Epigenetic Regulation of Adipocyte Differentiation by a Rho Guanine Nucleotide Exchange Factor, WGEF

Epigenetic regulation, including DNA methylation, plays an important role in several differentiation processes and possibly in adipocyte differentiation. To search for genes that show methylation change during adipogenesis, genome-wide DNA methylation analysis in insulin-induced adipogenesis of 3T3-L1 preadipocyte cells was performed using a method called microarray-based integrated analysis of methylation by isoschizomers (MIAMI). The MIAMI revealed that Hpa II sites of exon 1 in a Rho guanine nucleotide exchange factor 19 (ARHGEF19; WGEF) gene were demethylated during adipocyte differentiation of 3T3-L1 cells. Deletion of the region containing cytosine-guanine (CpG) sites that showed methylation change suppressed transcriptional activity in the reporter assay, indicating that this region regulates WGEF transcription. WGEF expression in 3T3-L1 cells was reduced during adipocyte differentiation, and high-fat diet-induced obese mice also showed lower expression of WGEF gene than control mice in white adipose tissue. Additionally, forced expression of WGEF in 3T3-L1 cells down-regulated the expression of adipogenic marker genes and inhibited the adipogenic program. This study clarified that adipogenesis was regulated by WGEF expression through DNA methylation change

One Argonaute family member, Eif2c2 (Ago2), is essential for development and appears not to be involved in DNA methylation

AbstractTo elucidate the epigenetic role of RNAi in mammals, we disrupted the gene for Eif2c2 (Ago2), which works as the sole slicer of RNAi in the Argonaute family. In mice, disruption of Eif2c2 leads to embryonic lethality early in development after the implantation stage. This phenotype is completely different from that in a previous report, but somewhat similar to the disruption of Dicer1, another important component of RNAi. We also show that Eif2c2 is not required for the maintenance of DNA methylation in imprinted genes, centromeric repeats, and Xist. This suggests that developmental defects in the Eif2c2-deficient mouse are caused not at the transcriptional level, but rather at the posttranscriptional level through the miRNA–protein complex

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

Microarray analysis of promoter methylation in lung cancers

Aberrant DNA methylation is an important event in carcinogenesis. Of the various regions of a gene that can be methylated in cancers, the promoter is the most important for the regulation of gene expression. Here, we describe a microarray analysis of DNA methylation in the promoter regions of genes using a newly developed promoter-associated methylated DNA amplification DNA chip (PMAD). For each sample, methylated Hpa II-resistant DNA fragments and Msp I-cleaved (unmethylated + methylated) DNA fragments were amplified and labeled with Cy3 and Cy5 respectively, then hybridized to a microarray containing the promoters of 288 cancer-related genes. Signals from Hpa II-resistant (methylated) DNA (Cy3) were normalized to signals from Msp I-cleaved (unmethylated + methylated) DNA fragments (Cy5). Normalized signals from lung cancer cell lines were compared to signals from normal lung cells. About 10.9% of the cancer-related genes were hypermethylated in lung cancer cell lines. Notably, HIC1, IRF7, ASC, RIPK3, RASSF1A, FABP3, PRKCDBP, and PAX3 genes were hypermethylated in most lung cancer cell lines examined. The expression profiles of these genes correlated to the methylation profiles of the genes, indicating that the microarray analysis of DNA methylation in the promoter region of the genes is convenient for epigenetic study. Further analysis of primary tumors indicated that the frequency of hypermethylation was high for ASC (82%) and PAX3 (86%) in all tumor types, and high for RIPK3 in small cell carcinoma (57%). This demonstrates that our PMAD method is effective at finding epigenetic changes during cancer

Genome-wide profiling of promoter methylation in human

DNA methylation in the promoter region of a gene is associated with a loss of that gene's expression and plays an important role in gene silencing. The inactivation of tumor-suppressor genes by aberrant methylation in the promoter region is well recognized in carcinogenesis. However, there has been little study in this area when it comes to genome-wide profiling of the promoter methylation. Here, we developed a genome-wide profiling method called Microarray-based Integrated Analysis of Methylation by Isoschizomers to analyse the DNA methylation of promoter regions of 8091 human genes. With this method, resistance to both the methylation-sensitive restriction enzyme HpaII and the methylation-insensitive isoschizomer MspI was compared between samples by using a microarray with promoter regions of the 8091 genes. The reliability of the difference in HpaII resistance was judged using the difference in MspI resistance. We demonstrated the utility of this method by finding epigenetic mutations in cancer. Aberrant hypermethylation is known to inactivate tumour suppressor genes. Using this method, we found that frequency of the aberrant promoter hypermethylation in cancer is higher than previously hypothesized. Aberrant hypomethylation is known to induce activation of oncogenes in cancer. Genome-wide analysis of hypomethylated promoter sequences in cancer demonstrated low CG/GC ratio of these sequences, suggesting that CpG-poor genes are sensitive to demethylation activity in cancer

"Matkalla jo jutellaan, mitähän mielenkiintoista taas saa kuulla" : Äänikirjalukupiiri Vaasan kaupunginkirjastossa

Tutkimuksen tavoitteena oli selvittää kvalitatiivisten menetelmien avulla lukupiiriläisten kokemuksellisuutta. Opinnäytetyössäni painotettiin äänikirjojen lukemisen kokemuksellisuutta. Vertailututkimuksessa käytettiin perinteisempää, painettuja teoksia lukevaa kirjallisuuspiiriä. Tutkimusongelmaan pyrittiin hakemaan vastauksia seuraavien kysymysten kautta: miksi halutaan kuulua lukupiirin, mitä luetaan, millaisista aiheista keskustellaan, kuinka lukupiirissä lukeminen eroaa yksinlukemisesta, mikä merkitys lukupiirin kuulumisella ja siihen liittyvällä kanssakäymisellä on, ja miten äänikirjalukeminen koetaan. Lisäksi selvitettiin, kuinka äänikirjalukupiirissä lukeminen eroaa niin sanotussa perinteisessä lukupiirissä lukemisesta. Kvalitatiivisina tutkimusmetodeina käytettiin havainnointia ja haastatteluja. Äänikirjalukupiirissä havainnointitapaamisia oli kaksi ja vertailulukupiirissä yksi kerta. Molemmista lukupiireistä haastattelin kuutta piiriläistä, eli yhteensä 12 jäsentä. Lukupiiriin kuulumisen perusteluja käsiteltiin seuraavien ulottuvuuksien kautta: lukemisen merkitys, kokemus lukijana, keskustelut lukukokemuksista ja muiden aiheiden merkitys, lukupiirin jäsenten välinen sosiaalinen kanssakäyminen sekä lukija yhteisöllisen lukupiirin jäsenenä. Tutkimustulosten mukaan lukupiireihin hakeuduttiin laajemman kirjallisuudentuntemuksen saamiseksi, yhteisen keskustelun ja lukukokemusten jakamisen vuoksi. Äänikirjalukupiiriin kuulumisessa korostettiin laajemman mahdollisuuden saamista kirjallisuuden harrastamiseen, aineiston tärkeää ja helppoa saamista kerhon kautta. Celian rinkiin pääsy kerhon kautta oli osa koettua merkitystä. Lukupiirien sosiaalisissa kontakteissa pyrittiin ” helppouteen perustuvaan ystävyyteen”, jossa ryhmään hakeudutaan viihtymään ja nauttimaan yhdessäolosta. Lukupiirien sosiaaliset suhteet koettiin työelämän suhteiden korvaajina. Koettiin oppimisen iloa, itsetuntemuksen lisääntymistä, antamisen ja auttamisen iloa. Muutostoiveet liitettiin keskustelun parantamiseen, tietoisuuden lisääntymiseen ja omaan oppimiseen.The aim of the study was to examine, through qualitative methods, experience among the literature circle. The thesis study emphasized experience during the reading of audio books. In addition, differences between an audio book circle and a traditional literature circle were studied. The research questions were the following: why the members want to participate in literature circles; what books are read; what kinds of issues are discussed; how reading in a literature circle differs from reading alone; what participating in a literature circle means to the participants, and what the participants thought about the listening of audio books. In addition, it was examined how the reading of audio books differs from reading in a traditional literature circle. I used qualitative methods, observation and interviews. I interviewed six members of an audio books circle and six members of the literature circle. Arguments for membership in a reading circle were analysed thought the following dimensions: the significance of reading, experience as a reader, discussions about reading experiences others methods meaning of the reading, experience as reader, discussions from read experiences and the significance of others topics, interaction between the members of the literature circles, and the reader as a member in a communal reading circle. According to the results, the interviewees joined the literature circles in order to improve their knowledge of literature, to have common discussions, and to share reading experiences. The members of the audio book circle emphasized the opportunity to take an interest in literature and easy access to materials through the club. Becoming a customer of Celia through the circle was part of its perceived significance. In the social contacts in the reading circles, “friendship based on easiness” was sought. The social relationships within the group were perceived as a substitute to relationships in working life

The N⁶-methyladenosine methyltransferase METTL16 enables erythropoiesis through safeguarding genome integrity

RNA修飾による赤血球造血制御機構を解明 --RNAのメチル化がDNA修復に必要--. 京都大学プレスリリース. 2022-11-10.Mice show METTL in DNA blood repair: RNA methylation shows important role in erythropoiesis. 京都大学プレスリリース. 2022-11-25.During erythroid differentiation, the maintenance of genome integrity is key for the success of multiple rounds of cell division. However, molecular mechanisms coordinating the expression of DNA repair machinery in erythroid progenitors are poorly understood. Here, we discover that an RNA N⁶-methyladenosine (m⁶A) methyltransferase, METTL16, plays an essential role in proper erythropoiesis by safeguarding genome integrity via the control of DNA-repair-related genes. METTL16-deficient erythroblasts exhibit defective differentiation capacity, DNA damage and activation of the apoptotic program. Mechanistically, METTL16 controls m⁶A deposition at the structured motifs in DNA-repair-related transcripts including Brca2 and Fancm mRNAs, thereby upregulating their expression. Furthermore, a pairwise CRISPRi screen revealed that the MTR4-nuclear RNA exosome complex is involved in the regulation of METTL16 substrate mRNAs in erythroblasts. Collectively, our study uncovers that METTL16 and the MTR4-nuclear RNA exosome act as essential regulatory machinery to maintain genome integrity and erythropoiesis

Epigenetic modulation of Fgf21 in the perinatal mouse liver ameliorates diet-induced obesity in adulthood

The nutritional environment to which animals are exposed in early life can lead to epigenetic changes in the genome that influence the risk of obesity in later life. Here, we demonstrate that the fibroblast growth factor-21 gene (Fgf21) is subject to peroxisome proliferator-activated receptor (PPAR) α–dependent DNA demethylation in the liver during the postnatal period. Reductions in Fgf21 methylation can be enhanced via pharmacologic activation of PPARα during the suckling period. We also reveal that the DNA methylation status of Fgf21, once established in early life, is relatively stable and persists into adulthood. Reduced DNA methylation is associated with enhanced induction of hepatic FGF21 expression after PPARα activation, which may partly explain the attenuation of diet-induced obesity in adulthood. We propose that Fgf21 methylation represents a form of epigenetic memory that persists into adulthood, and it may have a role in the developmental programming of obesity