Analysis of DNA Methylation and Transcription During Granulopoiesis Reveals Timed Methylation Changes in Low CpG Areas That Correlate with Changed Transcriptional Activity.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2334-2334
Author(s):  
Rönnerblad Michelle ◽  
Olofsson Tor ◽  
Iyadh Douagi ◽  
Sören Lehmann ◽  
Karl Ekwall ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Conflicts Of Interest ◽  
Cell Types ◽  
Cpg Methylation ◽  
Epigenetic Mechanism ◽  
450K Array ◽  
Chi Square ◽  
Heterochromatin Formation ◽  
Chi Square Test

Abstract Abstract 2334 Accumulating evidence demonstrates that epigenetic changes, including DNA methylation play a central role in differentiation, providing cellular memory and stabilizing lineage choice in hematopoiesis1–3. DNA methylation is an important epigenetic mechanism involved in transcriptional regulation, heterochromatin formation and the normal development of many organisms. In this study we investigated the DNA methylome and transcriptome of human cells in four separate differentiation stages in granulopoiesis, ranging from the multipotent Common Myeloid progenitor (CMP) to terminally differentiated bone marrow neutrophils (PMN). To this end we employed HumanMethylation 450 BeadChip (450K array) from Illumina with extensive genomic coverage and mRNA expression arrays (Illumina). Temporally distinct methylation changes during granulopoiesis Differential methylation between two cell stages was defined as an average difference in β value of at least 0.17 (p ≤ 0.05). We detected 12132 DMSs during granulopoiesis. Of these the majority showed decreased methylation during granulopoeisis (10771 CpGs) and a smaller set gained methylation (1658 CpGs). Strikingly, increases in methylation predominantly occur between CMP and GMP, the two least mature cell types. Some CpGs also show increased methylation in the GMP-PMC transition, while very few CpG sites increase at the final stage of differentiation from PMC to PMN. Although reduction of methylation occurs at all stages of granulopoiesis, the greatest change is between GMP and PMC. It is striking that the DNA methylation patterns preferentially change at points of lineage restriction, and that the greatest change occurs upon loss of oligopotency between GMP and PMC. DMSs within CGIs were greatly underrepresented (p<0.001 with chi-square test), while DMSs were overrepresented in shelves (p<0.001) and open sea (p<0.001). Thus, methylation appears to be more dynamic outside of CGIs during granulocytic development. For all regions the variation within enhancers was greater than outside of enhancers indicating greater methylation changes in enhancers compared to non-enhancers. In addition, CpGs in enhancer regions are significantly enriched in the list of DMSs (p<0.001, chi-square test) further supporting the observation that enhancer regions display dynamic DNA methylation changes during granulopoiesis. Changes in gene expression correlate with DNA methylation changes There was a significant overlap between genes showing decreased methylation and genes with increased expression as well as for the reverse comparison between genes with increased methylation and decreased expression. Thus, support a general anticorrelation between DNA methylation and gene expression. Azurophilic granule proteins showed increased expression peaking in PMC and a rapid decrease toward PMN. CpG methylation levels for those genes decreased concomitantly with the peak in expression. We report cell population specific changes of DNA methylation levels. The main reduction of CpG methylation coincides with the loss of oligopotency at the transition from GMP-PMC. This suggests a role of DNA methylation in regulating cell plasticity and lineage choice. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CpG traffic lights are markers of regulatory regions in humans

2016 ◽  
Author(s):  
Abdullah M. Khamis ◽  
Anna V. Lioznova ◽  
Artem V. Artemov ◽  
Vasily Ramensky ◽  
Vladimir B. Bajic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Regulation Of Gene Expression ◽  
Cell Types ◽  
Regulatory Elements ◽  
Cpg Methylation ◽  
Population Heterogeneity ◽  
Gene Body Methylation ◽  
Enhancer Activity ◽  
Traffic Lights

AbstractDNA methylation is involved in regulation of gene expression. Although modern methods profile DNA methylation at single CpG sites, methylation levels are usually averaged over genomic regions in the downstream analyses. In this study we demonstrate that single CpG methylation can serve as a more accurate predictor of gene expression compared to average promoter / gene body methylation. CpG positions with significant correlation between methylation and expression of a gene nearby (named CpG traffic lights) are evolutionary conserved and enriched for exact TSS positions and active enhancers. Among all promoter types, CpG traffic lights are especially enriched in poised promoters. Genes that harbor CpG traffic lights are associated with development and signal transduction. Methylation levels of individual CpG traffic lights vary between cell types dramatically with the increased frequency of intermediate methylation levels, indicating cell population heterogeneity in CpG methylation levels. Being in line with the concept of the inherited stochastic epigenetic variation, methylation of such CpG positions might contribute to transcriptional regulation. Alternatively, one can hypothesize that traffic lights are markers of absent gene expression resulting from inactivation of their regulatory elements. The CpG traffic lights provide a promising insight into mechanisms of enhancer activity and gene regulation linking methylation of single CpG to expression.

scholarly journals Substantial DNA methylation differences between two major neuronal subtypes in human brain

2015 ◽  
Vol 44 (6) ◽  
pp. 2593-2612 ◽  
Author(s):  
Alexey Kozlenkov ◽  
Minghui Wang ◽  
Panos Roussos ◽  
Sergei Rudchenko ◽  
Mihaela Barbu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Cell Types ◽  
Cpg Methylation ◽  
Projection Neurons ◽  
Specific Gene ◽  
Cpg Sites ◽  
Gaba Neurons ◽  
The Brain

Abstract The brain is built from a large number of cell types which have been historically classified using location, morphology and molecular markers. Recent research suggests an important role of epigenetics in shaping and maintaining cell identity in the brain. To elucidate the role of DNA methylation in neuronal differentiation, we developed a new protocol for separation of nuclei from the two major populations of human prefrontal cortex neurons—GABAergic interneurons and glutamatergic (GLU) projection neurons. Major differences between the neuronal subtypes were revealed in CpG, non-CpG and hydroxymethylation (hCpG). A dramatically greater number of undermethylated CpG sites in GLU versus GABA neurons were identified. These differences did not directly translate into differences in gene expression and did not stem from the differences in hCpG methylation, as more hCpG methylation was detected in GLU versus GABA neurons. Notably, a comparable number of undermethylated non-CpG sites were identified in GLU and GABA neurons, and non-CpG methylation was a better predictor of subtype-specific gene expression compared to CpG methylation. Regions that are differentially methylated in GABA and GLU neurons were significantly enriched for schizophrenia risk loci. Collectively, our findings suggest that functional differences between neuronal subtypes are linked to their epigenetic specification.

FAS Gene Expression Is Epigenetically Regulated and Predicts the Responsiveness to Azacitidine In High-Risk Myelodysplastic Syndromes

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 232-232 ◽  
Author(s):  
Sandrine Ettou ◽  
Catherine Humbrecht ◽  
Blandine Benet ◽  
Olivier Kosmider ◽  
Nathalie Droin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
High Risk ◽  
Protein Expression ◽  
Myelodysplastic Syndromes ◽  
Conflicts Of Interest ◽  
Epigenetic Mechanism ◽  
Rank Test ◽  
Cpg Dinucleotides ◽  
Fas Mrna

Abstract Abstract 232 Background: Low risk myelodysplastic syndromes (MDS) CD34-positive cells exhibit high level of the death receptor Fas at their surface and abnormal Fas-dependent apoptosis. Fas expression decreases when the disease progresses to acute myeloid leukemia (AML). Based on recent evidence of higher DNA methylation level in high-risk MDS (Figueroa M et al, Blood 2010), we explored the epigenetic regulation of FAS gene during MDS evolution to AML. Methods: We quantified FAS gene expression by RT-qPCR in bone marrow mononuclear cells (BMMNC) from 82 MDS, 37 AML and 17 controls, including 54 patients treated with azacitidine according to the FDA/EMEA schedule. Response was scored according to IWG2006 criteria for MDS and to Cheson et al (JCO, 2003) for AML. We used HL-60 and SW480 cell lines to set up DNA methylation and chromatin immunoprecipitation (ChIP) assays. DNA methylation of 36 CpG dinucleotides in the FAS promoter was explored by bisulfite-treatment of genomic DNA, PCR, cloning and sequencing. Histone modifications of FAS promoter and control genes (B2M, RAG1 and PAX6) were assessed in 4 AML-post MDS patients before and during azacitidine treatment, using acetylated H3 (AcH3) and trimethyl-lysine 9-H3 (H3K9me3) as transcriptionally active and dimethyl-lysine 4-H3 (H3K4me2) and trimethyl-lysine 27-H3 (H3K27me3) as transcriptionally repressive markers. Results: qRT-PCR identified a significant decrease in FAS mRNA in AML-post MDS compared to RAEB1 (P<0.05). We also noticed a trend to decreased expression of FAS mRNA in AML-post MDS compared to all-stage MDS (P=0.12). Fas protein expression at the surface of BM CD34-positive cells had no impact on overall survival in a cohort of 45 untreated patients. By contrast, a low Fas protein expression (cut-off, RFI<1.8) correlated with a higher response rate in a series of 54 MDS and AML-post MDS patients treated with azacitidine (P=0.043). In these patients, Kaplan-Meier analysis demonstrated that a low Fas protein expression also correlated with longer survival in high-risk MDS and AML-post MDS (Log-Rank test, HR 0.47 [CI95%: 0.23 – 0.96]; P=0.032), and a serial evaluation of Fas protein demonstrated a significant increase in responding patients after 6 cycles (P=0.015). HL60 leukemic cells express Fas at their surface whereas SW480 colon cancer cells do not. FAS mRNA was higher in HL-60 (nR=2.3±0.4) than in SW-480 (nR=0.5±0.4) cells. In HL-60 cells, only 2 of the 36 studied CpG in FAS promoter were methylated, compared to 20/36 in SW-480, and AcH3 and H3K9me3 active markers were highly enriched, suggesting opened chromatin in HL-60 FAS promoter. In BM CD34-positive cells, we observed a significant increase in the methylation of 2 CpG dinucleotides in 14 AML-post MDS compared to 18 MDS (CpG # 2, P=0.05; CpG # 9, P=0.02). In 5/6 AML-post MDS samples, in vitro treatment with azacitidine induced a 1.7-fold increase of FAS mRNA, and rescued Fas-mediated apoptosis. In 4 AML-post MDS patients, a series of 6 cycles of azacitidine induced a decrease in DNA methylation of the FAS promoter in CD34-positive cells (from 28% [range: 20 – 37] to 14% [range: 11 – 15] methylated CpG) and an enrichment for H3K9me3, not for H3K27me3 marker. Conclusions: An epigenetic mechanism is responsible for the down-regulation of Fas in AML-post MDS, which is corrected by azacitidine in responding patients. These results suggest that FAS gene reactivation could predict the responsiveness to azacitidine and could be a biomarker of azacitidine efficacy. Disclosures: No relevant conflicts of interest to declare.

Epigenetics In Autoimmune Disease

2018 ◽  
Author(s):  
Matlock A Jeffries
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Autoimmune Disease ◽  
Noncoding Rna ◽  
Specific Treatment ◽  
Cell Types ◽  
Response To Therapy ◽  
Human Immune System ◽  
Treatment Regimens ◽  
New Research

Autoimmunity refers to a pathologic state of immunologic dysregulation in which the human immune system turns inward, attacking healthy tissues. The key step in this process is a break of self-immune tolerance. Recent studies have implicated dysregulation of gene expression via altered epigenetic control as a key mechanism in the development and promotion of autoimmunity. Epigenetics is defined as heritable changes in gene expression as a result of modification of DNA methylation, histone side chains, and noncoding RNA. Studies examining identical twins discordant for lupus, for example, were among the first to identify alterations in DNA methylation leading to lupus. Histone side-chain changes have been studied extensively in rheumatoid arthritis (RA), and many pathogenic cell types in RA exhibit a hyperacetylation phenotype. Finally, new research in the noncoding RNA field has not only uncovered potentially targetable pathways (e.g., miR-155) but may lead to the development of new diagnostic and prognostic biomarkers, helping physicians better tailor specific treatment regimens to improve response to therapy in autoimmune disease.   This review contains 4 figures, 1 table and 47 references Key Words: autoimmunity, big data, biomarkers, computational biology, DNA methylation, epigenetics, histone acetylation, histone methylation, microRNA, noncoding RNA

scholarly journals GSDMA drives the most replicated association with asthma in naïve CD4+ T cells

2019 ◽  
Author(s):  
Anne-Marie Madore ◽  
Lucile Pain ◽  
Anne-Marie Boucher-Lafleur ◽  
Jolyane Meloche ◽  
Andréanne Morin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
T Cells ◽  
Genetic Variants ◽  
Immune Cell ◽  
Cell Types ◽  
Sequencing Data ◽  
Opposite Pattern ◽  
Genetic Mechanisms ◽  
New Gene

AbstractBackgroundThe 17q12-21 locus is the most replicated association with asthma. However, no study had described the genetic mechanisms underlying this association considering all genes of the locus in immune cell samples isolated from asthmatic and non-asthmatic individuals.ObjectiveThis study takes benefit of samples from naïve CD4+ T cells and eosinophils isolated from the same 200 individuals to describe specific interactions between genetic variants, gene expression and DNA methylation levels for the 17q12-21 asthma locus.Methods and ResultsAfter isolation of naïve CD4+ T cells and eosinophils from blood samples, next generation sequencing was used to measure DNA methylation levels and gene expression counts. Genetic interactions were then evaluated considering genetic variants from imputed genotype data. In naïve CD4+ T cells but not eosinophils, 20 SNPs in the fourth and fifth haplotype blocks modulated both GSDMA expression and methylation levels, showing an opposite pattern of allele frequencies and expression counts in asthmatics compared to controls. Moreover, negative correlations have been measured between methylation levels of CpG sites located within the 1.5 kb region from the transcription start site of GSDMA and its expression counts.ConclusionAvailability of sequencing data from two key cell types isolated from asthmatic and non-asthmatic individuals allowed identifying a new gene in naïve CD4+ T cells that drives the association with the 17q12-21 locus, leading to a better understanding of the genetic mechanisms taking place in it.

scholarly journals Reading the unique DNA methylation landscape of the brain: Non-CpG methylation, hydroxymethylation, and MeCP2

2015 ◽  
Vol 112 (22) ◽  
pp. 6800-6806 ◽  
Author(s):  
Benyam Kinde ◽  
Harrison W. Gabel ◽  
Caitlin S. Gilbert ◽  
Eric C. Griffith ◽  
Michael E. Greenberg
Keyword(s):  
Dna Methylation ◽  
Binding Protein ◽  
Cell Types ◽  
Cpg Methylation ◽  
Early Postnatal Development ◽  
Cpg Dinucleotides ◽  
Epigenetic Regulator ◽  
And Function ◽  
Syndrome Protein ◽  
The Brain

DNA methylation at CpG dinucleotides is an important epigenetic regulator common to virtually all mammalian cell types, but recent evidence indicates that during early postnatal development neuronal genomes also accumulate uniquely high levels of two alternative forms of methylation, non-CpG methylation and hydroxymethylation. Here we discuss the distinct landscape of DNA methylation in neurons, how it is established, and how it might affect the binding and function of protein readers of DNA methylation. We review studies of one critical reader of DNA methylation in the brain, the Rett syndrome protein methyl CpG-binding protein 2 (MeCP2), and discuss how differential binding affinity of MeCP2 for non-CpG and hydroxymethylation may affect the function of this methyl-binding protein in the nervous system.

scholarly journals Stress-induced gene expression and behavior are controlled by DNA methylation and methyl donor availability in the dentate gyrus

2016 ◽  
Vol 113 (17) ◽  
pp. 4830-4835 ◽  
Author(s):  
Emily A. Saunderson ◽  
Helen Spiers ◽  
Karen R. Mifsud ◽  
Maria Gutierrez-Mecinas ◽  
Alexandra F. Trollope ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dentate Gyrus ◽  
Crucial Role ◽  
Histone H3 ◽  
Behavioral Responses ◽  
Cpg Methylation ◽  
Granule Neurons ◽  
Methyl Donor ◽  
And Behavior

Stressful events evoke long-term changes in behavioral responses; however, the underlying mechanisms in the brain are not well understood. Previous work has shown that epigenetic changes and immediate-early gene (IEG) induction in stress-activated dentate gyrus (DG) granule neurons play a crucial role in these behavioral responses. Here, we show that an acute stressful challenge [i.e., forced swimming (FS)] results in DNA demethylation at specific CpG (5′-cytosine–phosphate–guanine-3′) sites close to the c-Fos (FBJ murine osteosarcoma viral oncogene homolog) transcriptional start site and within the gene promoter region of Egr-1 (early growth response protein 1) specifically in the DG. Administration of the (endogenous) methyl donor S-adenosyl methionine (SAM) did not affect CpG methylation and IEG gene expression at baseline. However, administration of SAM before the FS challenge resulted in an enhanced CpG methylation at the IEG loci and suppression of IEG induction specifically in the DG and an impaired behavioral immobility response 24 h later. The stressor also specifically increased the expression of the de novo DNA methyltransferase Dnmt3a [DNA (cytosine-5-)-methyltransferase 3 alpha] in this hippocampus region. Moreover, stress resulted in an increased association of Dnmt3a enzyme with the affected CpG loci within the IEG genes. No effects of SAM were observed on stress-evoked histone modifications, including H3S10p-K14ac (histone H3, phosphorylated serine 10 and acetylated lysine-14), H3K4me3 (histone H3, trimethylated lysine-4), H3K9me3 (histone H3, trimethylated lysine-9), and H3K27me3 (histone H3, trimethylated lysine-27). We conclude that the DNA methylation status of IEGs plays a crucial role in FS-induced IEG induction in DG granule neurons and associated behavioral responses. In addition, the concentration of available methyl donor, possibly in conjunction with Dnmt3a, is critical for the responsiveness of dentate neurons to environmental stimuli in terms of gene expression and behavior.

scholarly journals Specific Histone Tail Modification and Not DNA Methylation Is a Determinant of Herpes Simplex Virus Type 1 Latent Gene Expression

2004 ◽  
Vol 78 (3) ◽  
pp. 1139-1149 ◽  
Author(s):  
Nicole J. Kubat ◽  
Robert K. Tran ◽  
Peterjon McAnany ◽  
David C. Bloom
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Epigenetic Mechanism ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT During herpes simplex virus type 1 (HSV-1) latency, gene expression is tightly repressed except for the latency-associated transcript (LAT). The mechanistic basis for this repression is unknown, but its global nature suggests regulation by an epigenetic mechanism such as DNA methylation. Previous work demonstrated that latent HSV-1 genomes are not extensively methylated, but these studies lacked the resolution to examine methylation of individual CpGs that could repress transcription from individual promoters during latency. To address this point, we employed established models to predict genomic regions with the highest probability of being methylated and, using bisulfite sequencing, analyzed the methylation profiles of these regions. We found no significant methylation of latent DNA isolated from mouse dorsal root ganglia in any of the regions examined, including the ICP4 and LAT promoters. This analysis indicates that methylation is unlikely to play a major role in regulating HSV-1 latent gene expression. Subsequently we focused on differential histone modification as another epigenetic mechanism that could regulate latent transcription. Chromatin immunoprecipitation analysis of the latent HSV-1 DNA repeat regions demonstrated that a portion of the LAT region is associated with histone H3 acetylated at lysines 9 and 14, consistent with a euchromatic and nonrepressed structure. In contrast, the chromatin associated with the HSV-1 DNA polymerase gene located in the unique long segment was not enriched in H3 acetylated at lysines 9 and 14, suggesting a transcriptionally inactive structure. These data suggest that histone composition may be a major regulatory determinant of HSV latency.

Study on DNA Methylation Status of WT1 Gene in Its Promoter Region in Hematologic Neoplasm Cell Lines.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4386-4386
Author(s):  
Ye Zhao ◽  
Zi-xing Chen ◽  
Shao-yan Hu ◽  
Jian-nong Cen
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Cell Lines ◽  
Promoter Region ◽  
U937 Cell ◽  
Methylation Status ◽  
Gene Promoter ◽  
Epigenetic Mechanism ◽  
Wt1 Gene ◽  
Gene Promoter Region

Abstract The methylation at CpG island in the promoter region of a gene is one of the important epigenetic mechanism which regulates the gene activity. To study the DNA methylation pattern of WT1 gene promoter region within hematologic neoplastic cell lines and its correlation with WT1 gene expression by using the PCR-based methods. RT-PCR and Methylation-specific PCR were performed to study the WT1 gene expression in 8226, HL-60, Jurkat, K562, KG-1, NB4, Raji, SHI-1, U266 and U937 cell lines and the DNA methylation status in promoter region of WT1 gene. After treatment of U937 cell line by 5-aza-CdR, a demethylation inducing agent, the changes of WT1 gene expression level and the methylation status in its promter region in U937 cells was determined. Our Results showed that HL-60, K562, KG-1, NB4, SHI-1 cell lines demonstrated higher level of WT1 expression, while extremely low level was found in 8226, Jurkat, Raji, U266 and U937. The DNA hypermethylation in WT1 gene promoter region was identified in 8226, Jurkat, Raji, U266 and U937 cell lines. The WT1 gene expression in U937 was markedly enhanced after treatment with 5-aza-CdR in company with the decrease of methylated level and the increase of unmethylated level in its promoter region. These results indicate that modulation of the DNA methylation in WT1 promoter region is one of the epigenetic mechanisms to regulate its expression.

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