Gene_locus Report for: human-MEST

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.

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.

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.

BACKGROUND: A growing body of evidence suggests that many downstream pathologies of obesity are amplified or even initiated by molecular changes within the white adipose tissue (WAT). Such changes are the result of an excessive expansion of individual white adipocytes and could potentially be ameliorated via an increase in de novo adipocyte recruitment (adipogenesis). Mesoderm-specific transcript (MEST) is a protein with a putative yet unidentified enzymatic function and has previously been shown to correlate with adiposity and adipocyte size in mouse. OBJECTIVES: This study analysed WAT samples and employed a cell model of adipogenesis to characterise MEST expression and function in human. METHODS AND
RESULTS: MEST mRNA and protein levels increased during adipocyte differentiation of human multipotent adipose-derived stem cells. Further, obese individuals displayed significantly higher MEST levels in WAT compared with normal-weight subjects, and MEST was significantly correlated with adipocyte volume. In striking contrast to previous mouse studies, knockdown of MEST enhanced human adipocyte differentiation, most likely via a significant promotion of peroxisome proliferator-activated receptor signalling, glycolysis and fatty acid biosynthesis pathways at early stages. Correspondingly, overexpression of MEST impaired adipogenesis. We further found that silencing of MEST fully substitutes for the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) as an inducer of adipogenesis. Accordingly, phosphorylation of the pro-adipogenic transcription factors cyclic AMP responsive element binding protein (CREB) and activating transcription factor 1 (ATF1) were highly increased on MEST knockdown.
CONCLUSIONS: Although we found a similar association between MEST and adiposity as previously described for mouse, our functional analyses suggest that MEST acts as an inhibitor of human adipogenesis, contrary to previous murine studies. We have further established a novel link between MEST and CREB/ATF1 that could be of general relevance in regulation of metabolism, in particular obesity-associated diseases.

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.

Methyl CpG-binding protein 2 gene (MeCP2) mutations are implicated in Rett syndrome (RTT), one of the common causes of female mental retardation. Two MeCP2 isoforms have been reported: MeCP2_e2 (splicing of all four exons) and MeCP2_e1 (alternative splicing of exons 1, 3, and 4). Their relative expression levels vary among tissues, with MeCP2_e1 being more dominant in adult brain, whereas MeCP2_e2 is expressed more abundantly in placenta, liver, and skeletal muscle. In this study, we performed specific disruption of the MeCP2_e2-defining exon 2 using the Cre-loxP system and examined the consequences of selective loss of MeCP2_e2 function in vivo. We performed behavior evaluation, gene expression analysis, using RT-PCR and real-time quantitative PCR, and histological analysis. We demonstrate that selective deletion of MeCP2_e2 does not result in RTT-associated neurological phenotypes but confers a survival disadvantage to embryos carrying a MeCP2_e2 null allele of maternal origin. In addition, we reveal a specific requirement for MeCP2_e2 function in extraembryonic tissue, where selective loss of MeCP2_e2 results in placenta defects and up-regulation of peg-1, as determined by the parental origin of the mutant allele. Taken together, our findings suggest a novel role for MeCP2 in normal placenta development and illustrate how paternal X chromosome inactivation in extraembryonic tissues confers a survival disadvantage for carriers of a mutant maternal MeCP2_e2 allele. Moreover, our findings provide an explanation for the absence of reports on MeCP2_e2-specific exon 2 mutations in RTT. MeCP2_e2 mutations in humans may result in a phenotype that evades a diagnosis of RTT.

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.

BACKGROUND: With the completion of the human genome sequence the functional analysis and characterization of the encoded proteins has become the next urging challenge in the post-genome era. The lack of comprehensive ORFeome resources has thus far hampered systematic applications by protein gain-of-function analysis. Gene and ORF coverage with full-length ORF clones thus needs to be extended. In combination with a unique and versatile cloning system, these will provide the tools for genome-wide systematic functional analyses, to achieve a deeper insight into complex biological processes. RESULTS: Here we describe the generation of a full-ORF clone resource of human genes applying the Gateway cloning technology (Invitrogen). A pipeline for efficient cloning and sequencing was developed and a sample tracking database was implemented to streamline the clone production process targeting more than 2,200 different ORFs. In addition, a robust cloning strategy was established, permitting the simultaneous generation of two clone variants that contain a particular ORF with as well as without a stop codon by the implementation of only one additional working step into the cloning procedure. Up to 92 % of the targeted ORFs were successfully amplified by PCR and more than 93 % of the amplicons successfully cloned. CONCLUSION: The German cDNA Consortium ORFeome resource currently consists of more than 3,800 sequence-verified entry clones representing ORFs, cloned with and without stop codon, for about 1,700 different gene loci. 177 splice variants were cloned representing 121 of these genes. The entry clones have been used to generate over 5,000 different expression constructs, providing the basis for functional profiling applications. As a member of the recently formed international ORFeome collaboration we substantially contribute to generating and providing a whole genome human ORFeome collection in a unique cloning system that is made freely available in the community.

As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

Human chromosome 7 has historically received prominent attention in the human genetics community, primarily related to the search for the cystic fibrosis gene and the frequent cytogenetic changes associated with various forms of cancer. Here we present more than 153 million base pairs representing 99.4% of the euchromatic sequence of chromosome 7, the first metacentric chromosome completed so far. The sequence has excellent concordance with previously established physical and genetic maps, and it exhibits an unusual amount of segmentally duplicated sequence (8.2%), with marked differences between the two arms. Our initial analyses have identified 1,150 protein-coding genes, 605 of which have been confirmed by complementary DNA sequences, and an additional 941 pseudogenes. Of genes confirmed by transcript sequences, some are polymorphic for mutations that disrupt the reading frame.

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.

The mouse Peg1/Mest gene is an imprinted gene that is expressed particularly in mesodermal tissues in early embryonic stages. It was the most abundant imprinted gene among eight paternally expressed genes (Peg 1-8) isolated by a subtraction-hybridization method from a mouse embryonal cDNA library. It has been mapped to proximal mouse chromosome 6, maternal duplication of which causes early embryonic lethality. The human chromosomal region that shares syntenic homology with this is 7q21-qter, and human maternal uniparental disomy 7 (UPD 7) causes apparent growth deficiency and slight morphological abnormalities. Therefore, at least one paternally expressed imprinted gene seems to be present in this region. In this report, we demonstrate that human PEG1/MEST is an imprinted gene expressed from a paternal allele and located on chromosome 7q31-34, near D7S649. It is the first imprinted gene mapped to human chromosome 7 and a candidate for a gene responsible for primordial growth retardation including Silver-Russell syndrome (SRS).

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.

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.