Knockdown of CDKN1C (p57kip2) and PHLDA2 Results in Developmental Changes in Bovine Pre-implantation Embryos

Imprinted genes have been implicated in early embryonic, placental, and neonatal development and alterations in expression levels of these genes can lead to growth abnormalities and embryonic lethality. However, little is known about the functions of bovine imprinted genes during the pre-implantation period. Therefore, the objective of this study was to assess the influence of altered expression of imprinted genes on developmental progress of embryos using small interfering RNA (siRNA). Expression levels of 18 imprinted genes (MAGEL2, UBE3A, IGF2R, NAP1L5, TSSC4, PEG3, NDN, CDKN1C, PHLDA2, MKRN3, USP29, NNAT, PEG10, RTL1, IGF2, H19, MIM1, and XIST) were compared between embryos reaching the blastocyst stage and growth-arrested embryos (degenerates) using quantitative real-time PCR (qRT-PCR). Ten genes were found to be differentially expressed between blastocysts and degenerates. The CDKN1C gene showed the highest upregulation in blastocysts whereas PHLDA2 was highly expressed in degenerates. To assess whether the observed differential gene expression was causative or resultant of embryo degeneration, these genes were selected for functional analysis using siRNA. Injection of siRNA specific to PHLDA2 into one-cell zygotes resulted in a substantial increase in blastocyst development, whereas injection of CDKN1C-specific siRNA resulted in a 45% reduction (P = 0.0006) in blastocyst development. RNA-Seq analysis of CDKN1C-siRNA-injected vs. non-injected embryos revealed 51 differentially expressed genes with functions in apoptosis, lipid metabolism, differentiation, and cell cycle regulation. Gene ontology analysis revealed nine pathways related to cell signaling, metabolism, and nucleic acid processing. Overall, our results show that proper expression levels of the imprinted genes CDKN1C and PHLDA2 are critical for embryo development, which suggests that these genes can be used as markers for normal blastocyst formation.     

Culture of preimplantation mouse embryos affects fetal development and the expression of imprinted genes

Culture of preimplantation mammalian embryos and cells can influence their subsequent growth and differentiation. Previously, we reported that culture of mouse embryonic stem cells is associated with deregulation of genomic imprinting and affects the potential for these cells to develop into normal fetuses. The purpose of our current study was to determine whether culture of preimplantation mouse embryos in a chemically defined medium (M16) with or without fetal calf serum (FCS) can affect their subsequent development and imprinted gene expression. Only one third of the blastocysts that had been cultured from two-cell embryos in M16 medium complemented with FCS developed into viable Day 14 fetuses after transfer into recipients. These M16 + FCS fetuses were reduced in weight as compared with controls and M16 fetuses and had decreased expression of the imprinted H19 and insulin-like growth factor 2 genes associated with a gain of DNA methylation at an imprinting control region upstream of H19. They also displayed increased expression of the imprinted gene Grb10. The growth factor receptor binding gene Grb7, in contrast, was strongly reduced in its expression in most of the M16 + FCS fetuses. No alterations were detected for the imprinted gene MEST: Preimplantation culture in the presence of serum can influence the regulation of multiple growth-related imprinted genes, thus leading to aberrant fetal growth and development.     

Sequence polymorphisms, allelic expression status and chromosome locations of the chicken IGF2 and MPR1 genes

By screening 26 chicken breeds and lines, DNA polymorphisms were identified in the IGF2 and MPR1 genes, of which mammalian homologues are parentally imprinted, and the GAPD gene, a housekeeping control. Using the polymorphisms as genetic markers, we found that all three genes are expressed biallelically in embryonic tissues. IGF2 and MPR1 were mapped on chicken chromosomes 5 and 3, respectively, by fluorescence in situ hybridization, demonstrating conserved linkage homology between mammals and birds.     

Differential expression pattern of C4 bundle sheath expression genes in rice, a C3 plant

NADP-malic enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase(PCK) are specifically expressed in bundle sheath cells (BSCs)in NADP-ME-type and PCK-type C₄ plants, respectively. Unlikethe high activities of these enzymes in the green leaves ofC₄ plants, their low activities have been detected in the leavesof C₃ plants. In order to elucidate the differences in the geneexpression system between C₃ and C₄ plants, we have producedchimeric constructs with the ß-glucuronidase (GUS)reporter gene under the control of the maize NADP-Me (ZmMe)or Zoysia japonica Pck (ZjPck) promoter and introduced theseconstructs into rice. In leaves of transgenic rice, the ZmMepromoter directed GUS expression not only in mesophyll cells(MCs) but also in BSCs and vascular cells, whereas the ZjPckpromoter directed GUS expression only in BSCs and vascular cells.Neither the ZjPck nor ZmMe promoters induced GUS expressiondue to light. In rice leaves, the endogenous NADP-Me (OsMe1)was expressed in MCs, BSCs and vascular cells, whereas the ricePck (OsPck1) was expressed only in BSCs and vascular cells.Taken together, the results obtained from transgenic rice demonstratethat the expression pattern of ZmMe or ZjPck in transgenic ricewas reflected by that of its counterpart gene in rice. (Received August 8, 2004; Accepted February 20, 2005 )     

Synteny mapping of the bovine IGHG2, CRC and IGF1 genes

A panel of bovine-murine hybrid cell lines was analysed for 10 loci, including three (IGF1, IGHG2 and the calcium release channel gene [CRC]) that have previously been mapped in man, but not in cattle. The IGF and CRC genes were indirectly mapped to chromosomes 5 and 18 respectively and the syntenies of the HOX2 and GH genes and of the NP and FOS genes were confirmed. The results also show that the IGHG2 locus, which is linked to NP and FOS on human chromosome 14, is separated from these genes in cattle. By showing synteny of the IGHG2 and MPI loci, the IGHG2 locus has been indirectly mapped to chromosome 21.     

The role and interaction of imprinted genes in human fetal growth

Identifying the genetic input for fetal growth will help to understand common, serious complications of pregnancy such as fetal growth restriction. Genomic imprinting is an epigenetic process that silences one parental allele, resulting in monoallelic expression. Imprinted genes are important in mammalian fetal growth and development. Evidence has emerged showing that genes that are paternally expressed promote fetal growth, whereas maternally expressed genes suppress growth. We have assessed whether the expression levels of key imprinted genes correlate with fetal growth parameters during pregnancy, either early in gestation, using chorionic villus samples (CVS), or in term placenta. We have found that the expression of paternally expressing insulin-like growth factor 2 (IGF2), its receptor IGF2R, and the IGF2/IGF1R ratio in CVS tissues significantly correlate with crown–rump length and birthweight, whereas term placenta expression shows no correlation. For the maternally expressing pleckstrin homology-like domain family A, member 2 (PHLDA2), there is no correlation early in pregnancy in CVS but a highly significant negative relationship in term placenta. Analysis of the control of imprinted expression of PHLDA2 gave rise to a maternally and compounded grand-maternally controlled genetic effect with a birthweight increase of 93/155 g, respectively, when one copy of the PHLDA2 promoter variant is inherited. Expression of the growth factor receptor-bound protein 10 (GRB10) in term placenta is significantly negatively correlated with head circumference. Analysis of the paternally expressing delta-like 1 homologue (DLK1) shows that the paternal transmission of type 1 diabetes protective G allele of rs941576 single nucleotide polymorphism (SNP) results in significantly reduced birth weight (−132 g). In conclusion, we have found that the expression of key imprinted genes show a strong correlation with fetal growth and that for both genetic and genomics data analyses, it is important not to overlook parent-of-origin effects.     

Allelic gene expression imbalance of bovine IGF2, LEP and CCL2 genes in liver, kidney and pituitary

Allelic expression imbalance (AEI) is an important genetic factor being the cause of differences in phenotypic traits that can be heritable. Studying AEI can be useful in searching for factors that modulate gene expression and help to understand molecular mechanisms underlying phenotypic changes. Although it was commonly recognized in many species and we know many genes show allelic expression imbalance, this phenomena was not studied on a larger scale in cattle. Using the pyrosequencing method we analyzed a set of 29 bovine genes in order to find those that have preferential allelic expression. The study was conducted in three tissues: liver, pituitary and kindey. Out of the studied group of genes 3 of them—LEP (leptin), IGF2 (insulin-like growth factor 2), CCL2 (chemokine C–C motif ligand 2) showed allelic expression imbalance.     

Structure and expression of the zebrafish mest gene, an ortholog of mammalian imprinted gene PEG1/MEST

PEG1/MEST is a paternally expressed gene in placental mammals. Here, we report identification of zebrafish (Danio rerio) gene mest, an ortholog of mammalian PEG1/MEST. Zebrafish mest encodes a polypeptide of 344 amino acids and shows a significant similarity to mammalian orthologs. Zebrafish mest is present as a single copy in the zebrafish genome and is closely linked to copg2 as in mammals. It is notable that 10 of 11 intron positions in mest are conserved among mammalian PEG1/MEST genes, indicating that the genomic organization and linkage between mest and copg2 loci was established in ancient vertebrates. Zebrafish mest is expressed in blastula, segmentation, and larval stages, exhibiting gradually increased expression as the development proceeds. Allelic expression analysis in hybrid larvae shows that both parental alleles are transcribed. We also observed one-codon alternative splicing involving an alternative usage of the two consecutive splice acceptors of intron 1, generating two protein isoforms with different lengths of a single amino acid.     

Prohibitin-Mediated Lifespan and Mitochondrial Stress Implicate SGK-1, Insulin/IGF and mTORC2 in C. elegans

Lifespan regulation by mitochondrial proteins has been well described, however, the mechanism of this regulation is not fully understood. Amongst the mitochondrial proteins profoundly affecting ageing are prohibitins (PHB-1 and PHB-2). Paradoxically, in C. elegans prohibitin depletion shortens the lifespan of wild type animals while dramatically extending that of metabolically compromised animals, such as daf-2-insulin-receptor mutants. Here we show that amongst the three kinases known to act downstream of daf-2, only loss of function of sgk-1 recapitulates the ageing phenotype observed in daf-2 mutants upon prohibitin depletion. Interestingly, signalling through SGK-1 receives input from an additional pathway, parallel to DAF-2, for the prohibitin-mediated lifespan phenotype. We investigated the effect of prohibitin depletion on the mitochondrial unfolded protein response (UPR^mt). Remarkably, the lifespan extension upon prohibitin elimination, of both daf-2 and sgk-1 mutants, is accompanied by suppression of the UPR^mt induced by lack of prohibitin. On the contrary, gain of function of SGK-1 results in further shortening of lifespan and a further increase of the UPR^mt in prohibitin depleted animals. Moreover, SGK-1 interacts with RICT-1 for the regulation of the UPR^mt in a parallel pathway to DAF-2. Interestingly, prohibitin depletion in rict-1 loss of function mutant animals also causes lifespan extension. Finally, we reveal an unprecedented role for mTORC2-SGK-1 in the regulation of mitochodrial homeostasis. Together, these results give further insight into the mechanism of lifespan regulation by mitochondrial function and reveal a cross-talk of mitochondria with two key pathways, Insulin/IGF and mTORC2, for the regulation of ageing and stress response.     

Role of the CDKN1A/p21, CDKN1C/p57, and CDKN2A/p16 Genes in the Risk of Atherosclerosis and Myocardial Infarction

Atherosclerotic is characterised by excessive proliferation of neointimal leukocytes and vascular smooth muscle cells (VSMCs). In mice, the manipulation of cell cycle inhibitors such as CDKN1B (p27) and CDKN1A (p21) modifies the risk of developing atherosclerosis. In humans, CDKN1A, CDKN1B, and CDKN1C (p57) are differentially expressed in normal versus atherosclerotic vessels. A DNA-polymorphism within the CDKN1B promoter has been associated with myocardial infarction (MI). In the present study, we analysed the effect of CDKN1A, CDKN1C, and CDKN2A (p16) polymorphisms on MI-risk. A total of 316 patients (all male,     

Regulation of hepatic expression of IGF I and fetal IGF binding protein mRNA in streptozotocin-diabetic rats

Hepatic mRNA levels of insulin-like growth factor I (IGF I) and of the fetal, nonglycosylated 32 kDa IGF-binding protein (BP) were analysed in diabetic, diabetic insulin- and IGF I-treated rats as well as in age-matched, healthy control animals. IGF ImRNA levels are reduced in diabetic rats and increased by insulin treatment. In contrast, the infusion of IGF I does not significantly upregulate IGF I mRNA levels. Fetal IGF BP mRNA expression is very low in healthy control animals, but high levels are found in diabetic rats. Insulin therapy lowers fetal IGF BP mRNA levels, whereas IGF I has no effect. We propose that insulin is a major regulator of the 32 kDa IGF BP levels in adult rats.     

Characterization and expression pattern of IbPRP1 and IbPRP2 stress-related genes from sweetpotato

Two putative stress-related genes were isolated from sweet-potato and were designated as IbPRP1 and IbPRP2 (Ipomoea batatas proline-rich proteins 1 and 2). The deduced amino acid aligment of IbPRP1 and IbPRP2 shows that these two genes belong to the AAI_LTSS superfamily. Proteins in this family are known to play primary roles including defending plants from pathogens and insects, lipid transport between intracellular membranes, and nutrient storage. The mRNA expression of IbPRP1 and IbPRP2 were investigated and the results demonstrate that IbPRP2 has tissue-specific expression. Moreover, IbPRP1 and IbPRP2 may be involved in response to various stresses including drought, pathogen, and oxidative stress. In addition, when leaf disc test was performed, the IbPRP1 transgenic tobacco plants showed increase in tolerance to salt (100 mM, 200 mM, and 300 mM). Moreover, IbPRP1 and IbPRP2 may have some roles of transmembrane protein in sweetpotato when checked through GFP fusion cell localization and transmembrane analysis. All of these results indicate that IbPRP1 and IbPRP2 might play an important role in plant stress responses.     

Genomic organization of the IGF1, IGF2, MYF5, MYF6 and GRF/PACAP genes across Salmoninae genera

Whole-genome duplication in the ancient ray-finned fish and subsequent tetraploidization in the ancestor to the salmonids have complicated genomic and candidate gene studies in these organisms as many genes with multiple copies are present throughout their genomes. In an attempt to identify genes with a potential influence on growth and development, we investigated the genomic positions of insulin-like growth factors 1 and 2 (IGF1, IGF2), myogenic factors 5 and 6 (MYF5, MYF6) and growth hormone-releasing factor/pituitary adenylate cyclase-activating polypeptide (GRF/PACAP) in three salmonid species: rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar) and Arctic charr (Salvelinus alpinus). Our results suggest a tight association between the IGF1, MYF5 and MYF6 genes in all three species. We further localized the duplicated copies of IGF1 to the homeologous linkage groups RT-7/15 in rainbow trout and AC-3/24 in Arctic charr, and the two copies of MYF6 to homeologous linkage groups AS-22/24 in Atlantic salmon. Localization of GRF/PACAP to RT-7, AS-31 and AC-27 and IGF2 to RT-27, AS-2 and AC-4 in rainbow trout, Atlantic salmon and Arctic charr respectively is consistent with previously reported homologies among these chromosomal segments identified using other genetic markers. However, localization of the second copy of GRF/PACAP to RT-19 and AC-14 and the duplicated copy of IGF2 to AC-19 suggest a possible new homology/homeology between these chromosomes. These results might also be an indication of a more ancient polyploidization event that occurred deep in the ray-finned fish lineage.     

PPARG,KCNJ11, CDKAL1, CDKN2A-CDKN2B,IDE-KIF11-HHEX,IGF2BP2 and SLC30A8 Are Associated with Type 2 Diabetes in a Chinese Population

Background

Recent advance in genetic studies added the confirmed susceptible loci for type 2 diabetes to eighteen. In this study, we attempt to analyze the independent and joint effect of variants from these loci on type 2 diabetes and clinical phenotypes related to glucose metabolism.

Methods/Principal Findings

Twenty-one single nucleotide polymorphisms (SNPs) from fourteen loci were successfully genotyped in 1,849 subjects with type 2 diabetes and 1,785 subjects with normal glucose regulation. We analyzed the allele and genotype distribution between the cases and controls of these SNPs as well as the joint effects of the susceptible loci on type 2 diabetes risk. The associations between SNPs and type 2 diabetes were examined by logistic regression. The associations between SNPs and quantitative traits were examined by linear regression. The discriminative accuracy of the prediction models was assessed by area under the receiver operating characteristic curves. We confirmed the effects of SNPs from PPARG, KCNJ11, CDKAL1, CDKN2A-CDKN2B, IDE-KIF11-HHEX, IGF2BP2 and SLC30A8 on risk for type 2 diabetes, with odds ratios ranging from 1.114 to 1.406 (P value range from 0.0335 to 1.37E-12). But no significant association was detected between SNPs from WFS1, FTO, JAZF1, TSPAN8-LGR5, THADA, ADAMTS9, NOTCH2-ADAM30 and type 2 diabetes. Analyses on the quantitative traits in the control subjects showed that THADA SNP rs7578597 was association with 2-h insulin during oral glucose tolerance tests (P = 0.0005, empirical P = 0.0090). The joint effect analysis of SNPs from eleven loci showed the individual carrying more risk alleles had a significantly higher risk for type 2 diabetes. And the type 2 diabetes patients with more risk allele tended to have earlier diagnostic ages (P = 0.0006).

Conclusions/Significance

The current study confirmed the association between PPARG, KCNJ11, CDKAL1, CDKN2A-CDKN2B, IDE-KIF11-HHEX, IGF2BP2 and SLC30A8 and type 2 diabetes. These type 2 diabetes risk loci contributed to the disease additively.     

Gene expression profile of androgen modulated genes in the murine fetal developing lung

Background  

Accumulating evidences suggest that sex affects lung development. Indeed, a higher incidence of respiratory distress syndrome is observed in male compared to female preterm neonates at comparable developmental stage and experimental studies demonstrated an androgen-related delay in male lung maturation. However, the precise mechanisms underlying these deleterious effects of androgens in lung maturation are only partially understood.