Gene_locus Report for: mouse-MEST

Interindividual variation of white adipose tissue (WAT) expression of mesoderm specific transcript (Mest), a paternally-expressed imprinted gene belonging to the alpha/beta-hydrolase fold protein family, becomes apparent among genetically inbred mice fed high fat diet (HFD) and is positively associated with adipose tissue expansion (ATE). To elucidate a role for MEST in ATE, mice were developed with global and adipose tissue inactivation of Mest. Mice with homozygous (MestgKO) and paternal allelic (MestpKO) inactivation of Mest were born at expected Mendelian frequencies, showed no behavioral or physical abnormalities, and did not perturb expression of the Mest locus-derived microRNA miR-335. MestpKO mice fed HFD showed reduced ATE and adipocyte hypertrophy, improved glucose tolerance, and reduced WAT expression of genes associated with hypoxia and inflammation compared to littermate controls. Remarkably, caloric intake and energy expenditure were unchanged between genotypes. Mice with adipose tissue inactivation of Mest were phenotypically similar to MestpKO, supporting a role for WAT MEST in ATE. Global profiling of WAT gene expression of HFD-fed control and MestpKO mice detected few differences between genotypes; nevertheless, genes with reduced expression in MestpKO mice were associated with immune processes and consistent with improved glucose homeostasis. Ear-derived mesenchymal stem cells (EMSC) from MestgKO mice showed no differences in adipogenic differentiation compared to control cells unless challenged by shRNA knockdown of Gpat4, an enzyme that mediates lipid accumulation in adipocytes. Reduced adipogenic capacity of EMSC from MestgKO after Gpat4 knockdown suggests that MEST facilitates lipid accumulation in adipocytes. Our data suggests that reduced diet-induced ATE in MEST-deficient mice diminishes hypoxia and inflammation in WAT leading to improved glucose tolerance and insulin sensitivity. Since inactivation of Mest in mice has minimal additional effects aside from reduction of ATE, an intervention that mitigates MEST function in adipocytes is a plausible strategy to obviate obesity and type-2-diabetes.

Brain developmental disorders such as lissencephaly can result from faulty neuronal migration and differentiation during the formation of the mammalian neocortex. The cerebral cortex is a modular structure, where developmentally, newborn neurons are generated as a neuro-epithelial sheet and subsequently differentiate, migrate and organize into their final positions in the cerebral cortical plate via a process involving both tangential and radial migration. The specific role of Mest, an imprinted gene, in neuronal migration has not been previously studied. In this work, we reduced expression of Mest with in utero electroporation of neuronal progenitors in the developing embryonic mouse neocortex. Reduction of Mest levels by shRNA significantly reduced the number of neurons migrating to the cortical plate. Also, Mest-knockdown disrupted the transition of bipolar neurons into multipolar neurons migrating out of the sub-ventricular zone region. The migrating neurons also adopted a more tangential migration pattern upon knockdown of the Mest message, losing their potential to attach to radial glia cells, required for radial migration. The differentiation and migration properties of neurons via Wnt-Akt signaling were affected by Mest changes. In addition, miR-335, encoded in a Mest gene intron, was identified as being responsible for blocking the default tangential migration of the neurons. Our results suggest that Mest and its intron product, miR-335, play important roles in neuronal migration with Mest regulating the morphological transition of primary neurons required in the formation of the mammalian neocortex.

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.

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.

Interindividual variation of white adipose tissue (WAT) expression of mesoderm specific transcript (Mest), a paternally-expressed imprinted gene belonging to the alpha/beta-hydrolase fold protein family, becomes apparent among genetically inbred mice fed high fat diet (HFD) and is positively associated with adipose tissue expansion (ATE). To elucidate a role for MEST in ATE, mice were developed with global and adipose tissue inactivation of Mest. Mice with homozygous (MestgKO) and paternal allelic (MestpKO) inactivation of Mest were born at expected Mendelian frequencies, showed no behavioral or physical abnormalities, and did not perturb expression of the Mest locus-derived microRNA miR-335. MestpKO mice fed HFD showed reduced ATE and adipocyte hypertrophy, improved glucose tolerance, and reduced WAT expression of genes associated with hypoxia and inflammation compared to littermate controls. Remarkably, caloric intake and energy expenditure were unchanged between genotypes. Mice with adipose tissue inactivation of Mest were phenotypically similar to MestpKO, supporting a role for WAT MEST in ATE. Global profiling of WAT gene expression of HFD-fed control and MestpKO mice detected few differences between genotypes; nevertheless, genes with reduced expression in MestpKO mice were associated with immune processes and consistent with improved glucose homeostasis. Ear-derived mesenchymal stem cells (EMSC) from MestgKO mice showed no differences in adipogenic differentiation compared to control cells unless challenged by shRNA knockdown of Gpat4, an enzyme that mediates lipid accumulation in adipocytes. Reduced adipogenic capacity of EMSC from MestgKO after Gpat4 knockdown suggests that MEST facilitates lipid accumulation in adipocytes. Our data suggests that reduced diet-induced ATE in MEST-deficient mice diminishes hypoxia and inflammation in WAT leading to improved glucose tolerance and insulin sensitivity. Since inactivation of Mest in mice has minimal additional effects aside from reduction of ATE, an intervention that mitigates MEST function in adipocytes is a plausible strategy to obviate obesity and type-2-diabetes.

Brain developmental disorders such as lissencephaly can result from faulty neuronal migration and differentiation during the formation of the mammalian neocortex. The cerebral cortex is a modular structure, where developmentally, newborn neurons are generated as a neuro-epithelial sheet and subsequently differentiate, migrate and organize into their final positions in the cerebral cortical plate via a process involving both tangential and radial migration. The specific role of Mest, an imprinted gene, in neuronal migration has not been previously studied. In this work, we reduced expression of Mest with in utero electroporation of neuronal progenitors in the developing embryonic mouse neocortex. Reduction of Mest levels by shRNA significantly reduced the number of neurons migrating to the cortical plate. Also, Mest-knockdown disrupted the transition of bipolar neurons into multipolar neurons migrating out of the sub-ventricular zone region. The migrating neurons also adopted a more tangential migration pattern upon knockdown of the Mest message, losing their potential to attach to radial glia cells, required for radial migration. The differentiation and migration properties of neurons via Wnt-Akt signaling were affected by Mest changes. In addition, miR-335, encoded in a Mest gene intron, was identified as being responsible for blocking the default tangential migration of the neurons. Our results suggest that Mest and its intron product, miR-335, play important roles in neuronal migration with Mest regulating the morphological transition of primary neurons required in the formation of the mammalian neocortex.

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.

The mouse (Mus musculus) is the premier animal model for understanding human disease and development. Here we show that a comprehensive understanding of mouse biology is only possible with the availability of a finished, high-quality genome assembly. The finished clone-based assembly of the mouse strain C57BL/6J reported here has over 175,000 fewer gaps and over 139 Mb more of novel sequence, compared with the earlier MGSCv3 draft genome assembly. In a comprehensive analysis of this revised genome sequence, we are now able to define 20,210 protein-coding genes, over a thousand more than predicted in the human genome (19,042 genes). In addition, we identified 439 long, non-protein-coding RNAs with evidence for transcribed orthologs in human. We analyzed the complex and repetitive landscape of 267 Mb of sequence that was missing or misassembled in the previously published assembly, and we provide insights into the reasons for its resistance to sequencing and assembly by whole-genome shotgun approaches. Duplicated regions within newly assembled sequence tend to be of more recent ancestry than duplicates in the published draft, correcting our initial understanding of recent evolution on the mouse lineage. These duplicates appear to be largely composed of sequence regions containing transposable elements and duplicated protein-coding genes; of these, some may be fixed in the mouse population, but at least 40% of segmentally duplicated sequences are copy number variable even among laboratory mouse strains. Mouse lineage-specific regions contain 3,767 genes drawn mainly from rapidly-changing gene families associated with reproductive functions. The finished mouse genome assembly, therefore, greatly improves our understanding of rodent-specific biology and allows the delineation of ancestral biological functions that are shared with human from derived functions that are not.

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.

The expression of six imprinted genes (Dlk1, Gt12, Igf2r, Kcnq1, Nnat, and Peg1) was examined in brains of 21 mice derived from N2 x N2 intercrosses between C57BL/6 and MOLF/Ei strains. Imprinting of Igf2r, Kcnq1, Gt12, and Dlk1 varied among individuals. As three of these genes are implicated in cell-cell signaling or cell-environment interactions, variation in their imprinting may influence a wide range of biological processes from cell differentiation to behavior. To elucidate the mechanisms underlying the interindividual imprinting variation in the brain, we focused our effort on the paternally expressed gene Dlk1. We investigated expression of Dlk1 in the brains of animals from N9 and N10 backcrosses and found that reactivation of the normally silent maternal Dlk1 allele in the N9 and N10 mice occurred less often than in N2 x N2 animals. Our data suggest that trans-acting genetic factors of MOLF/Ei origin facilitate the reactivation of the normally silent maternal allele of Dlk1. We mapped one of these factors to the proximal part of Chr 7. The results of bisulfite sequencing methylation analysis show that reactivation of the maternal allele was also associated with hypermethylation of the intragenic differentially methylated region (IG DMR), which is the imprinting control region for the Dlk1-Gt12 domain. Thus, the imprinting status of Dlk1 in the brain depends upon trans-acting genetic influences and correlates with the methylation status of a specific subregion of the IG DMR.

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.

Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.

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.

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.