Genome-wide identification of genes regulating DNA methylation using genetic anchors for causal inference

BACKGROUND: DNA methylation is a key epigenetic modification in human development and disease, yet there is limited understanding of its highly coordinated regulation. Here, we identify 818 genes that affect DNA methylation patterns in blood using large-scale population genomics data. RESULTS: By employing genetic instruments as causal anchors, we establish directed associations between gene expression and distant DNA methylation levels, while ensuring specificity of the associations by correcting for linkage disequilibrium and pleiotropy among neighboring genes. The identified genes are enriched for transcription factors, of which many consistently increased or decreased DNA methylation levels at multiple CpG sites. In addition, we show that a substantial number of transcription factors affected DNA methylation at their experimentally determined binding sites. We also observe genes encoding proteins with heterogenous functions that have widespread effects on DNA methylation, e.g., NFKBIE, CDCA7(L), and NLRC5, and for several examples, we suggest plausible mechanisms underlying their effect on DNA methylation. CONCLUSION: We report hundreds of genes that affect DNA methylation and provide key insights in the principles underlying epigenetic regulation

A Restricted Role for FcγR in the Regulation of Adaptive Immunity.

By their interaction with IgG immune complexes, FcγR and complement link innate and adaptive immunity, showing functional redundancy. In complement-deficient mice, IgG downstream effector functions are often impaired, as well as adaptive immunity. Based on a variety of model systems using FcγR-knockout mice, it has been concluded that FcγRs are also key regulators of innate and adaptive immunity; however, several of the model systems underpinning these conclusions suffer from flawed experimental design. To address this issue, we generated a novel mouse model deficient for all FcγRs (FcγRI/II/III/IV-/- mice). These mice displayed normal development and lymphoid and myeloid ontogeny. Although IgG effector pathways were impaired, adaptive immune responses to a variety of challenges, including bacterial infection and IgG immune complexes, were not. Like FcγRIIb-deficient mice, FcγRI/II/III/IV-/- mice developed higher Ab titers but no autoantibodies. These observations indicate a redundant role for activating FcγRs in the modulation of the adaptive immune response in vivo. We conclude that FcγRs are downstream IgG effector molecules with a restricted role in the ontogeny and maintenance of the immune system, as well as the regulation of adaptive immunity

siRNA–Mediated Methylation of Arabidopsis Telomeres

Chromosome termini form a specialized type of heterochromatin that is important for chromosome stability. The recent discovery of telomeric RNA transcripts in yeast and vertebrates raised the question of whether RNA–based mechanisms are involved in the formation of telomeric heterochromatin. In this study, we performed detailed analysis of chromatin structure and RNA transcription at chromosome termini in Arabidopsis. Arabidopsis telomeres display features of intermediate heterochromatin that does not extensively spread to subtelomeric regions which encode transcriptionally active genes. We also found telomeric repeat–containing transcripts arising from telomeres and centromeric loci, a portion of which are processed into small interfering RNAs. These telomeric siRNAs contribute to the maintenance of telomeric chromatin through promoting methylation of asymmetric cytosines in telomeric (CCCTAAA)n repeats. The formation of telomeric siRNAs and methylation of telomeres relies on the RNA–dependent DNA methylation pathway. The loss of telomeric DNA methylation in rdr2 mutants is accompanied by only a modest effect on histone heterochromatic marks, indicating that maintenance of telomeric heterochromatin in Arabidopsis is reinforced by several independent mechanisms. In conclusion, this study provides evidence for an siRNA–directed mechanism of chromatin maintenance at telomeres in Arabidopsis

Absent B Cells, agammaglobulinemia, and Hypertrophic Cardiomyopathy in Folliculin-interacting Protein 1 Deficiency

Agammaglobulinemia is the most profound primary antibody deficiency that can occur due to an early termination of B-cell development. We here investigated 3 novel patients, including the first known adult, from unrelated families with agammaglobulinemia, recurrent infections, and hypertrophic cardiomyopathy (HCM). Two of them also presented with intermittent or severe chronic neutropenia. We identified homozygous or compound-heterozygous variants in the gene for folliculin interacting protein 1 (FNIP1), leading to loss of the FNIP1 protein. B-cell metabolism, including mitochondrial numbers and activity and phosphatidylinositol 3-kinase/AKT pathway, was impaired. These defects recapitulated the Fnip1-/- animal model. Moreover, we identified either uniparental disomy or copy-number variants (CNVs) in 2 patients, expanding the variant spectrum of this novel inborn error of immunity. The results indicate that FNIP1 deficiency can be caused by complex genetic mechanisms and support the clinical utility of exome sequencing and CNV analysis in patients with broad phenotypes, including agammaglobulinemia and HCM. FNIP1 deficiency is a novel inborn error of immunity characterized by early and severe B-cell development defect, agammaglobulinemia, variable neutropenia, and HCM. Our findings elucidate a functional and relevant role of FNIP1 in B-cell development and metabolism and potentially neutrophil activity

Autosomal genetic variation is associated with DNA methylation in regions variably escaping X-chromosome inactivation

X-chromosome inactivation (XCI), i.e., the inactivation of one of the female X chromosomes, restores equal expression of X-chromosomal genes between females and males. However, similar to 10% of genes show variable degrees of escape from XCI between females, although little is known about the causes of variable XCI. Using a discovery data-set of 1867 females and 1398 males and a replication sample of 3351 females, we show that genetic variation at three autosomal loci is associated with female-specific changes in X-chromosome methylation. Through cis-eQTL expression analysis, we map these loci to the genes SMCHD1/METTL4, TRIM6/HBG2, and ZSCAN9. Low-expression alleles of the loci are predominantly associated with mild hypomethylation of CpG islands near genes known to variably escape XCI, implicating the autosomal genes in variable XCI. Together, these results suggest a genetic basis for variable escape from XCI and highlight the potential of a population genomics approach to identify genes involved in XCI

Autosomal genetic variation is associated with DNA methylation in regions variably escaping X-chromosome inactivation

X-chromosome inactivation (XCI), i.e., the inactivation of one of the female X chromosomes, restores equal expression of X-chromosomal genes between females and males. However, ~10% of genes show variable degrees of escape from XCI between females, although little is known about the causes of variable XCI. Using a discovery data-set of 1867 females and 1398 males and a replication sample of 3351 females, we show that genetic variation at three autosomal loci is associated with female-specific changes in X-chromosome methylation. Through cis-eQTL expression analysis, we map these loci to the genes SMCHD1/METTL4, TRIM6/HBG2, and ZSCAN9. Low-expression alleles of the loci are predominantly associated with mild hypomethylation of CpG islands near genes known to variably escape XCI, implicating the autosomal genes in variable XCI. Together, these results suggest a genetic basis for variable escape from XCI and highlight the potential of a population genomics approach to identify genes involved in XCI

Heterochromatin Understanding transgenerational epigenetic inheritance via the gametes in mammals

For the past 60 years, human genetic research has focused on DNA as the heritable molecule that carries information about phenotype from the parent to the offspring. Mutations in single genes or a small number of genes have been tightly linked to some phenotypes, but for most phenotypes the situation is more complex and, in many cases, environmental factors are involved. In these instances, genome-wide association studies (GWASs) have enabled the identification of SNPs that are weakly associated with increased disease risk, but the odds ratios are generally small, and it remains impossible to predict phenotype at an individual level. In parallel, molecular biologists using animal models have realized that, in addition to DNA sequence, there are a number of other layers of information, termed epigenetic marks (BOX 1), that influence transcription. These epigenetic marks are fairly stable over the lifetime of an individual and have a role in determining phenotype. At some loci, the epigenetic marks are not tightly linked to the DNA sequence of the genome; both probabilistic and environmental events can influence the establishment of epigenetic states at these loci 1 . Considering the epigenome as well as the genome may allow us to develop better tools for predicting phenotype at an individual level. Moreover, there is evidence that epigenetic marks can sometimes be transmitted from parent to offspring via the gametes, and studies have been published in the past couple of years that support this idea. In this Review, we describe the evidence for this form of inheritance, focusing on mammals but also looking at informative examples from other species. The molecular nature of the epigenetic marks that are inherited is unknown in most cases, but the recent emergence of high-throughput sequencing technologies makes this problem tractable. An emerging theme in cases of transgenerational epigenetic inheritance via the gametes (BOX 1) is the involvement of repeats and transposable elements, and recent progress in our understanding of the establishment of heterochromatin at repeats reveals the importance of RNA; this raises the possibility that RNA may have a role in transgenerational epigenetic inheritance via the gametes. Evidence in mammals Reprogramming of the epigenome. The epigenetic marks that are established in most tissues during an organism's lifetime are irrelevant with respect to the next generation. Only those of the mature gametes have the potential to contribute to the phenotype of the offspring. Moreover, there is considerable reprogramming between generations -and, in particular, of the gametic epigenome immediately after fertilization -to endow the cells of the early pre-implantation embryo with the capacity to differentiate into all cell types of a fully developed organism. Studies carried out in mice more than 30 years ago found that global DNA methylation levels, which were analysed using methylation-sensitive restriction enzymes, were much lower just after fertilization compared to those found in mature gametes and after implantation 2 . The idea that DNA methylation erasure and resetting is the basis of epigenetic reprogramming emerged from this finding 2 . However, our understanding of the function of DNA methylation Heterochromatin The portion of the genome that stays highly condensed throughout the cell cycle. It contains a high proportion of repetitive sequences, is gene-poor overall and is enriched for histone marks, such as histone H3 lysine 9 trimethylation (H3K9me3) and H4K20me3, as well as DNA methylation. Heterochromatin is generally associated with gene silencing. Understanding transgenerational epigenetic inheritance via the gametes in mammals Lucia Daxinger and Emma Whitelaw Abstract | It is known that information that is not contained in the DNA sequenceepigenetic information -can be inherited from the parent to the offspring. However, many questions remain unanswered regarding the extent and mechanisms of such inheritance. In this Review, we consider the evidence for transgenerational epigenetic inheritance via the gametes, including cases of environmentally induced epigenetic changes. The molecular basis of this inheritance remains unclear, but recent evidence points towards diffusible factors, in particular RNA, rather than DNA methylation or chromatin. Interestingly, many cases of epigenetic inheritance seem to involve repeat sequences

Heterochromatin Understanding transgenerational epigenetic inheritance via the gametes in mammals

For the past 60 years, human genetic research has focused on DNA as the heritable molecule that carries information about phenotype from the parent to the offspring. Mutations in single genes or a small number of genes have been tightly linked to some phenotypes, but for most phenotypes the situation is more complex and, in many cases, environmental factors are involved. In these instances, genome-wide association studies (GWASs) have enabled the identification of SNPs that are weakly associated with increased disease risk, but the odds ratios are generally small, and it remains impossible to predict phenotype at an individual level. In parallel, molecular biologists using animal models have realized that, in addition to DNA sequence, there are a number of other layers of information, termed epigenetic marks (BOX 1), that influence transcription. These epigenetic marks are fairly stable over the lifetime of an individual and have a role in determining phenotype. At some loci, the epigenetic marks are not tightly linked to the DNA sequence of the genome; both probabilistic and environmental events can influence the establishment of epigenetic states at these loci 1 . Considering the epigenome as well as the genome may allow us to develop better tools for predicting phenotype at an individual level. Moreover, there is evidence that epigenetic marks can sometimes be transmitted from parent to offspring via the gametes, and studies have been published in the past couple of years that support this idea. In this Review, we describe the evidence for this form of inheritance, focusing on mammals but also looking at informative examples from other species. The molecular nature of the epigenetic marks that are inherited is unknown in most cases, but the recent emergence of high-throughput sequencing technologies makes this problem tractable. An emerging theme in cases of transgenerational epigenetic inheritance via the gametes (BOX 1) is the involvement of repeats and transposable elements, and recent progress in our understanding of the establishment of heterochromatin at repeats reveals the importance of RNA; this raises the possibility that RNA may have a role in transgenerational epigenetic inheritance via the gametes. Evidence in mammals Reprogramming of the epigenome. The epigenetic marks that are established in most tissues during an organism's lifetime are irrelevant with respect to the next generation. Only those of the mature gametes have the potential to contribute to the phenotype of the offspring. Moreover, there is considerable reprogramming between generations -and, in particular, of the gametic epigenome immediately after fertilization -to endow the cells of the early pre-implantation embryo with the capacity to differentiate into all cell types of a fully developed organism. Studies carried out in mice more than 30 years ago found that global DNA methylation levels, which were analysed using methylation-sensitive restriction enzymes, were much lower just after fertilization compared to those found in mature gametes and after implantation 2 . The idea that DNA methylation erasure and resetting is the basis of epigenetic reprogramming emerged from this finding 2 . However, our understanding of the function of DNA methylation Heterochromatin The portion of the genome that stays highly condensed throughout the cell cycle. It contains a high proportion of repetitive sequences, is gene-poor overall and is enriched for histone marks, such as histone H3 lysine 9 trimethylation (H3K9me3) and H4K20me3, as well as DNA methylation. Heterochromatin is generally associated with gene silencing. Understanding transgenerational epigenetic inheritance via the gametes in mammals Lucia Daxinger and Emma Whitelaw Abstract | It is known that information that is not contained in the DNA sequenceepigenetic information -can be inherited from the parent to the offspring. However, many questions remain unanswered regarding the extent and mechanisms of such inheritance. In this Review, we consider the evidence for transgenerational epigenetic inheritance via the gametes, including cases of environmentally induced epigenetic changes. The molecular basis of this inheritance remains unclear, but recent evidence points towards diffusible factors, in particular RNA, rather than DNA methylation or chromatin. Interestingly, many cases of epigenetic inheritance seem to involve repeat sequences. REVIEWS NATURE REVIEWS | GENETICS VOLUME 13 | MARCH 2012 | 15

Transgenerational epigenetic inheritance: More questions than answers

Epigenetic modifications are widely accepted as playing a critical role in the regulation of gene expression and thereby contributing to the determination of the phenotype of multicellular organisms. In general, these marks are cleared and re-established each generation, but there have been reports in a number of model organisms that at some loci in the genome this clearing is incomplete. This phenomenon is referred to as transgenerational epigenetic inheritance. Moreover, recent evidence shows that the environment can stably influence the establishment of the epigenome. Together, these findings suggest that an environmental event in one generation could affect the phenotype in subsequent generations, and these somewhat Lamarckian ideas are stimulating interest from a broad spectrum of biologists, from ecologists to health workers