mouse deletion studies, which failed to reveal an obvious abnormal
phenotype attributable solely to the loss of the H19 mRNA7. Nevertheless, this apparently functionless gene still shows conserved features among eutherian mammals, which argues in favor of a functional gene product. In addition to conserved gene structure, exon 1 contains a conserved stem-loop structure5. Recently, the stem-loop was shown to be the precursor for the microRNA miR-675, but its targets and function remain unknown8. Further, conserved short sequences that are central to the imprinting mechanism are located upstream to the transcription start site (TSS) in the imprinting control region (also designated as the differentially methylated domain (DMD)), which regulates the reciprocal imprinting of H19 and Igf2. The conserved sites (four in mouse, seven in human) bind to the insulator protein CTCF on the unmethylated maternal allele, allowing H19 exclusive access to shared downstream enhancers and associated Igf2 repression9. On the paternal allele, hypermethylated CTCF sites prevent CTCF from binding, thereby eliminating
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news and views
Discovery of marsupial H19 The demonstration that IGF2 is imprinted in eutherians and marsupials (that is, mammals with pouches) was straightforward and constituted the first evidence of genomic imprinting conservation in mammals12. However, many attempts relying on traditional hybridization and PCR strategies failed to yield a marsupial H19 ortholog. This was initially chalked up to the apparent dispensability of H19 to IGF2 imprinting, prompting the conclusion that H19 was specific to eutherians. Alternatively, H19 was present in marsupials, but because of relaxed evolutionary constraint on the non– protein-coding sequence, the marsupial gene was simply not recognizable. We now know the latter to be true. Smits and colleagues sequenced BAC clones from the tammar wallaby that covered the putative H19-IGF2 locus and defined evolutionary conserved regions (ECRs) among the human, mouse and wallaby loci2. Two ECR clusters flanked a 38-kb region that contained the eutherian H19 gene. The investigators ultimately used low-stringency Bl2seq comparisons to identify the wallaby 2.7-kb H19 genomic sequence. Sequence comparisons revealed 51% identity with human H19 and marked conservation of exon-intron structure and of the pre-miRNA, TSS and poly(A) sequence (Fig. 1). One surprising finding is that both the miRNA and the longer mRNA structure are highly conserved, suggesting that both are functional. It seems unlikely that the sole role of the mRNA is to provide a host for miR-675. Multiple RNA species emanating from this locus could provide a plausible explanation for why H19 appears in screens for growth and tumorpromoting factors in some cases and differentiation and tumor-suppressor factors in others. Finally, the wallaby H19 gene is imprinted, with an upstream paternally methylated DMD that shows insulator function and contains
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insulator activity and allowing Igf2 exclusive enhancer access. It is notable, however, that although the H19 and Igf2 insulatordependent imprinting mechanism seems well conserved among eutherian mammals (that is, placental mammals), other clusters of imprinted genes largely use long ncRNAs to imprint genes in cis10. The ncRNAs, which partially overlap genes that they repress, may act by influencing chromatin states in cis and may even directly participate in transcript degradation to effect imprinted gene silencing. Contrarily, the mouse H19 mRNA seems to have no role in Igf2 imprinted expression, as precise deletion of the H19 transcription unit has minimal effects on maternal Igf2 silencing11.
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Figure 1 Conserved H19-associated features in mouse and wallaby. (a) Mouse H19-Igf2 imprinted cluster. Paternally, maternally and biallelically expressed genes are in blue, red and gray, respectively. The eutherian enhancers are shown (black circles), but enhancers have not yet been identified in marsupials. (b) H19 exon structure, with miR-675 (yellow), differentially methylated domain (thick line) and CTCF binding sites (triangles). (c) miR-675 hairpin structure. Orange, 5p mature sequence; yellow, 3p mature sequence. Wallaby photograph courtesy of Marilyn Renfree.
three putative CTCF binding sites. These significant results show that the H19-IGF2 imprinting mechanism is the most ancient identified to date. Evolutionary implications Much has been made of the insights gained from the comparison of imprinted loci among eutherian species and between eutherians, marsupials, monotremes and nonmammalian vertebrates13. By identifying marsupial H19 and its insulating DMD, Smits and colleagues reveal the marked similarities in H19IGF2 imprinting between marsupials and eutherians and show that this imprinting axis likely evolved before therian divergence2. The similarities are particularly interesting when considering recent work showing that loss of methylation (LOM) at an upstream DMR in opossum (also a marsupial) IGF2 activates the maternal allele14 whereas LOM in mouse leads to biallelic H19 activation and Igf2 repression15. Hence, despite the conserved locus structure in therians, there has been apparent divergence of the epigenetic mechanism of allelic silencing. It will be interesting to determine the effect of LOM on H19-IGF2 expression and to assess
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long-range chromatin interactions between the H19 DMD and IGF2 control elements in marsupials. Finally, it will be important to resolve whether egg-laying monotremes have imprinting and H19, as current evidence suggests that both are absent. 1. Bartolomei, M.S., Zemel, S. & Tilghman, S.M. Nature 351, 153–155 (1991). 2. Smits, G. et al. Nat. Genet. 40, 971–976 (2008). 3. Pachnis, V., Belayew, A. & Tilghman, S.M. Proc. Natl. Acad. Sci. USA 81, 5523–5527 (1984). 4. Davis, R.L., Weintraub, H. & Lassar, A.B. Cell 51, 987– 1000 (1987). 5. Tilghman, S.M. et al. in Nuclear Processes and Oncogenes (ed. Sharp, P.A.) 188–200 (Academic Press, New York, 1992). 6. Hao, Y., Crenshaw, T., Moulton, T., Newcomb, E. & Tycko, B. Nature 365, 764–767 (1993). 7. Leighton, P.A., Ingram, R.S., Eggenschwiler, J., Efstratiadis, A. & Tilghman, S.M. Nature 375, 34–39 (1995). 8. Mineno, J. et al. Nucleic Acids Res. 34, 1765–1771 (2006). 9. Reik, W. & Murrell, A. Nature 405, 408–409 (2000). 10. Edwards, C.A. & Ferguson-Smith, A.C. Curr. Opin. Cell Biol. 19, 281–289 (2007). 11. Ripoche, M.-A., Chantal, K., Poirier, F. & Dandolo, L. Genes Dev. 11, 1596–1604 (1997). 12. O’Neill, M.J., Ingram, R.S., Vrana, P.B. & Tilghman, S.M. Dev. Genes Evol. 210, 18–20 (2000). 13. Edwards, C.A. et al. PLoS Biol. 6, e135 (2008). 14. Lawton, B.R. et al. BMC Genomics 9, 205 (2008). 15. Li, E., Beard, C. & Jaenisch, R. Nature 366, 362–365 (1993).
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