N4-acetylcytidine of Nop2 mRNA is required for the transition of morula-to-blastocyst.

N-acetyltransferase 10 (NAT10)-mediated N4-acetylcytidine (ac4C) modification is crucial for mRNA stability and translation efficiency, yet the underlying function in mammalian preimplantation embryos remains unclear. Here, we characterized the ac4C modification landscape in mouse early embryos and found that the majority of embryos deficient in ac4C writer-NAT10 failed to develop into normal blastocysts. Through single-cell sequencing, RNA-seq, acetylated RNA immunoprecipitation combined with PCR (acRIP-PCR), and embryonic phenotype monitoring, Nop2 was screened as a target gene of Nat10. Mechanistically, Nat10 knockdown decreases the ac4C modification on Nop2 mRNA and reduces RNA and protein abundance by affecting the mRNA stability of Nop2. Then, depletion of NOP2 may inhibit the translation of transcription factor TEAD4, resulting in defective expression of the downstream lineage-specific gene Cdx2, and ultimately preventing blastomeres from undergoing the trophectoderm (TE) fate. However, exogenous Nop2 mRNA partially reverses this abnormal development. In conclusion, our findings demonstrate that defective ac4C modification of Nop2 mRNA hinders the morula-to-blastocyst transition by influencing the first cell fate decision in mice.

Deletion of Meg8-DMR Enhances Migration and Invasion of MLTC-1 Depending on the CTCF Binding Sites.

The Dlk1-Dio3 imprinted domain on mouse chromosome 12 contains three well-characterized paternally methylated differentially methylated regions (DMRs): IG-DMR, Gtl2-DMR, and Dlk1-DMR. These DMRs control the expression of many genes involved in embryonic development, inherited diseases, and human cancer in this domain. The first maternal methylation DMR discovered in this domain was the Meg8-DMR, the targets and biological function of which are still unknown. Here, using an enhancer-blocking assay, we first dissected the functional parts of the Meg8-DMR and showed that its insulator activity is dependent on the CCCTC-binding factor (CTCF) in MLTC-1. Results from RNA-seq showed that the deletion of the Meg8-DMR and its compartment CTCF binding sites, but not GGCG repeats, lead to the downregulation of numerous genes on chromosome 12, in particular the drastically reduced expression of Dlk1 and Rtl1 in the Dlk1-Dio3 domain, while differentially expressed genes are enriched in the MAPK pathway. In vitro assays revealed that the deletion of the Meg8-DMR and CTCF binding sites enhances cell migration and invasion by decreasing Dlk1 and activating the Notch1-Rhoc-MAPK/ERK pathway. These findings enhance research into gene regulation in the Dlk1-Dio3 domain by indicating that the Meg8-DMR functions as a long-range regulatory element which is dependent on CTCF binding sites and affects multiple genes in this domain.

[Analysis of animal models of ulcerative colitis based on characteristics of clinical symptoms of traditional Chinese and Wes-tern medicine].

This article aims to provide a good experimental method for the study of drug treatment of ulcerative colitis. According to the characteristics of ulcerative colitis's clinical symptoms, common ulcerative colitis animal models were analyzed. Based on the characteristics of clinical symptoms of traditional Chinese medicine and Western medicine for ulcerative colitis disease, the existing commonly used animal models of ulcerative colitis were analyzed to summarize the current matching degree, advantages and disadvantages of the exi-sting animal models of ulcerative colitis and clinical symptoms. At present, studies on ulcerative colitis mainly adopt four types of induction modeling methods, such as immunization, chemical stimulation, compound method and gene model. There are many reported methods of colitis modeling, but no model can reflect the characteristics of clinical symptoms of ulcerative colitis treated with Western or Chinese medicine. This article summarizes the characteristics, clinically relevant symptoms and applicable scope of immunization, chemical stimulation, compound method, and gene model, so as to provide a reliable animal model for subsequent studies of prevention and treatment of colitis.

C/EBPß mediates palmitate-induced musclin expression via the regulation of PERK/ATF4 pathways in myotubes.

Musclin is a muscle-secreted cytokine that disrupts glucose uptake and glycogen synthesis in type 2 diabetes. The purpose of this study was to investigate the mechanisms responsible for the regulation of musclin gene expression in response to treatment with palmitate. RNA sequencing results showed that biological processes activated by palmitate are mainly enriched in endoplasmic reticulum (ER) stress. The protein kinase RNA-like ER kinase (PERK) signaling pathway is involved in the regulation of musclin expression induced by palmitate. Chromatin immunoprecipitation data showed that activating transcription factor 4 (ATF4)-downstream of PERK-bound to the promoter of the C/EBPß gene. Notably, C/EBPß also contains a binding site in the region -94~-52 of the musclin gene promoter. Knockdown or knockout of PERK and ATF4 using short hairpin RNA or CRISPR-Cas9 decreased the expression of C/EBPß and musclin induced by palmitate. Furthermore, knockdown and knockout of C/EBPß alleviated the high expression of musclin in response to treatment with palmitate. Moreover, CRISPR-Cas9 knockout of the region -94~-52 in which C/EBPß binds to the promoter of musclin abrogated the induction of high musclin expression caused by palmitate. Collectively, these findings suggest that treatment with palmitate activates the PERK/ATF4 signaling pathway, which in turn increases the expression of C/EBPß. C/EBPß binds directly to the promoter of the musclin gene and upregulates its expression.

The saturated fatty acid palmitate induces insulin resistance through Smad3-mediated down-regulation of FNDC5 in myotubes.

Elevated plasma free fatty acid (FFA) levels are associated with insulin resistance and can cause lipotoxicity in skeletal muscles. In response to FFAs, skeletal muscle can secrete a variety of cytokines. Irisin, one such muscle-secreted cytokine, can improve glucose tolerance, glucose uptake, and lipid metabolism. It is produced by the transmembrane protein fibronectin type Ⅲ domain containing 5 (FNDC5) by specific proteases. The purpose of this study was to investigate the regulatory mechanisms of the FNDC5 response to palmitate and their relationships with insulin resistance in C2C12 myotubes. RNA sequencing analysis results from C2C12 myotubes treated with palmitate showed that palmitate could activate the TGF-ß signaling pathway. Palmitate directly affected the expression of Smad3, but not its phosphorylation level, in C2C12 myotubes. Furthermore, knockdown and knockout of Smad3 alleviated the inhibitory effect of palmitate on the expression of FNDC5. In contrast, overexpression of Smad3 aggravated the inhibition of FNDC5 expression. There is a Smad3 binding motif in the -660 bp to -649 bp region of the Fndc5 promoter. CRISPR/Cas9 knockout of this region also alleviated the inhibition of FNDC5 expression in response to palmitate. More importantly, inhibition of FNDC5 expression mediated by Smad3 led to a decrease in insulin sensitivity in C2C12 myotubes. Collectively, these findings suggest that palmitate could induce insulin resistance through Smad3-mediated down-regulation of the Fndc5 gene.

Alteration of the tumor suppressor SARDH in sporadic colorectal cancer: A functional and transcriptome profiling-based study.

Sporadic colorectal cancer (sCRC) is one of the leading causes of cancer death worldwide. As a highly heterogeneous complex disease, the currently reported classical genetic markers for sCRC, including APC, KRAS, BRAF, and TP53 gene mutations and epigenetic alterations, can explain only some sCRC patients. Here, we first reported a deleterious c.551C>T mutation in SARDH in sCRC. SARDH was identified as a novel tumor suppressor gene and was abnormally decreased in sCRC at both the transcriptional and the translational level. SARDH mRNA levels were also down-regulated in oesophageal cancer, lung cancer, liver cancer, and pancreatic cancer in the TCGA database. SARDH overexpression inhibited the proliferation, migration, and invasion of CRC cell lines, whereas its depletion improved these processes. SARDH overexpression was down-regulated in multiple pathways, especially in the chemokine pathway. The SARDH transcript level was positively correlated with the methylation states of CXCL1 and CCL20. Therefore, we concluded that SARDH depletion is involved in the development of sCRC.

MicroRNA-3191 promotes migration and invasion by downregulating TGFBR2 in colorectal cancer.

Mutations in transforming growth factor beta receptor II (TGFBR2) are detected in up to 30% of overall colorectal cancer (CRC). Dysregulation of some microRNAs participated in the CRC pathogenesis. In this study, we used the gene ontology analyses, the Kyoto Encyclopedia of Genes and Genomes pathway analyses and gene set enrichment analysis to indicate that miR-3191 was involved in the regulation of transforming growth factor beta (TGF-BETA) signal pathway in CRC. These bioinformatics results were supported by data obtained from CRC samples and experiments in vitro. The luciferase reporter assay was used to confirm that miR-3191 modulates TGF-BETA signal pathway by targeting TGFBR2. By transwell migration and invasion assays, we showed that miR-3191 promoted CRC cell migration and invasion by downregulating TGFBR2. And it may serve as a novel therapeutic strategy for treating CRC patients.

l-Methionine activates Nrf2-ARE pathway to induce endogenous antioxidant activity for depressing ROS-derived oxidative stress in growing rats.

BACKGROUND: Methionine is an essential sulfur-containing amino acid. To elucidate the influence of l-methionine on activation of the nuclear factor erythroid 2-related factor 2-antioxidant responsive element (Nrf2-ARE) antioxidant pathway to stimulate the endogenous antioxidant activity for depressing reactive oxygen species (ROS)-derived oxidative stress, male Wistar rats were orally administered l-methionine daily for 14 days. RESULTS: With the intake of l-methionine, Nrf2 was activated by l-methionine through depressing Keap1 and Cul3, resulting in upregulation of ARE-driven antioxidant expression (glutamate cysteine ligase catalytic subunit, glutamate cysteine ligase modulatory subunit, glutathione synthase (GS), catalase (CAT), superoxide dismutase (SOD), heme oxygenase 1, NAD(P)H:quinone oxidoreductase 1, glutathione reductase (GR), glutathione S-transferase (GST), glutathione peroxidase (GPx)) with increasing l-methionine availability. Upon activation of Nrf2, glutathione synthesis was increased through upregulated expression of methionine adenosyltransferase, S-adenosylhomocysteine hydrolase, cystathionine ß-synthase, cystathionine γ-lyse, glutamate cysteine ligase (GCL) and GS, while hepatic expressions of methionine sulfoxide reductases (MsrA, MsrB2, MsrB3) and hepatic enzyme activities (CAT, SOD, GCL, GR, GST, GPx) were uniformly stimulated with increasing consumption of l-methionine. As a result, hepatic content of ROS and MDA were effectively reduced by l-methionine intake. CONCLUSION: The present study demonstrates that methionine availability plays a critical role in activation of the Nrf2-ARE pathway to induce an endogenous antioxidant response for depressing ROS-derived oxidative stress, which is primarily attributed to the stimulation of methionine sulfoxide reductase expression and glutathione synthesis. © 2019 Society of Chemical Industry.

Alteration of tumor suppressor BMP5 in sporadic colorectal cancer: a genomic and transcriptomic profiling based study.

BACKGROUND: Although the genetic spectrum of human colorectal cancer (CRC) is mainly characterized by APC, KRAS and TP53 mutations, driver genes in tumor initiation have not been conclusively demonstrated. In this study, we aimed to identify novel markers for CRC. METHODS: We performed exome analysis of sporadic colorectal cancer (sCRC) coding regions to screen loss of function (LoF) mutation genes, and carried out systems-level approaches to confirm top rank gene in this study. RESULTS: We identified loss of BMP5 is an early event in CRC. Deep sequencing identified BMP5 was mutated in 7.7% (8/104) of sCRC samples, with 37.5% truncating mutation frequency. Notably, BMP5 negative expression and its prognostic value is uniquely significant in sCRC but not in other tumor types. Furthermore, BMP5 expression was positively correlated with E-cadherin in CRC patients and its dysregulation play a vital role in epithelial-mesenchymal transition (EMT), thus triggering tumor initiation and development. RNA sequencing identified, independent of BMP/Smads pathway, BMP5 signaled though Jak-Stat pathways to inhibit the activation of oncogene EPSTI1. CONCLUSIONS: Our result support a novel concept that the importance of BMP5 in sCRC. The tumor suppressor role of BMP5 highlights its crucial role in CRC initiation and development.

Hypoxia in 3T3-L1 adipocytes suppresses adiponectin expression via the PERK and IRE1 unfolded protein response.

Adiponectin, an adipocytokine produced by adipocytes, functions as an anti-inflammatory and anti-apoptotic substance, while also enhancing insulin sensitivity. Patients or model animals with obesity or diabetes typically present attenuated expression of adiponectin. Moreover, obesity and diabetes are often accompanied with hypoxia in adipose tissue, which may result in endoplasmic reticulum (ER) stress as well as low expression of adiponectin. The purpose of this study was to investigate the specific role of the unfolded protein response (UPR) involved in the low expression of adiponectin induced by hypoxia. Subjecting 3T3-L1 adipocytes to hypoxia significantly reduced adiponectin expression and activated the PERK and IRE1 signaling pathways in a time-dependent manner. The ATF6 signaling pathway showed no obvious changes with hypoxia treatment under a similar time course. Moreover, the down-regulated expression of adiponectin induced by hypoxia was relieved once the PERK and IRE1 signaling pathways were suppressed by the inhibitors GSK2656157 and 4µ8C, respectively. Overall, these data demonstrate that hypoxia can suppress adiponectin expression and activate the PERK and IRE1 signaling pathways in differentiated adipocytes, and this two pathways are involved in the suppression of adiponectin expression induced by hypoxia.

Aquaporin-1, a New Maternally Expressed Gene, Regulates Placental Development in the Mouse.

Imprinted genes play an important role in placental and embryonic development. Abnormalities in their regulation can result in placental and embryonic dysplasia, leading to congenital diseases. The imprinting state, expression, and function of aquaporin-1 (Aqp1) were explored in knockout mice by imprinting analysis, real-time PCR, and immunohistochemistry. In the present study, Aqp1 was identified as a new, imprinted, and placenta-specific maternally expressed gene in the mouse. Compared with wild-type Aqp1(+/+) mice, there was significant placental and embryonic overgrowth in Aqp1(-/+) (loss of maternal allele) and Aqp1(-/-) mice, but not in Aqp1(+/-) (loss of paternal allele) mice at Embryonic Day (E) 12.5-E18.5. In addition, the masses of Postnatal Day 0 (P0) embryos (Aqp1(-/-) and Aqp1(-/+)) were highest among the four types. In Aqp1(-/+) and Aqp1(-/-) mice, phenotypic analysis indicated that the number and branching of blood vessels, as well as the labyrinth area, increased significantly in placentae of E12.5-E18.5 mice. Moreover, there were abnormalities in the placental junctional zone and the labyrinthine zone at E15.5. Quantitative analysis showed that Aqp1 expression decreased significantly in the placentae of Aqp1(-/+) and Aqp1(-/-) mice at E15.5, and that the AQP1 protein expression signals were detected weakly in the decidual and spongioblast layers. Our results demonstrate that Aqp1 is maternally expressed in the placenta, and that its deficiency resulted in placental abnormalities in the mouse. Aqp1 may have a specific inhibitory role in mouse placental development. These results provide new insights for the treatment of diseases relating to placental and embryonic development.

Juvenile hormone prevents 20-hydroxyecdysone-induced metamorphosis by regulating the phosphorylation of a newly identified broad protein.

The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis. By contrast, juvenile hormone (JH) prevents metamorphosis. However, the mechanism by which JH inhibits metamorphosis remains unclear. In this study, we propose that JH induces the phosphorylation of Broad isoform Z7 (BrZ7), a newly identified protein, to inhibit 20E-mediated metamorphosis in the lepidopteran insect Helicoverpa armigera. The knockdown of BrZ7 in larvae inhibited metamorphosis by repressing the expression of the 20E response gene. BrZ7 was weakly expressed and phosphorylated during larval growth but highly expressed and non-phosphorylated during metamorphosis. JH regulated the rapid phosphorylation of BrZ7 via a G-protein-coupled receptor-, phospholipase C-, and protein kinase C-triggered pathway. The phosphorylated BrZ7 bound to the 5'-regulatory region of calponin to regulate its expression in the JH pathway. Exogenous JH induced BrZ7 phosphorylation to prevent metamorphosis by suppressing 20E-related gene transcription. JH promoted non-phosphorylated calponin interacting with ultraspiracle protein to activate the JH pathway and antagonize the 20E pathway. This study reveals one of the possible mechanisms by which JH counteracts 20E-regulated metamorphosis by inducing the phosphorylation of BrZ7.

Palmitate increases musclin gene expression through activation of PERK signaling pathway in C2C12 myotubes.

Musclin is a type of muscle-secreted cytokine and its increased gene expression induces insulin resistance in type 2 diabetes. However, the mechanism underlying increased musclin gene expression is currently unclear. Excessive saturated fatty acids (SFA) can activate the secretion of several muscle-secreted cytokines as well as endoplasmic reticulum (ER) stress pathway, thereby contributing to the development of type 2 diabetes. The purpose of this study was to investigate the mechanisms responsible for the effect of palmitate, the most abundant SFA in the plasma, on the gene expression of musclin in C2C12 myotubes. Treatment of C2C12 myotubes with palmitate or tunicamycin significantly increased the expression of musclin as well as ER stress-related genes, but treatment with oleate did not. Pre-treatment of C2C12 myotubes with 4-phenyl butyrate suppressed the expression of ER stress-related genes, simultaneously, resulting in decreased expression of the musclin gene induced by palmitate or tunicamycin. These results indicate that ER stress is related to palmitate-induced musclin gene expression. Moreover, palmitate-induced musclin gene expression was significantly inhibited in C2C12 myotubes when PERK pathway signaling was suppressed by knockdown of the PERK gene or treatment with GSK2656157, a PERK autophosphorylation inhibitor. However, there was no difference in the palmitate-induced musclin gene expression when IRE1 and ATF6 signaling pathways were suppressed by knockdown of the IRE1 and ATF6 genes. These findings suggest that palmitate increases musclin gene expression via the activation of the PERK signaling pathway in C2C12 myotubes.

Long non-coding RNA identification over mouse brain development by integrative modeling of chromatin and genomic features.

In silico prediction of genomic long non-coding RNAs (lncRNAs) is prerequisite to the construction and elucidation of non-coding regulatory network. Chromatin modifications marked by chromatin regulators are important epigenetic features, which can be captured by prevailing high-throughput approaches such as ChIP sequencing. We demonstrate that the accuracy of lncRNA predictions can be greatly improved when incorporating high-throughput chromatin modifications over mouse embryonic stem differentiation toward adult Cerebellum by logistic regression with LASSO regularization. The discriminating features include H3K9me3, H3K27ac, H3K4me1, open reading frames and several repeat elements. Importantly, chromatin information is suggested to be complementary to genomic sequence information, highlighting the importance of an integrated model. Applying integrated model, we obtain a list of putative lncRNAs based on uncharacterized fragments from transcriptome assembly. We demonstrate that the putative lncRNAs have regulatory roles in vicinity of known gene loci by expression and Gene Ontology enrichment analysis. We also show that the lncRNA expression specificity can be efficiently modeled by the chromatin data with same developmental stage. The study not only supports the biological hypothesis that chromatin can regulate expression of tissue-specific or developmental stage-specific lncRNAs but also reveals the discriminating features between lncRNA and coding genes, which would guide further lncRNA identifications and characterizations.

Titanium dioxide nanoparticles increase plasma glucose via reactive oxygen species-induced insulin resistance in mice.

There have been few reports about the possible toxic effects of titanium dioxide (TiO2 ) nanoparticles on the endocrine system. We explored the endocrine effects of oral administration to mice of anatase TiO2 nanoparticles (0, 64 and 320 mg kg(-1) body weight per day to control, low-dose and high-dose groups, respectively, 7 days per week for 14 weeks). TiO2 nanoparticles were characterized by scanning and transmission electron microscopy (TEM) and dynamic light scattering (DLS), and their physiological distribution was investigated by inductively coupled plasma. Biochemical analyzes included plasma glucose, insulin, heart blood triglycerides (TG), free fatty acid (FFA), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6 and reactive oxygen species (ROS)-related markers (total SOD, GSH and MDA). Phosphorylation of IRS1, Akt, JNK1, and p38 MAPK were analyzed by western blotting. Increased titanium levels were found in the liver, spleen, small intestine, kidney and pancreas. Biochemical analyzes showed that plasma glucose significantly increased whereas there was no difference in plasma insulin secretion. Increased ROS levels were found in serum and the liver, as evidenced by reduced total SOD activity and GSH level and increased MDA content. Western blotting showed that oral administration of TiO2 nanoparticles induced insulin resistance (IR) in mouse liver, shown by increased phosphorylation of IRS1 (Ser307) and reduced phosphorylation of Akt (Ser473). The pathway by which TiO2 nanoparticles increase ROS-induced IR were included in the inflammatory response and phosphokinase, as shown by increased serum levels of TNF-α and IL-6 and increased phosphorylation of JNK1 and p38 MAPK in liver. These results show that oral administration of TiO2 nanoparticles increases ROS, resulting in IR and increasing plasma glucose in mice.

4-Phenylbutyric acid increases GLUT4 gene expression through suppression of HDAC5 but not endoplasmic reticulum stress.

BACKGROUND: The chemical chaperone 4-phenylbutyric acid (4-PBA) has been shown to relieve endoplasmic reticulum (ER) stress. Therefore, it improves insulin sensitivity and promotes glucose metabolism in skeletal muscle. Glucose transporter type 4 (GLUT4), as a major glucose transporter protein, plays a central role in glucose metabolism. Until now, it has been unclear whether 4-PBA affects GLUT4 gene expression and thus, contributes to glucose metabolism. METHODS: C2C12 myotubes were treated with 4-PBA, tunicamycin or butyrate and subjected to Western blot and RT-PCR. RESULTS: 4-PBA-treated C2C12 myotubes increased GLUT4 expression and promoted glucose metabolism. Most interestingly, GLUT4 gene expression induced by 4-PBA was not associated with ER stress even in the presence of tunicamycin, an ER stress inducer. Moreover, we also found that 4-PBA inhibited histonedeacetylase 5 (HDAC5) expression in C2C12 myotubes, resulting in hyperacetylation of the histone H3 at the myocyte enhancer factor 2 (MEF2) binding site. This increased the binding of MEF2A to the site on the GLUT4 promoter, resulting in increased GLUT4 expression. CONCLUSIONS: Our data indicate that 4-PBA increases GLUT4 expression by acetylating the MEF2 site to increase the MEF2A binding through a mechanism that involves suppression of the HDAC5 pathway, but without involving ER stress.

Identification and characterization of long intergenic non-coding RNAs related to mouse liver development.

Long non-coding RNAs (lncRNAs) have been studied extensively over the last few years. Liver is an important organ that plays a crucial role in glucose metabolism and homeostasis; however, there are few reports of the identification and functional characterization of lncRNAs with important roles in liver development. Therefore, it is necessary to systematically identify lncRNAs that are involved in liver development. In this paper, we assembled the transcriptome using published RNA-seq data across three mouse liver developmental stages and identified 4,882 putative long intergenic non-coding RNAs (lincRNAs) expressed in at least one of the investigated stages. Combining these with Ensembl lincRNAs, we established a reference catalog of 6,602 transcribed lincRNAs in the mouse liver. We then analyzed all the lincRNAs in this reference catalog systematically and revealed that liver lincRNAs carry different genomic signatures from protein-coding genes, while the putative lincRNAs are generally comparable with known Ensembl lincRNAs. In addition, putative lincRNAs are functionally associated with essential biological processes, including RNA splicing, protein localization and fatty acid metabolic process, implying that they may play an important role in regulating liver development. The validation of selected lincRNAs that are specifically expressed in developing liver tissues further suggested the effectiveness of our approach. Our study shows that lincRNAs that are differentially expressed during three liver developmental stages could have important regulatory roles in liver development. The identified putative lincRNAs are a valuable resource for further functional studies.

Maternal RNA transcription in Dlk1-Dio3 domain is critical for proper development of the mouse placental vasculature.

The placenta is a unique organ for ensuring normal embryonic growth in the uterine. Here, we found that maternal RNA transcription in Dlk1-Dio3 imprinted domain is essential for placentation. PolyA signals were inserted into Gtl2 to establish a mouse model to prevent the expression of maternal RNAs in the domain. The maternal allele knock-in (MKI) and homozygous (HOMO) placentas showed an expanded junctional zone, reduced labyrinth and poor vasculature impacting both fetal and maternal blood spaces. The MKI and HOMO models displayed dysregulated gene expression in the Dlk1-Dio3 domain. In situ hybridization detected Dlk1, Gtl2, Rtl1, miR-127 and Rian dysregulated in the labyrinth vasculature. MKI and HOMO induced Dlk1 to lose imprinting, and DNA methylation changes of IG-DMR and Gtl2-DMR, leading to abnormal gene expression, while the above changes didn't occur in paternal allele knock-in placentas. These findings demonstrate that maternal RNAs in the Dlk1-Dio3 domain are involved in placental vasculature, regulating gene expression, imprinting status and DNA methylation.

Dynamic expression pattern of Pde4d and its relationship with CpG methylation in the promoter during mouse embryo development.

Mouse Pde4d is located on chromosome 13 and serves many functions in important physiological processes involving cyclic adenosine monophosphate. In this study, imprinting analysis indicated that Pde4d exhibits a dynamic and specific allelic expression pattern during embryo development. This showed paternal-origin sex bias in embryonic day 9.5 (E9.5) whole embryos and placenta, and biallelic expression in the major embryonic organs and placenta at E15.5. In situ hybridization determined the spatiotemporal expression pattern of Pde4d in mouse embryos from the mid- to late-embryonic stages. This demonstrated that Pde4d was widely expressed in the neural tissues, including the forebrain, midbrain, hindbrain, and neural tube, at the mid-embryonic stage. By the late-embryonic stage, Pde4d was extensively detected throughout the developing organism, including in the liver, brain, lung, kidney, and tongue. In addition, methylation analyses indicated that tissue-specific CpG methylation of the Pde4d promoter was correlated with Pde4d mRNA expression in major E15.5 tissues. Furthermore, stage-specific CpG methylation of the Pde4d promoter was associated with gene expression in the liver at three developmental stages. Our results suggest that Pde4d might serve specific biological functions in regulating the development process of the mouse embryo, and that CpG methylation of the Pde4d promoter may play an important role in regulating Pde4d at a transcriptional level.

Rice protein exerts a hypocholesterolemic effect through regulating cholesterol metabolism-related gene expression and enzyme activity in adult rats fed a cholesterol-enriched diet.

The major aim of this study is to elucidate the hypocholesterolemic mechanism exerted by rice protein (RP) in adult rats under cholesterol-enriched dietary condition. Compared with casein, the cholesterol levels in plasma and the liver were significantly reduced by RP, accompanying significant inhibition of cholesterol absorption. RP increased the activity and mRNA level of cholesterol 7α-hydroxylase, whereas acyl-CoA:cholesterol acyltransferase activity and gene expression were significantly depressed with consumption of RP. Neither the activity nor gene expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase of RP differed from that of casein. The gene expression of the peroxisome proliferator-activated receptor α and liver X receptor α were significantly activated by consumption of RP. RP did not modify the mRNA level of sterol regulatory element-binding protein-2 with respect to casein. These results suggest RP can induce a cholesterol-lowering effect through modifying cholesterol metabolism-related gene expression and enzyme activity in adult rats.