Publications by authors named "Humaira Gowher"

34 Publications

Simplified MethylRAD Sequencing to Detect Changes in DNA Methylation at Enhancer Elements in Differentiating Embryonic Stem Cells.

Epigenomes 2020 Dec 1;4(4). Epub 2020 Oct 1.

Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA.

Differential DNA methylation is characteristic of gene regulatory regions, such as enhancers, which mostly constitute low or intermediate CpG content in their DNA sequence. Consequently, quantification of changes in DNA methylation at these sites is challenging. Given that DNA methylation across most of the mammalian genome is maintained, the use of genome-wide bisulfite sequencing to measure fractional changes in DNA methylation at specific sites is an overexertion which is both expensive and cumbersome. Here, we developed a MethylRAD technique with an improved experimental plan and bioinformatic analysis tool to examine regional DNA methylation changes in embryonic stem cells (ESCs) during differentiation. The transcriptional silencing of pluripotency genes (PpGs) during ESC differentiation is accompanied by PpG enhancer (PpGe) silencing mediated by the demethylation of H3K4me1 by LSD1. Our MethylRAD data show that in the presence of LSD1 inhibitor, a significant fraction of LSD1-bound PpGe fails to gain DNA methylation. We further show that this effect is mostly observed in PpGes with low/intermediate CpG content. Underscoring the sensitivity and accuracy of MethylRAD sequencing, our study demonstrates that this method can detect small changes in DNA methylation in regulatory regions, including those with low/intermediate CpG content, thus asserting its use as a method of choice for diagnostic purposes.
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http://dx.doi.org/10.3390/epigenomes4040024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8023688PMC
December 2020

Integrative genomic analysis of pediatric T- cell lymphoblastic lymphoma reveals candidates of clinical significance.

Blood 2020 Nov 5. Epub 2020 Nov 5.

University Hospital Münster, Münster, Germany.

T-cell lymphoblastic lymphoma (T-LBL) is a heterogeneous malignancy of lymphoblasts committed to T-cell lineage. Dismal outcomes (15-30%) in case of T-LBL relapses warrants for establishing risk-based treatment in future. This is a first comprehensive, systematic, integrated genome-wide analysis including relapse cases aimed towards identifying molecular markers of prognostic relevance for T-LBL. NOTCH1 was identified as putative driver for T-LBL. Activated NOTCH/PI3K-AKT signaling axis and alterations in cell cycle regulators constitutes the core oncogenic program for T-LBL. Mutated KMT2D was identified as a prognostic marker. The cumulative incidence of relapse was 47±17% in patients with KMT2D mutations compared with 14±3% in KMT2D wildtype. Structural analysis of the mutated domains of KMT2D revealed plausible impact on the structure and functional consequences. These findings provide new insights into the pathogenesis of T-LBL including high translational potential. The ongoing trial LBL 2018 (NCT04043494) allows prospective validation and subsequent fine-tuning of the stratification criteria for T-LBL risk groups to improve survival of the pediatric patients.
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http://dx.doi.org/10.1182/blood.2020005381DOI Listing
November 2020

Development of Biphenylthiazoles Exhibiting Improved Pharmacokinetics and Potent Activity Against Intracellular .

ACS Infect Dis 2020 11 8;6(11):2887-2900. Epub 2020 Oct 8.

Department of Pharmaceutical Organic Chemistry, College of Pharmacy, Al-Azhar University, 1-Elmokhayem Eldaem Street, Cairo 11884, Egypt.

Exploring the structure-activity relationship (SAR) at the cationic part of arylthiazole antibiotics revealed hydrazine as an active moiety. The main objective of the study is to overcome the inherited toxicity associated with the free hydrazine. A series of hydrocarbon bridges was inserted in between the groups, to separate the two amino groups. Hence, the aminomethylpiperidine-containing analog was identified as a new promising antibacterial agent with efficient antibacterial and pharmacokinetic profiles. Briefly, compound outperformed vancomycin in terms of the antibacterial spectrum against vancomycin-resistant staphylococcal and enterococcal strains with minimum inhibitory concentrations (MICs) ranging from 2 to 4 μg/mL, which is a faster bactericidal mode of action, completely eradicating the high staphylococcal burden within 6-8 h, and it has a unique ability to completely clear intracellular staphylococci. In addition, the initial pharmacokinetic assessment confirmed the high metabolic stability of compound (biological half-life >4 h); it had a good extravascular distribution and maintained a plasma concentration higher than the average MIC value for over 12 h. Moreover, compound significantly reduced MRSA burden in an MRSA skin infection mouse experiment. These attributes collectively suggest that compound is a good therapeutic candidate for invasive staphylococcal and enterococcal infections. From a mechanistic point of view, compound inhibited undecaprenyl diphosphate phosphatase (UppP) with an IC value of 29 μM.
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http://dx.doi.org/10.1021/acsinfecdis.0c00137DOI Listing
November 2020

The acute myeloid leukemia variant DNMT3A Arg882His is a DNMT3B-like enzyme.

Nucleic Acids Res 2020 04;48(7):3761-3775

Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA.

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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http://dx.doi.org/10.1093/nar/gkaa139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144950PMC
April 2020

Oct4-Mediated Inhibition of Lsd1 Activity Promotes the Active and Primed State of Pluripotency Enhancers.

Cell Rep 2020 02;30(5):1478-1490.e6

Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA; Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA. Electronic address:

An aberrant increase in pluripotency gene (PpG) expression due to enhancer reactivation could induce stemness and enhance the tumorigenicity of cancer stem cells. Silencing of PpG enhancers (PpGe) during embryonic stem cell differentiation involves Lsd1-mediated H3K4me1 demethylation and DNA methylation. Here, we observed retention of H3K4me1 and DNA hypomethylation at PpGe associated with a partial repression of PpGs in F9 embryonal carcinoma cells (ECCs) post-differentiation. H3K4me1 demethylation in F9 ECCs could not be rescued by Lsd1 overexpression. Given our observation that H3K4me1 demethylation is accompanied by strong Oct4 repression in P19 ECCs, we tested if Oct4 interaction with Lsd1 affects its catalytic activity. Our data show a dose-dependent inhibition of Lsd1 activity by Oct4 and retention of H3K4me1 at PpGe in Oct4-overexpressing P19 ECCs. These data suggest that Lsd1-Oct4 interaction in cancer stem cells could establish a "primed" enhancer state that is susceptible to reactivation, leading to aberrant PpG expression.
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http://dx.doi.org/10.1016/j.celrep.2019.11.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316367PMC
February 2020

From Phenylthiazoles to Phenylpyrazoles: Broadening the Antibacterial Spectrum toward Carbapenem-Resistant Bacteria.

J Med Chem 2019 09 19;62(17):7998-8010. Epub 2019 Aug 19.

Department of Pharmaceutical Organic Chemistry, College of Pharmacy , Al-Azhar University , Cairo 11884 , Egypt.

The narrow antibacterial spectrum of phenylthiazole antibiotics was expanded by replacing central thiazole with a pyrazole ring while maintaining its other pharmacophoric features. The most promising derivative, compound , was more potent than vancomycin against multidrug-resistant Gram-positive clinical isolates, including vancomycin- and linezolid-resistant methicillin-resistant MRSA), with a minimum inhibitory concentration (MIC) value as low as 0.5 μg/mL. Moreover, compound was superior to imipenem and meropenem against highly pathogenic carbapenem-resistant strains, such as , and . In addition to the notable biofilm inhibition activity, compound outperformed both vancomycin and kanamycin in reducing the intracellular burden of both Gram-positive and Gram-negative pathogenic bacteria. Compound cleared 90% of intracellular MRSA and 98% of at 2× the MIC. Moreover, preliminary pharmacokinetic investigations indicated that this class of novel antibacterial compounds is highly metabolically stable with a biological half-life of 10.5 h, suggesting a once-daily dosing regimen.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00720DOI Listing
September 2019

Editorial-Role of DNA Methyltransferases in the Epigenome.

Genes (Basel) 2019 07 30;10(8). Epub 2019 Jul 30.

Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA.

DNA methylation, a modification found in most species, regulates chromatin functions in conjunction with other epigenome modifications, such as histone post-translational modifications and non-coding RNAs. In mammals, DNA methylation has essential roles in development by orchestrating the generation and maintenance of the phenotypic diversity of human cell types. This Special Issue of Genes contains eight review articles, which cover several aspects of epigenome regulation by DNA methyltransferases (DNMTs), the enzymes responsible for the introduction of DNA methylation. The manuscripts present the most recent advances regarding the structure and function of DNMTs, their targeting and regulation by interacting factors and chromatin modifications, and the roles of DNMTs in mammalian development and human diseases. However, many aspects of these important enzymes are still insufficiently understood. Potential directions of future work are the regulation of DNMTs by post-translational modifications and their connection to cellular signaling and second messenger cascades on one hand and to large multifactorial epigenetic chromatin circuits on the other. Additionally, technical advancements, including the availability of designer nucleosomes and the rapid development of cryo-electron microscopy are expected to trigger breakthrough discoveries in this exciting field.
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http://dx.doi.org/10.3390/genes10080574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6723798PMC
July 2019

Effect of Disease-Associated Germline Mutations on Structure Function Relationship of DNA Methyltransferases.

Genes (Basel) 2019 05 14;10(5). Epub 2019 May 14.

Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA.

Despite a large body of evidence supporting the role of aberrant DNA methylation in etiology of several human diseases, the fundamental mechanisms that regulate the activity of mammalian DNA methyltransferases (DNMTs) are not fully understood. Recent advances in whole genome association studies have helped identify mutations and genetic alterations of DNMTs in various diseases that have a potential to affect the biological function and activity of these enzymes. Several of these mutations are germline-transmitted and associated with a number of hereditary disorders, which are potentially caused by aberrant DNA methylation patterns in the regulatory compartments of the genome. These hereditary disorders usually cause neurological dysfunction, growth defects, and inherited cancers. Biochemical and biological characterization of DNMT variants can reveal the molecular mechanism of these enzymes and give insights on their specific functions. In this review, we introduce roles and regulation of DNA methylation and DNMTs. We discuss DNMT mutations that are associated with rare diseases, the characterized effects of these mutations on enzyme activity and provide insights on their potential effects based on the known crystal structure of these proteins.
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http://dx.doi.org/10.3390/genes10050369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562416PMC
May 2019

Lipophilic efficient phenylthiazoles with potent undecaprenyl pyrophosphatase inhibitory activity.

Eur J Med Chem 2019 Aug 27;175:49-62. Epub 2019 Apr 27.

Department of Pharmaceutical Organic Chemistry, College of Pharmacy, Al-Azhar University, Cairo, 11884, Egypt; University of Science and Technology, Nanoscience Program, Zewail City of Science and Technology, October Gardens, 6th of October, Giza, 12578, Egypt. Electronic address:

Antibiotic resistance remains a pressing medical challenge for which novel antibacterial agents are urgently needed. The phenylthiazole scaffold represents a promising platform to develop novel antibacterial agents for drug-resistant infections. However, enhancing the physicochemical profile of this class of compounds remains a challenging endeavor to address to successfully translate these molecules into novel antibacterial agents in the clinic. We extended our understanding of the SAR of the phenylthiazoles' lipophilic moiety by exploring its ability to accommodate a hydrophilic group or a smaller sized hetero-ring with the objective of enhancing the physicochemical properties of this class of novel antimicrobials. Overall, the 2-thienyl derivative 20 and the hydroxyl-containing derivative 31 emerged as the most promising antibacterial agents inhibiting growth of drug-resistant Staphylococcus aureus at a concentration as low as 1 μg/mL. Remarkably, compound 20 suppressed bacterial undecaprenyl pyrophosphatase (UppP), the molecular target of the phenylthiazole compounds, in a sub nano-molar concentration range (almost 20,000 times more potent than the lead compounds 1a and 1b). Compound 31 possessed the most balanced antibacterial and physicochemical profile. The compound exhibited rapid bactericidal activity against S. aureus, and successfully cleared intracellular S. aureus within infected macrophages. Furthermore, insertion of the hydroxyl group enhanced the aqueous solubility of 31 by more than 50-fold relative to the first-generation lead 1c.
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http://dx.doi.org/10.1016/j.ejmech.2019.04.063DOI Listing
August 2019

DNMT3L facilitates DNA methylation partly by maintaining DNMT3A stability in mouse embryonic stem cells.

Nucleic Acids Res 2019 01;47(1):152-167

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX 78957, USA.

DNMT3L (DNMT3-like), a member of the DNMT3 family, has no DNA methyltransferase activity but regulates de novo DNA methylation. While biochemical studies show that DNMT3L is capable of interacting with both DNMT3A and DNMT3B and stimulating their enzymatic activities, genetic evidence suggests that DNMT3L is essential for DNMT3A-mediated de novo methylation in germ cells but is dispensable for de novo methylation during embryogenesis, which is mainly mediated by DNMT3B. How DNMT3L regulates DNA methylation and what determines its functional specificity are not well understood. Here we show that DNMT3L-deficient mouse embryonic stem cells (mESCs) exhibit downregulation of DNMT3A, especially DNMT3A2, the predominant DNMT3A isoform in mESCs. DNA methylation analysis of DNMT3L-deficient mESCs reveals hypomethylation at many DNMT3A target regions. These results confirm that DNMT3L is a positive regulator of DNA methylation, contrary to a previous report that, in mESCs, DNMT3L regulates DNA methylation positively or negatively, depending on genomic regions. Mechanistically, DNMT3L forms a complex with DNMT3A2 and prevents DNMT3A2 from being degraded. Restoring the DNMT3A protein level in DNMT3L-deficient mESCs partially recovers DNA methylation. Thus, our work uncovers a role for DNMT3L in maintaining DNMT3A stability, which contributes to the effect of DNMT3L on DNMT3A-dependent DNA methylation.
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http://dx.doi.org/10.1093/nar/gky947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326784PMC
January 2019

Mammalian DNA methyltransferases: new discoveries and open questions.

Biochem Soc Trans 2018 10 28;46(5):1191-1202. Epub 2018 Aug 28.

Institute of Biochemistry and Technical Biochemistry, Department of Biochemistry, University of Stuttgart, Stuttgart, Germany

As part of the epigenetic network, DNA methylation is a major regulator of chromatin structure and function. In mammals, it mainly occurs at palindromic CpG sites, but asymmetric methylation at non-CpG sites is also observed. Three enzymes are involved in the generation and maintenance of DNA methylation patterns. DNMT1 has high preference for hemimethylated CpG sites, and DNMT3A and DNMT3B equally methylate unmethylated and hemimethylated DNA, and also introduce non-CpG methylation. Here, we review recent observations and novel insights into the structure and function of mammalian DNMTs (DNA methyltransferases), including new structures of DNMT1 and DNMT3A, data on their mechanism, regulation by post-translational modifications and on the function of DNMTs in cells. In addition, we present news findings regarding the allosteric regulation and targeting of DNMTs by chromatin modifications and chromatin proteins. In combination, the recent publications summarized here impressively illustrate the intensity of ongoing research in this field. They provide a deeper understanding of key mechanistic properties of DNMTs, but they also document still unsolved issues, which need to be addressed in future research.
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http://dx.doi.org/10.1042/BST20170574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581191PMC
October 2018

Characterization of Small Molecules Inhibiting the Pro-Angiogenic Activity of the Zinc Finger Transcription Factor Vezf1.

Molecules 2018 07 3;23(7). Epub 2018 Jul 3.

Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA.

Discovery of inhibitors for endothelial-related transcription factors can contribute to the development of anti-angiogenic therapies that treat various diseases, including cancer. The role of transcription factor Vezf1 in vascular development and regulation of angiogenesis has been defined by several earlier studies. Through construction of a computational model for Vezf1, work here has identified a novel small molecule drug capable of inhibiting Vezf1 from binding to its cognate DNA binding site. Using structure-based design and virtual screening of the NCI Diversity Compound Library, 12 shortlisted compounds were tested for their ability to interfere with the binding of Vezf1 to DNA using electrophoretic gel mobility shift assays. We identified one compound, T4, which has an IC50 of 20 μM. Using murine endothelial cells, MSS31, we tested the effect of T4 on endothelial cell viability and angiogenesis by using tube formation assay. Our data show that addition of T4 in cell culture medium does not affect cell viability at concentrations lower or equal to its IC 50 but strongly inhibits the network formation by MSS31 in the tube formation assays. Given its potential efficacy, this inhibitor has significant therapeutic potential in several human diseases.
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http://dx.doi.org/10.3390/molecules23071615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6100598PMC
July 2018

The transcription factor Vezf1 represses the expression of the antiangiogenic factor Cited2 in endothelial cells.

J Biol Chem 2018 07 24;293(28):11109-11118. Epub 2018 May 24.

From the Department of Biochemistry and

Formation of the vasculature by angiogenesis is critical for proper development, but angiogenesis also contributes to the pathogenesis of various disorders, including cancer and cardiovascular diseases. Vascular endothelial zinc finger 1 (Vezf1), is a Krüppel-like zinc finger protein that plays a vital role in vascular development. However, the mechanism by which Vezf1 regulates this process is not fully understood. Here, we show that mouse embryonic stem cells (ESC) have significantly increased expression of a stem cell factor, Cbp/p300-interacting transactivator 2 (Cited2). Compared with WT ESCs, ESCs inefficiently differentiated into endothelial cells (ECs), which exhibited defects in the tube-formation assay. These defects were due to reduced activation of EC-specific genes concomitant with lower enrichment of histone 3 acetylation at Lys (H3K27) at their promoters. We hypothesized that overexpression of Cited2 in cells sequesters P300/CBP away from the promoters of proangiogenic genes and thereby contributes to defective angiogenesis in these cells. This idea was supported by the observation that shRNA-mediated depletion of Cited2 significantly reduces the angiogenic defects in the ECs. In contrast to previous studies that have focused on the role of Vezf1 as a transcriptional activator of proangiogenic genes, our findings have revealed a role for Vezf1 in modulating the expression of the antiangiogenic factor Cited2. Vezf1 previously has been characterized as an insulator protein, and our results now provide insights into the mechanism, indicating that Vezf1 can block inappropriate, nonspecific interactions of promoters with -located enhancers, preventing aberrant promoter activation.
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http://dx.doi.org/10.1074/jbc.RA118.002911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6052231PMC
July 2018

A refined DNA methylation detection method using MspJI coupled quantitative PCR.

Anal Biochem 2017 09 15;533:1-9. Epub 2017 Jun 15.

Department of Biochemistry, Purdue University, West Lafayette, IN 47907, United States; Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, United States. Electronic address:

DNA methylation is a highly conserved epigenetic modification with critical roles ranging from protection against phage infection in bacteria to the regulation of gene expression in mammals. DNA methylation at specific sequences can be measured by using methylation dependent or sensitive restriction enzymes coupled to semi- or quantitative PCR (MD-qPCR). This study reports a refined MD-qPCR method for detecting gain or loss of DNA methylation at specific sites through the specific use of MspJI or HpaII, respectively. By employing varying concentrations of DNA with methylation ranging from 0 to 100%, our data provide evidence that compared to HpaII, MspJI increases the sensitivity and accuracy of detecting relative DNA methylation gains by MD-qPCR. We also show that the MspJI-coupled MD-qPCR can accurately determine the percent gain in DNA methylation at the Sall4 enhancer and is more sensitive than HpaII in detecting relative gains in DNA methylation at the Oct4 proximal enhancer during embryonic stem cell (ESC) differentiation. The high specificity and sensitivity of this targeted approach increases its potential as a diagnostic tool to detect relatively smaller gains in DNA methylation at specific sites from limited amounts of sample.
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http://dx.doi.org/10.1016/j.ab.2017.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5972016PMC
September 2017

Dnmt3b Methylates DNA by a Noncooperative Mechanism, and Its Activity Is Unaffected by Manipulations at the Predicted Dimer Interface.

Biochemistry 2018 07 4;57(29):4312-4324. Epub 2016 Nov 4.

Department of Biochemistry, Purdue University Center for Cancer Research , Purdue University , West Lafayette , Indiana 47907 , United States.

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 not cooperative and confirm the processivity of Dnmt3b-C and the full length Dnmt3b enzyme. Whereas the R878H mutation (mouse homologue of R882H) led to the loss of cooperativity of Dnmt3a-C, the activity and processivity of the analogous Dnmt3b-C R829H variant were comparable to those of the wild-type enzyme. Additionally, buffer acidification that attenuates the dimer interface interactions of Dnmt3a-C had no effect on Dnmt3b-C activity. Taken together, these results demonstrate an important mechanistic difference between Dnmt3b and Dnmt3a and suggest that interactions at the dimer interface may play a limited role in regulating Dnmt3b-C activity. These new insights have potential implications for the distinct biological roles of Dnmt3a and Dnmt3b.
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http://dx.doi.org/10.1021/acs.biochem.6b00964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992102PMC
July 2018

Extra-coding RNAs regulate neuronal DNA methylation dynamics.

Nat Commun 2016 07 7;7:12091. Epub 2016 Jul 7.

Department of Neurobiology, McKnight Brain Institute, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

Epigenetic mechanisms such as DNA methylation are essential regulators of the function and information storage capacity of neurons. DNA methylation is highly dynamic in the developing and adult brain, and is actively regulated by neuronal activity and behavioural experiences. However, it is presently unclear how methylation status at individual genes is targeted for modification. Here, we report that extra-coding RNAs (ecRNAs) interact with DNA methyltransferases and regulate neuronal DNA methylation. Expression of ecRNA species is associated with gene promoter hypomethylation, is altered by neuronal activity, and is overrepresented at genes involved in neuronal function. Knockdown of the Fos ecRNA locus results in gene hypermethylation and mRNA silencing, and hippocampal expression of Fos ecRNA is required for long-term fear memory formation in rats. These results suggest that ecRNAs are fundamental regulators of DNA methylation patterns in neuronal systems, and reveal a promising avenue for therapeutic targeting in neuropsychiatric disease states.
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http://dx.doi.org/10.1038/ncomms12091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4941050PMC
July 2016

An epigenetic switch regulates de novo DNA methylation at a subset of pluripotency gene enhancers during embryonic stem cell differentiation.

Nucleic Acids Res 2016 09 13;44(16):7605-17. Epub 2016 May 13.

Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA

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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http://dx.doi.org/10.1093/nar/gkw426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5027477PMC
September 2016

Chromatin domains, insulators, and the regulation of gene expression.

Biochim Biophys Acta 2012 Jul 2;1819(7):644-51. Epub 2012 Feb 2.

Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Key Diseases, National Insitute of Health, 9000 Rockville Pike, Bethesda, MD 20892-0540, USA.

The DNA sequence elements called insulators have two basic kinds of properties. Barrier elements block the propagation of heterochromatic structures into adjacent euchromatin. Enhancer blocking elements interfere with interaction between an enhancer and promoter when placed between them. We have dissected a compound insulator element found at the 5' end of the chicken β-globin locus, which possesses both activities. Barrier insulation is mediated by two kinds of DNA binding proteins: USF1/USF2, a heterodimer which recruits multiple enzyme complexes capable of marking histone on adjacent nucleosomes with 'activating' marks, and Vezf1, which protects against DNA methylation. We have found that the heterochromatic region upstream of the insulator element is maintained in its silent state by a dicer-dependent mechanism, suggesting a mechanism for Vezf1 function in the insulator. Enhancer blocking function in the β-globin insulator element is conferred by a binding site for CTCF. Consistent with this property, CTCF binding was found some years ago to be essential for imprinted expression at the Igf2/H19 locus. Work in many laboratories has since demonstrated that CTCF helps stabilize long-range interactions in the nucleus. We have recently shown that in the case of the human insulin locus such an interaction, over a distance of ~300kb, can result in stimulation of a target gene which itself is important for insulin secretion. This article is part of a Special Issue entitled: Chromatin in time and space.
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http://dx.doi.org/10.1016/j.bbagrm.2012.01.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3484685PMC
July 2012

Vezf1 protein binding sites genome-wide are associated with pausing of elongating RNA polymerase II.

Proc Natl Acad Sci U S A 2012 Feb 30;109(7):2370-5. Epub 2012 Jan 30.

Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0540, USA.

The protein Vezf1 plays multiple roles important for embryonic development. In Vezf1(-/-) mouse embryonic stem (mES) cells, our earlier data showed widespread changes in gene-expression profiles, including decreased expression of the full-length active isoform of Dnmt3b methyltransferase and concomitant genome-wide reduction in DNA methylation. Here we show that in HeLaS3 cells there is a strong genome-wide correlation between Vezf1 binding and peaks of elongating Ser2-P RNA polymerase (Pol) ll, reflecting Vezf1-dependent slowing of elongation. In WT mES cells, the elongating form of RNA pol II accumulates near Vezf1 binding sites within the dnmt3b gene and at several other Vezf1 sites, and this accumulation is significantly reduced at these sites in Vezf1(-/-) mES cells. Depending upon genomic location, Vezf1-mediated Pol II pausing can have different regulatory roles in transcription and splicing. We find examples of genes in which Vezf1 binding sites are located near cassette exons, and in which loss of Vezf1 leads to a change in the relative abundance of alternatively spliced messages. We further show that Vezf1 interacts with Mrg15/Mrgbp, a protein that recognizes H3K36 trimethylation, consistent with the role of histone modifications at alternatively spliced sites.
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http://dx.doi.org/10.1073/pnas.1121538109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3289347PMC
February 2012

VEZF1 elements mediate protection from DNA methylation.

PLoS Genet 2010 Jan 8;6(1):e1000804. Epub 2010 Jan 8.

Section of Pathology and Gene Regulation, Faculty of Medicine, University of Glasgow, Western Infirmary, Glasgow, United Kingdom.

There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm beta-globin HS4 insulator to block the range of enhancers or the spread of repressive histone modifications is well established. Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin state.
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http://dx.doi.org/10.1371/journal.pgen.1000804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2795164PMC
January 2010

Vezf1 regulates genomic DNA methylation through its effects on expression of DNA methyltransferase Dnmt3b.

Genes Dev 2008 Aug;22(15):2075-84

Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.

The zinc finger protein vascular endothelial zinc finger 1 (Vezf1) has been implicated in the development of the blood vascular and lymphatic system in mice, and has been characterized as a transcriptional activator in some systems. The chicken homolog, BGP1, has binding sites in the beta-globin locus, including the upstream insulator element. We report that in a mouse embryonic stem cell line deletion of both copies of Vezf1 results in loss of DNA methylation at widespread sites in the genome, including Line1 elements and minor satellite repeats, some imprinted genes, and several CpG islands. Loss of methylation appears to arise from a substantial decrease in the abundance of the de novo DNA methyltransferase, Dnmt3b. These results suggest that naturally occurring mutations in Vezf1/BGP1 might have widespread effects on DNA methylation patterns and therefore on epigenetic regulation of gene expression.
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http://dx.doi.org/10.1101/gad.1658408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2492749PMC
August 2008

Phosphorylation of serine-515 activates the Mammalian maintenance methyltransferase Dnmt1.

Epigenetics 2007 Sep 20;2(3):155-60. Epub 2007 Jul 20.

Institut für Biochemie, Justus-Liebig Universität, Giessen, Germany.

DNA methyltransferase 1 methylates hemi-methylated CG sites generated during DNA replication. Serine 515 of this enzyme has been shown to be phosphorylated. To explore the importance of S515 phosphorylation, we generated mutants of Dnmt1 which removed the phosphorylation potential (S515A) or mimic phosphoserine (S515E), purified the proteins from insect cells and analyzed their DNA methylation activity in vitro. The S515E mutant was found to be active, while S515A mutant had severe loss in activity when compared to the wild type protein. The loss of activity of the S515A variant was not due to loss of DNA binding capacity. Furthermore, we show that a phosphorylated peptide whose sequence mimics the surrounding of Ser515 (EKIYIS(P)KIVVE) inhibited the activity of wild type Dnmt1 ten-fold more than the non-phosphorylated peptide. The inhibition was specific for Dnmt1 and for the particular peptide sequence. Our data suggest that phosphorylation of Ser515 is important for an interaction between the N-terminal domain of Dnmt1 and its catalytic domain that is necessary for activity and that this interaction is specifically disrupted by the phosphorylated peptide. We conclude that phosphorylation of Dnmt1 at Ser515 could be an important regulator of Dnmt1 activity during cell cycle and after proliferative stimuli.
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http://dx.doi.org/10.4161/epi.2.3.4768DOI Listing
September 2007

Mutations in DNA methyltransferase DNMT3B in ICF syndrome affect its regulation by DNMT3L.

Hum Mol Genet 2006 May 16;15(9):1375-85. Epub 2006 Mar 16.

The State Key Laboratory of Molecular Biology, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China.

Deficiency in DNA methyltransferase DNMT3B causes a recessive human disorder characterized by immunodeficiency, centromeric instability and facial anomalies (ICF) in association with defects in genomic methylation. The majority of ICF mutations are single amino acid substitutions in the conserved catalytic domain of DNMT3B, which are believed to impair its enzymatic activity directly. The establishment of intact genomic methylation patterns in development requires a fine regulation of the de novo methylation activity of the two related methyltransferases DNMT3A and DNMT3B by regulatory factors including DNMT3L which has a stimulatory effect. Here, we show that two DNMT3B mutant proteins with ICF-causing substitution (A766P and R840Q) displayed a methylation activity similar to the wild-type enzyme both in vitro and in vivo. However, their stimulation by DNMT3L was severely compromised due to deficient protein interaction. Our findings suggest that methylation defects in ICF syndrome may also result from impaired stimulation of DNMT3B activity by DNMT3L or other unknown regulatory factors as well as from a weakened basal catalytic activity of the mutant DNMT3B protein per se.
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http://dx.doi.org/10.1093/hmg/ddl059DOI Listing
May 2006

Mutational analysis of the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase.

J Mol Biol 2006 Mar 30;357(3):928-41. Epub 2006 Jan 30.

International University Bremen, Biochemistry, School of Engineering and Science, Campus Ring 1, 28759 Bremen, Germany.

On the basis of amino acid sequence alignments and structural data of related enzymes, we have performed a mutational analysis of 14 amino acid residues in the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase. The target residues are located within the ten conserved amino acid sequence motifs characteristic for cytosine-C5 methyltransferases and in the putative DNA recognition domain of the enzyme (TRD). Mutant proteins were purified and tested for their catalytic properties and their abilities to bind DNA and AdoMet. We prepared a structural model of Dnmt3a to interpret our results. We demonstrate that Phe50 (motif I) and Glu74 (motif II) are important for AdoMet binding and catalysis. D96A (motif III) showed reduced AdoMet binding but increased activity under conditions of saturation with S-adenosyl-L-methionine (AdoMet), indicating that the contact of Asp96 to AdoMet is not required for catalysis. R130A (following motif IV), R241A and R246A (in the TRD), R292A, and R297A (both located in front of motif X) showed reduced DNA binding. R130A displayed a strong reduction in catalytic activity and a complete change in flanking sequence preferences, indicating that Arg130 has an important role in the DNA interaction of Dnmt3a. R292A also displayed reduced activity and changes in the flanking sequence preferences, indicating a potential role in DNA contacts farther away from the CG target site. N167A (motif VI) and R202A (motif VIII) have normal AdoMet and DNA binding but reduced catalytic activity. While Asn167 might contribute to the positioning of residues from motif VI, according to structural data Arg202 has a role in catalysis of cytosine-C5 methyltransferases. The R295A variant was catalytically inactive most likely because of destabilization of the hinge sub-domain of the protein.
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http://dx.doi.org/10.1016/j.jmb.2006.01.035DOI Listing
March 2006

De novo methylation of nucleosomal DNA by the mammalian Dnmt1 and Dnmt3A DNA methyltransferases.

Biochemistry 2005 Jul;44(29):9899-904

School of Engineering and Science, International University Bremen, Campus Ring 1, D-28759 Bremen, Germany.

In the cell, DNA is wrapped on histone octamers, which reduces its accessibility for DNA interacting enzymes. We investigated de novo methylation of nucleosomal DNA in vitro and show that the Dnmt3a and Dnmt1 DNA methyltransferases efficiently methylate nucleosomal DNA without dissociation of the histone octamer from the DNA. In contrast, the prokaryotic SssI DNA methyltransferase and the catalytic domain of Dnmt3a are strongly inhibited by nucleosomes. We also found that full-length Dnmt1 and Dnmt3a bind to nucleosomes much stronger than their isolated catalytic domains, demonstrating that the N-terminal parts of the MTases are required for the interaction with nucleosomes. Variations of the DNA sequence or the histone tails did not significantly influence the methylation activity of Dnmt3a. The observation that mammalian methyltransferases directly modify nucleosomal DNA provides an insight into the mechanisms by which histone tail and DNA methylation patterns can influence each other because the DNA methylation pattern can be established while histones remain associated to the DNA.
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http://dx.doi.org/10.1021/bi047634tDOI Listing
July 2005

Avidin plate assay system for enzymatic characterization of a histone lysine methyltransferase.

Anal Biochem 2005 Jul;342(2):287-91

School of Engineering and Science, International University Bremen, Campus Ring 1, 28759 Bremen, Germany.

Modification of proteins by protein methyltransferases has several important biological functions. Here, we study the methylation of histone H3 tail at position Lys9 by the Dim-5 histone lysine methyltransferase, which is involved in epigenetic signaling and gene silencing and which triggers DNA methylation in Neurospora crassa. We have developed a new assay to detect protein methylation using a biotinylated synthetic peptide substrate and a radioactively labeled coenzyme. We show that the assay is linear with respect to time and enzyme concentration (under multiple turnover conditions) and that its background is very low. Data points were reproducible within 3%. At least 200 pmol of biotinylated peptide is bound completely to the microplate. We employed the assay system to determine the K(m) and k(cat) values of the Dim-5 enzyme for the methylation of a 20 mer peptide to be 7.4 microM and 2.3 min(-1), respectively. In addition, we determined the activity of four Dim-5 variants, ranging from full activity to less than 1% of residual activity. The microplate biotin/avidin peptide methylation assay developed here is convenient, very accurate, reproducible, and inexpensive. Because it yields quantitative results, it can be employed for a characterization of the enzymatic properties of histone lysine methyltransferases and other protein methyltransferases. The assay also is well suited for high-throughput applications.
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http://dx.doi.org/10.1016/j.ab.2005.04.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2696273PMC
July 2005

Mechanism of stimulation of catalytic activity of Dnmt3A and Dnmt3B DNA-(cytosine-C5)-methyltransferases by Dnmt3L.

J Biol Chem 2005 Apr 24;280(14):13341-8. Epub 2005 Jan 24.

International University Bremen, School of Engineering and Science, Campus Ring 1, 28759 Bremen, Germany.

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.
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http://dx.doi.org/10.1074/jbc.M413412200DOI Listing
April 2005

Mechanism of inhibition of DNA methyltransferases by cytidine analogs in cancer therapy.

Cancer Biol Ther 2004 Nov 12;3(11):1062-8. Epub 2004 Nov 12.

School of Engineering and Science, International University Bremen, Bremen 28759, Germany.

Hypermethylation of tumor suppressor genes caused by aberrant activity of DNA methyltransferases is an important mechanism that contributes to cancer. The reaction mechanism of DNA methyltransferases, which includes formation of a covalent intermediate between the enzyme and the target base, is the basis of the success of several anti-cancer drugs that are targeted against DNA methylation. These include 5-fluoro-2'-deoxycytidine, 5-aza-2'-deoxycytidine (Decitabine) and 2-H pyrimidinone-1-beta-D(2'-deoxyriboside) (Zebularine). This review provides an insight to how the chemistry of DNA methylation is involved in the performance of these drugs targeted against it.
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http://dx.doi.org/10.4161/cbt.3.11.1308DOI Listing
November 2004

Chromatin targeting of de novo DNA methyltransferases by the PWWP domain.

J Biol Chem 2004 Jun 3;279(24):25447-54. Epub 2004 Mar 3.

State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China.

DNA methylation patterns of mammalian genomes are generated in gametogenesis and early embryonic development. Two de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the process. Both enzymes contain a long N-terminal regulatory region linked to a conserved C-terminal domain responsible for the catalytic activity. Although a PWWP domain in the N-terminal region has been shown to bind DNA in vitro, it is unclear how the DNA methyltransferases access their substrate in chromatin in vivo. We show here that the two proteins are associated with chromatin including mitotic chromosomes in mammalian cells, and the PWWP domain is essential for the chromatin targeting of the enzymes. The functional significance of PWWP-mediated chromatin targeting is suggested by the fact that a missense mutation in this domain of human DNMT3B causes immunodeficiency, centromeric heterochromatin instability, facial anomalies (ICF) syndrome, which is characterized by loss of methylation in satellite DNA, pericentromeric instability, and immunodeficiency. We demonstrate that the mutant protein completely loses its chromatin targeting capacity. Our data establish the PWWP domain as a novel chromatin/chromosome-targeting module and suggest that the PWWP-mediated chromatin association is essential for the function of the de novo methyltransferases during development.
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http://dx.doi.org/10.1074/jbc.M312296200DOI Listing
June 2004