305 SPERM CHROMATIN STRUCTURE AND DNA METHYLATION BULLS

2006 ◽  
Vol 18 (2) ◽  
pp. 260
Author(s):  
K. Hengstberger ◽  
N. Phutikanit ◽  
O. Berking ◽  
M. D'Occhio ◽  
D. Sester ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Conformational Changes ◽  
Chromatin Structure ◽  
Methylation Status ◽  
Regulation Of Gene Expression ◽  
Bos Indicus ◽  
Methylation Pattern ◽  
Embryonic Mortality ◽  
Sperm Chromatin ◽  
Detergent Solution

Mammalian spermatogenesis is a complex process that involves genome-wide deprogramming and reprogramming of DNA methylation at different stages in order to produce sperm that are capable of fertilization and also TO support ongoing embryo development (Webster et al. 2005 PNAS 102, 4068-4073). Embryonic mortality occurs when there is inappropriate expression of developmentally regulated genes of both maternal and paternal origin. DNA methylation is associated with epigenetic regulation of gene expression and it was recently shown that aberrant DNA methylation causes a change in sperm chromatin structure (Webster et al. 2005). Studies, primarily in man, have indicated that sperm chromatin instability, as determined by the sperm chromatin structure assay (SCSA), does not influence fertilization but is linked with early pregnancy failure (Evenson and Jost 2000 Methods Cell Sci. 22, 169-189). The aim of the present study was to investigate the relationship between chromatin structure and DNA methylation in bull sperm. Chromatin structure was determined using the SCSA that involves the exposure of sperm to a low pH (1.2) detergent solution followed by the addition of acridine orange (AO). When exposed to a 488 nm laser, AO fluoresces green when bound to native (double-stranded) DNA and red when bound to denatured (single-stranded) DNA. The SCSA yields a value for the DNA fragmentation index (DFI), and in men a DFI > 27-30% is associated with early embryonic mortality (Larson-Cook et al. 2003 Fertil. Steril. 80, 895-902). Semen was obtained by electroejaculation from Zebu (Bos indicus) bulls (n = 12) in a subtropical environment; a proportion of bulls had a DFI < 15% (n = 4) and the remainder a DFI > 27% (n = 8). The DNA methylation pattern, as determined by the amplified methylation polymorphism (AMP) protocol (Webster et al. 2005), appeared to differ between sperm with a DFI < 15% and sperm with a DFI > 27% when assessed by principle coordinate analysis. Also, sperm with a DFI < 15% had a more consistent methylation pattern compared with apparent differences in methylation among sperm with a DFI > 27%. These preliminary findings could be interpreted to suggest that methylation status can contribute to chromatin structure in sperm of mature bulls. It remains to be determined whether the environment can influence sperm chromatin status in bulls by altering DNA methylation which then impacts on the conformational changes in DNA that occur during spermiogenesis. This work was supported, in part, by Meat and Livestock Australia.

EPCO-01. MOLECULAR PROFILING OF SPINAL CORD EPENDYMOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi1-vi1
Author(s):  
Erika Yamazawa ◽  
Shota Tanaka ◽  
Genta Nagae ◽  
Takayoshi Umeda ◽  
Taijun Hana ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Dna Methylation ◽  
Methylation Status ◽  
Neurofibromatosis Type ◽  
Methylation Pattern ◽  
Who Grade ◽  
Spinal Ependymoma ◽  
Fresh Frozen ◽  
Spinal Cord Ependymoma ◽  
The Difference

Abstract BACKGROUND Ependymomas are currently classified into 9 subgroups by DNA methylation profiles. Although spinal cord ependymoma (SP-EPN) is distinct from other tumors, diversity within SP-EPN is still unclear. Here, we used transcriptomic and epigenomic profiles to investigate the diversity among Japanese SP-EPN cases. MATERIALS AND METHODS We analyzed 57 SP-EPN patients (32 males and 25 females, aged from 18 to 78 years, median: 52), including two cases of neurofibromatosis type 2, five cases of grade 3 (WHO grade). We obtained transcriptome (RNA-seq) and DNA methylation (Infinium Methylation EPIC array) data from fresh frozen specimens of SP-EPN resected at the University of Tokyo Hospital and our collaborative groups. RESULTS Three cases had a previous intracranial ependymoma operation. Hierarchical clustering of the DNA methylation data showed that these three cases of intracranial origin as a different cluster from spinal origin. The 45 grade 2 spinal ependymoma showed a relatively homogenous methylation pattern. However, the methylation status of HOX gene cluster regions is compatible with the segment of origin, which reflects the cells of origins are derived after the determination of segment identity. RNA sequencing of 57 cases revealed two subgroups within grade 2. Gene ontology analysis of differentially expressed genes suggested the difference in metabolic state such as rRNA translation and mitochondrial respiration between the two expression subgroups. CONCLUSION Epigenetic analysis indicated the accurate body segment origin of SP-EPN. We observed that metabolic states could divide grade 2 spinal cord ependymoma into 2 subgroups and will present the relationship to clinicopathological information.

scholarly journals Signatures of polycomb repression and reduced H3K4 trimethylation are associated with p15INK4b DNA methylation in AML

Blood ◽  
2010 ◽  
Vol 115 (15) ◽  
pp. 3098-3108 ◽  
Author(s):  
Thomas A. Paul ◽  
Juraj Bies ◽  
Donald Small ◽  
Linda Wolff
Keyword(s):  
Dna Methylation ◽  
Histone Modifications ◽  
Transcriptional Repression ◽  
Trichostatin A ◽  
Methylation Status ◽  
Suppressor Gene ◽  
Methylation Pattern ◽  
Dna Hypermethylation ◽  
Polycomb Repression ◽  
On Chip

Abstract DNA hypermethylation of the p15INK4b tumor suppressor gene is commonly observed in acute myeloid leukemia (AML). Repressive histone modifications and their associated binding proteins have been implicated in the regulation of DNA methylation and the transcriptional repression of genes with DNA methylation. We have used high-density chromatin immunoprecipitation-on-chip to determine the histone modifications that normally regulate p15INK4b expression in AML cells and how these marks are altered in cells that have p15INK4b DNA methylation. In AML patient blasts without p15INK4b DNA methylation, a bivalent pattern of active (H3K4me3) and repressive (H3K27me3) modifications exist at the p15INK4b promoter. AML patient blasts with p15INK4b DNA methylation lose H3K4me3 at p15INK4b and become exclusively marked by H3K27me3. H3K27me3, as well as EZH2, extends throughout p14ARF and p16INK4a, indicating that polycomb repression of p15INK4b is a common feature in all AML blasts irrespective of the DNA methylation status of the gene. Reactivation of p15INK4b expression in AML cell lines and patient blasts using 5-aza-2′-deoxycytidine (decitabine) and trichostatin A increased H3K4me3 and maintained H3K27me3 enrichment at p15INK4b. These data indicate that AML cells with p15INK4b DNA methylation have an altered histone methylation pattern compared with unmethylated samples and that these changes are reversible by epigenetic drugs.

scholarly journals Deoxyribonucleic Acid Methylation Controls Cell Type-Specific Expression of Steroidogenic Factor 1

Endocrinology ◽  
2008 ◽  
Vol 149 (11) ◽  
pp. 5599-5609 ◽  
Author(s):  
Erling A. Hoivik ◽  
Linda Aumo ◽  
Reidun Aesoy ◽  
Haldis Lillefosse ◽  
Aurélia E. Lewis ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase Ii ◽  
Methylation Status ◽  
Endocrine System ◽  
Methylation Pattern ◽  
Proximal Promoter ◽  
Specific Expression ◽  
Steroidogenic Factor 1 ◽  
In Cells

Steroidogenic factor 1 (SF1) is expressed in a time- and cell-specific manner in the endocrine system. In this study we present evidence to support that methylation of CpG sites located in the proximal promoter of the gene encoding SF1 contributes to the restricted expression pattern of this nuclear receptor. DNA methylation analyses revealed a nearly perfect correlation between the methylation status of the proximal promoter and protein expression, such that it was hypomethylated in cells that express SF1 but hypermethylated in nonexpressing cells. Moreover, in vitro methylation of this region completely repressed reporter gene activity in transfected steroidogenic cells. Bisulfite sequencing of DNA from embryonic tissue demonstrated that the proximal promoter was unmethylated in the developing testis and ovary, whereas it was hypermethylated in tissues that do not express SF1. Together these results indicate that the DNA methylation pattern is established early in the embryo and stably inherited thereafter throughout development to confine SF1 expression to the appropriate tissues. Chromatin immunoprecipitation analyses revealed that the transcriptional activator upstream stimulatory factor 2 and RNA polymerase II were specifically recruited to this DNA region in cells in which the proximal promoter is hypomethylated, providing functional support for the fact that lack of methylation corresponds to a transcriptionally active gene. In conclusion, we identified a region within the SF1/Sf1 gene that epigenetically directs cell-specific expression of SF1.

scholarly journals "Same Difference": Comprehensive evaluation of three DNA methylation measurement platforms

2016 ◽  
Author(s):  
Thadeous J Kacmarczyk ◽  
Mame P. Fall ◽  
Xihui Zhang ◽  
Yuan Xin ◽  
Yushan Li ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Restriction Enzyme ◽  
Comprehensive Evaluation ◽  
Methylation Status ◽  
Regulation Of Gene Expression ◽  
Starting Material ◽  
Reduced Representation ◽  
Targeted Capture ◽  
Areas Of Interest

ABSTRACTBackgroundDNA methylation in CpG context is fundamental to the epigenetic regulation of gene expression in high eukaryotes. Disorganization of methylation status is implicated in many diseases, cellular differentiation, imprinting, and other biological processes. Techniques that enrich for biologically relevant genomic regions with high CpG content are desired, since, depending on the size of an organism’s methylome, the depth of sequencing required to cover all CpGs can be prohibitively expensive. Currently, restriction enzyme based reduced representation bisulfite sequencing and its modified protocols are widely used to study methylation differences. Recently, Agilent Technologies and Roche NimbleGen have ventured to both reduce sequencing costs and capture CpGs of known biological relevance by marketing in-solution custom-capture hybridization platforms. We aimed to evaluate the similarities and differences of these three methods considering each targets approximately 10-13% of the human methylome.ResultsOverall, the regions covered per platform were as expected: targeted capture based methods covered >95% of their designed regions whereas the restriction enzyme-based method covered >70% of the expected fragments. While the total number of CpG loci shared by all methods was low, ~30% of any platform, the methylation levels of CpGs common across platforms were concordant. Annotation of CpG loci with genomic features revealed roughly the same proportions of feature annotations across the three platforms. Targeted capture methods encompass similar amounts of annotations with the restriction enzyme based method covering fewer promoters (~9%) and shores (~8%) and more unannotated loci (7-14%).ConclusionsAlthough all methods are largely consistent in terms of covered CpG loci and cover similar proportions of annotated CpG loci, the restriction based enrichment results in more unannotated regions and the commercially available capture methods result in less off-target regions. Quality of DNA is very important for restriction based enrichment and starting material can be low. Conversely, quality of the starting material is less important for capture methods, and at least twice the amount of starting material is required. Pricing is marginally less for restriction based enrichment, and number of samples to be prepared is not restricted to the number of samples a kit supports. The one advantage of capture libraries is the ability to custom design areas of interest. The choice of the technique should be decided by the number of samples, the quality and quantity of DNA available and the biological areas of interest since comparable data are obtained from all platforms.

scholarly journals Genome-Wide DNA Methylation Pattern in Whole Blood Associated With Primary Intracerebral Hemorrhage

2021 ◽  
Vol 12 ◽  
Author(s):  
Yupeng Zhang ◽  
Hongyu Long ◽  
Sai Wang ◽  
Wenbiao Xiao ◽  
Meishan Xiong ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Intracerebral Hemorrhage ◽  
Whole Blood ◽  
Peripheral Blood ◽  
Methylation Status ◽  
Methylation Pattern ◽  
Healthy Controls ◽  
Differentially Methylated Genes ◽  
Dna Methylation Pattern ◽  
Methylation Patterns

Primary intracerebral hemorrhage (ICH) is a significant cause of morbidity and mortality throughout the world. ICH is a multifactorial disease that emerges from interactions among multiple genetic and environmental factors. DNA methylation plays an important role in the etiology of complex traits and diseases. We used the Illumina Infinium Human Methylation 850k BeadChip to detect changes in DNA methylation in peripheral blood samples from patients with ICH and healthy controls to explore DNA methylation patterns in ICH. Here, we compared genomic DNA methylation patterns in whole blood from ICH patients (n = 30) and controls (n = 34). The ICH and control groups showed significantly different DNA methylation patterns at 1530 sites (p-value &lt; 5.92E-08), with 1377 hypermethylated sites and 153 hypomethylated sites in ICH patients compared to the methylation status in healthy controls. A total of 371 hypermethylated sites and 35 hypomethylated sites were in promoters, while 738 hypermethylated sites and 67 hypomethylated sites were in coding regions. Furthermore, the differentially methylated genes between ICH patients and controls were largely related to inflammatory pathways. Abnormalities in the DNA methylation pattern identified in the peripheral blood of ICH patients may play an important role in the development of ICH and warranted further investigation.

scholarly journals ATM Mediates pRB Function To Control DNMT1 Protein Stability and DNA Methylation

2013 ◽  
Vol 33 (16) ◽  
pp. 3113-3124 ◽  
Author(s):  
Awad Shamma ◽  
Misa Suzuki ◽  
Naoyuki Hayashi ◽  
Masahiko Kobayashi ◽  
Nobunari Sasaki ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Protein Stability ◽  
Genetic Interaction ◽  
Methylation Status ◽  
Tumor Model ◽  
Suppressor Gene ◽  
Methylation Pattern ◽  
Control Of Gene Expression ◽  
Retinoblastoma Tumor Suppressor ◽  
Retinoblastoma Tumor

The retinoblastoma tumor suppressor gene (RB) product has been implicated in epigenetic control of gene expression owing to its ability to physically bind to many chromatin modifiers. However, the biological and clinical significance of this activity was not well elucidated. To address this, we performed genetic and epigenetic analyses in anRb-deficient mouse thyroid C cell tumor model. Here we report that the genetic interaction ofRbandATMregulates DNMT1 protein stability and hence controls the DNA methylation status in the promoters of at least theInk4a,Shc2,FoxO6, andNoggingenes. Furthermore, we demonstrate that inactivation of pRB promotes Tip60 (acetyltransferase)-dependent ATM activation; allows activated ATM to physically bind to DNMT1, forming a complex with Tip60 and UHRF1 (E3 ligase); and consequently accelerates DNMT1 ubiquitination driven by Tip60-dependent acetylation. Our results indicate that inactivation of the pRB pathway in coordination with aberration in the DNA damage response deregulates DNMT1 stability, leading to an abnormal DNA methylation pattern and malignant progression.

Search for methylation-sensitive amplification polymorphisms associated with the "mantled" variant phenotype in oil palm (Elaeis guineensis Jacq.)

Genome ◽  
2004 ◽  
Vol 47 (1) ◽  
pp. 224-228 ◽  
Author(s):  
E Jaligot ◽  
T Beulé ◽  
F-C Baurens ◽  
N Billotte ◽  
A Rival
Keyword(s):  
Dna Methylation ◽  
Somaclonal Variation ◽  
Oil Palm ◽  
Elaeis Guineensis ◽  
Methylation Status ◽  
Methylation Pattern ◽  
Variant Phenotype ◽  
Elaeis Guineensis Jacq ◽  
The Moment ◽  
Direct Use

The methylation-sensitive amplification polymorphism (MSAP) technique has been employed on somatic embryo-derived oil palms (Elaeis guineensis Jacq.) to identify methylation polymorphisms correlated with the "mantled" somaclonal variation. The variant phenotype displays an unstable feminization of male organs in both male and female flowers. Using MSAP, the methylation status of CCGG sites was compared in three normal versus three mantled regenerants sampled in clonal populations obtained through somatic embryogenesis from four genotypically distinct mother palms. Overall, 64 selective primer combinations were used and they have amplified 23 markers exhibiting a differential methylation pattern between the two phenotypes. Our results indicate that CCGG sites are poorly affected by the considerable decrease in global DNA methylation that has been previously associated with the mantled phenotype. Each of the 23 markers isolated in the present study could discriminate between the two phenotypes only when they were from the same genetic origin. This result hampers at the moment the direct use of MSAP markers for the early detection of variants, even though valuable information on putative target sequences will be obtained from a further characterization of these polymorphic markers.Key words: DNA methylation, epigenetics, MSAP, oil palm, somaclonal variation.

scholarly journals Methylation of AR locus does not always reflect X chromosome inactivation state

Blood ◽  
2012 ◽  
Vol 119 (13) ◽  
pp. e100-e109 ◽  
Author(s):  
Sabina I. Swierczek ◽  
Lucie Piterkova ◽  
Jaroslav Jelinek ◽  
Neeraj Agarwal ◽  
Sue Hammoud ◽  
...  
Keyword(s):  
Dna Methylation ◽  
X Chromosome ◽  
Elderly Women ◽  
Methylation Status ◽  
Methylation Pattern ◽  
Elderly Persons ◽  
Quantitative Expression ◽  
Clonal Hematopoiesis ◽  
Healthy Elderly ◽  
Gene Assay

Abstract Clonality can be established by a lack of mosaicism in a female because of random inactivation of either the maternal or paternal X chromosome early in embryogenesis. The methylation status of CpG sites close to the trinucleotide repeats in exon 1 of the human androgen receptor (AR) X chromosome gene assay (HUMARA) has been used to determine clonality. This HUMARA at times indicated clonal hematopoiesis in healthy elderly women, thus precluding its applicability. We used a clonality assay based on quantitative expression of polymorphic X chromosome genes (qTCA) and found no evidence of clonal hematopoiesis in healthy nonanemic elderly persons. We found instances of discordance between HUMARA results and those obtained by pyrosequencing and qTCA methods, as well as by directly quantifying AR gene expression. To determine the basis of this discrepancy we examined the methylation pattern of the AR locus subject to HUMARA. Notably, we found the extent of DNA methylation to be highly variable at the AR gene in granulocytes of persons with discordant results and also in erythroid burst-forming unit colonies but not in those with clonal hematopoiesis. These data provide the molecular basis of incomplete correlation with the pattern of DNA methylation of this X chromosome AR gene locus.

scholarly journals Drying and temperature induced conformational changes of nucleic acids and stallion sperm chromatin in trehalose preservation formulations

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Raffaele Brogna ◽  
Juezhu Fan ◽  
Harald Sieme ◽  
Willem F. Wolkers ◽  
Harriëtte Oldenhof
Keyword(s):  
Reactive Oxygen Species ◽  
Dna Damage ◽  
Nucleic Acids ◽  
Conformational Changes ◽  
Chromatin Structure ◽  
Reactive Oxygen ◽  
Sperm Chromatin ◽  
Oxygen Species ◽  
Spectral Changes ◽  
And Storage

AbstractEven though dried sperm is not viable, it can be used for fertilization as long as its chromatin remains intact. In this study, we investigated drying- and temperature-induced conformational changes of nucleic acids and stallion sperm chromatin. Sperm was diluted in preservation formulations with and without sugar/albumin and subjected to convective drying at elevated temperatures on glass substrates. Accumulation of reactive oxygen species was studied during storage at different temperatures, and the sperm chromatin structure assay was used to assess DNA damage. Fourier transform infrared spectroscopy was used to identify dehydration and storage induced conformational changes in isolated DNA and sperm chromatin. Furthermore, hydrogen bonding in the preservation solutions associated with storage stability were investigated. Reactive oxygen species and DNA damage in dried sperm samples were found to accumulate with increasing storage temperature and storage duration. Non-reducing disaccharides (i.e., trehalose, sucrose) and albumin counteracted oxidative stress and preserved sperm chromatin during dried storage, whereas glucose increased DNA damage during storage. When sperm was dried in the presence of trehalose and albumin, no spectral changes were detected during storage at refrigeration temperatures, whereas under accelerated aging conditions, i.e., storage at 37 °C, spectral changes were detected indicating alterations in sperm chromatin structure.

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