Thomas Haaf

Epigenetic reprogramming in mouse primordial germ cells

Genome-wide epigenetic reprogramming in mammalian germ cells, zygote and early embryos, plays a crucial role in regulating genome functions at critical stages of development. We show here that mouse primordial germ cells (PGCs) exhibit dynamic changes in epigenetic modifications between days 10.5 and 12.5 post coitum (dpc). First, contrary to previous suggestions, we show that PGCs do indeed acquire genome-wide de novo methylation during early development and migration into the genital ridge. However, following their entry into the genital ridge, there is rapid erasure of DNA methylation of regions within imprinted and non-imprinted loci. For most genes, the erasure commences simultaneously in PGCs in both male and female embryos, which is completed within 1 day of development. Based on the kinetics of this process, we suggest that this is an active demethylation process initiated upon the entry of PGCs into the gonadal anlagen. The timing of reprogramming in PGCs is crucial since it ensures that germ cells of both sexes acquire an equivalent epigenetic state prior to the differentiation of the definitive male and female germ cells in which new parental imprints are established subsequently. Some repetitive elements, however, show incomplete erasure, which may be essential for chromosome stability and for preventing activation of transposons to reduce the risk of germline mutations. Aberrant epigenetic reprogramming in the germ line would cause the inheritance of epimutations that may have consequences for human diseases as suggested by studies on mouse models.

Embryogenesis: Demethylation of the zygotic paternal genome

In mammals, both parental genomes undergo dramatic epigenetic changes after fertilization to form the diploid somatic genome. Here we show that the paternal genome in the mouse is significantly and actively demethylated within 6–8 hours of fertilization, before the onset of DNA replication, whereas the maternal genome is demethylated after several cleavage divisions. This active demethylation of the paternal genome may be associated with epigenetic remodelling of sperm chroma-tin, in order to establish parent-specific developmental programmes during early embryogenesis.

A duplicated copy of DMRT1 in the sex-determining region of the Y chromosome of the medaka, Oryzias latipes

The genes that determine the development of the male or female sex are known in Caenorhabditis elegans, Drosophila, and most mammals. In many other organisms the existence of sex-determining factors has been shown by genetic evidence but the genes are unknown. We have found that in the fish medaka the Y chromosome-specific region spans only about 280 kb. It contains a duplicated copy of the autosomal DMRT1 gene, named DMRT1Y. This is the only functional gene in this chromosome segment and maps precisely to the male sex-determining locus. The gene is expressed during male embryonic and larval development and in the Sertoli cells of the adult testes. These features make DMRT1Y a candidate for the medaka male sex-determining gene.

Conserved synteny between the chicken Z sex chromosome and human chromosome 9 includes the male regulatory gene DMRT1: a comparative (re)view on avian sex determination

Sex-determination mechanisms in birds and mammals evolved independently for more than 300 million years. Unlike mammals, sex determination in birds operates through a ZZ/ZW sex chromosome system, in which the female is the heterogametic sex. However, the molecular mechanism remains to be elucidated. Comparative gene mapping revealed that several genes on human chromosome 9 (HSA 9) have homologs on the chicken Z chromosome (GGA Z), indicating the common ancestry of large parts of GGA Z and HSA 9. Based on chromosome homology maps, we isolated a Z-linked chicken ortholog of DMRT1, which has been implicated in XY sex reversal in humans. Its location on the avian Z and within the sex-reversal region on HSA 9p suggests that DMRT1 represents an ancestral dosage-sensitive gene for vertebrate sex-determination. Z dosage may be crucial for male sexual differentiation/determination in birds.

Metabolic Programming of MEST DNA Methylation by Intrauterine Exposure to Gestational Diabetes Mellitus

Epigenetic processes are primary candidates when searching for mechanisms that can stably modulate gene expression and metabolic pathways according to early life conditions. To test the effects of gestational diabetes mellitus (GDM) on the epigenome of the next generation, cord blood and placenta tissue were obtained from 88 newborns of mothers with dietetically treated GDM, 98 with insulin-dependent GDM, and 65 without GDM. Bisulfite pyrosequencing was used to compare the methylation levels of seven imprinted genes involved in prenatal and postnatal growth, four genes involved in energy metabolism, one anti-inflammatory gene, one tumor suppressor gene, one pluripotency gene, and two repetitive DNA families. The maternally imprinted MEST gene, the nonimprinted glucocorticoid receptor NR3C1 gene, and interspersed ALU repeats showed significantly decreased methylation levels (4–7 percentage points for MEST, 1–2 for NR3C1, and one for ALUs) in both GDM groups, compared with controls, in both analyzed tissues. Significantly decreased blood MEST methylation (3 percentage points) also was observed in adults with morbid obesity compared with normal-weight controls. Our results support the idea that intrauterine exposure to GDM has long-lasting effects on the epigenome of the offspring. Specifically, epigenetic malprogramming of MEST may contribute to obesity predisposition throughout life.

Methylation Reprogramming and Chromosomal Aneuploidy in In Vivo Fertilized and Cloned Rabbit Preimplantation Embryos

Abstract Active demethylation of the paternal genome but not of the maternal genome occurs in fertilized mouse, rat, pig, and bovine zygotes. To study whether this early demethylation wave is important for embryonic development, we have analyzed the global methylation patterns of both in vivo-fertilized and cloned rabbit embryos. Anti-5-methylcytosine immunofluorescence of in vivo-fertilized rabbit embryos revealed that the equally high methylation levels of the paternal and maternal genomes are largely maintained from the zygote up to the 16-cell stage. The lack of detectable methylation changes in rabbit preimplantation embryos suggests that genome-wide demethylation is not an obligatory requirement for epigenetic reprogramming. The methylation patterns of embryos derived from fibroblast and cumulus cell nuclear transfer were similar to those of in vivo-fertilized rabbit embryos. Fluorescence in situ hybridization with chromosome-specific BACs demonstrated significantly increased chromosomal aneuploidy rates in cumulus cell nuclear transfer rabbit embryos and embryos derived from nuclear transfer of rabbit fibroblasts into bovine oocytes compared with in vivo-fertilized rabbit embryos. The incidence of chromosomal abnormalities was correlated with subsequent developmental failure. We propose that postzygotic mitotic errors are one important explanation of why mammalian cloning often fails.

Disruption of CNTNAP2 and additional structural genome changes in a boy with speech delay and autism spectrum disorder

Patients with autism spectrum disorder (ASD) frequently harbour chromosome rearrangements and segmental aneuploidies, which allow us to identify candidate genes. In a boy with mild facial dysmorphisms, speech delay and ASD, we reconstructed by karyotyping, FISH and SNP array-based segmental aneuploidy profiling a highly complex chromosomal rearrangement involving at least three breaks in chromosome 1 and seven breaks in chromosome 7. Chromosome banding revealed an inversion of region 7q32.1–7q35 on the derivative chromosome 7. FISH with region-specific BACs mapped both inversion breakpoints and revealed additional breaks and structural changes in the CNTNAP2 gene. Two gene segments were transposed and inserted into the 1q31.2 region, while the CNTNAP2 segment between the two transposed parts as well as intron 13 to the 5-UTR were retained on the der(7). SNP array analysis revealed an additional de novo deletion encompassing the distal part of intron1 and exon 2 of CNTNAP2, which contains FOXP2 binding sites. Second, we found another de novo deletion on chromosome 1q41, containing 15 annotated genes, including KCTD3 and USH2A. Disruptions of the CNTNAP2 gene have been associated with ASD and with Gilles de la Tourette syndrome (GTS). Comparison of disruptions of CNTNAP2 in patients with GTS and ASD suggests that large proximal disruptions result in either GTS or ASD, while relatively small distal disruptions may be phenotypically neutral. For full-blown ASD to develop, a proximal disruption of CNTNAP2 may have to occur concomitantly with additional genome mutations such as hemizygous deletions of the KCTD3 and USH2A genes.

Spatial, temporal and interindividual epigenetic variation of functionally important DNA methylation patterns

DNA methylation is an epigenetic modification that plays an important role in gene regulation. It can be influenced by stochastic events, environmental factors and developmental programs. However, little is known about the natural variation of gene-specific methylation patterns. In this study, we performed quantitative methylation analyses of six differentially methylated imprinted genes (H19, MEG3, LIT1, NESP55, PEG3 and SNRPN), one hypermethylated pluripotency gene (OCT4) and one hypomethylated tumor suppressor gene (APC) in chorionic villus, fetal and adult cortex, and adult blood samples. Both average methylation level and range of methylation variation depended on the gene locus, tissue type and/or developmental stage. We found considerable variability of functionally important methylation patterns among unrelated healthy individuals and a trend toward more similar methylation levels in monozygotic twins than in dizygotic twins. Imprinted genes showed relatively little methylation changes associated with aging in individuals who are >25 years. The relative differences in methylation among neighboring CpGs in the generally hypomethylated APC promoter may not only reflect stochastic fluctuations but also depend on the tissue type. Our results are consistent with the view that most methylation variation may arise after fertilization, leading to epigenetic mosaicism.

Distribution of telomeric (TTAGGG) (n) sequences in avian chromosomes

Abstract. The physical ends of mammalian and other vertebrate chromosomes consist of tandemly repeated (TTAGGG)n hexamers, nucleating a specialized telomeric structure. However, (TTAGGG)n sequences can also occur at non-telomeric sites, providing important insights into karyotypic evolution. By fluorescence in situ hybridization (FISH) we studied the chromosomal distribution of (TTAGGG)n sequences in 16 bird species, representing seven different orders. Many species, in particular the ratites, display (TTAGGG)n hybridization signals in interstitial and centromeric regions of their macrochromosomes in addition to the typical telomeric signals. In some but not all species these non-telomeric sites coincide with C-band-positive heterochromatin. The retention and/or amplification of telomeric (TTAGGG)n repeats at interstitial and centromeric sites may indicate the fusion of ancestral chromosomes. Compared with the macrochromosomes, the microchromosomes of most species are enriched with (TTAGGG)n sequences, displaying heterogeneous hybridization patterns. We propose that this high density of (TTAGGG)n repeats contributes to the exceptionally high meiotic recombination rate of avian microchromosomes.

5-Azacytidine-induced undercondensations in human chromosomes

SummaryThe cytosine analogue 5-azacytidine induces very distinct undercondensations in human chromosomes if applied to lymphocyte cultures. The number of induced undercondensations and their chromosomal localization can be varied by the 5-azacytidine dose and the treatment time. “Pulverized” chromosomes or undercondensations in the G-band-positive chromosome regions are produced with high doses and long treatment times. If applied in low doses during the last hours of culture, 5-azacytidine induces specific undercondensations in the heterochromatin of chromosomes 1, 9, 15, 16, and Y. Optimum conditions required for inducing the various types of undercondensation in the chromosomes were determined. Various examples of the use 5-azacytidine in the analysis of chromosome rearrangements involving heterochromatic regions are presented.