scholarly journals Robertson's Mutator transposons in A. thaliana are regulated by the chromatin-remodeling gene Decrease in DNA Methylation (DDM1)

2001 ◽  
Vol 15 (5) ◽  
pp. 591-602 ◽  
Author(s):  
T. Singer
Keyword(s):  
Dna Methylation ◽  
Chromatin Remodeling

100 ASSESSMENT OF LOCUS-SPECIFIC DNA METHYLATION: OPTIMIZATION FOR BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 150
Author(s):  
E. Wroclawska ◽  
J. O. Brant ◽  
T. P. Yang ◽  
K. Moore
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Chromatin Remodeling ◽  
Plasmid Dna ◽  
Dna Amplification ◽  
Early Embryo ◽  
Small Samples ◽  
Small Scale ◽  
Research Education

Assessment of chromatin remodeling in early embryos is a major focus of studies today, and evaluation of DNA methylation at specific loci is one approach to study these epigenetic modifications. Our objective was to optimize the bisulfite sequencing methodology for use with very small cell numbers originating from pre-implantation embryos, making the process more time- and cost-efficient. The optimized steps include bisulfite conversion of small samples, bisulfite primer design, high-throughput plasmid DNA amplification, and preparation for sequencing. Methylation at 2 loci, Satellite I and Oct4, was investigated in bovine in vitro-produced (IVP) embryos collected at the 2-cell, 8-cell, and blastocyst stages. Bovine skin fibroblasts were first used to optimize the particular steps of the process. All reactions were run in duplicate and no-template negative and somatic cell-positive controls were treated alongside samples. Incorporating the use of Methyl Primer Express (Applied Biosystems, Foster City, CA), MacVector (Oxford Molecular Ltd., Campbell, CA), and Mfold software (Mathews DH et al. 1999 J. Mol. Biol. 288, 911–940; Zuker M 2003 Nucleic Acids Res. 31, 3406–3415) improved the specificity of bisulfite primers by exclusion of secondary or tertiary structures. The DNA from bisulfite treatment for 15 to 16 h was of better quality than DNA treated for 18 h. After initial PCR optimization, different cell concentrations were used to establish that detectable PCR products and subsequent methylation data could be obtained from DNA isolated from as few as 8 cells. Treating single blastocysts and pools of ten 8-cell and forty 2-cell embryos was sufficient for the entire scope of the experiment, allowing use of the same samples across all loci. After molecular cloning, plasmid DNA was amplified by 3 different methods and evaluated for efficiency: miniprep, TempliPhi (GE Healthcare, Piscataway, NJ), or 96-well glycerol stocks and automated TempliPhi format. Although TempliPhi alone was better than miniprep for small-scale experiments, it was the 96-well format that saved weeks of time and was most cost-effective. Sequencing was performed on a minimum of 8 clones/sample using ABI Prism sequencers (Applied Biosystems), and results were analyzed using Chromas Pro software (Technelysium Pty. Ltd., Helensvale, Australia). Percentage methylation of bovine IVP 2-cell, 8-cell, and blastocyst stage embryos for Satellite I was 25, 10, and 22%, respectively, and for Oct4 was 88, 88, and 79%, respectively. However, somatic cell methylation was 74% for Satellite I and 88% for Oct4, implying that Satellite I is demethylated during early embryo development, whereas Oct4 remains hypermethylated. In conclusion, these improved methods will benefit further studies of chromatin remodeling in early bovine pre-implantation embryos. This project was supported by National Research Initiative Competitive Grant no. 2006-35203-16620 from the USDA Cooperative State Research, Education, and Extension Service.

scholarly journals Histone acetylation recruits the SWR1 complex to regulate active DNA demethylation in Arabidopsis

2019 ◽  
Vol 116 (33) ◽  
pp. 16641-16650 ◽  
Author(s):  
Wen-Feng Nie ◽  
Mingguang Lei ◽  
Mingxuan Zhang ◽  
Kai Tang ◽  
Huan Huang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Conceptual Framework ◽  
Histone Acetylation ◽  
Chromatin Remodeling ◽  
Nuclear Protein ◽  
Dna Demethylation ◽  
Specific Target ◽  
Active Dna Demethylation ◽  
Chromatin Remodeling Complex ◽  
Genomic Regions

Active DNA demethylation is critical for controlling the DNA methylomes in plants and mammals. However, little is known about how DNA demethylases are recruited to target loci, and the involvement of chromatin marks in this process. Here, we identify 2 components of the SWR1 chromatin-remodeling complex, PIE1 and ARP6, as required for ROS1-mediated DNA demethylation, and discover 2 SWR1-associated bromodomain-containing proteins, AtMBD9 and nuclear protein X1 (NPX1). AtMBD9 and NPX1 recognize histone acetylation marks established by increased DNA methylation 1 (IDM1), a known regulator of DNA demethylation, redundantly facilitating H2A.Z deposition at IDM1 target loci. We show that at some genomic regions, H2A.Z and DNA methylation marks coexist, and H2A.Z physically interacts with ROS1 to regulate DNA demethylation and antisilencing. Our results unveil a mechanism through which DNA demethylases can be recruited to specific target loci exhibiting particular histone marks, providing a conceptual framework to understand how chromatin marks regulate DNA demethylation.

scholarly journals Involvement of Putative SNF2 Chromatin Remodeling Protein DRD1 in RNA-Directed DNA Methylation

2004 ◽  
Vol 14 (9) ◽  
pp. 801-805 ◽  
Author(s):  
Tatsuo Kanno ◽  
M.Florian Mette ◽  
David P Kreil ◽  
Werner Aufsatz ◽  
Marjori Matzke ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Chromatin Remodeling

400 KARYOTYPIC ANALYSIS AND EPIGENETIC MODIFICATIONS OF CULTURED PORCINE ADIPOSE TISSUE-DERIVED STEM CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 356
Author(s):  
K. J. Williams ◽  
K. R. Bondioli ◽  
R. A. Godke
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Stem Cells ◽  
Chromatin Remodeling ◽  
Primary Cultures ◽  
Donor Cell ◽  
Global Methylation ◽  
Histone 3 ◽  
Donor Cells

The introduction of genetic modifications in donor cells for NT requires a significant number of population doublings (PD), and the deleterious effects, which may be attributed to aneuploidy or changes in DNA methylation and histone acetylation, are difficult at this time to circumvent. We hypothesize that the identification of a donor cell that is genetically stable for a long period of time in vitro such as somatic stem cells or those cells that demonstrate stem-like characteristics may be reprogrammed more completely, thus providing the key to increasing the efficiency of NT. Regulators of development in undifferentiated cells are suggested to be silenced by the presence of a bivalent domain modification pattern in which a large region of repressive histone 3 lysine 27 trimethylation (H3K27me3) contains smaller regions of activating histone 3 lysine 4 trimethylation (H3K4me3).The dual marks work to silence developmental genes in embryonic stem cells while simultaneously keeping them receptive to activation. The objectives of the current study were to determine the chromosomal stability of porcine adipose tissue-derived adult stem cells (pASC) through in vitro culture, to analyze pASC alongside fetal porcine fibroblasts (FPF) for gene expression profiles of chromatin remodeling proteins and global methylation and acetylation patterns, and to determine the presence of a co-enrichment of H3K27me3 and H3K4me3 within the promoter regions of developmentally important transcription factors. Metaphase spreads were prepared, and the presence of H3K27me3 and H3K4me3 was investigated in each of 3 individual pASC primary cultures for each analysis; whereas, gene expression and global methylation and acetylation were analyzed in each of 4 individual pASC and FPF primary cultures. Of 714 metaphases analyzed, 509 (71.3%) were aneuploid and only 205 (28.7%) were normal diploid porcine cells. For each cell population, we found a remarkable percentage of aneuploidies (43.7, 48.9, and 47.3, with a 46.6 ± 1.5 average) present immediately after the cultures were established. Chi-square analysis indicated that the percent of aneuploid cells during PD 1-10 was significantly less than that for PD 11-20 and PD 21-30. Also, porcine ASC demonstrated a consistently lower level of DNA methylation and histone acetylation through passages 2 through 7; whereas, the patterns for FPF varied. The expression levels of chromatin remodeling transcripts remained lower in pASC throughout culture when compared with FPF. Finally, porcine ASC possess a co-enrichment of H3K27me3 and H3K4me3 on the promoter region of the developmentally important transcription factor OCT-4. In vitro-cultured porcine ASC used as donor cells for NT should be chosen from early PD because of increased levels of aneuploidy at later PD. With a more complete characterization of porcine ASC, a donor cell population that can be more efficiently reprogrammed following fusion with the oocyte might be identified.

Low Frequency of Molecular Alterations of H3.3-Atrx-Daxx Chromatin Remodeling Component Genes in Myelodysplastic Syndromes (MDS)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3844-3844
Author(s):  
Youmna Attieh ◽  
Yue Wei ◽  
Hui Yang ◽  
Yu Jia ◽  
Hong Zheng ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Chromatin Remodeling ◽  
Epigenetic Regulation ◽  
Myelodysplastic Syndromes ◽  
Conflicts Of Interest ◽  
Cpg Island ◽  
Genetic Mutations ◽  
P Value ◽  
Bone Marrow Mononuclear Cell ◽  
Hematopoietic Stem

Abstract Abstract 3844 Novel sequencing technologies have allowed identification of a group of highly recurrent genetic mutations in myelodysplastic syndromes (MDS). Of importance, it has been noticed that a majority of these mutated genes in MDS encode important components of epigenetic regulation, including both DNA methylation and histone modifications. This phenomenon highlights the importance of epigenetic mechanisms in the pathogenesis of MDS. Recently, highly recurrent somatic mutations in the Histone H3.3-ATRX-DAXX chromatin remodeling pathway have been documented in pediatric glioblastoma (Schwartzentruber et al. Nature and Wu et al. Nature Genetics 2012), further supporting the importance of epigenetic regulation for tumorgenesis. We therefore examined potential genetic and epigenetic alterations of the same pathway in MDS. First, in a cohort containing 80 samples of MDS whole bone marrow mononuclear cell DNA (representative of both lower and higher risk disease), we performed Sanger sequencing covering genomic areas of reported mutations of H3F3A, H3F3B, ATRX, and DAXX in glioblastoma. Sequenced genomic areas included reported mutations in pediatric tumors: Lys27 and Gly34 of H3F3A and H3F3B; sequences upstream of and within the helices domain of ATRXX; and the whole coding sequence of DAXX. Overall, we only detected one mutation of H3F3A (K27N) in one MDS case (76 year old male with RA; INT-1; diploid). No other reported mutation of H3F3B, ATRX and DAXX genes was detected in any other patients of this MDS cohort. Because of the potential of epigenetic deregulation, we then examined status of DNA methylation for the promoters of ATRX and DAXX in MDS patients by bisulfite pyrosquencing. While no DNA hypermethylation of DAXX promoter was detected, 8 out of 40 (20%) patients had hypermethylation of the CpG island in the promoter region of ATRX. However, six of these eight patients were females. Based on reports of ATRX methylation in healthy females, it is likely that the 6 cases in female patients represent physiological × chromosome inactivation. Finally, we performed RT-PCR analysis using cDNA samples isolated from CD34+ hematopoietic stem cells of 40 MDS patients. Results indicated that expression of ATRX and DAXX were increased by 2 fold (p-value 0.07) and 5.2 fold (p-value 0.0003) respectively compared to control CD34+ cells. The implications of this phenomenon need to be studied further. Taken together, these results suggest that genetic mutations of the H3.3-ATRX-DAXX chromatin remodeling do not play a role in the pathogenesis of MDS. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document