Publications by authors named "Swanand Hardikar"

11 Publications

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The inactive Dnmt3b3 isoform preferentially enhances Dnmt3b-mediated DNA methylation.

Genes Dev 2020 Nov 1;34(21-22):1546-1558. Epub 2020 Oct 1.

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA.

The de novo DNA methyltransferases Dnmt3a and Dnmt3b play crucial roles in developmental and cellular processes. Their enzymatic activities are stimulated by a regulatory protein Dnmt3L (Dnmt3-like) in vitro. However, genetic evidence indicates that Dnmt3L functions predominantly as a regulator of Dnmt3a in germ cells. How Dnmt3a and Dnmt3b activities are regulated during embryonic development and in somatic cells remains largely unknown. Here we show that Dnmt3b3, a catalytically inactive Dnmt3b isoform expressed in differentiated cells, positively regulates de novo methylation by Dnmt3a and Dnmt3b with a preference for Dnmt3b. Dnmt3b3 is equally potent as Dnmt3L in stimulating the activities of Dnmt3a2 and Dnmt3b2 in vitro. Like Dnmt3L, Dnmt3b3 forms a complex with Dnmt3a2 with a stoichiometry of 2:2. However, rescue experiments in triple-knockout (TKO) mouse embryonic stem cells (mESCs) reveal that Dnmt3b3 prefers Dnmt3b2 over Dnmt3a2 in remethylating genomic sequences. Dnmt3a2, an active isoform that lacks the N-terminal uncharacterized region of Dnmt3a1 including a nuclear localization signal, has very low activity in TKO mESCs, indicating that an accessory protein is absolutely required for its function. Our results suggest that Dnmt3b3 and perhaps similar Dnmt3b isoforms facilitate de novo DNA methylation during embryonic development and in somatic cells.
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http://dx.doi.org/10.1101/gad.341925.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7608744PMC
November 2020

Germline DNMT3A mutation in familial acute myeloid leukaemia.

Epigenetics 2021 May 28;16(5):567-576. Epub 2020 Aug 28.

Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Acute myeloid leukaemia (AML) is a heterogeneous myeloid malignancy characterized by recurrent clonal events, including mutations in epigenetically relevant genes such as , and . Next-generation sequencing analysis of a mother and son pair who both developed adult-onset diploid AML identified a novel germline missense mutation p.P709S. The p.P709S protein-altering variant resides in the highly conserved catalytic DNMT3A methyltransferase domain. Functional studies demonstrate that the p.P709S variant confers dominant negative effects when interacting with wildtype . LINE-1 pyrosequencing and reduced representation bisulphite sequencing (RBBS) analysis demonstrated global DNA hypomethylation in germline samples, not present in the leukaemic samples. Somatic acquisition of p.R172K mutations, in concert with additional acquired clonal DNMT3A events in both patients at the time of AML diagnosis, confirms the important pathogenic interaction of epigenetically active genes, and implies a strong selection and regulation of methylation in leukaemogenesis. Improved characterization of germline mutations may enable us to better predict malignant clonal evolution, improving our ability to provide customized treatment or future preventative strategies.
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http://dx.doi.org/10.1080/15592294.2020.1809871DOI Listing
May 2021

The ZBTB24-CDCA7 axis regulates HELLS enrichment at centromeric satellite repeats to facilitate DNA methylation.

Protein Cell 2020 03;11(3):214-218

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

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http://dx.doi.org/10.1007/s13238-019-00682-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026229PMC
March 2020

Structural basis of specific DNA binding by the transcription factor ZBTB24.

Nucleic Acids Res 2019 09;47(16):8388-8398

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

ZBTB24, encoding a protein of the ZBTB family of transcriptional regulators, is one of four known genes-the other three being DNMT3B, CDCA7 and HELLS-that are mutated in immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome, a genetic disorder characterized by DNA hypomethylation and antibody deficiency. The molecular mechanisms by which ZBTB24 regulates gene expression and the biological functions of ZBTB24 are poorly understood. Here, we identified a 12-bp consensus sequence [CT(G/T)CCAGGACCT] occupied by ZBTB24 in the mouse genome. The sequence is present at multiple loci, including the Cdca7 promoter region, and ZBTB24 binding is mostly associated with gene activation. Crystallography and DNA-binding data revealed that the last four of the eight zinc fingers (ZFs) (i.e. ZF5-8) in ZBTB24 confer specificity of DNA binding. Two ICF missense mutations have been identified in the ZBTB24 ZF domain, which alter zinc-binding cysteine residues. We demonstrated that the corresponding C382Y and C407G mutations in mouse ZBTB24 abolish specific DNA binding and fail to induce Cdca7 expression. Our analyses indicate and suggest a structural basis for the sequence specific recognition by a transcription factor centrally important for the pathogenesis of ICF syndrome.
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http://dx.doi.org/10.1093/nar/gkz557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895263PMC
September 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

DNMT3A and TET1 cooperate to regulate promoter epigenetic landscapes in mouse embryonic stem cells.

Genome Biol 2018 07 12;19(1):88. Epub 2018 Jul 12.

Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, TX, 77030, USA.

Background: DNA methylation is a heritable epigenetic mark, enabling stable but reversible gene repression. In mammalian cells, DNA methyltransferases (DNMTs) are responsible for modifying cytosine to 5-methylcytosine (5mC), which can be further oxidized by the TET dioxygenases to ultimately cause DNA demethylation. However, the genome-wide cooperation and functions of these two families of proteins, especially at large under-methylated regions, called canyons, remain largely unknown.

Results: Here we demonstrate that DNMT3A and TET1 function in a complementary and competitive manner in mouse embryonic stem cells to mediate proper epigenetic landscapes and gene expression. The longer isoform of DNMT3A, DNMT3A1, exhibits significant enrichment at distal promoters and canyon edges, but is excluded from proximal promoters and canyons where TET1 shows prominent binding. Deletion of Tet1 increases DNMT3A1 binding capacity at and around genes with wild-type TET1 binding. However, deletion of Dnmt3a has a minor effect on TET1 binding on chromatin, indicating that TET1 may limit DNA methylation partially by protecting its targets from DNMT3A and establishing boundaries for DNA methylation. Local CpG density may determine their complementary binding patterns and therefore that the methylation landscape is encoded in the DNA sequence. Furthermore, DNMT3A and TET1 impact histone modifications which in turn regulate gene expression. In particular, they regulate Polycomb Repressive Complex 2 (PRC2)-mediated H3K27me3 enrichment to constrain gene expression from bivalent promoters.

Conclusions: We conclude that DNMT3A and TET1 regulate the epigenome and gene expression at specific targets via their functional interplay.
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http://dx.doi.org/10.1186/s13059-018-1464-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6042404PMC
July 2018

Identification of Rpl29 as a major substrate of the lysine methyltransferase Set7/9.

J Biol Chem 2018 08 29;293(33):12770-12780. Epub 2018 Jun 29.

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, Texas 78957; Center for Cancer Epigenetics, The University of Texas MD Anderson Cancer Center, Smithville, Texas 78957; Program in Genetics and Epigenetics, The University of Texas MD Anderson Cancer Center UT Health Graduate School of Biomedical Sciences, Houston, Texas 77030. Electronic address:

Set7/9 (also known as Set7, Set9, Setd7, and Kmt7) is a lysine methyltransferase that catalyzes the methylation of multiple substrates, including histone H3 and non-histone proteins. Although not essential for normal development and physiology, Set7/9-mediated methylation events play important roles in regulating cellular pathways involved in various human diseases, making Set7/9 a promising therapeutic target. Multiple Set7/9 inhibitors have been developed, which exhibit varying degrees of potency and selectivity However, validation of these compounds has been hampered by the lack of a reliable cellular biomarker for Set7/9 activity. Here, we report the identification of Rpl29, a ribosomal protein abundantly expressed in all cell types, as a major substrate of Set7/9. We show that Rpl29 lysine 5 (Rpl29K5) is methylated exclusively by Set7/9 and can be demethylated by Lsd1 (also known as Kdm1a). Rpl29 is not a core component of the ribosome translational machinery and plays a regulatory role in translation efficiency. Our results indicate that Rpl29 methylation has no effect on global protein synthesis but affects Rpl29 subcellular localization. Using an Rpl29 methylation-specific antibody, we demonstrate that Rpl29K5 methylation is present ubiquitously and validate that ()-PFI-2, a Set7/9 inhibitor, efficiently reduces Rpl29K5 methylation in cell lines. Thus, Rpl29 methylation can serve as a specific cellular biomarker for measuring Set7/9 activity.
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http://dx.doi.org/10.1074/jbc.RA118.002890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102145PMC
August 2018

The Arginine Methyltransferase PRMT6 Regulates DNA Methylation and Contributes to Global DNA Hypomethylation in Cancer.

Cell Rep 2017 Dec;21(12):3390-3397

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX 78957, USA; Center for Cancer Epigenetics, The University of Texas MD Anderson Cancer Center, Smithville, TX 78957, USA; Program in Genetics and Epigenetics, The University of Texas MD Anderson Cancer Center, UTHealth Graduate School of Biomedical Sciences, Houston, TX 77030, USA. Electronic address:

DNA methylation plays crucial roles in chromatin structure and gene expression. Aberrant DNA methylation patterns, including global hypomethylation and regional hypermethylation, are associated with cancer and implicated in oncogenic events. How DNA methylation is regulated in developmental and cellular processes and dysregulated in cancer is poorly understood. Here, we show that PRMT6, a protein arginine methyltransferase responsible for asymmetric dimethylation of histone H3 arginine 2 (H3R2me2a), negatively regulates DNA methylation and that PRMT6 upregulation contributes to global DNA hypomethylation in cancer. Mechanistically, PRMT6 overexpression impairs chromatin association of UHRF1, an accessory factor of DNMT1, resulting in passive DNA demethylation. The effect is likely due to elevated H3R2me2a, which inhibits the interaction between UHRF1 and histone H3. Our work identifies a mechanistic link between protein arginine methylation and DNA methylation, which is disrupted in cancer.
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http://dx.doi.org/10.1016/j.celrep.2017.11.082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753604PMC
December 2017

Zscan4 Inhibits Maintenance DNA Methylation to Facilitate Telomere Elongation in Mouse Embryonic Stem Cells.

Cell Rep 2017 Aug;20(8):1936-1949

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX 78957, USA; Center for Cancer Epigenetics, The University of Texas MD Anderson Cancer Center, Smithville, TX 78957, USA; The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX 77030, USA. Electronic address:

Proper telomere length is essential for embryonic stem cell (ESC) self-renewal and pluripotency. Mouse ESCs (mESCs) sporadically convert to a transient totipotent state similar to that of two-cell (2C) embryos to recover shortened telomeres. Zscan4, which exhibits a burst of expression in 2C-like mESCs, is required for telomere extension in these cells. However, the mechanism by which Zscan4 extends telomeres remains elusive. Here, we show that Zscan4 facilitates telomere elongation by inducing global DNA demethylation through downregulation of Uhrf1 and Dnmt1, major components of the maintenance DNA methylation machinery. Mechanistically, Zscan4 recruits Uhrf1 and Dnmt1 and promotes their degradation, which depends on the E3 ubiquitin ligase activity of Uhrf1. Blocking DNA demethylation prevents telomere elongation associated with Zscan4 expression, suggesting that DNA demethylation mediates the effect of Zscan4. Our results define a molecular pathway that contributes to the maintenance of telomere length homeostasis in mESCs.
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http://dx.doi.org/10.1016/j.celrep.2017.07.070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5595351PMC
August 2017

Maternal Setdb1 Is Required for Meiotic Progression and Preimplantation Development in Mouse.

PLoS Genet 2016 Apr 12;12(4):e1005970. Epub 2016 Apr 12.

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, Texas, United States of America.

Oocyte meiotic progression and maternal-to-zygote transition are accompanied by dynamic epigenetic changes. The functional significance of these changes and the key epigenetic regulators involved are largely unknown. Here we show that Setdb1, a lysine methyltransferase, controls the global level of histone H3 lysine 9 di-methyl (H3K9me2) mark in growing oocytes. Conditional deletion of Setdb1 in developing oocytes leads to meiotic arrest at the germinal vesicle and meiosis I stages, resulting in substantially fewer mature eggs. Embryos derived from these eggs exhibit severe defects in cell cycle progression, progressive delays in preimplantation development, and degeneration before reaching the blastocyst stage. Rescue experiments by expressing wild-type or inactive Setdb1 in Setdb1-deficient oocytes suggest that the catalytic activity of Setdb1 is essential for meiotic progression and early embryogenesis. Mechanistically, up-regulation of Cdc14b, a dual-specificity phosphatase that inhibits meiotic progression, greatly contributes to the meiotic arrest phenotype. Setdb1 deficiency also leads to derepression of transposons and increased DNA damage in oocytes, which likely also contribute to meiotic defects. Thus, Setdb1 is a maternal-effect gene that controls meiotic progression and is essential for early embryogenesis. Our results uncover an important link between the epigenetic machinery and the major signaling pathway governing meiotic progression.
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http://dx.doi.org/10.1371/journal.pgen.1005970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4829257PMC
April 2016

A DNMT3A mutation common in AML exhibits dominant-negative effects in murine ES cells.

Blood 2013 Dec 28;122(25):4086-9. Epub 2013 Oct 28.

Department of Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Smithville, TX;

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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http://dx.doi.org/10.1182/blood-2013-02-483487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3952368PMC
December 2013