Genome-wide DNA methylation analysis reveals phytoestrogen modification of promoter methylation patterns during embryonic stem cell differentiation.

BACKGROUND: Environmental challenges during development affect the fetal epigenome, but the period(s) vulnerable to epigenetic dysregulation is(are) not clear. By employing a soy phytoestrogen, genistein, that is known to alter the epigenetic states of the A(vy) allele during embryogenesis, we have explored the sensitive period for epigenetic regulation. The post-implantation period, when de novo DNA methylation actively proceeds, is amenable to in vitro analysis using a mouse embryonic stem (ES) cell differentiation system. METHODS AND FINDINGS: Mouse ES cells were differentiated in the presence or absence of genistein, and DNA methylation patterns on day 10 were compared by microarray-based promoter methylation analysis coupled with a methylation-sensitive endonuclease (HpaII/McrBC)-dependent enrichment procedure. Moderate changes in methylation levels were observed in a subset of promoters following genistein treatment. Detailed investigation of the Ucp1 and Sytl1 promoters further revealed that genistein does not affect de novo methylation occurring between day 0 and day 4, but interferes with subsequent regulatory processes and leads to a decrease in methylation level for both promoters. CONCLUSION: Genistein perturbed the methylation pattern of differentiated ES cells after de novo methylation. Our observations suggest that, for a subset of genes, regulation after de novo DNA methylation in the early embryo may be sensitive to genistein.

Increasing the number of SNP loci does not necessarily improve prediction power at least in the comparison of MTHFR SNP and haplotypes.

BACKGROUND: Rapid advances in genotyping technology have made it possible to easily utilize a large number of genetic markers. According to information theory, an increase in the number of markers provides more information; however, the clinical usefulness does not increase linearly. This study aimed to assess the effect of folic acid supplementation quantitatively in MTHFR haplotypes, and compare its prediction power with that of the C677T single nucleotide polymorphism (SNP) alone. METHODS: The study was a randomized, double-blind, placebo-controlled trial, designed in accordance with the CONSORT statement. The participants were 202 healthy Japanese males who were administered either folic acid at 1 mg/day or a placebo postoperatively for 3 months. The primary endpoint was the total plasma homocysteine levels (tHcy). Stratified analysis by HapMap-based tag SNPs was performed. RESULTS: Of 52 SNPs on the MTHFR gene, 4 SNP loci covering more than 80% of the information were selected, and the haplotypes were estimated. The haplotypes were classified into 3 groups (Hap0, Hap1, and Hap2), on the basis of the number of times the most frequent haplotype was present. The greatest decrease was observed in Hap2 (6.61 micromol/L), compared with the other haplotypes (Hap0, 2.67; Hap1, 2.60) (trend test, P < 0.01). The haplotype information obtained was not more informative than that obtained with grouping by a single SNP, C677T, which strongly influences enzyme activity. CONCLUSIONS: Grouping by the C677T SNP alone was almost as good a predictor of the homocysteine-lowering effects as was grouping by the 4 best SNPs. This shows that increasing the number of typed SNPs does not necessarily provide more information, at least for this gene. A more efficient, cost-informative method for analyzing genomic data is required.

A functional polymorphism of the TNF-alpha gene that is associated with type 2 DM.

To examine the association of the tumor necrosis factor-alpha (TNF-alpha) gene region with type 2 diabetes (DM), 11 single-nucleotide polymorphisms (SNPs) of the region were analyzed. The initial study using a sample set (148 cases vs. 227 controls) showed a significant association of the SNP IVS1G+123A of the TNF-alpha gene with DM (p=0.0056). Multiple logistic regression analysis using an enlarged sample set (225 vs. 716) revealed the significant association of the SNP with DM independently of any clinical traits examined (OR: 1.49, p=0.014). The functional relevance of the SNP were examined by the electrophoretic mobility shift assays using nuclear extracts from the U937 and NIH3T3 cells and luciferase assays in these cells with Simian virus 40 promoter- and TNF-alpha promoter-reporter gene constructs. The functional analyses showed that YY1 transcription factor bound allele-specifically to the SNP region and, the IVS1+123A allele had an increase in luciferase expression compared with the G allele.

The role of G-protein-coupled receptor kinase 5 in pathogenesis of sporadic Parkinson's disease.

Sporadic Parkinson's disease (sPD) is a common neurodegenerative disorder, characterized by selective degeneration of dopaminergic neurons in the substantia nigra. Although the pathogenesis of the disease remains undetermined, phosphorylation of alpha-synuclein and its oligomer formation seem to play a key role. However, the protein kinase(s) involved in the phosphorylation in the pathogenesis of sPD has not been identified. Here, we found that G-protein-coupled receptor kinase 5 (GRK5) accumulated in Lewy bodies and colocalized with alpha-synuclein in the pathological structures of the brains of sPD patients. In cotransfected cells, GRK5 phosphorylated Ser-129 of alpha-synuclein at the plasma membrane and induced translocation of phosphorylated alpha-synuclein to the perikaryal area. GRK5-catalyzed phosphorylation also promoted the formation of soluble oligomers and aggregates of alpha-synuclein. Genetic association study revealed haplotypic association of the GRK5 gene with susceptibility to sPD. The haplotype contained two functional single-nucleotide polymorphisms, m22.1 and m24, in introns of the GRK5 gene, which bound to YY1 (Yin Yang-1) and CREB-1 (cAMP response element-binding protein 1), respectively, and increased transcriptional activity of the reporter gene. The results suggest that phosphorylation of alpha-synuclein by GRK5 plays a crucial role in the pathogenesis of sPD.

A CaMK cascade activates CRE-mediated transcription in neurons of Caenorhabditis elegans.

Calcium (Ca2+) signals regulate a diverse set of cellular responses, from proliferation to muscular contraction and neuro-endocrine secretion. The ubiquitous Ca2+ sensor, calmodulin (CaM), translates changes in local intracellular Ca2+ concentrations into changes in enzyme activities. Among its targets, the Ca2+/CaM-dependent protein kinases I and IV (CaMKs) are capable of transducing intraneuronal signals, and these kinases are implicated in neuronal gene regulation that mediates synaptic plasticity in mammals. Recently, the cyclic AMP response element binding protein (CREB) has been proposed as a target for a CaMK cascade involving not only CaMKI or CaMKIV, but also an upstream kinase kinase that is also CaM regulated (CaMKK). Here, we report that all components of this pathway are coexpressed in head neurons of Caenorhabditis elegans. Utilizing a transgenic approach to visualize CREB-dependent transcription in vivo, we show that this CaMK cascade regulates CRE-mediated transcription in a subset of head neurons in living nematodes.

Stimulation of 3T3-L1 adipogenesis by signal transducer and activator of transcription 5.

Signal transducer and activator of transcription 5 (Stat5) mediates signaling of many cytokines and growth factors. Here we show that Stat5 functions as an initial mediator of adipogenesis. The preadipocyte cell line 3T3-L1 undergoes adipocyte differentiation upon appropriate hormonal induction. We found that Stat5A and Stat5B were strongly activated at an early stage of 3T3-L1 differentiation. To investigate physiological roles of Stat5 in adipogenesis, we have constructed 3T3-L1 cell lines in which either an exogenous wild type (wt) or dominant negative (dn) form of Stat5A expression was controlled under the doxycycline-regulatable promoter. Precocious induction of wt-Stat5A in adipocyte differentiation promoted accumulation of triglycerides within the cells. In contrast, induction of dn-Stat5A attenuated lipid accumulation. Northern blot analyses revealed that the expression of proadipogenic transcription factors was influenced in a complementary fashion by ectopic expression of either wt- or dn-Stat5A. Notably, Stat5 regulated expression of peroxisome proliferator-activated receptor-gamma, which plays crucial roles in adipogenesis. We have also generated transgenic mice in which dn-Stat5A is expressed in an adipose tissue-specific fashion and found attenuation of peroxisome proliferator-activated receptor-gamma and of many adipocyte-related genes. These results highlight a novel role of Stat5 in adipocyte differentiation.