Two overlapping reading frames in a single exon encode interacting proteins--a novel way of gene usage.

The >1 kb XL-exon of the rat XLalphas/Galphas gene encodes the 37 kDa XL-domain, the N-terminal half of the 78 kDa neuroendocrine-specific G-protein alpha-subunit XLalphas. Here, we describe a novel feature of the XL-exon, the presence of an alternative >1 kb open reading frame (ORF) that completely overlaps with the ORF encoding the XL-domain. The alternative ORF starts 32 nucleotides downstream of the start codon for the XL-domain and is terminated by a stop codon exactly at the end of the XL-exon. The alternative ORF encodes ALEX, a very basic (pI 11.8), proline-rich protein of 356 amino acids. Both XLalphas and ALEX are translated from the same mRNA. Like XLalphas, ALEX is expressed in neuroendocrine cells and tightly associated with the cytoplasmic leaflet of the plasma membrane. Remarkably, ALEX binds to the XL-domain of XLalphas. Our results reveal a mechanism of gene usage that is without precedent in mammalian genomes.     

Identification of a methylation imprint mark within the mouse Gnas locus

The imprinted mouse gene Gnas produces the G protein alpha-subunit G(S)alpha and several other gene products by using alternative promoters and first exons. G(S)alpha is maternally expressed in some tissues and biallelically expressed in most other tissues, while the gene products NESP55 and XLalphas are maternally and paternally expressed, respectively. We investigated the mechanisms of Gnas imprinting. The G(S)alpha promoter and first exon are not methylated on either allele. A further upstream region (approximately from positions -3400 to -939 relative to the G(S)alpha translational start site) is methylated only on the maternal allele in all adult somatic tissues and in early postimplantation development. Within this region lies a fourth promoter and first exon (exon 1A) that generates paternal-specific mRNAs of unknown function. Exon 1A and G(S)alpha mRNAs have similar expression patterns, making competition between their promoters unlikely. Differential methylation in this region is established during gametogenesis, being present in oocytes and absent in spermatozoa, and is maintained in preimplantation E3. 5d blastocysts. Therefore, this region is a methylation imprint mark. In contrast, differential methylation of the NESP55 and XLalphas promoter regions (Nesp and Gnasxl) is not established during gametogenesis. The methylation imprint mark that we identified may be important for the tissue-specific imprinting of G(S)alpha.     

New mutations at the imprinted Gnas cluster show gene dosage effects of Gsα in postnatal growth and implicate XLαs in bone and fat metabolism but not in suckling

The imprinted Gnas cluster is involved in obesity, energy metabolism, feeding behavior, and viability. Relative contribution of paternally expressed proteins XLαs, XLN1, and ALEX or a double dose of maternally expressed Gsα to phenotype has not been established. In this study, we have generated two new mutants (Ex1A-T-CON and Ex1A-T) at the Gnas cluster. Paternal inheritance of Ex1A-T-CON leads to loss of imprinting of Gsα, resulting in preweaning growth retardation followed by catch-up growth. Paternal inheritance of Ex1A-T leads to loss of imprinting of Gsα and loss of expression of XLαs and XLN1. These mice have severe preweaning growth retardation and incomplete catch-up growth. They are fully viable probably because suckling is unimpaired, unlike mutants in which the expression of all the known paternally expressed Gnasxl proteins (XLαs, XLN1 and ALEX) is compromised. We suggest that loss of ALEX is most likely responsible for the suckling defects previously observed. In adults, paternal inheritance of Ex1A-T results in an increased metabolic rate and reductions in fat mass, leptin, and bone mineral density attributable to loss of XLαs. This is, to our knowledge, the first report describing a role for XLαs in bone metabolism. We propose that XLαs is involved in the regulation of bone and adipocyte metabolism.     

The origin and evolution of genomic imprinting and viviparity in mammals

Genomic imprinting is widespread in eutherian mammals. Marsupial mammals also have genomic imprinting, but in fewer loci. It has long been thought that genomic imprinting is somehow related to placentation and/or viviparity in mammals, although neither is restricted to mammals. Most imprinted genes are expressed in the placenta. There is no evidence for genomic imprinting in the egg-laying monotreme mammals, despite their short-lived placenta that transfers nutrients from mother to embryo. Post natal genomic imprinting also occurs, especially in the brain. However, little attention has been paid to the primary source of nutrition in the neonate in all mammals, the mammary gland. Differentially methylated regions (DMRs) play an important role as imprinting control centres in each imprinted region which usually comprises both paternally and maternally expressed genes (PEGs and MEGs). The DMR is established in the male or female germline (the gDMR). Comprehensive comparative genome studies demonstrated that two imprinted regions, PEG10 and IGF2-H19, are conserved in both marsupials and eutherians and that PEG10 and H19 DMRs emerged in the therian ancestor at least 160 Ma, indicating the ancestral origin of genomic imprinting during therian mammal evolution. Importantly, these regions are known to be deeply involved in placental and embryonic growth. It appears that most maternal gDMRs are always associated with imprinting in eutherian mammals, but emerged at differing times during mammalian evolution. Thus, genomic imprinting could evolve from a defence mechanism against transposable elements that depended on DNA methylation established in germ cells.     

The alternative stimulatory G protein alpha-subunit XLalphas is a critical regulator of energy and glucose metabolism and sympathetic nerve activity in adult mice

The complex imprinted Gnas locus encodes several gene products including G(s)alpha, the ubiquitously expressed G protein alpha-subunit required for receptor-stimulated cAMP generation, and the neuroendocrine-specific G(s)alpha isoform XLalphas. XLalphas is only expressed from the paternal allele, whereas G(s)alpha is biallelically expressed in most tissues. XLalphas knock-out mice (Gnasxl(m+/p-)) have poor suckling and perinatal lethality, implicating XLalphas as critical for postnatal feeding. We have now examined the metabolic phenotype of adult Gnasxl(m+/p-) mice. Gnasxl(m+/p-) mice had reduced fat mass and lipid accumulation in adipose tissue, with increased food intake and metabolic rates. Gene expression profiling was consistent with increased lipid metabolism in adipose tissue. These changes likely result from increased sympathetic nervous system activity rather than adipose cell-autonomous effects, as we found that XLalphas is not normally expressed in adult adipose tissue, and Gnasxl(m+/p-) mice had increased urinary norepinephrine levels but not increased metabolic responsiveness to a beta3-adrenergic agonist. Gnasxl(m+/p-) mice were hypolipidemic and had increased glucose tolerance and insulin sensitivity. The similar metabolic profile observed in some prior paternal Gnas knock-out models results from XLalphas deficiency (or deficiency of the related alternative truncated protein XLN1). XLalphas (or XLN1) is a negative regulator of sympathetic nervous system activity in mice.     

Loss of Gnas imprinting differentially affects REM/NREM sleep and cognition in mice

It has been suggested that imprinted genes are important in the regulation of sleep. However, the fundamental question of whether genomic imprinting has a role in sleep has remained elusive up to now. In this work we show that REM and NREM sleep states are differentially modulated by the maternally expressed imprinted gene Gnas. In particular, in mice with loss of imprinting of Gnas, NREM and complex cognitive processes are enhanced while REM and REM-linked behaviors are inhibited. This is the first demonstration that a specific overexpression of an imprinted gene affects sleep states and related complex behavioral traits. Furthermore, in parallel to the Gnas overexpression, we have observed an overexpression of Ucp1 in interscapular brown adipose tissue (BAT) and a significant increase in thermoregulation that may account for the REM/NREM sleep phenotypes. We conclude that there must be significant evolutionary advantages in the monoallelic expression of Gnas for REM sleep and for the consolidation of REM-dependent memories. Conversely, biallelic expression of Gnas reinforces slow wave activity in NREM sleep, and this results in a reduction of uncertainty in temporal decision-making processes.     

The evolution of genomic imprinting via variance minimization: an evolutionary genetic model

A small number of mammalian loci exhibit genomic imprinting, in which only one copy of a gene is expressed while the other is silenced. At some such loci, the maternally inherited allele is inactivated; others show paternal inactivation. Several hypotheses have been put forward to explain how this genetic system could have evolved in the face of the selective advantages of diploidy. In this study, we examine the variance-minimization hypothesis, which proposes that imprinting arose through selection for reduced variation in levels of gene expression. We present an evolutionary genetic model incorporating both this selection pressure and deleterious mutations to elucidate the conditions under which imprinting could evolve. Our analysis implies that additional mechanisms such as genetic drift are required for imprinting to evolve from an initial nonimprinting state. Other predictions of this hypothesis do not appear to fit the available data as well as predictions for two alternative hypotheses, genetic conflict and the ovarian time bomb. On the basis of this evidence, we conclude that the variance-minimization hypothesis appears less adequate to explain the evolution of genomic imprinting.     

Evolutionary genetic models of the ovarian time bomb hypothesis for the evolution of genomic imprinting

At a small number of loci in eutherian mammals, only one of the two copies of a gene is expressed; the other is silenced. Such loci are said to be "imprinted," with some having the maternally inherited allele inactivated and others showing paternal inactivation. Several hypotheses have been proposed to explain how such a genetic system could evolve in the face of the selective advantages of diploidy. In this study, we examine the "ovarian time bomb" hypothesis, which proposes that imprinting arose through selection for reduced risk of ovarian trophoblastic disease in females. We present three evolutionary genetic models that incorporate both this selection pressure and the effect of deleterious mutations to elucidate the conditions under which imprinting could evolve. Our findings suggest that the ovarian time bomb hypothesis can explain why some growth-enhancing genes active in early embryogenesis [e.g., mouse insulin-like growth factor 2 (Igf2)] have evolved to be maternally rather than paternally inactive and why the opposite imprinting status has evolved at some growth-inhibiting loci [e.g., mouse insulin-like growth factor 2 receptor (Igf2r)].     

Genomic imprinting mechanisms in mammals

Genomic imprinting is a form of epigenetic gene regulation that results in expression from a single allele in a parent-of-origin-dependent manner. This form of monoallelic expression affects a small but growing number of genes and is essential to normal mammalian development. Despite extensive studies and some major breakthroughs regarding this intriguing phenomenon, we have not yet fully characterized the underlying molecular mechanisms of genomic imprinting. This is in part due to the complexity of the system in that the epigenetic markings required for proper imprinting must be established in the germline, maintained throughout development, and then erased before being re-established in the next generation's germline. Furthermore, imprinted gene expression is often tissue or stage-specific. It has also become clear that while imprinted loci across the genome seem to rely consistently on epigenetic markings of DNA methylation and/or histone modifications to discern parental alleles, the regulatory activities underlying these markings vary among loci. Here, we discuss different modes of imprinting regulation in mammals and how perturbations of these systems result in human disease. We focus on the mechanism of genomic imprinting mediated by insulators as is present at the H19/Igf2 locus, and by non-coding RNA present at the Igf2r and Kcnq1 loci. In addition to imprinting mechanisms at autosomal loci, what is known about imprinted X-chromosome inactivation and how it compares to autosomal imprinting is also discussed. Overall, this review summarizes many years of imprinting research, while pointing out exciting new discoveries that further elucidate the mechanism of genomic imprinting, and speculating on areas that require further investigation.     

Conflict theory of genomic imprinting in mammals

In mammals, some embryonic genes are expressed differently depending on whether they are inherited from the sperm or egg, a phenomenon known as genomic imprinting. The information on the parental origin is transmitted by an epigenetic mark. Both the molecular mechanisms and evolutionary processes of genomic imprinting have been studied extensively. Here, I illustrate the simplest evolutionary dynamics of imprinting evolution based on the “conflict theory,” by considering the evolution of a gene encoding an embryonic growth factor controlling the maternal resource supply. It demonstrates that (a) the autosomal genes controlling placenta development to modify maternal resource acquisition may evolve a strong asymmetry of gene expression, provided the mother has some chance of accepting multiple males. (b) The genomic imprinting may not evolve if there is a small fraction of recessive deleterious mutations on the gene. (c) The growth-enhancing genes should evolve to paternally expressed, while the growth-suppressing genes should evolve to maternally expressed. (d) The X-linked genes also evolve genomic imprinting, but the main evolutionary force is the sex difference in the optimal embryonic size. I discuss other aberrations that can be explained by the modified versions of the basic model.     

Imprinting in Plants and Its Underlying Mechanisms

Genomic imprinting (or imprinting) refers to an epigenetic phenomenon by which the allelic expression of a gene depends on the parent of origin. It has evolved independently in placental mammals and flowering plants. In plants, imprinting is mainly found in endosperm. Recent genome-wide surveys in Arabidopsis, rice, and maize identified hundreds of imprinted genes in endosperm. Since these genes are of diverse functions, endosperm development is regulated at different regulatory levels. The imprinted expression of only a few genes is conserved between Arabidopsis and monocots, suggesting that imprinting evolved quickly during speciation. In Arabidopsis, DEMETER (DME) mediates hypomethylation in the maternal genome at numerous loci (mainly transposons and repeats) in the central cell and results in many differentially methylated regions between parental genomes in the endosperm, and subsequent imprinted expression of some genes. In addition, histone modification mediated by Polycomb group (PcG) proteins is also involved in regulating imprinting. DME-induced hypomethylated alleles in the central cell are considered to produce small interfering RNAs (siRNAs) which are imported to the egg to reinforce DNA methylation. In parallel, the activity of DME in the vegetative cell of the male gametophyte demethylates many regions which overlap with the demethylated regions in the central cell. siRNAs from the demethylated regions are hypothesized to be also transferred into sperm to reinforce DNA methylation. Imprinting is partly the result of genome-wide epigenetic reprogramming in the central cell and vegetative cell and evolved under different selective pressures.     

Mapping the epistatic network underlying murine reproductive fatpad variation

Genome-wide mapping analyses are now commonplace in many species and several networks of interacting loci have been reported. However, relatively few details regarding epistatic interactions and their contribution to complex trait variation in multicellular organisms are available and the identification of positional candidate loci for epistatic QTL (epiQTL) is hampered, especially in mammals, by the limited genetic resolution inherent in most study designs. Here we further investigate the genetic architecture of reproductive fatpad weight in mice using the F(10) generation of the LG,SM advanced intercross (AI) line. We apply multiple mapping techniques including a single-locus model, locus-specific composite interval mapping (CIM), and tests for multiple QTL per chromosome to the 12 chromosomes known to harbor single-locus QTL (slQTL) affecting obesity in this cross. We also perform a genome-wide scan for pairwise epistasis. Using this combination of approaches we detect 199 peaks spread over all 19 autosomes, which potentially contribute to trait variation including all eight original F(2) loci (Adip1-8), novel slQTL peaks on chromosomes 7 and 9, and several novel epistatic loci. Extensive epistasis is confirmed involving both slQTL confidence intervals (C.I.) as well as regions that show no significant additive or dominance effects. These results provide important new insights into mapping complex genetic architectures and the role of epistasis in complex trait variation.