Prenatal correction of IGF2 to rescue the growth phenotypes in mouse models of Beckwith-Wiedemann and Silver-Russell syndromes.

Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) are imprinting disorders manifesting as aberrant fetal growth and severe postnatal-growth-related complications. Based on the insulator model, one-third of BWS cases and two-thirds of SRS cases are consistent with misexpression of insulin-like growth factor 2 (IGF2), an important facilitator of fetal growth. We propose that the IGF2-dependent BWS and SRS cases can be identified by prenatal diagnosis and can be prevented by prenatal intervention targeting IGF2. We test this hypothesis using our mouse models of IGF2-dependent BWS and SRS. We find that genetically normalizing IGF2 levels in a double rescue experiment corrects the fetal overgrowth phenotype in the BWS model and the growth retardation in the SRS model. In addition, we pharmacologically rescue the BWS growth phenotype by reducing IGF2 signaling during late gestation. This animal study encourages clinical investigations to target IGF2 for prenatal diagnosis and prenatal prevention in human BWS and SRS.

High type I error and misrepresentations in search for transgenerational epigenetic inheritance: response to Guerrero-Bosagna.

In a recent paper, we described our efforts in search for evidence supporting epigenetic transgenerational inheritance caused by endocrine disrupter chemicals. One aspect of our study was to compare genome-wide DNA methylation changes in the vinclozolin-exposed fetal male germ cells (n = 3) to control samples (n = 3), their counterparts in the next, unexposed, generation (n = 3 + 3) and also in adult spermatozoa (n = 2 + 2) in both generations. We reported finding zero common hits in the intersection of these four comparisons. In our interpretation, this result did not support the notion that DNA methylation provides a mechanism for a vinclozolin-induced transgenerational male infertility phenotype. In response to criticism by Guerrero-Bosagna regarding our statistical power in the above study, here we provide power calculations to clarify the statistical power of our study and to show the validity of our conclusions. We also explain here how our data is misinterpreted in the commentary by Guerrero-Bosagna by leaving out important data points from consideration.Please see related Correspondence article: xxx (13059_2016_982) and related Research article: http://genomebiology.biomedcentral.com/articles/10.1186/s13059-015-0619-z.

Deleterious effects of endocrine disruptors are corrected in the mammalian germline by epigenome reprogramming.

BACKGROUND: Exposure to environmental endocrine-disrupting chemicals during pregnancy reportedly causes transgenerationally inherited reproductive defects. We hypothesized that to affect the grandchild, endocrine-disrupting chemicals must alter the epigenome of the germ cells of the in utero-exposed G1 male fetus. Additionally, to affect the great-grandchild, the aberration must persist in the germ cells of the unexposed G2 grandchild. RESULTS: Here, we treat gestating female mice with vinclozolin, bisphenol A, or di-(2-ethylhexyl)phthalate during the time when global de novo DNA methylation and imprint establishment occurs in the germ cells of the G1 male fetus. We map genome-wide features in purified G1 and G2 prospermatogonia, in order to detect immediate and persistent epigenetic aberrations, respectively. We detect changes in transcription and methylation in the G1 germline immediately after endocrine-disrupting chemicals exposure, but changes do not persist into the G2 germline. Additional analysis of genomic imprints shows no persistent aberrations in DNA methylation at the differentially methylated regions of imprinted genes between the G1 and G2 prospermatogonia, or in the allele-specific transcription of imprinted genes between the G2 and G3 soma. CONCLUSIONS: Our results suggest that endocrine-disrupting chemicals exert direct epigenetic effects in exposed fetal germ cells, which are corrected by reprogramming events in the next generation. Avoiding transgenerational inheritance of environmentally-caused epigenetic aberrations may have played an evolutionary role in the development of dual waves of global epigenome reprogramming in mammals.

Characterization of the imprinting signature of mouse embryo fibroblasts by RNA deep sequencing.

Mouse embryo fibroblasts (MEFs) are convenient sources for biochemical studies when cell number in mouse embryos is limiting. To derive the imprinting signature of MEFs and potentially detect novel imprinted genes we performed strand- and allele-specific RNA deep sequencing. We used sequenom allelotyping in embryo and adult organs to verify parental allele-specific expression. Thirty-two known ubiquitously imprinted genes displayed correct parental allele-specific transcripts in MEFs. Our analysis did not reveal any novel imprinted genes, but detected extended parental allele-specific transcripts in several known imprinted domains: maternal allele-specific transcripts downstream of Grb10 and downstream of Meg3, Rtl1as and Rian in the Dlk1-Dio3 cluster, an imprinted domain implicated in development and pluripotency. We detected paternal allele-specific transcripts downstream of Nespas, Peg3, Peg12 and Snurf/Snrpn. These imprinted transcript extensions were not unique to MEFs, but were also present in other somatic cells. The 5' end points of the imprinted transcript extensions did not carry opposing chromatin marks or parental allele-specific DNA methylation, suggesting that their parental allele-specific transcription is under the control of the extended imprinted genes. Based on the imprinting signature of MEFs, these cells provide valid models for understanding the biochemical aspects of genomic imprinting.

De novo DNA methylation in the male germ line occurs by default but is excluded at sites of H3K4 methylation.

To understand what dictates the emerging patterns of de novo DNA methylation in the male germline, we mapped DNA methylation, chromatin, and transcription changes in purified fetal mouse germ cells by using methylated CpG island recovery assay (MIRA)-chip, chromatin immunoprecipitation (ChIP)-chip, and strand-specific RNA deep sequencing, respectively. Global de novo methylation occurred by default in prospermatogonia without any apparent trigger from preexisting repressive chromatin marks but was preceded by broad, low-level transcription along the chromosomes, including the four known paternally imprinted differentially methylated regions (DMRs). Default methylation was excluded only at precisely aligned constitutive or emerging peaks of H3K4me2, including most CpG islands and some intracisternal A particles (IAPs). Similarly, each maternally imprinted DMR was protected from default DNA methylation among highly methylated DNA by an H3K4me2 peak and transcription initiation at least in one strand. Our results suggest that the pattern of de novo DNA methylation in prospermatogonia is dictated by opposing actions of broad, low-level transcription and dynamic patterns of active chromatin.

Transcriptional repression by the proximal exonic region at the human TERT gene.

In humans, the enzyme telomerase (hTERT) is responsible for the synthesis of new repeat sequences at the telomeres of chromosomes. Although active in early embryogenesis, the hTERT gene is transcriptionally silenced in almost all somatic cells in the adult, but is aberrantly re-activated in over 90% of human cancers. The molecular mechanisms responsible for repression of this gene are thought to involve the transcription factor CTCF. In this study, we bioinformatically identify putative CTCF binding sites in the hTERT proximal exonic region (PER) and determine their functional relevance in mediating transcriptional silencing at this gene. Tests using a reporter gene assay in HeLa cancer cells demonstrate that a sub-region of the PER exhibits strong transcriptional repressive activity. This repression is independent of the previously identified CTCF binding site near the transcriptional start site of the hTERT gene. In addition, site directed mutagenesis of three predicted CTCF binding sites, including a previously characterized in vivo site in exon 2, does not result in a loss of the repression mediated by the PER. The results from this study indicate that expression of the hTERT gene in HeLa cells is regulated by sequences in the PER. This transcriptional control is mediated through additional regulatory molecular mechanisms, independent of CTCF binding.

Effects of endocrine disruptors on imprinted gene expression in the mouse embryo.

Environmental endocrine disruptors (EDs) are synthetic chemicals that resemble natural hormones and are known to cause epigenetic perturbations. EDs have profound effects on development and fertility. Imprinted genes had been identified as susceptible loci to environmental insults by EDs because they are functionally haploid, and because the imprints undergo epigenetic resetting between generations. To screen for possible epigenetic perturbations caused by EDs at imprinted loci, we treated pregnant mice daily between 8.5 and 12.5 days post coitum (dpc) with di-(2-ethylhexyl)-phthalate (DEHP), bisphenol A (BPA), vinclozolin (VZ), or control oil vehicle. After isolating RNA from the placenta, yolk sac, amnion, head, body, heart, liver, lung, stomach, and intestines of 13.5 dpc embryos we measured the allele-specific expression of 38 imprinted transcripts using multiplex single nucleotide primer extension (SNuPE) assays. In this representative data set we identified only a small number of transcripts that exhibited a substantial relaxation of imprinted expression with statistical significance: Slc22a18 with 10% relaxation in the embryo after BPA treatment; Rtl1as with 11 and 16% relaxation in the lung and placenta, respectively after BPA treatment; and Rtl1 with 12% relaxation in the yolk sac after DEHP treatment. Additionally, the standard deviation of allele-specificity increased in various organs after ED treatment for several transcripts including Igf2r, Rasgrf1, Usp29, Slc38a4, and Xist. Our data suggest that the maintenance of strongly biased monoallelic expression of imprinted genes is generally insensitive to EDs in the 13.5 dpc embryo and extra-embryonic organs, but is not immune to those effects.

MIRA-SNuPE, a quantitative, multiplex method for measuring allele-specific DNA methylation.

5-methyl-C (5mC) and 5-hydroxymethyl-C (5hmC) are epigenetic marks with well known and putative roles in gene regulation, respectively. These two DNA covalent modifications cannot be distinguished by bisulfite sequencing or restriction digestion, the standard methods of 5mC detection. The methylated CpG island recovery assay (MIRA), however, specifically detects 5mC but not 5hmC. We further developed MIRA for the analysis of allele-specific CpG methylation at differentially methylated regions (DMRs) of imprinted genes. MIRA specifically distinguished between the parental alleles by capturing the paternally methylated H19/Igf2 DMR and maternally methylated KvDMR1 in mouse embryo fibroblasts (MEFs) carrying paternal and maternal duplication of mouse distal Chr7, respectively. MIRA in combination with multiplex single nucleotide primer extension (SNuPE) assays specifically captured the methylated parental allele from normal cells at a set of maternally and paternally methylated DMRs. The assay correctly recognized aberrant biallelic methylation in a case of loss-of imprinting. The MIRA-SNuPE assays revealed that placenta exhibited less DNA methylation bias at DMRs compared to yolk sac, amnion, brain, heart, kidney, liver and muscle. This method should be useful for the analysis of allele-specific methylation events related to genomic imprinting, X chromosome inactivation and for verifying and screening haplotype-associated methylation differences in the human population.

Characterization of an ultra-conserved putative cis-regulatory module at the mammalian telomerase reverse transcriptase gene.

Telomeres are regions of repeated DNA sequence that cap the ends of eukaryotic chromosomes. They act as disposable safeguards to prevent the loss of important genetic information during DNA replication due to the inability of DNA polymerase to replicate DNA to the ends of linear chromosomes. The synthesis of new telomeric repeats using an RNA molecule as a template is catalyzed by the enzyme telomerase. In embryonic stem cells, the gene encoding the catalytic protein subunit of the telomerase complex (telomere reverse transcriptase [TERT]) is transcriptionally active and critical for telomere elongation, allowing for continued cellular differentiation during development. The TERT gene is down-regulated as embryogenesis progresses to limit the proliferative capacity of cells. As a result, in normal human adult somatic cells the TERT gene is silenced. However, in over 90% of cancers, the TERT gene is reactivated, allowing cells to bypass senescence and become immortalized. In this study, we explore the molecular mechanisms that regulate transcriptional expression of the TERT gene. Bioinformatic analysis of the noncoding genomic regions around the human TERT gene identified a TERT ultra-conserved (TUC) module located 5 kb upstream of the transcription start site. This 308 bp region is over 75% conserved between distantly related mammalian species and over 91% conserved among primate species. The cis-regulatory potential of the TUC region was tested in cell-based reporter gene assays. Transient transfections into HeLa and lung fibroblast cells demonstrated that the TUC module has transcriptional enhancer activity. Further bioinformatic analysis revealed that the TUC region is highly enriched in putative transcription factor binding sites for proteins involved during hematopoiesis, indicating that the TUC module may be an enhancer for the TERT gene in specific cell lineages.

Dissecting the regulatory switches of development: lessons from enhancer evolution in Drosophila.

Cis-regulatory modules are non-protein-coding regions of DNA essential for the control of gene expression. One class of regulatory modules is embryonic enhancers, which drive gene expression during development as a result of transcription factor protein binding at the enhancer sequences. Recent comparative studies have begun to investigate the evolution of the sequence architecture within enhancers. These analyses are illuminating the way that developmental biologists think about enhancers by revealing their molecular mechanism of function.

Functional evolution of cis-regulatory modules at a homeotic gene in Drosophila.

It is a long-held belief in evolutionary biology that the rate of molecular evolution for a given DNA sequence is inversely related to the level of functional constraint. This belief holds true for the protein-coding homeotic (Hox) genes originally discovered in Drosophila melanogaster. Expression of the Hox genes in Drosophila embryos is essential for body patterning and is controlled by an extensive array of cis-regulatory modules (CRMs). How the regulatory modules functionally evolve in different species is not clear. A comparison of the CRMs for the Abdominal-B gene from different Drosophila species reveals relatively low levels of overall sequence conservation. However, embryonic enhancer CRMs from other Drosophila species direct transgenic reporter gene expression in the same spatial and temporal patterns during development as their D. melanogaster orthologs. Bioinformatic analysis reveals the presence of short conserved sequences within defined CRMs, representing gap and pair-rule transcription factor binding sites. One predicted binding site for the gap transcription factor KRUPPEL in the IAB5 CRM was found to be altered in Superabdominal (Sab) mutations. In Sab mutant flies, the third abdominal segment is transformed into a copy of the fifth abdominal segment. A model for KRUPPEL-mediated repression at this binding site is presented. These findings challenge our current understanding of the relationship between sequence evolution at the molecular level and functional activity of a CRM. While the overall sequence conservation at Drosophila CRMs is not distinctive from neighboring genomic regions, functionally critical transcription factor binding sites within embryonic enhancer CRMs are highly conserved. These results have implications for understanding mechanisms of gene expression during embryonic development, enhancer function, and the molecular evolution of eukaryotic regulatory modules.