Mesoderm-specific transcript (Mest) predominantly expressed in mesoderm and its derivatives is also an imprinted gene whose expression is dependent on parental origin and is designated Peg1 (paternally expressed gene-1). Curiously orthologues seem present in vertebrates, few invertebrates (molluscs, ticks, amphioxus) but not in insects or nematodes. Absent in plants and present in a limited number of bacteria
A 50-fold variation in mRNA and protein levels of the mesoderm-specific transcript gene (Mest) in white fat of C57BL/6J (B6) mice fed an obesogenic diet is positively correlated with expansion of fat mass. MEST protein was detected only in adipocytes, in which its induction occurred with both unsaturated and saturated dietary fat. To test the hypothesis that MEST modulates fat mass expansion, its expression was compared to that of stearoyl CoA desaturase (Scd1) in B6 mice exposed to diets and environmental temperatures that generated conditions separating the effects of food intake and adiposity. Under a range of conditions, Mest expression was always associated with variations in adiposity, whereas Scd1 expression was associated with the amount of saturated fat in the diet. Mest mRNA was expressed at its highest levels during early postnatal growth at the onset of the most rapid phase of fat mass expansion. MEST is localized to the endoplasmic reticulum/Golgi apparatus where its putative enzymatic properties as a lipase or acyltransferase, predicted from sequence homology with members of the alpha/beta fold hydrolase superfamily, can enable it to function in lipid accumulation under conditions of positive energy balance. Variations in adiposity and Mest expression in genetically identical mice also provides a model of epigenetic regulation.
Mest (also known as Peg1), an imprinted gene expressed only from the paternal allele during development, was disrupted by gene targeting in embryonic stem (ES) cells. The targeted mutation is imprinted and reversibly silenced by passage through the female germ line. Paternal transmission activates the targeted allele and causes embryonic growth retardation associated with reduced postnatal survival rates in mutant progeny. More significantly, Mest-deficient females show abnormal maternal behaviour and impaired placentophagia, a distinctive mammalian behaviour. Our results provide evidence for the involvement of an imprinted gene in the control of adult behaviour.
We previously identified Peg1/Mest as a novel paternally expressed gene in the developing mouse embryo. The human PEG1 gene was recently assigned to 7q32 and shown to be imprinted and paternally expressed. Therefore, PEG1 deficiency could participate in the aetiology of pre- and post-natal growth retardation associated with maternal uniparental disomy 7 in humans. We have now initiated the characterization of the Peg1 locus in order to identify and dissect cis-acting elements implicated in its imprinted monoallelic expression. The genomic structure of Peg1 as well as the DNA sequence of the 5'-end of the gene, including 2.4 kb of promoter sequences and covering the first 2 exons, have been determined. Important sequence elements, such as a CpG island spanning exon 1 and direct repeats, are identified and discussed. To address the role of epigenetic modifications in the imprinting of Peg1, a methylation analysis of the Peg1 gene is presented. Partially methylated cytosine residues in 13.5 d.p.c. embryos and undifferentiated ES cells were identified. Using embryos carrying a targetted mutation at the Peg1 locus, we show that this partial promoter methylation pattern reflects a strict parent-of-origin-specific differential methylation: the expressed paternal allele is unmethylated, whereas the silenced maternal allele is fully methylated at the CpG sites studied. That the gametes carry the epigenetic information necessary to lay down this allele-specific methylation pattern is suggested by analysis of DNA isolated from sperm and parthenogenetic embryos.
Highly variable expression of mesoderm-specific transcript (Mest) in adipose tissue among genetically homogeneous mice fed an obesogenic diet, and its positive association with fat mass expansion, suggests that Mest is an epigenetic determinant for the development of obesity. Although the mechanisms by which MEST augments fat accumulation in adipocytes have not been elucidated, it has sequence homology and catalytic peptide motifs which suggests that it functions as an epoxide hydrolase or as a glycerol- or acylglycerol-3-phosphate acyltransferase. To better understand MEST function, detailed studies were performed to precisely define the intracellular organelle localization of MEST using immunofluorescence confocal microscopy. Lentiviral-mediated expression of a C-terminus Myc-DDK-tagged MEST fusion protein expressed in 3T3-L1 preadipocytes/adipocytes, and ear-derived mesenchymal stem cells (EMSC) from mice was observed in the endoplasmic reticulum (ER) membranes and is consistent with previous studies showing endogenous MEST in the membrane fraction of adipose tissue. MEST was not associated with the Golgi apparatus or mitochondria; however, frequent contacts were observed between MEST-positive ER and mitochondria. MEST-positive domains were also shown on the plasma membrane (PM) of non-permeabilized cells but they did not co-localize with ER-PM bridges. Post-adipogenic differentiated 3T3-L1 adipocytes and EMSC showed significant co-localization of MEST with the lipid droplet surface marker perilipin at contact points between the ER and lipid droplet. Identification of MEST as an ER-specific protein that co-localizes with lipid droplets in cells undergoing adipogenic differentiation supports a function for MEST in the facilitation of lipid accumulation and storage in adipocytes.
        
Title: Adipose tissue Mest and Sfrp5 are concomitant with variations of adiposity among inbred mouse strains fed a non-obesogenic diet Anunciado-Koza RP, Higgins DC, Koza RA Ref: Biochimie, 124:134, 2016 : PubMed
The expression of a subset of genes including mesoderm specific transcript (Mest), secreted frizzled-related protein 5 (Sfrp5) and bone morphogenetic protein 3 (Bmp3) in adipose tissue biopsies of C57BL/6J mice before exposure to an obesogenic diet were shown to be predictive for the development of obesity in mice after feeding a high fat diet for 8 weeks. This observation led to the supposition that adipose tissue expression of this subset of genes within inbred strains of mice could be associated with their susceptibility in the development of adiposity when fed a low fat diet. The analyses of male mice from 5 inbred strains showed average bodyweights ranging from 25.82 to 36.58 g at 16 weeks of age. Bodyweight was highest for AKR/J and adiposity correlated highly with bodyweight for all strains. Analyses of epididymal fat gene expression showed Mest, Sfrp5 and Bmp3 to be highly concomitant with adiposity across all strains of mice. Naked 1 (Nkd1), a gene previously shown to be associated with variations of adiposity in mice fed a high fat diet, but not predictive for the development of adiposity, showed no correlation with adiposity. In addition, the expression of Mest and Sfrp5 were tightly associated across the 5 mouse strains with the highest and lowest expression occurring in DBA/2J and C57BL/6J (B6) respectively suggesting a common mechanism for their regulation. Surprisingly, when independent cohorts for these 2 strains were fed high fat diet for 8 weeks, DBA/2J showed no further increase in Sfrp5 expression whereas expression levels for B6 mice were induced almost 20-fold. Analyses of (B6 x DBA2/J) F1 mice fed a low fat diet for 8 weeks showed intermediate levels of adiposity and gene expression for Sfrp5 and Mest suggesting a strong genetic basis for these differences.
INTRODUCTION: Monoallelic expression of imprinted genes is necessary for placental development and normal fetal growth. Differentially methylated domains (DMDs) largely determine the parental-specific monoallelic expression of imprinted genes. Maternally derived DNA (cytosine-5-) -methyltransferase 1o (DNMT1o) maintains DMDs during the eight-cell stage of development. DNMT1o-deficient mouse placentas have a generalized disruption of genomic imprints. Previous studies have demonstrated that DNMT1o deficiency alters placental morphology and broadens the embryonic weight distribution in late gestation. Lipids are critical for fetal growth. Thus, we assessed the impact of disrupted imprinting on placental lipids. METHODS: Lipids were quantified from DNMT1o-deficient mouse placentas and embryos at E17.5 using a modified Folch method. Expression of select genes critical for lipid metabolism was quantified with RT-qPCR. Mitochondrial morphology was assessed by TEM and mitochondrial aconitase and cytoplasmic citrate concentrations quantified. DMD methylation was determined by EpiTYPER. RESULTS: We found that DNMT1o deficiency is associated with increased placental triacylglycerol levels. Neither fetal triacylglycerol concentrations nor expression of select genes that mediate placental lipid transport were different from wild type. Placental triacylglycerol accumulation was associated with impaired beta-oxidation and abnormal citrate metabolism with decreased mitochondrial aconitase activity and increased cytoplasmic citrate concentrations. Loss of methylation at the MEST DMD was strongly associated with placental triacylglycerol accumulation. DISCUSSION: A generalized disruption of genomic imprints leads to triacylglycerol accumulation and abnormal mitochondrial function. This could stem directly from a loss of methylation at a given DMD, such as MEST, or represent a consequence of abnormal placental development.
        
Title: Identification of Mest/Peg1 gene expression as a predictive biomarker of adipose tissue expansion sensitive to dietary anti-obesity interventions Voigt A, Ribot J, Sabater AG, Palou A, Bonet ML, Klaus S Ref: Genes Nutr, 10:477, 2015 : PubMed
Food components with anti-obesity properties are commonly evaluated using mouse models of diet-induced obesity. The ability of these components to reduce or prevent white adipose tissue (WAT) accumulation is usually tested in feeding trials of several weeks duration in order to detect significant effects on fat mass expansion. Here, we aimed to identify early, predictive biomarkers for WAT expansion. We performed a 5-day high-fat diet (HFD) feeding trial with C57BL/6J mice using different established anti-obesity interventions: epigallocatechin gallate, replacing dietary lipids by n-3 PUFA, and increasing dietary protein. WAT gene expression was analyzed of genes known to be similarly affected by short- and long-term HFD. Gene expression of Leptin and Mest (mesoderm-specific transcript) was increased by HFD and normalized by all anti-obesity interventions. In a second experiment, translatability to whole blood-based expression data was assessed. Mice were challenged for 21 days with a HFD without or with simultaneous treatment with anti-obesity bioactives, hydroxytyrosol or resveratrol, and compared for parameters including Leptin and Mest expression in whole blood at day 5. While Leptin mRNA could not be detected in mouse whole blood, there was an induction of Mest mRNA by HFD which was suppressed by hydroxytyrosol. Moreover, Mest expression in whole blood at day 5 positively correlated with adiposity and negatively with lean body mass and the subcutaneous/visceral fat ratio at day 21. We conclude that gene expression of Leptin and Mest in WAT and of Mest in whole blood represent early, predictive markers of adipose tissue expansion of potential usefulness in nutritional studies and trials.
There is evidence that expression and methylation of the imprinted paternally expressed gene 1/mesoderm-specific transcript homologue (PEG1/MEST) gene may be affected by assisted reproductive technologies (ARTs) and infertility. In this study, we sought to assess the imprinting status of the MEST gene in a large cohort of in vitro-derived human preimplantation embryos, in order to characterise potentially adverse effects of ART and infertility on this locus in early human development. Embryonic genomic DNA from morula or blastocyst stage embryos was screened for a transcribed AflIII polymorphism in MEST and imprinting analysis was then performed in cDNA libraries derived from these embryos. In 10 heterozygous embryos, MEST expression was monoallelic in seven embryos, predominantly monoallelic in two embryos, and biallelic in one embryo. Screening of cDNA derived from 61 additional human preimplantation embryos, for which DNA for genotyping was unavailable, identified eight embryos with expression originating from both alleles (biallelic or predominantly monoallelic). In some embryos, therefore, the onset of imprinted MEST expression occurs during late preimplantation development. Variability in MEST imprinting was observed in both in vitro fertilization and intracytoplasmic sperm injection-derived embryos. Biallelic or predominantly monoallelic MEST expression was not associated with any one cause of infertility. Characterisation of the main MEST isoforms revealed that isoform 2 was detected in early development and was itself variably imprinted between embryos. To our knowledge, this report constitutes the largest expression study to date of genomic imprinting in human preimplantation embryos and reveals that for some imprinted genes, contrasting imprinting states exist between embryos.
        
Title: Mest/Peg1 inhibits Wnt signalling through regulation of LRP6 glycosylation Jung H, Lee SK, Jho EH Ref: Biochemical Journal, 436:263, 2011 : PubMed
Mest (mesoderm-specific transcript)/Peg1 (paternally expressed gene 1) is an imprinted gene that plays important roles in embryo development, although its biochemical role has not been determined. Ectopic expression of Mest/Peg1 inhibited Wnt-mediated reporter activity by enhancing the ubiquitination of beta-catenin. The maturation and plasma membrane localization of the Wnt co-receptor LRP6 [LDLR (low-density lipoprotein receptor)-related protein 6], which are both necessary for Wnt signalling, were blocked by the expression of Mest/Peg1. Mest/Peg1 inhibited maturation of LRP6 by controlling the glycosylation of LRP6. Knockdown of Mest/Peg1, which might enhance Wnt signalling, blocked adipogenic differentiation of 3T3-L1 cells. Overall, our results suggest that Mest/Peg1 is a novel regulator of Wnt/beta-catenin signalling during adipogenic differentiation.
A 50-fold variation in mRNA and protein levels of the mesoderm-specific transcript gene (Mest) in white fat of C57BL/6J (B6) mice fed an obesogenic diet is positively correlated with expansion of fat mass. MEST protein was detected only in adipocytes, in which its induction occurred with both unsaturated and saturated dietary fat. To test the hypothesis that MEST modulates fat mass expansion, its expression was compared to that of stearoyl CoA desaturase (Scd1) in B6 mice exposed to diets and environmental temperatures that generated conditions separating the effects of food intake and adiposity. Under a range of conditions, Mest expression was always associated with variations in adiposity, whereas Scd1 expression was associated with the amount of saturated fat in the diet. Mest mRNA was expressed at its highest levels during early postnatal growth at the onset of the most rapid phase of fat mass expansion. MEST is localized to the endoplasmic reticulum/Golgi apparatus where its putative enzymatic properties as a lipase or acyltransferase, predicted from sequence homology with members of the alpha/beta fold hydrolase superfamily, can enable it to function in lipid accumulation under conditions of positive energy balance. Variations in adiposity and Mest expression in genetically identical mice also provides a model of epigenetic regulation.
Imprinted gene(s) on human chromosome 7 are thought to be involved in Russell-Silver syndrome (RSS), based on the fact that approximately 10% of patients have maternal uniparental disomy of chromosome 7. However, involvement of the known imprinted genes (GRB10 at 7p12, PEG10 at 7q21.3 and MEST at 7q32) in RSS has yet to be established. To screen for new imprinted genes, we are initially using somatic cell hybrids containing a paternal or maternal human chromosome 7. Transcripts located between D7S530 and D7S649 (a 1.5 Mb interval encompassing MEST ) were subjected to RT-PCR analysis using somatic cell hybrids. One transcript named MESTIT1 (for MEST intronic transcript 1) reproducibly showed paternal-specific expression. Upon further analysis, we found MESTIT1 to be (1) paternally (and not maternally) expressed in all fetal tissues and fibroblasts examined, (2) to be located in an intron of one of the two isoforms of MEST but transcribed in the opposite direction, (3) to be composed of at least two exons without any significant open reading frame, and (4) to exist as a 4.2 kb transcript in many fetal and adult tissues. We could also identify two isoforms of the mouse Mest gene as observed in humans, but it is still unknown if a murine ortholog of MESTIT1 exists. We also examined the imprinting status of MEST isoforms as a first step in assessing whether MESTIT1 might influence the allelic expression pattern of the sense transcript. MEST isoform 1 was determined to be exclusively expressed from the paternal allele in all fetal tissues and cell lines examined, whereas MEST isoform 2 was only preferentially expressed from the paternal allele in a tissue/cell-type-specific manner. Our results suggest that MESTIT1 is a paternally expressed non-coding RNA that may be involved in the regulation of MEST expression during development. MESTIT1 (also known as PEG1-AS) is now the third independent transcript (with MEST and COPG2IT1) identified at human chromosome 7q32 demonstrating paternal chromosome-specific expression.
Mest (also known as Peg1), an imprinted gene expressed only from the paternal allele during development, was disrupted by gene targeting in embryonic stem (ES) cells. The targeted mutation is imprinted and reversibly silenced by passage through the female germ line. Paternal transmission activates the targeted allele and causes embryonic growth retardation associated with reduced postnatal survival rates in mutant progeny. More significantly, Mest-deficient females show abnormal maternal behaviour and impaired placentophagia, a distinctive mammalian behaviour. Our results provide evidence for the involvement of an imprinted gene in the control of adult behaviour.
We previously identified Peg1/Mest as a novel paternally expressed gene in the developing mouse embryo. The human PEG1 gene was recently assigned to 7q32 and shown to be imprinted and paternally expressed. Therefore, PEG1 deficiency could participate in the aetiology of pre- and post-natal growth retardation associated with maternal uniparental disomy 7 in humans. We have now initiated the characterization of the Peg1 locus in order to identify and dissect cis-acting elements implicated in its imprinted monoallelic expression. The genomic structure of Peg1 as well as the DNA sequence of the 5'-end of the gene, including 2.4 kb of promoter sequences and covering the first 2 exons, have been determined. Important sequence elements, such as a CpG island spanning exon 1 and direct repeats, are identified and discussed. To address the role of epigenetic modifications in the imprinting of Peg1, a methylation analysis of the Peg1 gene is presented. Partially methylated cytosine residues in 13.5 d.p.c. embryos and undifferentiated ES cells were identified. Using embryos carrying a targetted mutation at the Peg1 locus, we show that this partial promoter methylation pattern reflects a strict parent-of-origin-specific differential methylation: the expressed paternal allele is unmethylated, whereas the silenced maternal allele is fully methylated at the CpG sites studied. That the gametes carry the epigenetic information necessary to lay down this allele-specific methylation pattern is suggested by analysis of DNA isolated from sperm and parthenogenetic embryos.
We have isolated the human PEG1/MEST gene and have investigated its imprinting status and parental-specific methylation. FISH mapping assigned the gene to chromosome 7q32, and homologous sequences were identified on the short arm of human chromosomes 3 and 5. Through the use of a newly identified intragenic polymorphism, expression analysis revealed that PEG1/MEST is monoallelically transcribed in all fetal tissues examined. In two informative cases, expression was shown to be confined to the paternally derived allele. In contrast to the monoallelic expression observed in fetal tissues, biallelic expression was evident in adult blood lymphocytes. Biallelic expression in blood is supported by the demonstration of PEG1/MEST transcripts in a lymphoblastoid cell line with maternal uniparental disomy 7. The human PEG1/MEST gene spans a genomic region of approximately 13 kb. Sequence analysis of the 5' region of PEG1/MEST revealed the existence of a 620-bp-long CpG island that extends from the putative promoter region into intron 1. We demonstrate that this CpG island is methylated in a parent-of-origin-specific manner. All MspI/HpaII sites were unmethylated on the active paternal allele but methylated on the inactive maternal one.
        
Title: Genomic imprinting and chromosomal localization of the human MEST gene Nishita Y, Yoshida I, Sado T, Takagi N Ref: Genomics, 36:539, 1996 : PubMed
We have isolated a human homologue (MEST) of the mouse mesoderm-specific transcript (Mest) gene that shares about 70% nucleotide sequence homology. Northern blot analysis showed that the MEST gene was expressed in all major fetal organs and tissues so far examined, i.e., amnion, brain, heart, lung, stomach, gut, adrenal, kidney, muscle, and liver, which does not contradict with mesoderm-specific expression. MEST was abundantly expressed in hydatidiform moles of androgenetic origin, whereas it was barely detectable in dermoid cysts of parthenogenetic origin. Thus, it seems likely that the MEST gene, mapped to 7q32 by fluorescence in situ hybridization, is maternally repressed as the mouse homologue.
Parthenogenesis in the mouse is embryonic lethal partly because of imprinted genes that are expressed only from the paternal genome. In a systematic screen using subtraction hybridization between cDNAs from normal and parthenogenetic embryos, we initially identified two apparently novel imprinted genes, Peg1 and Peg3. Peg1 (paternally expressed gene 1) or Mest, the first imprinted gene found on the mouse chromosome 6, may contribute to the lethality of parthenogenones and of embryos with a maternal duplication for the proximal chromosome 6. Peg1/Mest is widely expressed in mesodermal tissues and belongs to the alpha/beta hydrolase fold family. A similar approach with androgenones can be used to identify imprinted genes that are expressed from the maternal genome only.
        
Title: A novel mesoderm-specific cDNA isolated from a mouse embryonal carcinoma cell line Sado T, Nakajima N, Tada M, Takagi N Ref: Dev Growth Differ, 35:551, 1993 : PubMed