Colin V. Beechey

Regulation of alternative polyadenylation by genomic imprinting

Maternally and paternally derived alleles can utilize different promoters, but allele-specific differences in cotranscriptional processes have not been reported. We show that alternative polyadenylation sites at a novel murine imprinted gene (H13) are utilized in an allele-specific manner. A differentially methylated CpG island separates polyA sites utilized on maternal and paternal alleles, and contains an internal promoter. Two genetic systems show that alleles lacking methylation generate truncated H13 transcripts that undergo internal polyadenylation. On methylated alleles, the internal promoter is inactive and elongation proceeds to downstream polyadenylation sites. This demonstrates that epigenetic modifications can influence utilization of alternative polyadenylation sites.

Genome imprinting phenomena on mouse chromosome 7

Heterozygotes for the reciprocal translocation T(7;15)9H were intercrossed, with albino (c) and underwhite (uw) as genetic markers, in order to study genetic complementation in mouse chromosome 7. Chromosome 15 is known to show normal complementation. Neither reciprocal cross in which one parent was c/c and the other wild type yielded albino progeny at birth although about 17% would be expected, but albino foetuses were recovered when the mother was c/c and father wild type. These products of maternal duplication/paternal deficiency for distal 7 were markedly retarded with small placentae. No albino foetuses were found when the father was c/c and mother wild type, which suggested earlier lethality. Equivalent crosses with uw (chromosome 15) as proximal marker gave normal underwhite progeny when the mother was uw/uw but small placentae, retardation and neonatal death of presumptive underwhites in the reciprocal cross. These abnormal newborn would have had a maternal duplication/paternal deficiency for proximal 7. These and other findings indicate that one region of defective complementation probably lies distal to the breakpoint of T(7;18)50H at 7E2-F2, while another is between the centromere and 7B3. Examination of man-mouse homologies suggests that the loci for three pathological human conditions (Beckwith-Weidemann syndrome, dystrophia myotonia and rhabdomyosarcoma) with differential parental transmission may be located in homologous regions to those affected by imprinting phenomena on mouse chromosome 7.

A candidate model for Angelman syndrome in the mouse

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are well-recognized examples of imprinting in humans. They occur most commonly with paternal and maternal 15ql 1-13 deletions, but also with maternal and paternal disomy. Both syndromes have also occurred more rarely in association with smaller deletions seemingly causing abnormal imprinting. A putative mouse model of PWS, occurring with maternal duplication (partial maternal disomy) for the homologous region, has been described in a previous paper but, although a second imprinting effect that could have provided a mouse model of AS was found, it appeared to be associated with a slightly different region of the chromosome. Here, we provide evidence that the same region is in fact involved and further demonstrate that animals with paternal duplication for the region exhibit characteristics of AS patients. A mouse model of AS is, therefore, strongly indicated.

Uncoupling antisense-mediated silencing and DNA methylation in the imprinted Gnas cluster.

There is increasing evidence that non-coding macroRNAs are major elements for silencing imprinted genes, but their mechanism of action is poorly understood. Within the imprinted Gnas cluster on mouse chromosome 2, Nespas is a paternally expressed macroRNA that arises from an imprinting control region and runs antisense to Nesp, a paternally repressed protein coding transcript. Here we report a knock-in mouse allele that behaves as a Nespas hypomorph. The hypomorph mediates down-regulation of Nesp in cis through chromatin modification at the Nesp promoter but in the absence of somatic DNA methylation. Notably there is reduced demethylation of H3K4me3, sufficient for down-regulation of Nesp, but insufficient for DNA methylation; in addition, there is depletion of the H3K36me3 mark permissive for DNA methylation. We propose an order of events for the regulation of a somatic imprint on the wild-type allele whereby Nespas modulates demethylation of H3K4me3 resulting in repression of Nesp followed by DNA methylation. This study demonstrates that a non-coding antisense transcript or its transcription is associated with silencing an overlapping protein-coding gene by a mechanism independent of DNA methylation. These results have broad implications for understanding the hierarchy of events in epigenetic silencing by macroRNAs.

The mouse Zac1 locus: basis for imprinting and comparison with human ZAC

We identified a maternally methylated CpG island at the mouse Zac1 locus on chromosome (Chr.) 10 in a screen for imprinted genes. The homologous human gene ZAC (also known as LOT1 and PLAGLI) is a candidate gene for transient neonatal diabetes (TNDM), an imprinted disorder associated with paternal duplication for 6q24 and characterized by intrauterine growth retardation and insulin dependence. A mouse model would be indispensable to investigate the basis of the disorder, however, there is apparently no similar phenotype in mice with the corresponding chromosome anomaly. To begin to understand this difference, we have undertaken a comparative analysis of the mouse and human genes. We show that the CpG island is far upstream of the coding body of mouse Zac1, that Zac1 transcripts initiate in a conserved region in the CpG island, and transcripts undergo complex splicing--all properties shared with the human gene. CpG island methylation is present in oocyte DNA and constitutes a germline-specific epigenetic mark. Mice with uniparental disomy (UPD) for Chr. 10 exhibit appropriate parent-of-origin dependent expression of Zac1, indicating that the absence of phenotypes comparable to aspects of human TNDM is not because imprinting of Zac1 is relaxed in these UPD mice.

Time of initiation and site of action of the mouse chromosome 11 imprinting effects

Previous studies have shown that mice with paternal disomy for chromosome 11 are consistently larger at birth than their normal sibs, whereas mice with the maternal disomy are consistently smaller. An imprinting effect with monoallelic expression of some gene/s affecting growth was indicated. Here we show that the size differences become established prior to birth and are only maintained subsequently, indicating that the gene repression is limited to prenatal development. Fetal analysis was limited to 12.5-17.5 days post coitum. However by extrapolating the data backwards it could be calculated that both the maternal and paternal size effects might commence as early as 7 days post coitum, although possibly slightly later. It may be deduced that initiation of expression of the gene/s responsible may occur at about this time in development. The two disomy growth rates were mirror-images of each other, suggesting that expressed gene dosage is the underlying cause. Differential growth of the placentas of the two disomies was also found, and extrapolation of these data backwards suggested that the placental size differences were initiated later in development than those for the fetuses. The differential placental growth of the maternal and paternal disomies may therefore have developed independently or emerged as a consequence of the differential fetal growth. In either event it would seem that the expression of the responsible gene occurs in the fetus itself to cause the anomalies of growth. The data therefore provide information on the temporal and tissue specificity of the gene/s responsible for the chromosome 11 imprinting effects. Possible candidate genes are discussed.

Two new X-autosome translocations in the mouse

The reciprocal translocations T(X;4)37H and T(X;11)38H were induced by acute X-irradiation of spermatozoa. Male heterozygotes are completely aspermic with a spermatogenic block at pachytene and testis masses about one third of normal, though metaphase I is very occasionally reached in T37H. For both translocations the X chromosome breakpoints are in band XA2, and the autosomal breakpoints are in 4D3 for T37H and 11E1 for T38H, leading to long and short marker chromosomes. Chain quadrivalents predominated in oocytes at MI, with no rings; there were 32% trivalent + univalent configurations in T37H and 40% in T38H. These generated (1) XO mice, (2) tertiary trisomics carrying 20 bivalents and the small X4 (T37H) or X11 (T38H) markers. These trisomics were apparently lethal in T37H but sub-viable in T38H and sometimes fertile as XX11 and trisomic XXX11 females, though sterile as XX11Y males. However, many developed exencephaly in utero, probably because of the distal duplication on chromosome 11. No tertiary monosomics were found in 12 to 14 day embryos. Seven percent of all female progeny were XO, with a higher than expected frequency in T37H and a lower than expected one in T38H. T37H is about two units and T38H about five units from the spf locus on the X chromosome with evidence for some crossover suppression between the T38H breakpoint and Ta. Autosomal linkages found were T37H-m-b (R.F. of 16 +/- 4% between T37H and m) and T38H-Rewc (R.F. of 5 +/- 3%). T37H/+ females weighed about 10% less than normal females at birth and about 30% less at weaning; there was little if any effect in T38H/+ females or in males carrying either translocation. Neither translocation had any marked effect on viability. T37H/+ females showed variegation when heterozygous for b with the wild type allele in the long 4X marker. On average, about 20% of the coat was brown rather than black. However, no clear evidence for m variegation was found.

Imprinted genes: identification by chromosome rearrangements and post-genomic strategies

Imprinted genes are differentially expressed from the maternally and paternally inherited alleles. Accordingly, inheritance of both copies of an imprinted chromosome or region from a single parent leads to the mis-expression of the imprinted genes present in the selected region. Strains of mice with reciprocal and Robertsonian chromosomal translocations or mice with engineered chromosomal rearrangements can be used to produce progeny where both copies of a chromosomal region are inherited from one parent. In combination with systematic differential expression and methylation-based approaches, these mice can be used to identify novel imprinted genes. Advances in genome sequencing and computer-based technologies have facilitated this approach to finding imprinted genes.

DDC and COBL, flanking the imprinted GRB10 gene on 7p12, are biallelically expressed

Maternal duplication of human 7p11.2-p13 has been associated with Silver-Russell syndrome (SRS) in two familial cases. GRB10 is the only imprinted gene identified within this region to date. GRB10 demonstrates an intricate tissue- and isoform-specific imprinting profile in humans, with paternal expression in fetal brain and maternal expression of one isoform in skeletal muscle. The mouse homolog is maternally transcribed. The GRB10 protein is a potent growth inhibitor and represents a candidate for SRS, which is characterized by pre- and postnatal growth retardation and a spectrum of additional dysmorphic features. Since imprinted genes tend to be grouped in clusters, we investigated the imprinting status of the dopa-decarboxylase gene (DDC) and the Cordon-bleu gene (COBL) which flank GRB10 within the 7p11.2-p13 SRS duplicated region. Although both genes were found to replicate asynchronously, suggestive of imprinting, SNP expression analyses showed that neither gene was imprinted in multiple human fetal tissues. The mouse homologues, Ddc and Cobl, which map to the homologous imprinted region on proximal Chr 11, were also biallelically expressed in mice with uniparental maternal or paternal inheritance of this region. With the intent of using mouse Grb10 as an imprinted control, biallelic expression was consistently observed in fetal, postnatal, and adult brain of these mice, in contrast to the maternal-specific transcription previously demonstrated in brain in inter-specific F1 progeny. This may be a further example of over-expression of maternally derived transcripts in inter-specific mouse crosses. GRB10 remains the only imprinted gene identified within 7p11.2-p13.

Interactions between imprinting effects: summary and review

Mice with uniparental disomies (uniparental duplications) for defined regions of certain chromosomes, or certain disomies, show a range of developmental abnormalities most of which affect growth. These defects can be attributed to incorrect dosages of maternal or paternal copies of imprinted genes lying within the regions involved. Combinations of certain partial disomies result in interactions between the imprinting effects that seemingly independently affect foetal and/or placental growth in different ways or modify neonatal and postnatal development. The findings are generally in accord with the ‘conflict hypothesis’ for the evolution of genomic imprinting but do not demonstrate common growth axes within which imprinted genes may interact. Instead, it would seem that any gene that favours embryonic/foetal development, at consequent cost to the mother, will have been subject to evolutionary selection for only paternal allele expression. Reciprocally, any gene that reduces embryonic/foetal growth to limit disadvantage to the mother will have been selected for only maternal allele expression. It is concluded that survival of the placenta is core to the evolution of imprinting.