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Introduction
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May 1992 - March 1997
January 2017 - present
June 1984 - April 1992
Education
September 1984 - April 1992
August 1980 - June 1984
Publications
Publications (148)
Epigenetics is one of the fastest moving fields in drug discovery, with almost every large pharmaceutical company, and a substantial number of biotechnology companies, targeting epigenetic processes to treat diseases ranging from cancer to Huntington’s disease and from inflammation to sickle cell anaemia. This book aims to provide an overview of th...
CDCA7 , encoding a protein with a carboxyl-terminal cysteine-rich domain (CRD), is mutated in immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, a disease related to hypomethylation of juxtacentromeric satellite DNA. How CDCA7 directs DNA methylation to juxtacentromeric regions is unknown. Here, we show that the CDCA7 C...
DNA methylation, as exemplified by cytosine-C5 methylation in mammals and adenine-N6 methylation in bacteria, is a crucial epigenetic mechanism driving numerous vital biological processes. Developing non-nucleoside inhibitors to cause DNA hypomethylation is a high priority, in order to treat a variety of significant medical conditions without the t...
CDCA7, encoding a protein with a C-terminal cysteine-rich domain (CRD), is mutated in immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome, a disease related to hypomethylation of juxtacentromeric satellite DNA. How CDCA7 directs DNA methylation to juxtacentromeric regions is unknown. Here, we show that the CDCA7 CRD adopts...
The CCCTC-binding factor (CTCF) binds tens of thousands of enhancers and promoters on mammalian chromosomes by means of its 11 tandem zinc finger (ZF) DNA-binding domain. In addition to the 12-15-bp CORE sequence, some of the CTCF binding sites contain 5' upstream and/or 3' downstream motifs. Here, we describe two structures for overlapping portion...
Much is known about the generation, removal, and roles of 5-methylcytosine (5mC) in eukaryote DNA, and there is a growing body of evidence regarding N6-methyladenine, but very little is known about N4-methylcytosine (4mC) in the DNA of eukaryotes. The gene for the first metazoan DNA methyltransferase generating 4mC (N4CMT) was reported and characte...
S-Adenosyl-l-methionine (SAM) analogs are adaptable tools for studying and therapeutically inhibiting SAM-dependent methyltransferases (MTases). Some MTases play significant roles in host-pathogen interactions, one of which is Clostridioides difficile-specific DNA adenine MTase (CamA). CamA is needed for efficient sporulation and alters persistence...
ZNF410 is a highly-conserved transcription factor, remarkable in that it recognizes a 15-base pair DNA element but has just a single responsive target gene in mammalian erythroid cells. ZNF410 includes a tandem array of five zinc-fingers (ZFs), surrounded by uncharacterized N- and C-terminal regions. Unexpectedly, full-length ZNF410 has reduced DNA...
ZBTB7A belongs to a small family of transcription factors having three members in humans (7A, 7B, and 7C). They share a BTB/POZ protein interaction domain at the amino end and a zinc-finger DNA-binding domain at the carboxyl end. They control the transcription of a wide range of genes, having varied functions in hematopoiesis, oncogenesis, and meta...
Antivirulence agents targeting endospore-transmitted Clostridioides difficile infections are urgently needed. C. difficile-specific DNA adenine methyltransferase (CamA) is required for efficient sporulation and affects persistence in the colon. The active site of CamA is conserved and closely resembles those of hundreds of related S-adenosyl-l-meth...
The N-terminal half of PHF2 harbors both a plant homeodomain (PHD) and a Jumonji domain. The PHD recognizes both histone H3 trimethylated at lysine 4 (H3K4me3) and methylated non-histone proteins including vaccinia-related kinase 1 (VRK1). The Jumonji domain erases the repressive dimethylation mark from histone H3 lysine 9 (H3K9me2) at select promo...
The modification of DNA bases is a classic hallmark of epigenetics. Four forms of modified cytosine-5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine-have been discovered in eukaryotic DNA. In addition to cytosine carbon-5 modifications, cytosine and adenine methylated in the exocyclic amine-N4-methylcytosine and N...
The fetal-to-adult switch in hemoglobin production is a model of developmental gene control with relevance to the treatment of hemoglobinopathies. The expression of transcription factor BCL11A, which represses fetal β-type globin (HBG) genes in adult erythroid cells, is predominantly controlled at the transcriptional level but the underlying mechan...
DNMT1 maintains the parental DNA methylation pattern on newly replicated hemimethylated DNA. The failure of this maintenance process causes aberrant DNA methylation that affects transcription and contributes to the development and progression of cancers such as acute myeloid leukemia. Here, we structurally characterized a set of newly discovered DN...
Cytosine to thymine (C>T) somatic mutation is highly enriched in certain types of cancer, and most commonly occurs via deamination of a 5-methylcytosine (5mC) to thymine, in the context of a CpG dinucleotide. In theory, deamination should occur at equal rates to both 5mC nucleotides on opposite strands. In most cases, the resulting T:G or G:T misma...
DNA methylation, a key epigenetic driver of transcriptional silencing, is universally dysregulated in cancer. Reversal of DNA methylation by hypomethylating agents, such as the cytidine analogs decitabine or azacytidine, has demonstrated clinical benefit in hematologic malignancies. These nucleoside analogs are incorporated into replicating DNA whe...
Epigenetically targeted therapeutic development, particularly for SAM-dependent methylations of DNA, mRNA and histones has been proceeding rapidly for cancer treatments over the past few years. However, this approach has barely begun to be exploited for developing new antibiotics, despite an overwhelming global need to counter antimicrobial resista...
Elevated levels of fetal globin protect against β-hemoglobinopathies, such as sickle cell disease and β-thalassemia. Two zinc-finger (ZF) repressors, BCL11A and ZBTB7A/LRF, bind directly to the fetal globin promoter elements positioned at −115 and −200, respectively. Here, we describe X-ray structures of the ZBTB7A DNA-binding domain, consisting of...
Clostridioides difficile infections are an urgent medical problem. The newly discovered C. difficile a denine m ethyltransferase A (CamA) is specified by all C. difficile genomes sequenced to date (>300), but is rare among other bacteria. CamA is an orphan methyltransferase, unassociated with a restriction endonuclease. CamA-mediated methylation at...
MettL3-MettL14 methyltransferase complex has been studied widely for its role in RNA adenine methylation. This complex is also recruited to UV- and X-ray exposed DNA damaged sites, and its methyltransfer activity is required for subsequent DNA repair, though in theory this could result from RNA methylation of short transcripts made at the site of d...
The reader ability of PHD fingers is largely limited to the recognition of the histone H3 N-terminal tail. Distinct subsets of PHDs bind either H3K4me3 (a transcriptional activator mark) or H3K4me0 (a transcriptional repressor state). Structural studies have identified common features among the different H3K4me3 effector PHDs, including 1) removal...
DNA cytosine methylation in mammals modulates gene expression and chromatin accessibility. It also impacts mutation rates, via spontaneous oxidative deamination of 5-methylcytosine (5mC) to thymine. In most cases the resulting T:G mismatches are repaired, following T excision by one of the thymine DNA glycosylases, TDG or MBD4. We found that C-to-T...
Metazoan transcription factors typically regulate large numbers of genes. Here we identify via a CRISPR-Cas9 genetic screen ZNF410, a pentadactyl DNA-binding protein that in human erythroid cells directly activates only a single gene, the NuRD component CHD4. Specificity is conveyed by two highly evolutionarily conserved clusters of ZNF410 binding...
Transcription factors typically regulate a large number of genes. Here we found that transcription factor ZNF410 binds and activates the expression of a single direct target gene, CHD4, to enforce the silencing of the fetal hemoglobin genes (HBG1 and HBG2) in adult erythroid cells.
ZNF410 is a pentadactyl DNA binding protein that emerged from a DNA...
Metazoan transcription factors typically regulate large numbers of genes. Here we identify via a CRISPR-Cas9 genetic screen ZNF410, a pentadactyl DNA binding protein that in human erythroid cells directly and measurably activates only one gene, the NuRD component CHD4. Specificity is conveyed by two highly evolutionarily conserved clusters of ZNF41...
The recently characterized mammalian writer (methyltransferase) and eraser (demethylase) of the DNA N6-methyladenine (N6mA) methyl mark act on single-stranded (ss) and transiently-unpaired DNA. As YTH domain-containing proteins bind N6mA-containing RNA in mammalian cells, we investigated whether mammalian YTH domains are also methyl mark readers of...
Most characterized protein methylation events encompass arginine and lysine N-methylation, and only a few cases of protein methionine thiomethylation have been reported. Newly discovered oncohistone mutations include lysine-to-methionine substitutions at positions 27 and 36 of histone H3.3. In these instances, the methionine substitution localizes...
S-adenosyl-l-methionine dependent methyltransferases catalyze methyl transfers onto a wide variety of target molecules, including DNA and RNA. We discuss a family of methyltransferases, those that act on the amino groups of adenine or cytosine in DNA, have conserved motifs in a particular order in their amino acid sequence, and are referred to as c...
Most characterized SET domain (SETD) proteins are protein lysine methyltransferases, but SETD3 was recently demonstrated to be a protein (i.e.actin) histidine-N3methyltransferase. Human SETD3 shares a high structural homology with two known protein lysine methyltransferases—human SETD6 and the plant LSMT—but differs in the residues constituting the...
HhaI, a Type II restriction endonuclease, recognizes the symmetric sequence 5'-GCG↓C-3' in duplex DNA and cleaves ('↓') to produce fragments with 2-base, 3'-overhangs. We determined the structure of HhaI in complex with cognate DNA at an ultra-high atomic resolution of 1.0 Å. Most restriction enzymes act as dimers with two catalytic sites, and clea...
The establishment of polyubiquitin conjugates with distinct linkages play important roles in the DNA damage response. Much remains unknown about the regulation of linkage-specific ubiquitin signaling at sites of DNA damage. Here we reveal that Cezanne (also known as Otud7B) deubiquitinating enzyme promotes the recruitment of Rap80/BRCA1-A complex b...
The Caulobacter crescentus cell cycle-regulated DNA methyltransferase (CcrM) methylates the adenine of hemimethylated GANTC after replication. Here we present the structure of CcrM in complex with double-stranded DNA containing the recognition sequence. CcrM contains an N-terminal methyltransferase domain and a C-terminal nonspecific DNA-binding do...
Collective invasion, the coordinated movement of cohesive packs of cells, has become recognized as a major mode of metastasis for solid tumors. These packs are phenotypically heterogeneous and include specialized cells that lead the invasive pack and others that follow behind. To better understand how these unique cell types cooperate to facilitate...
SETD3 is an actin histidine-N3 methyltransferase, whereas other characterized SET-domain enzymes are protein lysine methyltransferases. We report that in a pre-reactive complex SETD3 binds the N3-protonated form (N3-H) of actin His73, and in a post-reactive product complex, SETD3 generates the methylated histidine in an N1-protonated (N1-H) and N3-...
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 mechan...
The psychiatric risk-associated transcription factor 4 (TCF4) is linked to schizophrenia. Rare TCF4 coding variants are found in individuals with Pitt-Hopkins syndrome-an intellectual disability and autism spectrum disorder. TCF4 contains a C-terminal basic-helix-loop-helix (bHLH) DNA binding domain which recognizes the enhancer-box (E-box) element...
Since the discovery of compound BIX01294 over 10 years ago, only a very limited number of non-quinazoline inhibitors of H3K9-specific methyltransferases G9a and GLP have been reported. Herein we report the identification of a novel chemotype for G9a/GLP inhibitors, based on the under-investigated 2-alkyl-5-amino- and 2-aryl-5-amino-substituted 3H-b...
For more than 50 years, the methylation of mammalian actin at histidine 73 has been known to occur¹. Despite the pervasiveness of His73 methylation, which we find is conserved in several model animals and plants, its function remains unclear and the enzyme that generates this modification is unknown. Here we identify SET domain protein 3 (SETD3) as...
Cyclic GMP-AMP (cGAMP) synthase (cGAS) stimulator of interferon genes (STING) senses pathogen-derived or abnormal self-DNA in the cytosol and triggers an innate immune defense against microbial infection and cancer. STING agonists induce both innate and adaptive immune responses and are a new class of cancer immunotherapy agents tested in multiple...
Activation of ISGs by KDM5 inhibition is dependent on the cGAS-STING-TBK1-IRF3 pathway.
(A) T7 endonuclease assays showing the genome editing efficiency of MCF7 cells with CRISPR/Cas9-mediated TLR3 knockout. (B, C) RT-qPCR (panel B) and western blot (panel C) analyses of control or IRF3 knockout MCF7 cells generated by 3 independent sgRNAs against...
KDM5 represses interferon response by inhibiting STING expression.
(A–D) Western blot analysis of the indicated cell lines after treatment with DMSO or 1 μM KDM5-C70 for 6 days. (E, F) RT-qPCR (panel E) and western blot (panel F) analyses of control or KDM5B/KDM5C double KO MCF7 cells. (G) RT-qPCR analysis of MCF7 cells treated with control or KDM5...
List of primers used for RT-qPCR.
RT-qPCR, reverse transcription followed by quantitative PCR.
(DOCX)
KDM5-C70, Dong-A 167, GDC-50, and CPI-48 are potent KDM5 demethylase inhibitors that bind in the active site of KDM5A.
(A) KDM5-C70 was designed as a cell-permeable prodrug that is hydrolyzed by intracellular esterase(s) to generate KDM5-C49, which contains an isonicotinic acid moiety with a carboxylic acid (PCT WO 2014053491) [30, 32]. (B) KDM5-C4...
Induced resistance to virus infection by KDM5 inhibition is dependent on the cGAS-STING-TBK1-IRF3 pathway.
(A) Flow cytometry plots (left panel) and quantification of GFP-positive cells (right panel) in MCF7 cells with knockout of the indicated genes 24 hours after infection with VSV-GFP at MOI 0.5. Cells were pretreated with DMSO or 1 μM KDM5-C70...
List of genes with log2FC ≥1 or ≤−1 and p < 0.05 in MCF7 cells after treatment with KDM5-C70 for 6 days.
(XLSX)
KDM5B and KDM5C bind to the promoter of STING.
(A, B) H3K4me3 ChIP-qPCR analysis of MCF7 cells (panel A) or BT474 cells (panel B) treated with DMSO or 1 μM KDM5-C70 for 1 day. (C) Analysis of ChIP-seq data for KDM5B binding at the STING genomic region in K562 cells (GSE29611, upper panel) and KDM5C in ZR-75-30 cells (GSE71327, lower panel) [42]. He...
KDM5-C70 does not affect cytosolic DNA in MCF7 cells, and components of the PRR pathway are efficiently deleted in SKBR3 cells.
(A, B) RT-qPCR analysis of MCF7 cells with the indicated treatment. MCF7 cells were treated with 10 μM VE821 for 3 days (panel A) or 1 μM KDM5-C70 for 4 days (panel B), followed by 1-day treatment with 0.2 μM LMB. (C) dsDN...
KDM5B is negatively associated with CD8+ T-cell infiltration and clinical outcome in cervical cancer.
(A) Correlation between KDM5B and STING in TCGA CESC. n = 302. (B) Correlation between KDM5B and CXCL10 or STING and CXCL10 in TCGA CESC. (C) Correlation between KDM5B expression and CD8+ T-cell infiltration in TCGA CESC. CESC, Cervical Squamous Ce...
List of primers used for ChIP-qPCR.
ChIP-qPCR, chromatin immunoprecipitation followed by quantitative PCR.
(DOCX)
Inhibition of KDM5 activates ISGs.
(A) Western blot analyses of MCF7 cells treated with 1 μM KDM5-C70 for 3 days. (B) Pathways that were up-regulated by 3 μM KDM5-C70 or CPI-48 treatment, revealed by GSEA. The gene set database of h. all. v6.1. symbols. gmt (Hallmarks) was used. All the up-regulated pathways in inhibitor-treated cells with FDR q va...
List of sgRNA targeting sequences.
sgRNA, single guide RNA.
(DOCX)
List of genes with log2FC ≥1 or ≤−1 and p < 0.05 in MCF7 cells after treatment with CPI-48 for 6 days.
(XLSX)
The generation, alteration, recognition, and erasure of epigenetic modifications of DNA are fundamental to controlling gene expression in mammals. These covalent DNA modifications include cytosine methylation by AdoMet-dependent methyltransferases and 5-methylcytosine oxidation by Fe(II)-dependent and α-ketoglutarate-dependent dioxygenases. Sequenc...
Isomers of chiral drugs can exhibit marked differences in biological activities. We studied the binding and inhibitory activities of 12 compounds against KDM5A. Among them are two pairs of enantiomers representing two distinct inhibitor chemotypes, namely (R)- and (S)-2-((2-chlorophenyl)(2-(piperidin-1-yl)ethoxy)methyl)-1H-pyrrolo[3,2-b]pyridine-7-...
Tandem zinc finger (ZF) proteins are the largest and most rapidly diverging family of DNA-binding transcription regulators in mammals. ZFP568 represses a transcript of placental-specific insulin like growth factor 2 (Igf2-P0) in mice. ZFP568 binds a 24-base pair sequence-specific element upstream of Igf2-P0 via the eleven-ZF array. Both DNA and pro...
Wilms tumor protein (WT1) is a Cys2-His2 zinc-finger transcription factor vital for embryonic development of the genitourinary system. The protein contains a C-terminal DNA binding domain with four tandem zinc-fingers (ZF1-4). An alternative splicing of Wt1 can add three additional amino acids-lysine (K), threonine (T) and serine (S)-between ZF3 an...
The methylation of lysine residues on histone tails--and their subsequent demethylation--is among some of the most important covalent post-translational modifications controlling gene expression. When gene expression goes awry, disease ensues and often this disease is some form of cancer. Thus, an understanding of histone tail modification in nucle...
The multidomain CCCTC-binding factor (CTCF), containing a tandem array of 11 zinc fingers (ZFs), modulates the three-dimensional organization of chromatin. We crystallized the human CTCF DNA-binding domain in complex with a known CTCF-binding site. While ZF2 does not make sequence-specific contacts, each finger of ZF3-7 contacts three bases of the...
