S137 DNA METHYLATION DYNAMICS IDENTIFY LINEAGE-SPECIFIC REGULATION PATTERNS OF HEMATOPOIETIC DIFFERENTIATION

De novo DNA methyltransferase 3A (DNMT3A) plays pivotal roles in hematopoietic differentiation. In this study, we followed the hypothesis that alternative splicing ofDNMT3Ahas characteristic epigenetic and functional sequels. SpecificDNMT3Atranscripts were either down-regulated or overexpressed in human hematopoietic stem and progenitor cells, and this resulted in complementary and transcript-specific DNA methylation and gene expression changes. Functional analysis indicated that, particularly, transcript 2 (coding for DNMT3A2) activates proliferation and induces loss of a primitive immunophenotype, whereas transcript 4 interferes with colony formation of the erythroid lineage. Notably, in acute myeloid leukemia expression of transcript 2 correlates with its in vitro DNA methylation and gene expression signatures and is associated with overall survival, indicating thatDNMT3Avariants also affect malignancies. Our results demonstrate that specificDNMT3Avariants have a distinct epigenetic and functional impact. Particularly, DNMT3A2 triggers hematopoietic differentiation and the corresponding signatures are reflected in acute myeloid leukemia.

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Role of the DNA repair genes H2AX and HMGB1 in human fat distribution and lipid profiles

BMJ Open Diabetes Research & Care ◽

10.1136/bmjdrc-2019-000831 ◽

2020 ◽

Vol 8 (1) ◽

pp. e000831

Author(s):

Kerstin Rohde ◽

Torunn Rønningen ◽

Lars la Cour Poulsen ◽

Maria Keller ◽

Matthias Blüher ◽

...

Keyword(s):

Dna Methylation ◽

Dna Repair ◽

Density Lipoprotein ◽

Fat Distribution ◽

Lipoprotein Cholesterol ◽

Mrna Levels ◽

Dna Repair Genes ◽

Specific Regulation ◽

Repair Genes

IntroductionRegional fat distribution strongly relates to metabolic comorbidities. We identified the DNA repair genes H2AX and HMGB1 to be differentially expressed between human subcutaneous (SAT) and omental visceral adipose tissue (OVAT) depots. As increased DNA damage is linked to metabolic disease, we here sought to analyze whether depot-specific H2AX and HMGB1 expression is related to anthropometric and metabolic profiles of obesity. We further tested for different H2AX mRNA regulatory mechanisms by analyzing promoter DNA methylation and genotyped rs7350 in the H2AX locus.Research design and methodsGene expression (OVAT n=48; SAT n=55) and DNA promoter methylation data (OVAT and SAT n=77) were extracted from an existing dataset as described elsewhere. Genotype data for the 3’untranslated region (3’UTR) H2AX variant rs7350 were generated by using the TaqMan genotyping system in 243 subjects of the same cohort. Statistical analyses were done using SPSS statistics software 24 and GraphPad Prism 6.ResultsWe identified H2AX being higher (p=0.002) and HMGB1 being less expressed (p=0.0001) in OVAT compared with SAT. Further, we observed positive interdepot correlations of OVAT and SAT for both HMGB1 (p=1×10–6) and H2AX mRNA levels (p=0.024). Depot-specific associations were observed for both genes’ methylation levels with either high density lipoprotein cholesterol, low density lipoprotein cholesterol, triglycerides and/or with OVAT/SAT-ratio (all p<0.05). A significantly lower level of total cholesterol in minor A-Allele carriers of rs7350 compared with AG and GG carriers (p=0.001) was observed. Additionally, subjects carrying the A-allele showed lower SAT HMGB1 expression level (p=0.030).ConclusionOur results suggest a fat depot-specific regulation of H2AX and HMGB1 potentially mediated by both DNA methylation and genetic variation. Rs7350, DNA methylation and/or mRNA levels of H2AX and HMGB1 are related to lipid parameters. Further studies are warranted to evaluate the functional role of the DNA repair genes H2AX and HMGB1 in obesity and fat distribution.

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Stage-specific regulation of DNA methylation by TET enzymes during human cardiac differentiation

Cell Reports ◽

10.1016/j.celrep.2021.110095 ◽

2021 ◽

Vol 37 (10) ◽

pp. 110095

Author(s):

Yahui Lan ◽

Kelly M. Banks ◽

Heng Pan ◽

Nipun Verma ◽

Gary R. Dixon ◽

...

Keyword(s):

Dna Methylation ◽

Cardiac Differentiation ◽

Specific Regulation ◽

Tet Enzymes

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Abstract 15945: Temporal Analyses of Chromatin Accessibility, Dna Methylation and Epigenomic Structure Identify Mechanisms of Locus-specific Regulation in the Heart

Circulation ◽

10.1161/circ.142.suppl_3.15945 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Manuel Rosa-garrido ◽

Douglas J Chapski ◽

Maximilian Cabaj ◽

Marco Morselli ◽

Shuxun Ren ◽

...

Keyword(s):

Gene Expression ◽

Heart Failure ◽

Dna Methylation ◽

Transcription Factors ◽

Pressure Overload ◽

Structural Data ◽

Chromatin Accessibility ◽

Noncoding Regions ◽

Specific Regulation ◽

Time Point

Heart failure can be induced or ameliorated in animal models by regulation of chromatin modifying enzymes, yet the chromatin level actions of these enzymes during pathogenesis is unknown. Because many histone modifiers and transcription factors regulate gene expression, we sought to directly measure chromatin accessibility through an unbiased method (ATAC-seq) that reports the status of a given locus at any time—the sum total of all epigenetic modifiers—in a mouse model of pressure overload hypertrophy. Early compensation of pressure overload at 3 days was associated with widespread changes in chromatin accessibility and DNA methylation, primarily in noncoding regions. The majority of changes that persisted to the decompensated phase (3weeks) were already established at the earlier time point, revealing a temporal nature of epigenomic compensation to pathologic stimuli. A cardiac-specific CTCF depletion model was used to examine basal cardiac chromatin function and revealed that disruption of this structure by loss of CTCF causes widespread changes in accessibility and methylation distinct from those in pressure overload. Less than half of the gene expression changes occurring at either time point after pressure overload were explained by DNA methylation alone and accessibility was likewise an imperfect predictor of transcription. Distal enhancers were paired with genes based on chromatin structural data and the regulatory actions of these elements examined in the context of DNA methylation and accessibility: enhancer actions require specific combinations of transcription factors and histone modifications at different stages of disease and to execute aspecific transcriptional event (methylation or accessibility alone was insufficient to predict the behavior). For example, the subset of differentially accessible enhancers in both 3 weeks TACand CTCF depletion significantly overlaps with cardiac transcription factors Gata4 (p=4.13x10 -6 ),Nkx2-5 (p=2.49x10 -5 ) and P300 (p=8.38x10 -7 ). In summary, these studies characterize the logic employed at coding, regulatory, and noncoding regions to regulate chromatin accessibility and transcription, providing a resource of epigenomic data at distinct temporal stages of heart failure.

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Exportin 4, a Potential Amplifier of TGF-β Signaling, Is Increased in Primary Baboon Bone Marrow Erythroblasts Following Decitabine Treatment.

Blood ◽

10.1182/blood.v108.11.1582.1582 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1582-1582

Author(s):

Donald Lavelle ◽

Tatiana Kouznetsova ◽

Kestis Vaitkus ◽

Peter Larsen ◽

Maria Hankewych ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Bone Marrow ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Globin Gene ◽

Splicing Factor ◽

Hematopoietic Differentiation ◽

Cell Disease ◽

Human Genechip

Abstract The DNA demethylating drug decitabine increased fetal hemoglobin (HbF) to therapeutic levels and reduced the level of DNA methylation of the γ-globin gene promoter in patients with sickle cell disease and in experimental baboons. Whether decreased DNA methylation of the γ-globin gene is solely responsible for increased HbF following decitabine treatment is unknown. Increased platelet counts in patients with sickle cell disease and myelodysplastic syndrome and in experimental baboons following decitabine treatment suggest that decitabine also affects hematopoietic differentiation. To investigate to what extent the mechanism responsible for the ability of decitabine to reactivate HbF and alter hematopoietic differentiation may involve the induction of other unknown genes, the global pattern of gene expression in purified primary bone marrow erythroblasts pre- and post-decitabine treament was analyzed. Baboons were phlebotomized for ten days (Hct 20) followed by adminstration of decitabine for ten days (0.52mg/kg/d; sc). RNA was isolated from nucleated erythroblasts purified from bone marrow aspirates obtained pre- and post-decitabine treatment. Purification of erythroblasts was performed by sedimentation in Percoll gradients followed by immunomagnetic column purification using an anti-baboon RBC antibody (Pharmingen). To assess the feasibility of using human Genechip arrays to detect differences in expression of baboon transcripts, RNA isolated from purified erythroblasts of a single baboon pre- and post-decitabine was hybridized in triplicate to human Genechip Focus arrays (Affymetrix) containing over 8500 genes. The expression of 48 genes was increased >2 fold in the post-decitabine treated sample compared to the pre-treatment sample. Among the more highly induced genes were HLA-A (3 fold), HLA-B (5.7 fold), exportin 4 (3.9 fold), and splicing factor 3b1 (3.7 fold). Reverse transcriptase PCR using human primer sets was performed to analyze the expression of these genes in pre- and post-decitabine treated bone marrow erythroblasts in independent samples from three additional baboons. Induction of HLA-A, exportin 4, and splicing factor 3b1 was confirmed in all three post-decitabine treated samples. The exportin 4 gene encodes a protein involved in nuclear export of the Smad3 protein. Activated TGF-β receptors phosphorylate Smad3 and induce its nuclear import to affect gene transcription. Following dephosphorylation of Smad3 in the nucleus, transport of the protein to the cytoplasm mediated by exportin 4 has been proposed to allow the propagation of multiple rounds of activation by activated TGF-β receptors thus amplifying TGF-β signaling (Kurisaki et al; Mol Cell Biol26:1318, 2006). Because TGF-β increases HbF synthesis in cultured erythroid progenitors and also induces erythroid and megakaryocytic differentiation, we suggest that induction of exportin 4 by decitabine may play a role in the ability of this drug to increase HbF synthesis and alter hematopoietic differentiation. Our results thus confirm the feasibility of using human Genechip arrays to assess gene expression levels in baboons. Furthermore, we have indentified a gene induced by decitabine that potentially amplifies TGf-β signaling and thus may play a role in the ability of this drug to increase HbF and alter hematopoietic differentiation.

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Comprehensive Comparison Of Gene Expression, Genomic DNA Methylation, and In Vitro Hematopoietic Differentiation Among Many Human iPS and ES Cell Lines

Blood ◽

10.1182/blood.v122.21.1187.1187 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1187-1187

Author(s):

Masatoshi Nishizawa ◽

Kazuhisa Chonabayashi ◽

Akiko Oishi ◽

Ikue Takei ◽

Misato Nishikawa ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Cell Lines ◽

Genomic Dna ◽

Ips Cells ◽

Hematopoietic Differentiation ◽

Es Cell ◽

Differentiation Potential ◽

Genome Wide ◽

Differentiation Efficiency

Abstract Objective Hematopoietic differentiation from human induced pluripotent stem (iPS)/embryonic stem (ES) cell attracts much attention due to its huge potential for regenerative medicine. As indicated by some earlier papers, there is large variation in differentiation potential among pluripotent stem cell (PSC) lines, and this is one of major concerns in clinical application of PSCs. If it becomes possible to predict which PSC line has high differentiation potential without real differentiation experiment, it would greatly contribute to clinical application of PSCs. Although some papers reported about presence of epigenetic memories of parental somatic cells in iPS cells, the amount of the influence on differentiation potential remains to be known. Furthermore, especially in studies using human PSCs, genetic difference among individual donors of iPS/ES cells seems to be large, thus the study using many PSC lines from many donors is warranted. To address these issues, we planned to collect data of many iPS/ES cell lines on genome-wide gene expression and genomic DNA methylation, and differentiation potentials of individual lines, and identify the factors which affected difference in differentiation potential among PSC lines. The final goal of this study is to create data base about gene expression and DNA methylation profile and differentiation potentials of many PSC lines. We believe that this dataset will allow us to predict differentiation potentials of individual PSC lines, and accelerate clinical application of PSC lines in hematology field. Method We utilized 39 iPS/ES lines (iPS 35 lines, ES 4 lines) in this study. The iPS cell lines were derived from dermal fibroblast (n = 16), cord blood (n = 3), peripheral blood (n = 10), keratinocyte (n = 3), and dental pulp cell (n = 3), and were generated by retrovirus vector (n = 9), episomal vector (n = 25), and sendai virus vector (n = 1). The iPS cells were derived from 15 donors, and the ES cells were derived from 4 donors. We assessed hematopoietic differentiation potential by investigating hematopoietic differentiation efficiency for the first 15 days from start of differentiation, and colony forming potential of hematopoietic precursor cells (CD34+CD38-CD43+lineage marker- population) generated from PSC lines using semi-solid methylcellulose based-media. In addition, we collected genome-wide mRNA expression and DNA methylation profile of PSC lines, parental lines of iPS cells, hematopoietic precursor cells generated from PSCs by using mRNA microarray, genomic methylation beads array, and next generation sequencers, and analyzed correlation of these data with differentiation potentials of individual PSC lines. Result We have found that there is large variation in hematopoietic differentiation efficiency and colony forming ability as reported previously. Genome-wide investigation of gene expression and genomic DNA methylation revealed that expression of some genes or some factors were significantly correlated with hematopoietic differentiation efficiency or colony forming ability of hematopoietic precursor cells. Importantly, the factors affecting differentiation efficiency for first 15 days and those affecting colony-forming ability were absolutely different. More importantly, by combining several factors discovered in this analysis, we can predict hematopoietic differentiation potential of individual iPS/ES cell lines regardless of what parental cell lines iPS cells are derived or whether it is an iPS cell or ES cell. Conclusion From genome-wide analysis of gene expression and genomic DNA methylation, and hematopoietic differentiation experiments, we discovered the factors that were associated with difference in differentiation potential among PSC lines. Now, we are focusing on investigating molecular mechanisms by which the discovered factors are responsible for the difference in hematopoietic differentiation potentials among PSC lines. We believe that our findings will contribute not only to clinical application of hematopoietic cells generated from human PSCs, but also to further understanding of human developmental hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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DNA methylation disruption reshapes the hematopoietic differentiation landscape

Nature Genetics ◽

10.1038/s41588-020-0595-4 ◽

2020 ◽

Vol 52 (4) ◽

pp. 378-387 ◽

Cited By ~ 19

Author(s):

Franco Izzo ◽

Stanley C. Lee ◽

Asaf Poran ◽

Ronan Chaligne ◽

Federico Gaiti ◽

...

Keyword(s):

Dna Methylation ◽

Hematopoietic Differentiation

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Developmental and tissue-specific regulation of the Q10 class I gene by DNA methylation.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.83.20.7598 ◽

1986 ◽

Vol 83 (20) ◽

pp. 7598-7602 ◽

Cited By ~ 6

Author(s):

K. Tanaka ◽

Y. Barra ◽

K. J. Isselbacher ◽

G. Khoury ◽

G. Jay

Keyword(s):

Dna Methylation ◽

Class I ◽

Tissue Specific ◽

Specific Regulation ◽

I Gene

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DNMT3A knockouts in human iPSCs prevent de novo DNA methylation and reveal growth advantage during hematopoietic differentiation

10.1101/2021.12.15.472782 ◽

2021 ◽

Author(s):

Olivia Cypris ◽

Julia Franzen ◽

Joana Frobel ◽

Philipp Glueck ◽

Chao-Chung Kuo ◽

...

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

De Novo ◽

Competitive Growth ◽

Hematopoietic Differentiation ◽

Exon 2 ◽

Human Ipscs ◽

Functional Relevance ◽

Induced Pluripotent ◽

Exon 19

DNA methyltransferase 3A (DNMT3A) is a frequently mutated gene in many hematological malignancies, indicating that it may be essential for hematopoietic differentiation. Here, we addressed the functional relevance of DNMT3A for differentiation of human induced pluripotent stem cells (iPSCs) by knocking out exon 2, 19, or 23. Exon 19-/- and 23-/- lines revealed absence of almost the entire de novo DNA methylation during mesenchymal and hematopoietic differentiation. Yet, differentiation was only slightly reduced in exon 19-/- and increased in exon 23-/- lines, whereas there was no significant impact on gene expression in hematopoietic progenitors (iHPCs). Notably, DNMT3A-/- iHPCs recapitulate some DNA methylation differences of acute myeloid leukemia with DNMT3A mutations. Furthermore, multicolor genetic barcoding revealed competitive growth advantage of exon 23-/- iHPCs. Our results demonstrate that de novo DNA methylation during hematopoietic differentiation of iPSCs is almost entirely dependent on DNMT3A and exon 23-/- iHPCs even gained growth advantage.

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Variants ofDNMT3Acause transcript-specific DNA methylation patterns and affect hematopoietic differentiation

10.1101/318188 ◽

2018 ◽

Author(s):

Tanja Božić ◽

Joana Frobel ◽

Annamarija Raic ◽

Fabio Ticconi ◽

Chao-Chung Kuo ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Myeloid Leukemia ◽

Dna Methyltransferase ◽

De Novo ◽

Hematopoietic Differentiation ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Methylation Patterns

AbstractThede novoDNA methyltransferase 3A (DNMT3A) plays pivotal roles in hematopoietic differentiation. In this study, we followed the hypothesis that alternative splicing ofDNMT3Ahas characteristic epigenetic and functional sequels. SpecificDNMT3Atranscripts were either downregulated or overexpressed in human hematopoietic stem and progenitor cells and this resulted in complementary and transcript-specific DNA methylation and gene expression changes. Functional analysis indicated that particularly transcript 2 (coding for DNMT3A2) activates proliferation and induces loss of a primitive immunophenotype, whereas transcript 4 interferes with colony formation of the erythroid lineage. Notably, in acute myeloid leukemia (AML) expression of transcript 2 correlates with itsin vitroDNA methylation and gene expression signatures and is associated with overall survival, indicating thatDNMT3Avariants impact also on malignancies. Our results demonstrate that specificDNMT3Avariants have distinct epigenetic and functional impact. Particularly DNMT3A2 triggers hematopoietic differentiation and the corresponding signatures are reflected in AML.

Download Full-text