Genome Editing and Myocardial Development.

Congenital heart disease (CHD) has a strong genetic etiology, making it a likely candidate for therapeutic intervention using genetic editing. Complex genetics involving an orchestrated series of genetic events and over 400 genes are responsible for myocardial development. Cooperation is required from a vast series of genetic networks, and mutations in such can lead to CHD and cardiovascular abnormalities, affecting up to 1% of all live births. Genome editing technologies are becoming better studied and with time and improved logistics, CHD could be a prime therapeutic target. Syndromic, nonsyndromic, and cases of familial inheritance all involve identifiable causative mutations and thus have the potential for genome editing therapy. Mouse models are well-suited to study and predict clinical outcome. This review summarizes the anatomical and genetic timeline of myocardial development in both mice and humans, the potential of gene editing in typical CHD categories, as well as the use of mice thus far in reproducing models of human CHD and correcting the mutations that create them.

Compromised oocyte quality and assisted reproduction contribute to sex-specific effects on offspring outcomes and epigenetic patterning.

Clinical studies have revealed an increased incidence of growth and genomic imprinting disorders in children conceived using assisted reproductive technologies (ARTs), and aberrant DNA methylation has been implicated. We propose that compromised oocyte quality associated with female infertility may make embryos more susceptible to the induction of epigenetic defects by ART. DNA methylation patterns in the preimplantation embryo are dependent on the oocyte-specific DNA methyltransferase 1o (DNMT1o), levels of which are decreased in mature oocytes of aging females. Here, we assessed the effects of maternal deficiency in DNMT1o (Dnmt1Δ1o/+) in combination with superovulation and embryo transfer on offspring DNA methylation and development. We demonstrated a significant increase in the rates of morphological abnormalities in offspring collected from Dnmt1Δ1o/+ females only when combined with ART. Together, maternal oocyte DNMT1o deficiency and ART resulted in an accentuation of placental imprinting defects and the induction of genome-wide DNA methylation alterations, which were exacerbated in the placenta compared to the embryo. Significant sex-specific trends were also apparent, with a preponderance of DNA hypomethylation in females. Among genic regions affected, a significant enrichment for neurodevelopmental pathways was observed. Taken together, our results demonstrate that oocyte DNMT1o-deficiency exacerbates genome-wide DNA methylation abnormalities induced by ART in a sex-specific manner and plays a role in mediating poor embryonic outcome.

Transient DNMT1 suppression reveals hidden heritable marks in the genome.

Genome-wide demethylation and remethylation of DNA during early embryogenesis is essential for development. Imprinted germline differentially methylated domains (gDMDs) established by sex-specific methylation in either male or female germ cells, must escape these dynamic changes and sustain precise inheritance of both methylated and unmethylated parental alleles. To identify other, gDMD-like sequences with the same epigenetic inheritance properties, we used a modified embryonic stem (ES) cell line that emulates the early embryonic demethylation and remethylation waves. Transient DNMT1 suppression revealed gDMD-like sequences requiring continuous DNMT1 activity to sustain a highly methylated state. Remethylation of these sequences was also compromised in vivo in a mouse model of transient DNMT1 loss in the preimplantation embryo. These novel regions, possessing heritable epigenetic features similar to imprinted-gDMDs are required for normal physiological and developmental processes and when disrupted are associated with disorders such as cancer and autism spectrum disorders. This study presents new perspectives on DNA methylation heritability during early embryo development that extend beyond conventional imprinted-gDMDs.

Liver-specific deletion of augmenter of liver regeneration accelerates development of steatohepatitis and hepatocellular carcinoma in mice.

BACKGROUND & AIMS: Augmenter of liver regeneration (ALR, encoded by GFER) is a widely distributed pleiotropic protein originally identified as a hepatic growth factor. However, little is known about its roles in hepatic physiology and pathology. We created mice with liver-specific deletion of ALR to study its function. METHODS: We developed mice with liver-specific deletion of ALR (ALR-L-KO) using the albumin-Cre/LoxP system. Liver tissues were collected from ALR-L-KO mice and ALR(floxed/floxed) mice (controls) and analyzed by histology, reverse-transcription polymerase chain reaction, immunohistochemistry, electron microscopy, and techniques to measure fibrosis and lipids. Liver tissues from patients with and without advanced liver disease were determined by immunoblot analysis. RESULTS: Two weeks after birth, livers of ALR-L-KO mice contained low levels of ALR and adenosine triphosphate (ATP); they had reduced mitochondrial respiratory function and increased oxidative stress, compared with livers from control mice, and had excessive steatosis, and hepatocyte apoptosis. Levels of carbamyl-palmitoyl transferase 1a and ATP synthase subunit ATP5G1 were reduced in livers of ALR-L-KO mice, indicating defects in mitochondrial fatty acid transport and ATP synthesis. Electron microscopy showed mitochondrial swelling with abnormalities in shapes and numbers of cristae. From weeks 2-4 after birth, levels of steatosis and apoptosis decreased in ALR-L-KO mice, and numbers of ALR-expressing cells increased, along with ATP levels. However, at weeks 4-8 after birth, livers became inflamed, with hepatocellular necrosis, ductular proliferation, and fibrosis; hepatocellular carcinoma developed by 1 year after birth in nearly 60% of the mice. Hepatic levels of ALR were also low in ob/ob mice and alcohol-fed mice with liver steatosis, compared with controls. Levels of ALR were lower in liver tissues from patients with advanced alcoholic liver disease and nonalcoholic steatohepatitis than in control liver tissues. CONCLUSIONS: We developed mice with liver-specific deletion of ALR, and showed that it is required for mitochondrial function and lipid homeostasis in the liver. ALR-L-KO mice provide a useful model for investigating the pathogenesis of steatohepatitis and its complications.

Loss of DNMT1o disrupts imprinted X chromosome inactivation and accentuates placental defects in females.

The maintenance of key germline derived DNA methylation patterns during preimplantation development depends on stores of DNA cytosine methyltransferase-1o (DNMT1o) provided by the oocyte. Dnmt1o(mat-/-) mouse embryos born to Dnmt1(Δ1o/Δ1o) female mice lack DNMT1o protein and have disrupted genomic imprinting and associated phenotypic abnormalities. Here, we describe additional female-specific morphological abnormalities and DNA hypomethylation defects outside imprinted loci, restricted to extraembryonic tissue. Compared to male offspring, the placentae of female offspring of Dnmt1(Δ1o/Δ1o) mothers displayed a higher incidence of genic and intergenic hypomethylation and more frequent and extreme placental dysmorphology. The majority of the affected loci were concentrated on the X chromosome and associated with aberrant biallelic expression, indicating that imprinted X-inactivation was perturbed. Hypomethylation of a key regulatory region of Xite within the X-inactivation center was present in female blastocysts shortly after the absence of methylation maintenance by DNMT1o at the 8-cell stage. The female preponderance of placental DNA hypomethylation associated with maternal DNMT1o deficiency provides evidence of additional roles beyond the maintenance of genomic imprints for DNA methylation events in the preimplantation embryo, including a role in imprinted X chromosome inactivation.

Role of the inositol polyphosphate-4-phosphatase type II Inpp4b in the generation of ovarian teratomas.

Teratomas are a unique class of tumors composed of ecto-, meso- and endodermal tissues, all foreign to the site of origin. In humans, the most common teratoma is the ovarian teratoma. Not much is known about the molecular and genetic etiologies of these tumors. Female carriers of the Tgkd transgene are highly susceptible to developing teratomas. Ovaries of Tgkd/+ hemizygous female mice exhibit defects in luteinization, with numerous corpora lutea, some of which contain central trapped, fully-grown oocytes. Genetically, Tgkd teratomas originate from mature oocytes that have completed meiosis I, suggesting that Tgkd teratomas originate from these trapped oocytes. The insertion of Tgkd 3' of the Inpp4b gene is associated with decreased expression of Inpp4b and changes in intracellular PI3 Kinase/AKT signaling in follicular granulosa cells. Because Inpp4b is not expressed in fully-grown wild-type or Tgkd oocytes, these findings suggest that hyperactivation of the PI3K/AKT pathway caused by the decrease in INPP4B in granulosa cells promotes an ovarian environment defective in folliculogenesis and conducive to teratoma formation.

Generalized disruption of inherited genomic imprints leads to wide-ranging placental defects and dysregulated fetal growth.

Monoallelic expression of imprinted genes, including ones solely expressed in the placenta, is essential for normal placental development and fetal growth. To better understand the role of placental imprinting in placental development and fetal growth, we examined conceptuses developing in the absence of maternally derived DNA (cytosine-5-)-methyltransferase 1o (DNMT1o). Absence of DNMT1o results in the partial loss of methylation at imprinted differentially methylated domain (DMD) sequences in the embryo and the placenta. Mid-gestation E9.5 DNMT1o-deficient placentas exhibited structural abnormalities of all tissue layers. At E17.5, all examined placentas had aberrant placental morphology, most notably in the spongiotrophoblast and labyrinth layers. Abnormalities included an expanded volume fraction of spongiotrophoblast tissue with extension of the spongiotrophoblast layer into the labyrinth. Many mutant placentas also demonstrated migration abnormalities of glycogen cells. Additionally, the volume fraction of the labyrinth was reduced, as was the surface area for maternal fetal gas exchange. Despite these placental morphologic abnormalities, approximately one-half of DNMT1o-deficient fetuses survived to late gestation (E17.5). Furthermore, DNMT1o-deficient placentas supported a broad range of fetal growth. The ability of some DNMT1o-deficient and morphologically abnormal placentas to support fetal growth in excess of wild type demonstrates the importance of differential methylation of DMDs and proper imprinting of discrete gene clusters to placental morphogenesis and fetal growth.

Identification of a region of the DNMT1 methyltransferase that regulates the maintenance of genomic imprints.

Reprogramming of DNA methylation patterns during mammalian preimplantation development involves the concurrent maintenance of methylation on differentially methylated domains (DMDs) of imprinted genes and a marked reduction of global (non-DMD) genomic methylation. In the developing mammalian embryo, one allele of a DMD is unmethylated, and the opposite parental allele is methylated, having inherited this methylation from the parental gamete. The maintenance of DMDs is important for monoallelic imprinted gene expression and normal development of the embryo. Because the DNMT1 cytosine methyltransferase governs maintenance methylation in mammals, rearrangements of non-DMD, but not DMD methylation in preimplantation embryos suggest that the preimplantation DNMT1-dependent maintenance mechanism specifically targets DMD sequences. We explored this possibility using an engineered mouse ES cell line to screen for mutant DNMT1 proteins that protect against the loss of DMD and/or global (non-DMD) methylation in the absence of the wild-type endogenous DNMT1 methyltransferase. We identified DNMT1 mutants that were defective in maintenance of either DMD and/or non-DMD methylation. Among these, one mutant maintained non-DMD methylation but not imprinted DMD methylation and another mutant maintained just DMD methylation. The mutated amino acids of these mutants reside in a mammal-specific, disordered region near the amino terminus of DNMT1. These findings suggest that DNMT1 participates in epigenetic reprogramming through its ability to distinguish different categories of methylated sequences.

Mouse ES cells overexpressing DNMT1 produce abnormal neurons with upregulated NMDA/NR1 subunit.

High levels of DNA methyltransferase 1 (DNMT1), hypermethylation, and downregulation of GAD(67) and reelin have been described in GABAergic interneurons of patients with schizophrenia (SZ) and bipolar (BP) disorders. However, overexpression of DNMT1 is lethal, making it difficult to assess the direct effect of high levels of DNMT1 on neuronal development in vivo. We therefore used Dnmt1(tet/tet) mouse ES cells that overexpress DNMT1 as an in vitro model to investigate the impact of high levels of DNMT1 on neuronal differentiation. Although there is down-regulation of DNMT1 during early stages of differentiation in wild type and Dnmt1(tet/tet) ES cell lines, neurons derived from Dnmt1(tet/tet) cells showed abnormal dendritic arborization and branching. The Dnmt1(tet/tet) neuronal cells also showed elevated levels of functional N-methyl d-aspartate receptor (NMDAR), a feature also reported in some neurological and neurodegenerative disorders. Considering the roles of reelin and GAD(67) in neuronal networking and excitatory/inhibitory balance, respectively, we studied methylation of these genes' promoters in Dnmt1(tet/tet) ES cells and neurons. Both reelin and GAD(67) promoters were not hypermethylated in the Dnmt1(tet/tet) ES cells and neurons, suggesting that overexpression of DNMT1 may not directly result in methylation-mediated repression of these two genes. Taken together, our results suggest that overexpression of DNMT1 in ES cells results in an epigenetic change prior to the onset of differentiation. This epigenetic change in turn results in abnormal neuronal differentiation and upregulation of functional NMDA receptor.

DNA methyltransferase 1o functions during preimplantation development to preclude a profound level of epigenetic variation.

Most mouse embryos developing in the absence of the oocyte-derived DNA methyltransferase 1o (DNMT1o-deficient embryos) have significant delays in development and a wide range of anatomical abnormalities. To understand the timing and molecular basis of such variation, we studied pre- and post-implantation DNA methylation as a gauge of epigenetic variation among these embryos. DNMT1o-deficient embryos showed extensive differences in the levels of methylation in differentially methylated domains (DMDs) of imprinted genes at the 8-cell stage. Because of independent assortment of the methylated and unmethylated chromatids created by the loss of DNMT1o, the deficient embryos were found to be mosaics of cells with different, but stable epigenotypes (DNA methylation patterns). Our results suggest that loss of DNMT1o in just one cell cycle is responsible for the extensive variation in the epigenotypes in both embryos and their associated extraembryonic tissues. Thus, the maternal-effect DNMT1o protein is uniquely poised during development to normally ensure uniform parental methylation patterns at DMDs.

Specific differentially methylated domain sequences direct the maintenance of methylation at imprinted genes.

Landmark features of imprinted genes are differentially methylated domains (DMDs), in which one parental allele is methylated on CpG dinucleotides and the opposite allele is unmethylated. Genetic experiments in the mouse have shown that DMDs are required for the parent-specific expression of linked clusters of imprinted genes. To understand the mechanism whereby the differential methylation is established and maintained, we analyzed a series of transgenes containing DMD sequences and showed that imperfect tandem repeats from DMDs associated with the Snurf/Snrpn, Kcnq1, and Igf2r gene clusters govern transgene imprinting. For the Igf2r DMD the minimal imprinting signal is two unit copies of the tandem repeat. This imprinted transgene behaves identically to endogenous imprinted genes in Dnmt1o and Dnmt3L mutant mouse backgrounds. The primary function of the imprinting signal within the transgene DMD is to maintain, during embryogenesis and a critical period of genomic reprogramming, parent-specific DNA methylation states established in the germ line. This work advances our understanding of the imprinting mechanism by defining a genomic signal that dependably perpetuates an epigenetic state during postzygotic development.

Klf14 is an imprinted transcription factor that regulates placental growth.

INTRODUCTION: Imprinted genes are preferentially expressed from one parentally inherited allele, and many are crucial to the regulation of placental function and fetal growth. Murine Krüppel-like factor 14 (Klf14) is a maternally expressed imprinted transcription factor that is a component of the Mest imprinted gene cluster on mouse chromosome 6. We sought to determine if loss of Klf14 expression alters the course of normal mouse extraembryonic development. We also used high-throughput RNA sequencing (RNAseq) to identify a set of differentially expressed genes (DEGs) in placentas with loss of Klf14. METHODS: We generated a Klf14 knockout (Klf14null) mouse using recombineering and transgenic approaches. To identify DEGs in the mouse placenta we compared mRNA transcriptomes derived from 17.5dpc Klf14matKO and wild-type littermate placentas by RNAseq. Candidate DEGs were confirmed with quantitative reverse transcription PCR (qPCR) on an independent cohort of male and female gestational age matched Klf14matKO placentas. RESULTS: We found that 17.5dpc placentas inheriting a maternal null allele (Klf14matKO) had a modest overgrowth phenotype and a near complete ablation of Klf14 expression. However, there was no effect on fetal growth. We identified 20 DEGs differentially expressed in Klf14matKO placentas by RNAseq, and subsequently validated five that are highly upregulated (Begain, Col26a1, Fbln5, Gdf10, and Nell1) by qPCR. The most enriched functional gene-networks included those classified as regulating cellular development and metabolism. CONCLUSION: These results suggest that loss of the maternal Klf14 locus in the mouse placenta acts results in changes in gene expression patterns that modulate placental growth.

Preimplantation expression of the somatic form of Dnmt1 suggests a role in the inheritance of genomic imprints.

BACKGROUND: Identical DNA methylation differences between maternal and paternal alleles in gametes and adults suggest that the inheritance of genomic imprints is strictly due to the embryonic maintenance of DNA methylation. Such maintenance would occur in association with every cycle of DNA replication, including those of preimplantation embryos. RESULTS: The expression of the somatic form of the Dnmt1 cytosine methyltransferase (Dnmt1s) was examined in cleavage-stage preimplantation mouse embryos. Low concentrations of Dnmt1s are found in 1-, 2-, 4-, and 8-cell embryos, as well as in morulae and blastocysts. Dnmt1s is present in the cytoplasm at all stages, and in the nuclei of all stages except the 1-cell, pronuclear-stage embryo. The related oocyte-derived Dnmt1o protein is also present in nuclei of 8-cell embryos, along with embryo-synthesized Dnmt1s. Dnmt1s protein expressed in 1-cell and 2-cell embryos is derived from the oocyte, whereas the embryo synthesizes its own Dnmt1s from the 2-cell stage onward. CONCLUSION: These observations suggest that Dnmt1s provides maintenance methyltransferase activity for the inheritance of methylation imprints in the early mouse embryo. Moreover, the ability of Dnmt1o and Dnmt1s proteins synthesized at the same time to substitute for one another's maintenance function, but the lack of functional interchange between oocyte- and embryo-synthesized Dnmt1 proteins, suggests that the developmental source is the critical determinant of Dnmt1 function during preimplantation development.

Human IL-12 p40 as a reporter gene for high-throughput screening of engineered mouse embryonic stem cells.

BACKGROUND: Establishing a suitable level of exogenous gene expression in mammalian cells in general, and embryonic stem (ES) cells in particular, is an important aspect of understanding pathways of cell differentiation, signal transduction and cell physiology. Despite its importance, this process remains challenging because of the poor correlation between the presence of introduced exogenous DNA and its transcription. Consequently, many transfected cells must be screened to identify those with an appropriate level of expression. To improve the screening process, we investigated the utility of the human interleukin 12 (IL-12) p40 cDNA as a reporter gene for studies of mammalian gene expression and for high-throughput screening of engineered mouse embryonic stem cells. RESULTS: A series of expression plasmids were used to study the utility of IL-12 p40 as an accurate reporter of gene activity. These studies included a characterization of the IL-12 p40 expression system in terms of: (i) a time course of IL-12 p40 accumulation in the medium of transfected cells; (ii) the dose-response relationship between the input DNA and IL-12 p40 mRNA levels and IL-12 p40 protein secretion; (iii) the utility of IL-12 p40 as a reporter gene for analyzing the activity of cis-acting genetic elements; (iv) expression of the IL-12 p40 reporter protein driven by an IRES element in a bicistronic mRNA; (v) utility of IL-12 p40 as a reporter gene in a high-throughput screening strategy to identify successful transformed mouse embryonic stem cells; (vi) demonstration of pluripotency of IL-12 p40 expressing ES cells in vitro and in vivo; and (vii) germline transmission of the IL-12 p40 reporter gene. CONCLUSION: IL-12 p40 showed several advantages as a reporter gene in terms of sensitivity and ease of the detection procedure. The IL-12 p40 assay was rapid and simple, in as much as the reporter protein secreted from the transfected cells was accurately measured by ELISA using a small aliquot of the culture medium. Remarkably, expression of Il-12 p40 does not affect the pluripotency of mouse ES cells. To our knowledge, human IL-12 p40 is the first secreted reporter protein suitable for high-throughput screening of mouse ES cells. In comparison to other secreted reporters, such as the widely used alkaline phosphatase (SEAP) reporter, the IL-12 p40 reporter system offers other real advantages.

Profound phenotypic variation among mice deficient in the maintenance of genomic imprints.

BACKGROUND: An alteration in the mechanism that maintains the monoallelic, imprinted expression of genes can result in their biallelic expression and lead to disruptions in fetal development. Here, we examined the consequences of a loss of maintenance methylation at one specific stage of preimplantation, induced by a deficiency of the oocyte-derived Dnmt1o protein and known to produce biallelic expression of imprinted genes. METHODS: Phenotypes of mid-gestation Dnmt1o-deficient mouse embryos were assessed by a scoring system based on the developmental stage of 17 anatomical features and by magnetic resonance microscopy. RESULTS: Many mid-gestation embryos developing without Dnmt1o protein exhibited significant developmental delays of multiple organ systems (P < 0.05) and a wide variety of morphologic anomalies compared with wild-type embryos. Most of the remaining mid-gestation Dnmt1o-deficient embryos appeared normal. CONCLUSIONS: These findings indicate that a profound range of gestational phenotypes can be induced by the loss of a single protein at a specific preimplantation developmental stage. This is best explained by the formation of epigenetic mosaic early embryos, composed of somatic cells with different spectra of normal intact genomic imprints. These findings have important implications for understanding the types of embryonic phenotypes related to the disruption of inherited imprints, and thus may provide a model of altered imprinting in humans. In particular, because Dnmt1o functions in the preimplantation embryo, a complete or partial loss of Dnmt1o function may play a role in epigenetic abnormalities seen in assisted reproduction technology births.

Conserved features of imprinted differentially methylated domains.

Genomic imprinting is a conserved epigenetic phenomenon in eutherian mammals, with regards both to the genes that are imprinted and the mechanism underlying the expression of just one of the parental alleles. Epigenetic modifications of alleles of imprinted genes are established during oogenesis and spermatogenesis, and these modifications are then inherited. Differentially methylated domains (DMDs) of imprinted genes are the genomic sites of these inherited epigenetic imprints. We previously showed that CpG-rich imperfect tandem direct repeats within three different mouse DMDs (Snurf/Snrpn, Kcnq1 and Igf2r), each with a unique sequence, play a central role in maintaining the differential methylation. This finding implicates repeat-related DNA structure, not sequence, in the imprinting mechanism. To better define the important features of this signal, we compared sequences of these three DMD tandem repeats among mammalian species. All DMD repeats contain short indirect repeats, many of which are organized into larger unit repeats. Even though the larger repeat units undergo deletion and addition during evolution (most likely through unequal crossovers during meiosis), the size of DMD tandem repeated regions has remained remarkably stable during mammalian evolution. Moreover, all three DMD tandem repeats have a high-CpG content, an ordered arrangement of CpG dinucleotides, and similar predicted secondary structures. These observations suggest that a structural feature or features of these DMD tandem repeats is the conserved DMD imprinting signal.

Shared role for differentially methylated domains of imprinted genes.

For most imprinted genes, a difference in expression between the maternal and paternal alleles is associated with a corresponding difference in DNA methylation that is localized to a differentially methylated domain (DMD). Removal of a gene's DMD leads to a loss of imprinting. These observations suggest that DMDs have a determinative role in genomic imprinting. To examine this possibility, we introduced sequences from the DMDs of the imprinted Igf2r, H19, and Snrpn genes into a nonimprinted derivative of the normally imprinted RSVIgmyc transgene, created by excising its own DMD. Hybrid transgenes with sequences from the Igf2r DMD2 were consistently imprinted, with the maternal allele being more methylated than the paternal allele. Only the repeated sequences within DMD2 were required for imprinting these transgenes. Hybrid transgenes containing H19 and Snrpn DMD sequences and ones containing sequences from the long terminal repeat of a murine intracisternal A particle retrotransposon were not imprinted. The Igf2r hybrid transgenes are comprised entirely of mouse genomic DNA and behave as endogenous imprinted genes in inbred wild-type and mutant mouse strains. These types of hybrid transgenes can be used to elucidate the functions of DMD sequences in genomic imprinting.

Genomic imprinting: cis-acting sequences and regional control.

This review explores the features of imprinted loci that have been uncovered by genetic experiments in the mouse. Imprinted genes are expressed from one parental allele and often contain parent-specific differences in DNA methylation within genomic regions known as differentially methylated domains (DMDs). The precise erasure, establishment, and propagation of methylation on the alleles of imprinted genes during development suggest that parental differences in methylation at DMD sequences are a fundamental distinguishing feature of imprinted loci. Furthermore, targeted mutations of many DMDs have shown that they are essential for the imprinting of single genes or large gene clusters. An essential role of DNA methylation in genomic imprinting is also shown by studies of methyltransferase-deficient embryos. Many of the DMDs known to be required for imprinting contain imprinted promoters, tandem repeats, and CpG-rich regions that may be important for regulating parent-specific gene expression.

Evidence of the capability of the CMV enhancer to activate in trans gene expression in mammalian cells.

Studies have reported that an enhancer can act in trans when artificially, noncovalently bridged to the promoter by a protein-linked biotin:streptavidin complex, or when an enhancer and a promoter are located on separate concatenated plasmids. To investigate such transactivation in mammalian cells, we constructed CMV promoter-enhancer mutants driving the expression of the EGFP reporter gene and transfected cultured cells with various combinations of the mutant PCR products; results were analyzed using fluorescence microscopy and flow cytometry. Our results show that the CMV enhancer can stimulate transcription in trans, even in the absence of physical association of the enhancer and promoter. Furthermore, we show that the transactivation of the CMV enhancer can be strengthened by the histone deacetylase inhibitor sodium butyrate. Finally, we provide evidence that the CMV enhancer can influence, in trans, the activity of heterologous promoters. Although different mechanisms may lead to transcriptional activation when the CMV enhancer is not covalently linked to the promoter, our results suggest that the main mechanism resembles the process of transfection and may be important for gene regulation. These findings may have implications in understanding the processes that underlie gene therapy because of the potential alteration of endogenous gene expression.

Partial Loss of Genomic Imprinting Reveals Important Roles for Kcnq1 and Peg10 Imprinted Domains in Placental Development.

Mutations in imprinted genes or their imprint control regions (ICRs) produce changes in imprinted gene expression and distinct abnormalities in placental structure, indicating the importance of genomic imprinting to placental development. We have recently shown that a very broad spectrum of placental abnormalities associated with altered imprinted gene expression occurs in the absence of the oocyte-derived DNMT1o cytosine methyltransferase, which normally maintains parent-specific imprinted methylation during preimplantation. The absence of DNMT1o partially reduces inherited imprinted methylation while retaining the genetic integrity of imprinted genes and their ICRs. Using this novel system, we undertook a broad and inclusive approach to identifying key ICRs involved in placental development by correlating loss of imprinted DNA methylation with abnormal placental phenotypes in a mid-gestation window (E12.5-E15.5). To these ends we measured DNA CpG methylation at 15 imprinted gametic differentially methylated domains (gDMDs) that overlap known ICRs using EpiTYPER-mass array technology, and linked these epigenetic measurements to histomorphological defects. Methylation of some imprinted gDMDs, most notably Dlk1, was nearly normal in mid-gestation DNMT1o-deficient placentas, consistent with the notion that cells having lost methylation on these DMDs do not contribute significantly to placental development. Most imprinted gDMDs however showed a wide range of methylation loss among DNMT1o-deficient placentas. Two striking associations were observed. First, loss of DNA methylation at the Peg10 imprinted gDMD associated with decreased embryonic viability and decreased labyrinthine volume. Second, loss of methylation at the Kcnq1 imprinted gDMD was strongly associated with trophoblast giant cell (TGC) expansion. We conclude that the Peg10 and Kcnq1 ICRs are key regulators of mid-gestation placental function.