Imprinted gene expression in hybrids: perturbed mechanisms and evolutionary implications.

Diverse mechanisms contribute to the evolution of reproductive barriers, a process that is critical in speciation. Amongst these are alterations in gene products and in gene dosage that affect development and reproductive success in hybrid offspring. Because of its strict parent-of-origin dependence, genomic imprinting is thought to contribute to the aberrant phenotypes observed in interspecies hybrids in mammals and flowering plants, when the abnormalities depend on the directionality of the cross. In different groups of mammals, hybrid incompatibility has indeed been linked to loss of imprinting. Aberrant expression levels have been reported as well, including imprinted genes involved in development and growth. Recent studies in humans emphasize that genetic diversity within a species can readily perturb imprinted gene expression and phenotype as well. Despite novel insights into the underlying mechanisms, the full extent of imprinted gene perturbation still remains to be determined in the different hybrid systems. Here we review imprinted gene expression in intra- and interspecies hybrids and examine the evolutionary scenarios under which imprinting could contribute to hybrid incompatibilities. We discuss effects on development and reproduction and possible evolutionary implications.

Mdm2, but not Mdm4, protects terminally differentiated smooth muscle cells from p53-mediated caspase-3-independent cell death.

p53 is a potent inhibitor of cell growth and an inducer of apoptosis. During embryonic development, Mdm2 and Mdm4 inhibit the growth suppressive activities of p53. However, whether tight surveillance of p53 activity is required in quiescent cells is unknown. To test this, conditional inactivation of mdm2 and mdm4 was carried out in smooth muscle cells (SMCs). Upon SMC-specific inactivation of mdm2, and not of mdm4, mice rapidly became ill and died. Necropsy showed small intestinal dilation, and histological analyses indicated a severe reduction in the number of intestinal SMCs. Increased p53 levels and activity were detected in the remaining SMCs, and the phenotype was completely rescued on a p53-null background. Interestingly, intestinal SMCs are caspase-3-negative and therefore did not undergo caspase-3-dependent apoptotic cell death. Together, Mdm2, but not Mdm4, prevents accumulation of active p53 in quiescent SMCs and thereby the induction of p53-mediated caspase-3-independent cell death.

Genomic imprinting in mammals: an interplay between chromatin and DNA methylation?

Most imprinted loci have key regulatory elements that are methylated on only one of the parental chromosomes. For several of these 'differentially methylated regions', recent studies establish that the unmethylated chromosome has a specialized chromatin organization that is characterized by nuclease hypersensitivity. The novel data raise the question of whether specific proteins and associated chromatin features regulate the allele-specificity of DNA methylation at these imprinting control elements.

Parental allele-specific chromatin configuration in a boundary-imprinting-control element upstream of the mouse H19 gene.

The mouse H19 gene is expressed from the maternal chromosome exclusively. A 2-kb region at 2 to 4 kb upstream of H19 is paternally methylated throughout development, and these sequences are necessary for the imprinted expression of both H19 and the 5'-neighboring Igf2 gene. In particular, on the maternal chromosome this element appears to insulate the Igf2 gene from enhancers located downstream of H19. We analyzed the chromatin organization of this element by assaying its sensitivity to nucleases in nuclei. Six DNase I hypersensitive sites (HS sites) were detected on the unmethylated maternal chromosome exclusively, the two most prominent of which mapped 2.25 and 2.75 kb 5' to the H19 transcription initiation site. Five of the maternal HS sites were present in expressing and nonexpressing tissues and in embryonic stem (ES) cells. They seem, therefore, to reflect the maternal origin of the chromosome rather than the expression of H19. A sixth maternal HS site, at 3.45 kb upstream of H19, was detected in ES cells only. The nucleosomal organization of this element was analyzed in tissues and ES cells by micrococcal nuclease digestion. Specifically on the maternal chromosome, an unusual and strong banding pattern was obtained, suggestive of a nonnucleosomal organization. From our studies, it appears that the unusual chromatin organization with the presence of HS sites (maternal chromosome) and DNA methylation (paternal chromosome) in this element are mutually exclusive and reflect alternate epigenetic states. In addition, our data suggest that nonhistone proteins are associated with the maternal chromosome and that these might be involved in its boundary function.

DNA methylation is linked to deacetylation of histone H3, but not H4, on the imprinted genes Snrpn and U2af1-rs1.

The relationship between DNA methylation and histone acetylation at the imprinted mouse genes U2af1-rs1 and Snrpn is explored by chromatin immunoprecipitation (ChIP) and resolution of parental alleles using single-strand conformational polymorphisms. The U2af1-rs1 gene lies within a differentially methylated region (DMR), while Snrpn has a 5' DMR (DMR1) with sequences homologous to the imprinting control center of the Prader-Willi/Angelman region. For both DMR1 of Snrpn and the 5' untranslated region (5'-UTR) and 3'-UTR of U2af1-rs1, the methylated and nonexpressed maternal allele was underacetylated, relative to the paternal allele, at all H3 lysines tested (K14, K9, and K18). For H4, underacetylation of the maternal allele was exclusively (U2af1-rs1) or predominantly (Snrpn) at lysine 5. Essentially the same patterns of differential acetylation were found in embryonic stem (ES) cells, embryo fibroblasts, and adult liver from F1 mice and in ES cells from mice that were dipaternal or dimaternal for U2af1-rs1. In contrast, in a region within Snrpn that has biallelic methylation in the cells and tissues analyzed, the paternal (expressed) allele showed relatively increased acetylation of H4 but not of H3. The methyl-CpG-binding-domain (MBD) protein MeCP2 was found, by ChIP, to be associated exclusively with the maternal U2af1-rs1 allele. To ask whether DNA methylation is associated with histone deacetylation, we produced mice with transgene-induced methylation at the paternal allele of U2af1-rs1. In these mice, H3 was underacetylated across both the parental U2af1-rs1 alleles whereas H4 acetylation was unaltered. Collectively, these data are consistent with the hypothesis that CpG methylation leads to deacetylation of histone H3, but not H4, through a process that involves selective binding of MBD proteins.

Conditional somatic mutagenesis in the mouse using site-specific recombinases.

In the last decade, site-specific recombinases (SSRs), such as Cre and Flp, have emerged as indispensable tools for the precise in vivo manipulation of the mouse genome. It is now feasible to control, in space and time, the onset of gene knockouts in almost any tissue of the mouse, thus greatly facilitating the creation of sophisticated animal models for human disease and drug development. This review describes the basic principles and current status of the SSR technology, with a focus on strategies for conditional somatic mutagenesis using the Cre/lox system and ligand-activated Cre recombinases. Practical hints for generating and analysing conditional mouse mutants will be given and exciting novel applications of the SSR technology will be discussed, such as cell fate mapping and the combined use of Cre, Flp and other biotechnological tools. It will be shown how genetic manipulation of the mouse by site-specific recombination can provide new solutions to old problems in the analysis of human physiology and pathophysiology and how it can be employed for drug discovery and development.

Genomic imprinting in the placenta.

Genomic imprinting is an epigenetic mechanism that is important for the development and function of the extra-embryonic tissues in the mouse. Remarkably all the autosomal genes which were found to be imprinted in the trophoblast (placenta) only are active on the maternal and repressed on the paternal allele. It was shown for several of these genes that their paternal silencing is not dependent on DNA methylation, at least not in its somatic maintenance. Rather, recent studies in the mouse suggest that placenta-specific imprinting involves repressive histone modifications and non-coding RNAs. This mechanism of autosomal imprinting is similar to imprinted X chromosome inactivation in the placenta. Although the underlying reasons remain to be explored, this suggests that imprinting in the placenta and imprinted X inactivation are evolutionarily related.

Analysis of chromatin in limited numbers of cells: a PCR-SSCP based assay of allele-specific nuclease sensitivity.

Chromatin can be analysed by assaying its sensitivity to DNase I or other nucleases in purified nuclei. Usually, this is performed by Southern analysis of genomic DNA extracted from nuclease-treated nuclei, a methodology that requires many cells. Applying restriction fragment length polymorphisms (RFLPs), this methodology has been used for parental allele-specific chromatin studies on imprinted mammalian genes. However, such allelic studies are limited by the availability of suitable RFLPs. We therefore developed an alternative, PCR and single strand conformation polymorphism (SSCP)-based assay with which allelic sensitivity to nucleases can be determined in virtually all localised regions that have nucleotide polymorphisms. We also demonstrate that analysis of DNase I sensitivity can be performed on permeabilised cells. Combining the two approaches, in the imprinted mouse U2af1-rs1 gene we analysed parental allele-specific chromatin conformation in limited numbers of cultured cells. We also applied the PCR-SSCP approach to assay allelic DNA methylation at specific restriction enzyme sites. In summary, we developed an allele-specific assay that should be useful for biochemical and developmental investigation of chromatin, in particular for studies on genomic imprinting and X-chromosome inactivation.

Distribution of cGMP-dependent protein kinase type I and its isoforms in the mouse brain and retina.

Nitric oxide (NO) modulates a variety of processes in the mammalian brain, but the mechanisms of neuronal NO signaling are poorly understood. In the periphery, many effects of NO are mediated via the generation of the second messenger cyclic guanosine-3',5'-monophosphate (cGMP) and activation of the cGMP-dependent protein kinase type I (cGKI). However, previous studies suggested that the expression of cGKI in the nervous system is rather restricted, thus, questioning the functional significance of the cGMP/cGKI pathway as a mediator of NO signaling in the brain. Here we have performed a detailed immunohistochemical study to elucidate the distribution of cGKI in the CNS and eye of the mouse. Expression of cGKI protein was detected not only in the previously described areas (cerebellum, hippocampus, dorsomedial hypothalamus) but also in a number of additional regions, such as medulla, subcommissural organ, cerebral cortex, amygdala, habenulae, various hypothalamic regions, olfactory bulb, pituitary gland, and retina. Immunoblotting with isoform-specific antibodies indicated that the cGKIalpha isoform is prominent in the cerebellum and medulla, whereas the cGKIbeta isoform is predominant in the cortex, hippocampus, hypothalamus, and olfactory bulb. Similar levels of the isoforms were detected in the pituitary gland and eye. Thus, it appears that distinct brain regions express distinct cGKI isoforms that signal via distinct pathways. Together, these results improve our understanding of the cellular and molecular mechanisms of NO/cGMP/cGKI signaling and indicate that the distribution and functional relevance of this pathway in the mammalian brain is broader than previously thought.

Engineering the mouse genome by site-specific recombination.

Site-specific recombination systems are powerful tools for introducing predetermined modifications into eukaryotic genomes. Recent advances allow the manipulation of chromosomal DNA in a spatially and temporally controlled manner in mice, offering unprecedented possibilities for studying mammalian genome function and for generating animal models for human diseases.

Igf2 imprinting in development and disease.

Igf2 is one of the first imprinted genes discovered and occupies a centre stage in the study of imprinting. This is because it has dramatic effects on the control of fetal growth, it is involved in growth disorders and in cancer, it interacts with products of other imprinted genes, and its imprinting status is under complex regulation in a cluster of tightly linked imprinted genes. Here we review briefly the key features of Igf2 imprinting in normal development and in disease, and hope to show what a fascinating subject of study this gene and its biology provides.

[The German Cartilage Registry (KnorpelRegister DGOU) for evaluation of surgical treatment for cartilage defects: experience after six months including first demographic data]. / Das KnorpelRegister DGOU zur Erfassung von Behandlungsergebnissen nach Knorpeloperationen: Erfahrungen nach 6 Monaten und erste epidemiologische Daten.

The German Cartilage Registry (KnorpelRegister DGOU) has been introduced in October 2013 and aims on the evaluation of patients who underwent cartilage repair for symptomatic cartilage defects. It represents a nation-wide cohort study which has been introduced by the working group "Tissue Regeneration" of the Germany Society of Orthopaedic Surgery and Traumatology and is technically based upon a web-based remote data entry (RDE) system. The present article describes first experiences with the registry including patient and treatment characteristics. Between October 2013 and April 2014, a total of 230 patients who had undergone surgical cartilage repair for symptomatic full-thickness cartilage defects of the knee has been included in the German Cartilage Registry from 23 cartilage repair centres. Mean age was 37.11 years (SD 13.61) and mean defect size was 3.68 cm(2) (SD 0.23). Since the introduction of the KnorpelRegister DGOU the total number of registered patients has increased steadily up to the most recent figure of 72 patients within one month. Patients were treated mainly according to the recommended therapies. The highest percentage in therapy is represented by the bone marrow stimulation techniques (55.02 %) as well as by the autologous chondrocyte transplantation (34.92 %). Unlike the patient collective in the majority of prospective randomised controlled trials, the patient population within the registry shows a high proportion of patients with accompanying pathologies, with an age of more than 50 years at the time of treatment and with unfavourably assessed accompanying pathologies such as an affection of the opposite cartilage surface or a previously resected meniscus. In summary, the technical platform and forms of documentation of the KnorpelRegister DGOU have proved to be very promising within the first six months. Unlike data from other clinical trials, the previous analysis of the patients' data and therapies reflects successfully the actual medical care situation of patients with cartilage defects of the knee joint. This analysis also provides new information on subgroups of patients that have not yet been recorded in the scientific literature. This will be part of the first analysis of clinical treatment data. An expansion of the KnorpelRegister DGOU to patients with cartilage defects of the ankle and hip joints is already decided upon and initialised.

Nucleotide sequence of a 28-kb mouse genomic region comprising the imprinted Igf2 gene.

The mouse insulin-like growth factor II gene (Igf2) is physically linked to the insulin II gene (Ins2) and both are subject to tissue-specific genomic imprinting. The paternal-specific expression of Igf2 has been associated with hypermethylation of some CpG sites in the 5' flanking region and in the body of the gene. As a first step in analyzing the structural features of this imprinted locus, we here report the complete nucleotide sequence of Igf2, including all introns and the intergenic region adjacent to Ins2. This 28-kb segment of mouse chromosome 7 exhibits 80% overall identity with the corresponding rat sequence and has a high GC content of 52%. In addition to the known CpG island within the second Igf2 promoter, another island was identified approximately 2 kb 5' to the first exon. Other features of this locus include a 35-fold tandem repeat of an 11-bp sequence that overlaps Igf2 pseudo-exon 2, and a B2 repeat element in the intergenic region between Ins2 and Igf2. The GC-richness and the presence of CpG islands associated with tandem repeats are common features of imprinted genes and thus may play a role in the imprinting mechanism.

High-level expression of functional cGMP-dependent protein kinase using the baculovirus system.

The understanding of the structure and function of cGMP-dependent protein kinase (cGMP kinase) has been hindered by the difficulty to obtain large quantities of functional enzyme. A recombinant baculovirus encoding bovine cGMP kinase I alpha was constructed and purified. Infected insect cells synthesized large amounts of soluble and biologically active cGMP kinase I alpha representing up to 10% of the total cell extract protein. The recombinant enzyme had an identical apparent molecular mass, cGMP affinity and kinase activity as the native bovine lung enzyme. The high-level expression of functional cGMP kinase I alpha should provide an excellent tool to study further the structure and function of cGMP kinase.

Differential patterns of histone methylation and acetylation distinguish active and repressed alleles at X-linked genes.

In female mammals, one of the two X chromosomes is inactivated to compensate for the difference in dosage of X-linked genes between males and females. X inactivation involves sequential alterations to the chromatin that ultimately lead to the transcriptional repression of genes on the X chromosome. Here, histone methylation and acetylation along X-linked genes are investigated by chromatin immunoprecipitation (ChIP) of adult fibroblast cell lines. At PGK1 and HPRT, chromatin on the active X chromosome reveals H3 lysine 4 methylation and acetylation of histones H3 and H4. These modifications are absent on the repressed allele, which is marked by H3 lysine 9 methylation. On the expressed allele of XIST (on the inactive X chromosome), we found that H3 acetylation is confined to the promoter, whereas H3 lysine 4 methylation and H4 acetylation are present along the entire gene. On the repressed XIST allele, in contrast, the promoter and gene exhibit H3 lysine 9 methylation. At only 1.5 kb upstream of the XIST gene, chromatin on the inactive X chromosome has strongly reduced levels of H4 acetylation and is marked by both H3 lysine 9 and H3 lysine 4 methylation. These data demonstrate that patterns of histone methylation and acetylation are distinct along and upstream of XIST and suggest that the inactive X chromatin configuration occurs at a region close to the 5' end of the gene.

Environmental effects on genomic imprinting in mammals.

Genomic imprinting is an epigenetic marking mechanism by which certain genes become repressed on one of the two parental alleles. Imprinting plays important roles in mammalian development, and in humans its deregulation may result in disease and carcinogenesis. During different medical, technological and scientific interventions, pre-implantation embryos and cells are taken from their natural environment and subjected to culture in artificial media. Studies in the mouse demonstrate that environmental stress, such as in vitro culture, can affect the somatic maintenance of epigenetic marks at imprinted loci. These effects are associated with aberrant growth and morphology at fetal and perinatal stages of development.

Attitudes toward abortion as a means of sex selection.

A questionnaire was administered to 317 unmarried male and female college students in order to determine the acceptability of abortion as a means of achieving offspring of a desired sex. Respondents were given three hypothetical situations (first, second, and last child) to rate, comparing the present method of amniocentesis followed by abortion with proposed simpler methods. Acceptance ranged from 4.2% to 40.3%, being greatest among the less religious, male, and Protestant respondents. Proposed methods were only slightly more acceptable than the present one. Thus, simplifying the abortion procedure for the purpose of sex selection would not appear to increase its acceptability or utilization.

Upregulation of non-ß cell-derived vascular endothelial growth factor A increases small clusters of insulin-producing cells in the pancreas.

Pancreatic ß cell-derived vascular endothelial growth factor A (VEGF-A) contributes to normal ß cell function. We therefore hypothesized that non-ß cell-derived VEGF-A may affect its properties in adult mice.We generated transgenic mice expressing human VEGF-A (hVEGF-A) in a visceral smooth muscle cell (SMC)-dominant manner under the control of the transgelin (Tagln/SM22α) promoter via a tamoxifen-induced Cre/loxP recombination system (SM-CreER(T2)/hVEGF mice).SM-CreER(T2)/hVEGF mice received tamoxifen orally followed by microscopic examination of their pancreas 4 weeks after the hVEGF-A induction. The number of clusters of insulin-producing cells (IPCs) in islets, pancreatic ducts, and individual IPCs were counted.The number of small IPC clusters (100-215 µm(2)) in the pancreas increased significantly in SM-CreER(T2)/hVEGF mice compared with SM-CreER(T2)(Ki) mice (473 out of 1 992 counts vs. 199 out of 976 counts, p<0.05), although total IPC area and the number of pancreatic duct IPCs, in proportion to exocrine area, were similar between the 2 groups. Although most small IPC clusters observed in SM-CreER(T2)/hVEGF mice were not accompanied by α and/or δ cells, some were attached to a single or a few α cells. An STZ-induced diabetic state in SM-CreER(T2)/hVEGF mice was slightly ameliorated, with only one point of significance 12 weeks after STZ administration, compared with SM-CreER(T2)(Ki) mice.Upregulation of non-ß cell-derived VEGF-A may alter the composition of pancreatic IPCs by increasing the number of small IPC clusters. These findings provide new information on the role of non-ß cell-derived VEGF-A to IPC regeneration and insulin production.

Partial loss of contractile marker proteins in human testicular peritubular cells in infertility patients.

Fibrotic remodelling of the testicular tubular wall is common in human male infertility caused by impaired spermatogenesis. We hypothesized that this morphological change bears witness of an underlying fundamentally altered state of the cells building this wall, that is, peritubular smooth muscle-like cells. This could include a loss of the contractile abilities of these cells and thus be a factor in male infertility. Immune cells are increased in the tubular wall in these cases, hence local immune cell-related factors, including a prostaglandin (PG) metabolite may be involved. To explore these points in the human, we used testicular biopsies, in which tubules with normal spermatogenesis and impaired spermatogenesis are next to each other [mixed atrophy (MA)], normal biopsies and cultured human testicular peritubular cells. Proteins essential for contraction, myosin heavy chain (MYH11), calponin (Cal) and relaxation, cGMP-dependent protein kinase 1 (cGKI), were readily detected by immunohistochemistry and were equally distributed in all peritubular cells of biopsies with normal spermatogenesis. In all biopsies, vascular smooth muscle cells also stained and served as important intrinsic controls, which showed that in MA samples when spermatogenesis was impaired, staining was restricted to only few peritubular cells or was absent. When spermatogenesis was normal, regular peritubular staining became obvious. This pattern suggests complex regulatory influences, which in face of the identical systemic hormonal situation in MA patients, are likely caused by the local testicular micromilieu. The PG metabolite 15dPGJ2 may represent such a factor and it reduced Cal protein levels in peritubular cells from patients with/without impaired spermatogenesis. The documented phenotypic switch of peritubular, smooth muscle-like cells in MA patients may impair the abilities of the afflicted seminiferous tubules to contract and relax and must now be considered as a part of the complex events in male infertility.

Child health, developmental plasticity, and epigenetic programming.

Plasticity in developmental programming has evolved in order to provide the best chances of survival and reproductive success to the organism under changing environments. Environmental conditions that are experienced in early life can profoundly influence human biology and long-term health. Developmental origins of health and disease and life-history transitions are purported to use placental, nutritional, and endocrine cues for setting long-term biological, mental, and behavioral strategies in response to local ecological and/or social conditions. The window of developmental plasticity extends from preconception to early childhood and involves epigenetic responses to environmental changes, which exert their effects during life-history phase transitions. These epigenetic responses influence development, cell- and tissue-specific gene expression, and sexual dimorphism, and, in exceptional cases, could be transmitted transgenerationally. Translational epigenetic research in child health is a reiterative process that ranges from research in the basic sciences, preclinical research, and pediatric clinical research. Identifying the epigenetic consequences of fetal programming creates potential applications in clinical practice: the development of epigenetic biomarkers for early diagnosis of disease, the ability to identify susceptible individuals at risk for adult diseases, and the development of novel preventive and curative measures that are based on diet and/or novel epigenetic drugs.