Robert Feil

Epigenetics and the environment: emerging patterns and implications

Epigenetic phenomena in animals and plants are mediated by DNA methylation and stable chromatin modifications. There has been considerable interest in whether environmental factors modulate the establishment and maintenance of epigenetic modifications, and could thereby influence gene expression and phenotype. Chemical pollutants, dietary components, temperature changes and other external stresses can indeed have long-lasting effects on development, metabolism and health, sometimes even in subsequent generations. Although the underlying mechanisms remain largely unknown, particularly in humans, mechanistic insights are emerging from experimental model systems. These have implications for structuring future research and understanding disease and development.

The Air Noncoding RNA Epigenetically Silences Transcription by Targeting G9a to Chromatin

A number of large noncoding RNAs (ncRNAs) epigenetically silence genes through unknown mechanisms. The Air ncRNA is imprinted—monoallelically expressed from the paternal allele. Air is required for allele-specific silencing of the cis-linked Slc22a3, Slc22a2, and Igf2r genes in mouse placenta. We show that Air interacts with the Slc22a3 promoter chromatin and the H3K9 histone methyltransferase G9a in placenta. Air accumulates at the Slc22a3 promoter in correlation with localized H3K9 methylation and transcriptional repression. Genetic ablation of G9a results in nonimprinted, biallelic transcription of Slc22a3. Truncated Air fails to accumulate at the Slc22a3 promoter, which results in reduced G9a recruitment and biallelic transcription. Our results suggest that Air, and potentially other large ncRNAs, target repressive histone-modifying activities through molecular interaction with specific chromatin domains to epigenetically silence transcription.

Culture of Preimplantation Mouse Embryos Affects Fetal Development and the Expression of Imprinted Genes

Abstract Culture of preimplantation mammalian embryos and cells can influence their subsequent growth and differentiation. Previously, we reported that culture of mouse embryonic stem cells is associated with deregulation of genomic imprinting and affects the potential for these cells to develop into normal fetuses. The purpose of our current study was to determine whether culture of preimplantation mouse embryos in a chemically defined medium (M16) with or without fetal calf serum (FCS) can affect their subsequent development and imprinted gene expression. Only one third of the blastocysts that had been cultured from two-cell embryos in M16 medium complemented with FCS developed into viable Day 14 fetuses after transfer into recipients. These M16 + FCS fetuses were reduced in weight as compared with controls and M16 fetuses and had decreased expression of the imprinted H19 and insulin-like growth factor 2 genes associated with a gain of DNA methylation at an imprinting control region upstream of H19. They also displayed increased expression of the imprinted gene Grb10. The growth factor receptor binding gene Grb7, in contrast, was strongly reduced in its expression in most of the M16 + FCS fetuses. No alterations were detected for the imprinted gene Mest. Preimplantation culture in the presence of serum can influence the regulation of multiple growth-related imprinted genes, thus leading to aberrant fetal growth and development.

Imprinting on distal chromosome 7 in the placenta involves repressive histone methylation independent of DNA methylation.

Imprinted genes are expressed from only one of the parental chromosomes and are marked epigenetically by DNA methylation and histone modifications. The imprinting center 2 (IC2) on mouse distal chromosome 7 is flanked by several paternally repressed genes, with the more distant ones imprinted exclusively in the placenta. We found that most of these genes lack parent-specific DNA methylation, and genetic ablation of methylation does not lead to loss of their imprinting in the trophoblast (placenta). The silent paternal alleles of the genes are marked in the trophoblast by repressive histone modifications (dimethylation at Lys9 of histone H3 and trimethylation at Lys27 of histone H3), which are disrupted when IC2 is deleted, leading to reactivation of the paternal alleles. Thus, repressive histone methylation is recruited by IC2 (potentially through a noncoding antisense RNA) to the paternal chromosome in a region of at least 700 kb and maintains imprinting in this cluster in the placenta, independently of DNA methylation. We propose that an evolutionarily older imprinting mechanism limited to extraembryonic tissues was based on histone modifications, and that this mechanism was subsequently made more stable for use in embryonic lineages by the recruitment of DNA methylation.

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.

Imprinting along the Kcnq1 domain on mouse chromosome 7 involves repressive histone methylation and recruitment of Polycomb group complexes

Imprinted genes are clustered in domains, and their allelic repression is mediated by imprinting control regions. These imprinting control regions are marked by DNA methylation, which is essential to maintain imprinting in the embryo. To explore how imprinting is regulated in placenta, we studied the Kcnq1 domain on mouse distal chromosome 7. This large domain is controlled by an intronic imprinting control region and comprises multiple genes that are imprinted in placenta, without the involvement of promoter DNA methylation. We found that the paternal repression along the domain involves acquisition of trimethylation at Lys27 and dimethylation at Lys9 of histone H3. Eed-Ezh2 Polycomb complexes are recruited to the paternal chromosome and potentially regulate its repressive histone methylation. Studies on embryonic stem cells and early embryos support our proposal that chromatin repression is established early in development and is maintained in the placenta. In the embryo, however, imprinting is stably maintained only at genes that have promoter DNA methylation. These data underscore the importance of histone methylation in placental imprinting and identify mechanistic similarities with X-chromosome inactivation in extraembryonic tissues, suggesting that the two epigenetic mechanisms are evolutionarily linked.

The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart

Hyperpolarization-activated, cyclic nucleotide-gated cation currents, termed If or Ih, are generated by four members of the hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channel family. These currents have been proposed to contribute to several functions including pacemaker activity in heart and brain, control of resting potential, and neuronal plasticity. Transcripts of the HCN4 isoform have been found in cardiomyocytes and neurons, but the physiological role of this channel is unknown. Here we show that HCN4 is essential for the proper function of the developing cardiac conduction system. In wild-type embryos, HCN4 is highly expressed in the cardiac region where the early sinoatrial node develops. Mice lacking HCN4 channels globally, as well as mice with a selective deletion of HCN4 in cardiomyocytes, died between embryonic days 9.5 and 11.5. On average, If in cardiomyocytes from mutant embryos is reduced by 85%. Hearts from HCN4-deficient embryos contracted significantly slower compared with wild type and could not be stimulated by cAMP. In both wild-type and HCN4-/- mice, cardiac cells with “primitive” pacemaker action potentials could be found. However, cardiac cells with “mature” pacemaker potentials, observed in wild-type embryos starting at day 9.0, were not detected in HCN4-deficient embryos. Thus, HCN4 channels are essential for the proper generation of pacemaker potentials in the emerging sinoatrial node.

Physiology and Pathophysiology of Vascular Signaling Controlled by Cyclic Guanosine 3′,5′-Cyclic Monophosphate–Dependent Protein Kinase

Despite impressive medical advances1 that have led to diminished cardiovascular death rates in some countries over the past 20 years, cardiovascular disease remains the leading cause of death in developed countries such as the United States. This promises to worsen as a result of aging populations; the incipient obesity and type II diabetes epidemic; sedentary lifestyle; and continued abuse of tobacco, alcohol, and other substances. Cardiovascular disease is clearly multifactorial, and the approach to its prevention necessarily likewise. Candidates for prevention include cyclic guanosine 3′,5′-cyclic monophosphate (cGMP)–dependent signaling networks initiated by natriuretic peptides (NPs) and nitric oxide (NO), which demonstrate characteristics deemed worthy of diagnostic and therapeutic exploitation. cGMP signaling contributes to the function and interaction of several vascular cell types, and its dysfunction could be involved in major destructive processes such as atherosclerosis, hypertension, diabetic complications, (re)stenosis, and tissue infarction, as well as the undermining of clinical therapy like in the case of nitrate tolerance. This review takes a focused look at key elements of the cGMP signaling cascade in vascular tissue, particularly recent advances in our knowledge of cGMP-dependent protein kinase (cGK, also known as PKG) function. Finally, we discuss the potential of clinical monitoring of cGK activity for assessing the functional status of cGMP signaling and for guiding the design of therapeutic strategies to improve vascular function. One of the 2 major synthetic pathways for cGMP generation from guanosine 5′-triphosphate (GTP) is directed by NPs (Figure 1), consisting of atrial (ANP), B-type (BNP), and C-type (CNP) natriuretic peptides, which act via the membrane receptor guanylate cyclases GC-A (highest affinity for ANP, BNP) and GC-B (highest affinity for CNP). ANP and BNP, released from the heart by mechanical stretch in response to increased atrial pressure/volume, and CNP, released from the endothelium, can all cause smooth muscle (SM) …

Allele‐specific histone lysine methylation marks regulatory regions at imprinted mouse genes

In different eukaryotic model systems, chromatin and gene expression are modulated by post‐translational modification of histone tails. In this in vivo study, histone methylation and acetylation are investigated along the imprinted mouse genes Snrpn, Igf2r and U2af1‐rs1. These imprinted genes all have a CpG‐rich regulatory element at which methylation is present on the maternal allele, and originates from the female germ line. At these ‘differentially methylated regions’ (DMRs), histone H3 on the paternal allele has lysine‐4 methylation and is acetylated. On the maternally inherited allele, in contrast, chromatin is marked by hypermethylation on lysine‐9 of H3. Allele‐specific patterns of lysine‐4 and lysine‐9 methylation are also detected at other regions of the imprinted loci. For the DMR at the U2af1‐rs1 gene, we establish that the methyl‐CpG‐binding‐domain (MBD) proteins MeCP2, MBD1 and MBD3 are associated with the maternal allele. These data support the hypothesis that MBD protein‐associated histone deacetylase/chromatin‐remodelling complexes are recruited to the parental allele that has methylated DNA and H3‐K9 methylation, and are prevented from binding to the opposite allele by H3 lysine‐4 methylation.

Global hypomethylation of the genome in XX embryonic stem cells

Embryonic stem (ES) cells are important tools in the study of gene function and may also become important in cell therapy applications. Establishment of stable XX ES cell lines from mouse blastocysts is relatively problematic owing to frequent loss of one of the two X chromosomes. Here we show that DNA methylation is globally reduced in XX ES cell lines and that this is attributable to the presence of two active X chromosomes. Hypomethylation affects both repetitive and unique sequences, the latter including differentially methylated regions that regulate expression of parentally imprinted genes. Methylation of differentially methylated regions can be restored coincident with elimination of an X chromosome in early-passage parthenogenetic ES cells, suggesting that selection against loss of methylation may provide the basis for X-chromosome instability. Finally, we show that hypomethylation is associated with reduced levels of the de novo DNA methyltransferases Dnmt3a and Dnmt3b and that ectopic expression of these factors restores global methylation levels.