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Humaira Gowher - One of the best experts on this subject based on the ideXlab platform.
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The acute myeloid leukemia variant DNMT3A Arg882His is a DNMT3B-like enzyme.
Nucleic acids research, 2020Co-Authors: Allison B Norvil, Taiping Chen, Bigang Liu, Nicole E Forstoffer, Amie R Michie, Alabdi, Humaira GowherAbstract:We have previously shown that the highly prevalent acute myeloid leukemia (AML) mutation, Arg882His, in DNMT3A disrupts its cooperative mechanism and leads to reduced enzymatic activity, thus explaining the genomic hypomethylation in AML cells. However, the underlying cause of the oncogenic effect of Arg882His in DNMT3A is not fully understood. Here, we discovered that DNMT3A WT enzyme under conditions that favor non-cooperative kinetic mechanism as well as DNMT3A Arg882His variant acquire CpG flanking sequence preference akin to that of DNMT3B, which is non-cooperative. We tested if DNMT3A Arg882His could preferably methylate DNMT3B-specific target sites in vivo. Rescue experiments in Dnmt3a/3b double knockout mouse embryonic stem cells show that the corresponding Arg878His mutation in mouse DNMT3A severely impairs its ability to methylate major satellite DNA, a DNMT3A-preferred target, but has no overt effect on the ability to methylate minor satellite DNA, a DNMT3B-preferred target. We also observed a previously unappreciated CpG flanking sequence bias in major and minor satellite repeats that is consistent with DNMT3A and DNMT3B specificity suggesting that DNA methylation patterns are guided by the sequence preference of these enzymes. We speculate that aberrant methylation of DNMT3B target sites could contribute to the oncogenic potential of DNMT3A AML variant.
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the acute myeloid leukemia variant dnmt3a arg882his is a dnmt3b like enzyme
bioRxiv, 2019Co-Authors: Allison B Norvil, Taiping Chen, Lama Alabdi, Bigang Liu, Nicole E Forstoffer, Amie R Michie, Humaira GowherAbstract:Mutations in DNMT3A, particularly the Arg882His substitution is highly prevalent in acute myeloid leukemia. Although the reduced activity of DNMT3A Arg882His variant alters DNA methylation, the underlying cause of its oncogenic effect is not fully understood. Our data show that DNMT3A Arg882His variant acquires CpG flanking sequence preference highly similar to that of DNMT3B. Interestingly, a similar substrate preference was observed in DNMT3A WT enzyme upon the loss of cooperative kinetic mechanism. We tested if DNMT3A Arg882His could preferably methylate DNMT3B-specific target sites. Rescue experiments in Dnmt3a/3b double knockout mouse embryonic stem cells show that the corresponding Arg878His mutation in mouse DNMT3A severely impairs its ability to methylate major satellite DNA, a DNMT3A-preferred target, but has no overt effect on the ability to methylate minor satellite DNA, a DNMT3B-preferred target. Our data suggest that methylation of DNMT3B target sites by DNMT3A Arg882His variant could contribute to its oncogenic potential.
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dnmt3b methylates dna by a noncooperative mechanism and its activity is unaffected by manipulations at the predicted dimer interface
Biochemistry, 2016Co-Authors: Allison B Norvil, Lama Alabdi, Christopher J Petell, Sandra Rossie, Humaira GowherAbstract:The catalytic domains of the de novo DNA methyltransferases Dnmt3a-C and Dnmt3b-C are highly homologous. However, their unique biochemical properties could potentially contribute to differences in the substrate preferences or biological functions of these enzymes. Dnmt3a-C forms tetramers through interactions at the dimer interface, which also promote multimerization on DNA and cooperativity. Similar to the case for processive enzymes, cooperativity allows Dnmt3a-C to methylate multiple sites on the same DNA molecule; however, it is unclear whether Dnmt3b-C methylates DNA by a cooperative or processive mechanism. The importance of the tetramer structure and cooperative mechanism is emphasized by the observation that the R882H mutation in the dimer interface of DNMT3A is highly prevalent in acute myeloid leukemia and leads to a substantial loss of its activity. Under conditions that distinguish between cooperativity and processivity, we show that in contrast to that of Dnmt3a-C, the activity of Dnmt3b-C is...
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an epigenetic switch regulates de novo dna methylation at a subset of pluripotency gene enhancers during embryonic stem cell differentiation
Nucleic Acids Research, 2016Co-Authors: Christopher J Petell, Lama Alabdi, San Miguel P, R Rose, Humaira GowherAbstract:Coordinated regulation of gene expression that involves activation of lineage specific genes and repression of pluripotency genes drives differentiation of embryonic stem cells (ESC). For complete repression of pluripotency genes during ESC differentiation, chromatin at their enhancers is silenced by the activity of the Lsd1-Mi2/NuRD complex. The mechanism/s that regulate DNA methylation at these enhancers are largely unknown. Here, we investigated the affect of the Lsd1-Mi2/NuRD complex on the dynamic regulatory switch that induces the local interaction of histone tails with the Dnmt3 ATRX-DNMT3-DNMT3L (ADD) domain, thus promoting DNA methylation at the enhancers of a subset of pluripotency genes. This is supported by previous structural studies showing a specific interaction between Dnmt3-ADD domain with H3K4 unmethylated histone tails that is disrupted by histone H3K4 methylation and histone acetylation. Our data suggest that Dnmt3a activity is triggered by Lsd1-Mi2/NuRD-mediated histone deacetylation and demethylation at these pluripotency gene enhancers when they are inactivated during mouse ESC differentiation. Using Dnmt3 knockout ESCs and the inhibitors of Lsd1 and p300 histone modifying enzymes during differentiation of E14Tg2A and ZHBTc4 ESCs, our study systematically reveals this mechanism and establishes that Dnmt3a is both reader and effector of the epigenetic state at these target sites.
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mechanism of stimulation of catalytic activity of dnmt3a and dnmt3b dna cytosine c5 methyltransferases by DNMT3L
Journal of Biological Chemistry, 2005Co-Authors: Humaira Gowher, Kirsten Liebert, Andrea Hermann, Guoliang Xu, Albert JeltschAbstract:DNMT3L has been identified as a stimulator of the catalytic activity of de novo DNA methyltransferases. It is essential in the development of germ cells in mammals. We show here that DNMT3L stimulates the catalytic activity of the Dnmt3A and Dnmt3B enzymes by directly binding to their respective catalytic domains via its own C-terminal domain. The catalytic activity of Dnmt3A and -3B was stimulated approximately 15-fold, and DNMT3L directly binds to DNA but not to S-adenosyl-L-methionine (AdoMet). Complex formation between Dnmt3A and DNMT3L accelerates DNA binding by Dnmt3A 20-fold and lowers its K(m) for DNA. Interaction of DNMT3L with Dnmt3A increases the binding of the coenzyme AdoMet to Dnmt3A, and it lowers the K(m) of Dnmt3A for AdoMet. On the basis of our data we propose a model in which the interaction of Dnmt3A with DNMT3L induces a conformational change of Dnmt3A that opens the active site of the enzyme and promotes binding of DNA and the AdoMet. We demonstrate that the interaction of Dnmt3A and DNMT3L is transient, and after DNA binding to Dnmt3A, DNMT3L dissociates from the complex. Following dissociation of DNMT3L, Dnmt3A adopts a closed conformation leading to slow rates of DNA release. Therefore, DNMT3L acts as a substrate exchange factor that accelerates DNA and AdoMet binding to de novo DNA methyltransferases.
Frederic Chedin - One of the best experts on this subject based on the ideXlab platform.
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inactive dnmt3b splice variants modulate de novo dna methylation
PLOS ONE, 2013Co-Authors: Catherine A Gordon, Stella R Hartono, Frederic ChedinAbstract:Inactive DNA methyltransferase (DNMT) 3B splice isoforms are associated with changes in DNA methylation, yet the mechanisms by which they act remain largely unknown. Using biochemical and cell culture assays, we show here that the inactive DNMT3B3 and DNMT3B4 isoforms bind to and regulate the activity of catalytically competent DNMT3A or DNMT3B molecules. DNMT3B3 modestly stimulated the de novo methylation activity of DNMT3A and also counteracted the stimulatory effects of DNMT3L, therefore leading to subtle and contrasting effects on activity. DNMT3B4, by contrast, significantly inhibited de novo DNA methylation by active DNMT3 molecules, most likely due to its ability to reduce the DNA binding affinity of co-complexes, thereby sequestering them away from their substrate. Immunocytochemistry experiments revealed that in addition to their effects on the intrinsic catalytic function of active DNMT3 enzymes, DNMT3B3 and DNMT34 drive distinct types of chromatin compaction and patterns of histone 3 lysine 9 tri-methylation (H3K9me3) deposition. Our findings suggest that regulation of active DNMT3 members through the formation of co-complexes with inactive DNMT3 variants is a general mechanism by which DNMT3 variants function. This may account for some of the changes in DNA methylation patterns observed during development and disease.
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icf syndrome mutations cause a broad spectrum of biochemical defects in dnmt3b mediated de novo dna methylation
Journal of Molecular Biology, 2011Co-Authors: Amir H Moarefi, Frederic ChedinAbstract:The DNMT3B de novo DNA methyltransferase (DNMT) plays a major role in establishing DNA methylation patterns in early mammalian development, but its catalytic mechanism remains poorly characterized. Here, we provide a comprehensive biochemical analysis of human DNMT3B function through the characterization of a series of site-directed DNMT3B variants associated with immunodeficiency, centromere instability, and facial anomalies (ICF) syndrome. Our data reveal several novel and important aspects of DNMT3B function. First, DNMT3B, unlike DNMT3A, requires a DNA cofactor in order to stably bind to S-adenosyl-l-methionine (SAM), suggesting that it proceeds according to an ordered catalytic scheme. Second, ICF mutations cause a broad spectrum of biochemical defects in DNMT3B function, including defects in homo-oligomerization, SAM binding, SAM utilization, and DNA binding. Third, all tested ICF mutations, including the A766P and R840Q variants, result in altered catalytic properties without interfering with DNMT3L-mediated stimulation; this indicates that DNMT3L is not involved in the pathogenesis of ICF syndrome. Finally, our study reveals a novel level of coupling between substrate binding, oligomerization, and catalysis that is likely conserved within the DNMT3 family of enzymes.
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DNMT3L modulates significant and distinct flanking sequence preference for dna methylation by dnmt3a and dnmt3b in vivo
PLOS Genetics, 2010Co-Authors: Bethany L Wienholz, Catherine A Gordon, Amir H Moarefi, Michael S Kareta, Paul A Ginno, Frederic ChedinAbstract:The DNTM3A and DNMT3B de novo DNA methyltransferases (DNMTs) are responsible for setting genomic DNA methylation patterns, a key layer of epigenetic information. Here, using an in vivo episomal methylation assay and extensive bisulfite methylation sequencing, we show that human DNMT3A and DNMT3B possess significant and distinct flanking sequence preferences for target CpG sites. Selection for high or low efficiency sites is mediated by the base composition at the −2 and +2 positions flanking the CpG site for DNMT3A, and at the −1 and +1 positions for DNMT3B. This intrinsic preference reproducibly leads to the formation of specific de novo methylation patterns characterized by up to 34-fold variations in the efficiency of DNA methylation at individual sites. Furthermore, analysis of the distribution of signature methylation hotspot and coldspot motifs suggests that DNMT flanking sequence preference has contributed to shaping the composition of CpG islands in the human genome. Our results also show that the DNMT3L stimulatory factor modulates the formation of de novo methylation patterns in two ways. First, DNMT3L selectively focuses the DNA methylation machinery on properly chromatinized DNA templates. Second, DNMT3L attenuates the impact of the intrinsic DNMT flanking sequence preference by providing a much greater boost to the methylation of poorly methylated sites, thus promoting the formation of broader and more uniform methylation patterns. This study offers insights into the manner by which DNA methylation patterns are deposited and reveals a new level of interplay between members of the de novo DNMT family.
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physical and functional interactions between the human DNMT3L protein and members of the de novo methyltransferase family
Journal of Cellular Biochemistry, 2005Co-Authors: Zhao Xia Chen, Frederic Chedin, Jeffrey R Mann, Chihlin Hsieh, Arthur D RiggsAbstract:The de novo methyltransferase-like protein, DNMT3L, is required for methylation of imprinted genes in germ cells. Although enzymatically inactive, human DNMT3L was shown to act as a general stimulatory factor for de novo methylation by murine Dnmt3a. Several isoforms of DNMT3A and DNMT3B with development-stage and tissue-specific expression patterns have been described in mouse and human, thus bringing into question the identity of the physiological partner(s) for stimulation by DNMT3L. Here, we used an episome-based in vivo methyltransferase assay to systematically analyze five isoforms of human DNMT3A and DNMT3B for activity and stimulation by human DNMT3L. Our results show that human DNMT3A, DNMT3A2, DNMT3B1, and DNMT3B2 are catalytically competent, while DNMT3B3 is inactive in our assay. We also report that the activity of all four active isoforms is significantly increased upon co-expression with DNMT3L, albeit to varying extents. This is the first comprehensive description of the in vivo activities of the poorly characterized human DNMT3A and DNMT3B isoforms and of their functional interactions with DNMT3L. To further elucidate the mechanism by which DNMT3L stimulates DNA methylation, we have mapped in detail the domains that mediate interaction of human DNMT3L with human DNMT3A and DNMT3B. Our results show that the C-terminus of DNMT3L is the only region required for interaction with DNMT3A and DNMT3B and that interaction takes place through the C-terminal catalytic domain of DNMT3A and DNMT3B. The implications of these findings for the regulation of de novo methyltransferases and genomic imprinting are discussed. This article contains Supplementary Material available at http://www.mrw.interscience.wiley.com/suppmat/0730-2312/suppmat/2005/95/chen.html.
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the dna methyltransferase like protein DNMT3L stimulates de novo methylation by dnmt3a
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Frederic Chedin, Michael R Lieber, Chihlin HsiehAbstract:DNMT3L is required for the establishment of maternal methylation imprints at imprinting centers (ICs). DNMT3L, however, lacks the conserved catalytic domain common to DNA methyltransferases. In an attempt to define its function, we coexpressed DNMT3L with each of the two known de novo methyltransferases, Dnmt3a and DNMT3B, in human cells and monitored de novo methylation by using replicating minichromosomes carrying various ICs as targets. Coexpression of DNMT3L with DNMT3B led to little or no change in target methylation. However, coexpression of DNMT3L with Dnmt3a resulted in a striking stimulation of de novo methylation by Dnmt3a. Stimulation was observed at maternally methylated ICs such as small nuclear ribonucleoprotein polypeptide N (SNRPN), Snrpn, and Igf2r/Air, as well as at various nonimprinted sequences present on the episomes. Stimulation of Dnmt3a by DNMT3L was also observed at endogenous sequences in the genome. Therefore, DNMT3L acts as a general stimulatory factor for de novo methylation by Dnmt3a. The implications of these findings for the function of DNMT3L and Dnmt3a in DNA methylation and genomic imprinting are discussed.
Taiping Chen - One of the best experts on this subject based on the ideXlab platform.
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The acute myeloid leukemia variant DNMT3A Arg882His is a DNMT3B-like enzyme.
Nucleic acids research, 2020Co-Authors: Allison B Norvil, Taiping Chen, Bigang Liu, Nicole E Forstoffer, Amie R Michie, Alabdi, Humaira GowherAbstract:We have previously shown that the highly prevalent acute myeloid leukemia (AML) mutation, Arg882His, in DNMT3A disrupts its cooperative mechanism and leads to reduced enzymatic activity, thus explaining the genomic hypomethylation in AML cells. However, the underlying cause of the oncogenic effect of Arg882His in DNMT3A is not fully understood. Here, we discovered that DNMT3A WT enzyme under conditions that favor non-cooperative kinetic mechanism as well as DNMT3A Arg882His variant acquire CpG flanking sequence preference akin to that of DNMT3B, which is non-cooperative. We tested if DNMT3A Arg882His could preferably methylate DNMT3B-specific target sites in vivo. Rescue experiments in Dnmt3a/3b double knockout mouse embryonic stem cells show that the corresponding Arg878His mutation in mouse DNMT3A severely impairs its ability to methylate major satellite DNA, a DNMT3A-preferred target, but has no overt effect on the ability to methylate minor satellite DNA, a DNMT3B-preferred target. We also observed a previously unappreciated CpG flanking sequence bias in major and minor satellite repeats that is consistent with DNMT3A and DNMT3B specificity suggesting that DNA methylation patterns are guided by the sequence preference of these enzymes. We speculate that aberrant methylation of DNMT3B target sites could contribute to the oncogenic potential of DNMT3A AML variant.
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the acute myeloid leukemia variant dnmt3a arg882his is a dnmt3b like enzyme
bioRxiv, 2019Co-Authors: Allison B Norvil, Taiping Chen, Lama Alabdi, Bigang Liu, Nicole E Forstoffer, Amie R Michie, Humaira GowherAbstract:Mutations in DNMT3A, particularly the Arg882His substitution is highly prevalent in acute myeloid leukemia. Although the reduced activity of DNMT3A Arg882His variant alters DNA methylation, the underlying cause of its oncogenic effect is not fully understood. Our data show that DNMT3A Arg882His variant acquires CpG flanking sequence preference highly similar to that of DNMT3B. Interestingly, a similar substrate preference was observed in DNMT3A WT enzyme upon the loss of cooperative kinetic mechanism. We tested if DNMT3A Arg882His could preferably methylate DNMT3B-specific target sites. Rescue experiments in Dnmt3a/3b double knockout mouse embryonic stem cells show that the corresponding Arg878His mutation in mouse DNMT3A severely impairs its ability to methylate major satellite DNA, a DNMT3A-preferred target, but has no overt effect on the ability to methylate minor satellite DNA, a DNMT3B-preferred target. Our data suggest that methylation of DNMT3B target sites by DNMT3A Arg882His variant could contribute to its oncogenic potential.
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a dnmt3a mutation common in aml exhibits dominant negative effects in murine es cells
Blood, 2013Co-Authors: Soo Jin Kim, Swanand Hardikar, Taiping Chen, Margaret A Goodell, Hongbo Zhao, Anup Kumar SinghAbstract:Somatic heterozygous mutations of the DNA methyltransferase gene DNMT3A occur frequently in acute myeloid leukemia and other hematological malignancies, with the majority (∼60%) of mutations affecting a single amino acid, Arg882 (R882), in the catalytic domain. Although the mutations impair DNMT3A catalytic activity in vitro, their effects on DNA methylation in cells have not been explored. Here, we show that exogenously expressed mouse Dnmt3a proteins harboring the corresponding R878 mutations largely fail to mediate DNA methylation in murine embryonic stem (ES) cells but are capable of interacting with wild-type Dnmt3a and Dnmt3b. Coexpression of the Dnmt3a R878H (histidine) mutant protein results in inhibition of the ability of wild-type Dnmt3a and Dnmt3b to methylate DNA in murine ES cells. Furthermore, expression of Dnmt3a R878H in ES cells containing endogenous Dnmt3a or Dnmt3b induces hypomethylation. These results suggest that the DNMT3A R882 mutations, in addition to being hypomorphic, have dominant-negative effects.
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Synergistic function of DNA methyltransferases Dnmt3a and Dnmt3b in the methylation of Oct4 and Nanog
Molecular and cellular biology, 2007Co-Authors: Ryutaro Hirasawa, Taiping Chen, Yan-nv Huang, Rong Zeng, Naihe Jing, Hiroyuki SasakiAbstract:DNA methylation plays an important role in gene silencing in mammals. Two de novo methyltransferases, Dnmt3a and Dnmt3b, are required for the establishment of genomic methylation patterns in development. However, little is known about their coordinate function in the silencing of genes critical for embryonic development and how their activity is regulated. Here we show that Dnmt3a and Dnmt3b are the major components of a native complex purified from embryonic stem cells. The two enzymes directly interact and mutually stimulate each other both in vitro and in vivo. The stimulatory effect is independent of the catalytic activity of the enzyme. In differentiating embryonic carcinoma or embryonic stem cells and mouse postimplantation embryos, they function synergistically to methylate the promoters of the Oct4 and Nanog genes. Inadequate methylation caused by ablating Dnmt3a and Dnmt3b is associated with dysregulated expression of Oct4 and Nanog during the differentiation of pluripotent cells and mouse embryonic development. These results suggest that Dnmt3a and Dnmt3b form a complex through direct contact in living cells and cooperate in the methylation of the promoters of Oct4 and Nanog during cell differentiation. The physical and functional interaction between Dnmt3a and Dnmt3b represents a novel regulatory mechanism to ensure the proper establishment of genomic methylation patterns for gene silencing in development.
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Inactivation of Dnmt3b in mouse embryonic fibroblasts results in DNA hypomethylation, chromosomal instability, and spontaneous immortalization.
The Journal of biological chemistry, 2005Co-Authors: Jonathan E Dodge, Taiping Chen, Masaki Okano, Naomi Tsujimoto, Yoshihide Ueda, Frederick A. Dick, Shumei Wang, Nicholas J. DysonAbstract:Abstract DNA hypomethylation is a hallmark of many types of solid tumors. However, it remains elusive how DNA hypomethylation may contribute to tumorigenesis. In this study, we have investigated how targeted disruption of the DNA methyltransferases Dnmt3a and Dnmt3b affects the growth of mouse embryonic fibroblasts (MEFs). Our studies led to the following observations. 1) Constitutive or conditional deletion of Dnmt3b, but not Dnmt3a, resulted in partial loss of DNA methylation throughout the genome, suggesting that Dnmt3b, in addition to the major maintenance methyltransferase Dnmt1, is required for maintaining DNA methylation in MEF cells. 2) Dnmt3b-deficient MEF cells showed aneuploidy and polyploidy, chromosomal breaks, and fusions. 3) Inactivation of Dnmt3b resulted in either premature senescence or spontaneous immortalization of MEF cells. 4) The G1 to S-phase checkpoint was intact in primary and spontaneously immortalized Dnmt3b-deficient MEFs because the p53 protein was inducible by DNA damage. Interestingly, protein levels of the cyclindependent kinase inhibitor p21 were increased in immortalized Dnmt3b-deficient MEFs even in the absence of p53 induction. These results suggest that DNA hypomethylation may induce genomic instability, which in turn leads to spontaneous immortalization or premature senescence of Dnmt3b-deficient MEFs via a p53-independent mechanism.
Albert Jeltsch - One of the best experts on this subject based on the ideXlab platform.
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mutations of r882 change flanking sequence preferences of the dna methyltransferase dnmt3a and cellular methylation patterns
Nucleic Acids Research, 2019Co-Authors: Max Emperle, Philipp Rathert, Sabrina Adam, Stefan Kunert, Michael Dukatz, Annika Baude, Christoph Plass, Pavel Bashtrykov, Albert JeltschAbstract:Somatic DNMT3A mutations at R882 are frequently observed in AML patients including the very abundant R882H, but also R882C, R882P and R882S. Using deep enzymology, we show here that DNMT3A-R882H has more than 70-fold altered flanking sequence preferences when compared with wildtype DNMT3A. The R882H flanking sequence preferences mainly differ on the 3' side of the CpG site, where they resemble DNMT3B, while 5' flanking sequence preferences resemble wildtype DNMT3A, indicating that R882H behaves like a DNMT3A/DNMT3B chimera. Investigation of the activity and flanking sequence preferences of other mutations of R882 revealed that they cause similar effects. Bioinformatic analyses of genomic methylation patterns focusing on flanking sequence effects after expression of wildtype DNMT3A and R882H in human cells revealed that genomic methylation patterns reflect the details of the altered flanking sequence preferences of R882H. Concordantly, R882H specific hypermethylation in AML patients was strongly correlated with the R882H flanking sequence preferences. R882H specific DNA hypermethylation events in AML patients were accompanied by R882H specific mis-regulation of several genes with strong cancer connection, which are potential downstream targets of R882H. In conclusion, our data provide novel and detailed mechanistic understanding of the pathogenic mechanism of the DNMT3A R882H somatic cancer mutation.
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efficient targeted dna methylation with chimeric dcas9 dnmt3a DNMT3L methyltransferase
Nucleic Acids Research, 2017Co-Authors: Peter Stepper, Albert Jeltsch, Renata Z Jurkowska, Richard Reinhardt, Felix Krueger, Goran Kungulovski, Tamir Chandra, Wolf Reik, Tomasz P JurkowskiAbstract:DNA methylation plays a critical role in the regulation and maintenance of cell-type specific transcriptional programs. Targeted epigenome editing is an emerging technology to specifically regulate cellular gene expression in order to modulate cell phenotypes or dissect the epigenetic mechanisms involved in their control. In this work, we employed a DNA methyltransferase Dnmt3a-DNMT3L construct fused to the nuclease-inactivated dCas9 programmable targeting domain to introduce DNA methylation into the human genome specifically at the EpCAM, CXCR4 and TFRC gene promoters. We show that targeting of these loci with single gRNAs leads to efficient and widespread methylation of the promoters. Multiplexing of several guide RNAs does not increase the efficiency of methylation. Peaks of targeted methylation were observed around 25 bp upstream and 40 bp downstream of the PAM site, while 20-30 bp of the binding site itself are protected against methylation. Potent methylation is dependent on the multimerization of Dnmt3a/DNMT3L complexes on the DNA. Furthermore, the introduced methylation causes transcriptional repression of the targeted genes. These new programmable epigenetic editors allow unprecedented control of the DNA methylation status in cells and will lead to further advances in the understanding of epigenetic signaling.
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formation of nucleoprotein filaments by mammalian dna methyltransferase dnmt3a in complex with regulator DNMT3L
Nucleic Acids Research, 2008Co-Authors: Renata Z Jurkowska, Richard Reinhardt, Da Jia, Xiaodong Cheng, Nils Anspach, Claus Urbanke, Wolfgang Nellen, Albert JeltschAbstract:The C-terminal domains of Dnmt3a and DNMT3L form elongated heterotetramers (3L-3a-3a-3L). Analytical ultracentrifugation confirmed the Dnmt3a-C/3L-C complex exists as a 2:2 heterotetramer in solution. The 3a-3a interface is the DNA-binding site, while both interfaces are essential for AdoMet binding and catalytic activity. Hairpin bisulfite analysis shows correlated methylation of two CG sites in a distance of approximately 8-10 bp in the opposite DNA strands, which corresponds to the geometry of the two active sites in one Dnmt3a-C/3L-C tetramer. Correlated methylation was also observed for two CG sites at similar distances in the same DNA strand, which can be attributed to the binding of two tetramers next to each other. DNA-binding experiments show that Dnmt3a-C/3L-C complexes multimerize on the DNA. Scanning force microscopy demonstrates filament formation rather than binding of single tetramers and shows that protein-DNA filament formation leads to a 1.5-fold shortening of the DNA length.
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structure of dnmt3a bound to DNMT3L suggests a model for de novo dna methylation
Nature, 2007Co-Authors: Da Jia, Albert Jeltsch, Renata Z Jurkowska, Xing Zhang, Xiaodong ChengAbstract:Genetic imprinting, found in flowering plants and placental mammals, uses DNA methylation to yield gene expression that is dependent on the parent of origin1. DNA methyltransferase 3a (Dnmt3a) and its regulatory factor, DNA methyltransferase 3-like protein (DNMT3L), are both required for the de novo DNA methylation of imprinted genes in mammalian germ cells. DNMT3L interacts specifically with unmethylated lysine 4 of histone H3 through its amino-terminal PHD (plant homeodomain)-like domain2. Here we show, with the use of crystallography, that the carboxy-terminal domain of human DNMT3L interacts with the catalytic domain of Dnmt3a, demonstrating that DNMT3L has dual functions of binding the unmethylated histone tail and activating DNA methyltransferase. The complexed C-terminal domains of Dnmt3a and DNMT3L showed further dimerization through Dnmt3a–Dnmt3a interaction, forming a tetrameric complex with two active sites. Substitution of key non-catalytic residues at the Dnmt3a–DNMT3L interface or the Dnmt3a–Dnmt3a interface eliminated enzymatic activity. Molecular modelling of a DNA–Dnmt3a dimer indicated that the two active sites are separated by about one DNA helical turn. The C-terminal domain of Dnmt3a oligomerizes on DNA to form a nucleoprotein filament. A periodicity in the activity of Dnmt3a on long DNA revealed a correlation of methylated CpG sites at distances of eight to ten base pairs, indicating that oligomerization leads Dnmt3a to methylate DNA in a periodic pattern. A similar periodicity is observed for the frequency of CpG sites in the differentially methylated regions of 12 maternally imprinted mouse genes. These results suggest a basis for the recognition and methylation of differentially methylated regions in imprinted genes, involving the detection of both nucleosome modification and CpG spacing.
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structure of dnmt3a bound to DNMT3L suggests a model for de novo dna methylation
Nature, 2007Co-Authors: Renata Z Jurkowska, Albert Jeltsch, Xing Zhang, Xiaodong ChengAbstract:A crystal structure of a complex between the DNA methyltransferase regulatory factor DNMT3L and the catalytic domain of Dnmt3a leads to a model being proposed for the preferential methylation of DNA on maternally imprinted genes.
Masaki Okano - One of the best experts on this subject based on the ideXlab platform.
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genetic evidence for dnmt3a dependent imprinting during oocyte growth obtained by conditional knockout with zp3 cre and complete exclusion of dnmt3b by chimera formation
Genes to Cells, 2010Co-Authors: Masahiro Kaneda, Masaki Okano, Ryutaro Hirasawa, Hatsune Chiba, Hiroyuki SasakiAbstract:In the male and female germ-lines of mice, both of the two de novo DNA methyltransferases Dnmt3a and Dnmt3b are expressed. By the conditional knockout experiments using the Tnap-Cre gene, we previously showed that deletion of Dnmt3a in primordial germ cells disrupts paternal and maternal imprinting, however, Dnmt3b mutants did not show any defect. Here, we have knocked out Dnmt3a after birth in growing oocytes by using the Zp3-Cre gene and obtained genetic evidence that de novo methylation by Dnmt3a during the oocyte growth stage is indispensable for maternal imprinting. We also carried out DNA methylation analysis in the mutant oocytes and embryos and found that hypomethylation of imprinted genes in Dnmt3a-deficient oocytes was directly inherited to the embryos, but repetitive elements were re-methylated during development. Furthermore, we show that Dnmt3b-deficient cells can contribute to the male and female germ-lines in chimeric mice and can produce normal progeny, establishing that Dnmt3b is dispensable for mouse gametogenesis and imprinting. Finally, Dnmt3-related protein DNMT3L is not only essential for methylation of imprinted genes but also enhances de novo methylation of repetitive elements in growing oocytes.
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role of the dnmt3 family in de novo methylation of imprinted and repetitive sequences during male germ cell development in the mouse
Human Molecular Genetics, 2007Co-Authors: Yuzuru Kato, Masahiro Kaneda, Kenichiro Hata, Kenji Kumaki, Mizue Hisano, Yuji Kohara, Masaki OkanoAbstract:DNA methylation is an important epigenetic modification regulating various biological phenomena, including genomic imprinting and transposon silencing. It is known that methylation of the differentially methylated regions (DMRs) associated with paternally imprinted genes and of some repetitive elements occurs during male germ cell development in the mouse. We have performed a detailed methylation analysis of the paternally methylated DMRs (H19, Dlk1/Gtl2 and Rasgrf1), interspersed repeats [SineB1, intracisternal A particle (IAP) and Line1] and satellite repeats (major and minor) to determine the timing of this de novo methylation in male germ cells. Furthermore, we have examined the roles of the de novo methyltransferases (Dnmt3a and Dnmt3b) and related protein (DNMT3L) in this process. We found that methylation of all DMRs and repeats occurred progressively in fetal prospermatogonia and was completed by the newborn stage. Analysis of newborn prospermatogonia from germline-specific Dnmt3a and Dnmt3b knockout mice revealed that Dnmt3a mainly methylates the H19 and Dlk1/Gtl2 DMRs and a short interspersed repeat SineB1. Both Dnmt3a and Dnmt3b were involved in the methylation of Rasgrf1 DMR and long interspersed repeats IAP and Line1. Only Dnmt3b was required for the methylation of the satellite repeats. These results indicate both common and differential target specificities of Dnmt3a and Dnmt3b in vivo. Finally, all these sequences showed moderate to severe hypomethylation in DNMT3L-deficient prospermatogonia, indicating the critical function and broad specificity of this factor in de novo methylation.
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de novo dna methyltransferase is essential for self renewal but not for differentiation in hematopoietic stem cells
Journal of Experimental Medicine, 2007Co-Authors: Yuko Tadokoro, Masaki Okano, Hideo Ema, Hiromitsu NakauchiAbstract:DNA methylation is an epigenetic modification essential for development. The DNA methyltransferases Dnmt3a and Dnmt3b execute de novo DNA methylation in gastrulating embryos and differentiating germline cells. It has been assumed that these enzymes generally play a role in regulating cell differentiation. To test this hypothesis, we examined the role of Dnmt3a and Dnmt3b in adult stem cells. CD34(-/low), c-Kit(+), Sca-1(+), lineage marker(-) (CD34(-) KSL) cells, a fraction of mouse bone marrow cells highly enriched in hematopoietic stem cells (HSCs), expressed both Dnmt3a and Dnmt3b. Using retroviral Cre gene transduction, we conditionally disrupted Dnmt3a, Dnmt3b, or both Dnmt3a and Dnmt3b (Dnmt3a/Dnmt3b) in CD34(-) KSL cells purified from mice in which the functional domains of these genes are flanked by two loxP sites. We found that Dnmt3a and Dnmt3b function as de novo DNA methyltransferases during differentiation of hematopoietic cells. Unexpectedly, in vitro colony assays and in vivo transplantation assays showed that both myeloid and lymphoid lineage differentiation potentials were maintained in Dnmt3a-, Dnmt3b-, and Dnmt3a/Dnmt3b-deficient HSCs. However, Dnmt3a/Dnmt3b-deficient HSCs, but not Dnmt3a- or Dnmt3b-deficient HSCs, were incapable of long-term reconstitution in transplantation assays. These findings establish a critical role for DNA methylation by Dnmt3a and Dnmt3b in HSC self-renewal.
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Inactivation of Dnmt3b in mouse embryonic fibroblasts results in DNA hypomethylation, chromosomal instability, and spontaneous immortalization.
The Journal of biological chemistry, 2005Co-Authors: Jonathan E Dodge, Taiping Chen, Masaki Okano, Naomi Tsujimoto, Yoshihide Ueda, Frederick A. Dick, Shumei Wang, Nicholas J. DysonAbstract:Abstract DNA hypomethylation is a hallmark of many types of solid tumors. However, it remains elusive how DNA hypomethylation may contribute to tumorigenesis. In this study, we have investigated how targeted disruption of the DNA methyltransferases Dnmt3a and Dnmt3b affects the growth of mouse embryonic fibroblasts (MEFs). Our studies led to the following observations. 1) Constitutive or conditional deletion of Dnmt3b, but not Dnmt3a, resulted in partial loss of DNA methylation throughout the genome, suggesting that Dnmt3b, in addition to the major maintenance methyltransferase Dnmt1, is required for maintaining DNA methylation in MEF cells. 2) Dnmt3b-deficient MEF cells showed aneuploidy and polyploidy, chromosomal breaks, and fusions. 3) Inactivation of Dnmt3b resulted in either premature senescence or spontaneous immortalization of MEF cells. 4) The G1 to S-phase checkpoint was intact in primary and spontaneously immortalized Dnmt3b-deficient MEFs because the p53 protein was inducible by DNA damage. Interestingly, protein levels of the cyclindependent kinase inhibitor p21 were increased in immortalized Dnmt3b-deficient MEFs even in the absence of p53 induction. These results suggest that DNA hypomethylation may induce genomic instability, which in turn leads to spontaneous immortalization or premature senescence of Dnmt3b-deficient MEFs via a p53-independent mechanism.
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essential role for de novo dna methyltransferase dnmt3a in paternal and maternal imprinting
Nature, 2004Co-Authors: Masahiro Kaneda, Masaki Okano, Kenichiro Hata, Takashi Sado, Naomi TsujimotoAbstract:Imprinted genes are epigenetically marked during gametogenesis so that they are exclusively expressed from either the paternal or the maternal allele in offspring1. Imprinting prevents parthenogenesis in mammals and is often disrupted in congenital malformation syndromes, tumours and cloned animals1. Although de novo DNA methyltransferases of the Dnmt3 family are implicated in maternal imprinting2, the lethality of Dnmt3a and Dnmt3b knockout mice3 has precluded further studies. We here report the disruption of Dnmt3a and Dnmt3b in germ cells, with their preservation in somatic cells, by conditional knockout technology4. Offspring from Dnmt3a conditional mutant females die in utero and lack methylation and allele-specific expression at all maternally imprinted loci examined. Dnmt3a conditional mutant males show impaired spermatogenesis and lack methylation at two of three paternally imprinted loci examined in spermatogonia. By contrast, Dnmt3b conditional mutants and their offspring show no apparent phenotype. The phenotype of Dnmt3a conditional mutants is indistinguishable from that of DNMT3L knockout mice2,5, except for the discrepancy in methylation at one locus. These results indicate that both Dnmt3a and DNMT3L are required for methylation of most imprinted loci in germ cells, but also suggest the involvement of other factors.