Publications by authors named "John R Horton"

84 Publications

Preferential CEBP binding to T:G mismatches and increased C-to-T human somatic mutations.

Nucleic Acids Res 2021 Apr 20. Epub 2021 Apr 20.

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

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 mutations are enriched in the binding sites of CCAAT/enhancer binding proteins (CEBP). Within a CEBP site, the presence of a T:G mismatch increased CEBPβ binding affinity by a factor of >60 relative to the normal C:G base pair. This enhanced binding to a mismatch inhibits its repair by both TDG and MBD4 in vitro. Furthermore, repair of the deamination product of unmethylated cytosine, which yields a U:G DNA mismatch that is normally repaired via uracil DNA glycosylase, is also inhibited by CEBPβ binding. Passage of a replication fork over either a T:G or U:G mismatch, before repair can occur, results in a C-to-T mutation in one of the daughter duplexes. Our study thus provides a plausible mechanism for accumulation of C-to-T human somatic mutations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkab276DOI Listing
April 2021

ZNF410 Uniquely Activates the NuRD Component CHD4 to Silence Fetal Hemoglobin Expression.

Mol Cell 2021 01 9;81(2):239-254.e8. Epub 2020 Dec 9.

Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA; Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

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 sites near the CHD4 gene with no counterparts elsewhere in the genome. Loss of ZNF410 in adult-type human erythroid cell culture systems and xenotransplantation settings diminishes CHD4 levels and derepresses the fetal hemoglobin genes. While previously known to be silenced by CHD4, the fetal globin genes are exposed here as among the most sensitive to reduced CHD4 levels.. In vitro DNA binding assays and crystallographic studies reveal the ZNF410-DNA binding mode. ZNF410 is a remarkably selective transcriptional activator in erythroid cells, and its perturbation might offer new opportunities for treatment of hemoglobinopathies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2020.11.006DOI Listing
January 2021

Biochemical and structural basis for YTH domain of human YTHDC1 binding to methylated adenine in DNA.

Nucleic Acids Res 2020 10;48(18):10329-10341

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

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 N6mA DNA. Here, we show that the YTH domain of YTHDC1 (known to localize in the nucleus) binds ssDNA containing N6mA, with a 10 nM dissociation constant. This binding is stronger by a factor of 5 than in an RNA context, tested under the same conditions. However, the YTH domains of YTHDF2 and YTHDF1 (predominantly cytoplasmic) exhibited the opposite effect with ∼1.5-2× stronger binding to ssRNA containing N6mA than to the corresponding DNA. We determined two structures of the YTH domain of YTHDC1 in complex with N6mA-containing ssDNA, which illustrated that YTHDC1 binds the methylated adenine in a single-stranded region flanked by duplexed DNA. We discuss the hypothesis that the writer-reader-eraser of N6mA-containining ssDNA is associated with maintaining genome stability. Structural comparison of YTH and SRA domains (the latter a DNA 5-methylcytosine reader) revealed them to be diverse members of a larger family of DNA/RNA modification readers, apparently having originated from bacterial modification-dependent restriction enzymes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkaa604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544203PMC
October 2020

Characterization of SETD3 methyltransferase-mediated protein methionine methylation.

J Biol Chem 2020 08 5;295(32):10901-10910. Epub 2020 Jun 5.

Department of Epigenetics and Molecular Carcinogenesis, University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA

Most characterized protein methylation events encompass arginine and lysine -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 to the active-site pocket of the corresponding histone lysine methyltransferase, thereby inhibiting the respective transmethylation activity. SET domain-containing 3 (SETD3) is a protein ( actin) histidine methyltransferase. Here, we generated an actin variant in which the histidine target of SETD3 was substituted with methionine. As for previously characterized histone SET domain proteins, the methionine substitution substantially (76-fold) increased binding affinity for SETD3 and inhibited SETD3 activity on histidine. Unexpectedly, SETD3 was active on the substituted methionine, generating -methylmethionine in the context of actin peptide. The ternary structure of SETD3 in complex with the methionine-containing actin peptide at 1.9 Å resolution revealed that the hydrophobic thioether side chain is packed by the aromatic rings of Tyr and Trp, as well as the hydrocarbon side chain of Ile Our results suggest that placing methionine properly in the active site-within close proximity to and in line with the incoming methyl group of SAM-would allow some SET domain proteins to selectively methylate methionine in proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA120.014072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415985PMC
August 2020

Beta class amino methyltransferases from bacteria to humans: evolution and structural consequences.

Nucleic Acids Res 2020 10;48(18):10034-10044

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

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 class beta MTases. Members of this class include M.EcoGII and M.EcoP15I from Escherichia coli, Caulobacter crescentus cell cycle-regulated DNA methyltransferase (CcrM), the MTA1-MTA9 complex from the ciliate Oxytricha, and the mammalian MettL3-MettL14 complex. These methyltransferases all generate N6-methyladenine in DNA, with some members having activity on single-stranded DNA as well as RNA. The beta class of methyltransferases has a unique multimeric feature, forming either homo- or hetero-dimers, allowing the enzyme to use division of labor between two subunits in terms of substrate recognition and methylation. We suggest that M.EcoGII may represent an ancestral form of these enzymes, as its activity is independent of the nucleic acid type (RNA or DNA), its strandedness (single or double), and its sequence (aside from the target adenine).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkaa446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544214PMC
October 2020

Metabolic and cardiorespiratory effects of decreasing lung hyperinflation with budesonide/formoterol in COPD: a randomized, double-crossover, placebo-controlled, multicenter trial.

Respir Res 2020 Jan 20;21(1):26. Epub 2020 Jan 20.

Pulmonary and Critical Care Division, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, MA, 02115, USA.

Background: Studies suggest that acute decreases in lung hyperinflation at rest improves cardiac function and increases lung vascular perfusion from decompression of a compromised heart. In those studies, changes in resting oxygen uptake induced by medications, an alternative explanation for compensatory increased cardiac function, were not explored.

Methods: This double-blind, multicenter, double-crossover study enrolled adults with chronic obstructive pulmonary disease, resting hyperinflation, and > 10% improvement in inspiratory capacity after 2 inhalations of budesonide/formoterol 160/4.5 μg. Metabolic, cardiac, and ventilatory function were measured 60 min pre-/post-dose at each visit. Primary endpoint was change in resting oxygen uptake for budesonide/formoterol versus placebo.

Results: Fifty-one patients (median age: 63 years) received treatment. Compared with placebo, budesonide/formoterol significantly increased resting oxygen uptake (mean change from baseline: 1.25 vs 11.37 mL/min; P = 0.007) as well as tidal volume and minute ventilation. This occurred despite improvements in the inspiratory capacity, forced vital capacity, and expiratory volume in 1 s. No significant treatment differences were seen for oxygen saturation, respiratory rate, and resting dyspnea. There was a numerical increase in oxygen pulse (oxygen uptake/heart rate). Correlations between inspiratory capacity and oxygen pulse were weak.

Conclusions: Budesonide/formoterol treatment in resting hyperinflated patients with COPD results in significant deflation. The increase in oxygen uptake and minute ventilation at lower lung volumes, without changes in heart rate and with minimal improvement in oxygen pulse, suggests increased oxygen demand as a contributor to increased cardiac function.

Trial Registration: ClinicalTrials.gov identifier: NCT02533505.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12931-020-1288-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6972029PMC
January 2020

An engineered variant of SETD3 methyltransferase alters target specificity from histidine to lysine methylation.

J Biol Chem 2020 02 7;295(9):2582-2589. Epub 2020 Jan 7.

Department of Epigenetics and Molecular Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030. Electronic address:

Most characterized SET domain (SETD) proteins are protein lysine methyltransferases, but SETD3 was recently demonstrated to be a protein ( actin) histidine-N methyltransferase. 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 active site. In the SETD3 active site, Asn engages in a unique hydrogen-bonding interaction with the target histidine of actin that likely contributes to its >1300-fold greater catalytic efficiency (/ ) on histidine than on lysine. Here, we engineered active-site variants to switch the SETD3 target specificity from histidine to lysine. Substitution of Asn with phenylalanine (N255F), together with substitution of Trp with alanine (W273A), generated an active site mimicking that of known lysine methyltransferases. The doubly substituted SETD3 variant exhibited a 13-fold preference for lysine over histidine. We show, by means of X-ray crystallography, that the two target nitrogen atoms-the N atom of histidine and the terminal ϵ-amino nitrogen of lysine-occupy the same position and point toward and are within a short distance of the incoming methyl group of SAM for a direct methyl transfer during catalysis. In contrast, SETD3 and its Asn substituted derivatives did not methylate glutamine (another potentially methylated amino acid). However, the glutamine-containing peptide competed with the substrate peptide, and glutamine bound in the active site, but too far away from SAM to be methylated. Our results provide insight into the structural parameters defining the target amino acid specificity of SET enzymes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA119.012319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049955PMC
February 2020

Structure of HhaI endonuclease with cognate DNA at an atomic resolution of 1.0 Å.

Nucleic Acids Res 2020 02;48(3):1466-1478

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

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 cleave the two strands of duplex DNA simultaneously, in a single binding event. HhaI, in contrast, acts as a monomer with only one catalytic site, and cleaves the DNA strands sequentially, one after the other. HhaI comprises three domains, each consisting of a mixed five-stranded β sheet with a defined function. The first domain contains the catalytic-site; the second contains residues for sequence recognition; and the third contributes to non-specific DNA binding. The active-site belongs to the 'PD-D/EXK' superfamily of nucleases and contains the motif SD-X11-EAK. The first two domains are similar in structure to two other monomeric restriction enzymes, HinP1I (G↓CGC) and MspI (C↓CGG), which produce fragments with 5'-overhangs. The third domain, present only in HhaI, shifts the positions of the recognition residues relative to the catalytic site enabling this enzyme to cleave the recognition sequence at a different position. The structure of M.HhaI, the biological methyltransferase partner of HhaI, was determined earlier. Together, these two structures represent the first natural pair of restriction-modification enzymes to be characterized in atomic detail.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkz1195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026639PMC
February 2020

Crosstalk between Lys63- and Lys11-polyubiquitin signaling at DNA damage sites is driven by Cezanne.

Genes Dev 2019 12 7;33(23-24):1702-1717. Epub 2019 Nov 7.

Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA.

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 by binding to Lys63-polyubiquitin and targeting Lys11-polyubiquitin. Using a ubiquitin binding domain protein array screen, we identify that the UBA domains of Cezanne and Cezanne2 (also known as Otud7A) selectively bind to Lys63-linked polyubiquitin. Increased Lys11-linkage ubiquitination due to lack of Cezanne DUB activity compromises the recruitment of Rap80/BRCA1-A. Cezanne2 interacts with Cezanne, facilitating Cezanne in the recruitment of Rap80/BRCA1-A, Rad18, and 53BP1, in cellular resistance to ionizing radiation and DNA repair. Our work presents a model that Cezanne serves as a "reader" of the Lys63-linkage polyubiquitin at DNA damage sites and an "eraser" of the Lys11-linkage ubiquitination, indicating a crosstalk between linkage-specific ubiquitination at DNA damage sites.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.332395.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6942045PMC
December 2019

The cell cycle-regulated DNA adenine methyltransferase CcrM opens a bubble at its DNA recognition site.

Nat Commun 2019 10 10;10(1):4600. Epub 2019 Oct 10.

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

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 domain. CcrM is a dimer, with each monomer contacting primarily one DNA strand: the methyltransferase domain of one molecule binds the target strand, recognizes the target sequence, and catalyzes methyl transfer, while the C-terminal domain of the second molecule binds the non-target strand. The DNA contacts at the 5-base pair recognition site results in dramatic DNA distortions including bending, unwinding and base flipping. The two DNA strands are pulled apart, creating a bubble comprising four recognized base pairs. The five bases of the target strand are recognized meticulously by stacking contacts, van der Waals interactions and specific Watson-Crick polar hydrogen bonds to ensure high enzymatic specificity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-12498-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6787082PMC
October 2019

Genetic heterogeneity within collective invasion packs drives leader and follower cell phenotypes.

J Cell Sci 2019 10 9;132(19). Epub 2019 Oct 9.

Winship Cancer Institute, Emory University, Atlanta, GA 30322, USA

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 collective invasion, we analyzed transcriptomic sequence variation between leader and follower populations isolated from the H1299 non-small cell lung cancer cell line using an image-guided selection technique. We now identify 14 expressed mutations that are selectively enriched in leader or follower cells, suggesting a novel link between genomic and phenotypic heterogeneity within a collectively invading tumor cell population. Functional characterization of two phenotype-specific candidate mutations showed that ARP3 enhances collective invasion by promoting the leader cell phenotype and that wild-type KDM5B suppresses chain-like cooperative behavior. These results demonstrate an important role for distinct genetic variants in establishing leader and follower phenotypes and highlight the necessity of maintaining a capacity for phenotypic plasticity during collective cancer invasion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.231514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6803364PMC
October 2019

Structural basis for the target specificity of actin histidine methyltransferase SETD3.

Nat Commun 2019 08 6;10(1):3541. Epub 2019 Aug 6.

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

SETD3 is an actin histidine-N methyltransferase, whereas other characterized SET-domain enzymes are protein lysine methyltransferases. We report that in a pre-reactive complex SETD3 binds the N-protonated form (N-H) of actin His73, and in a post-reactive product complex, SETD3 generates the methylated histidine in an N-protonated (N-H) and N-methylated form. During the reaction, the imidazole ring of His73 rotates ~105°, which shifts the proton from N to N, thus ensuring that the target atom N is deprotonated prior to the methyl transfer. Under the conditions optimized for lysine deprotonation, SETD3 has weak lysine methylation activity on an actin peptide in which the target His73 is substituted by a lysine. The structure of SETD3 with Lys73-containing peptide reveals a bent conformation of Lys73, with its side chain aliphatic carbons tracing along the edge of imidazole ring and the terminal ε-amino group occupying a position nearly identical to the N atom of unmethylated histidine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-11554-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684798PMC
August 2019

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkz557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895263PMC
September 2019

Structural basis for preferential binding of human TCF4 to DNA containing 5-carboxylcytosine.

Nucleic Acids Res 2019 09;47(16):8375-8387

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

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 5'-CANNTG-3' (where N = any nucleotide). A subset of the TCF4-occupancy sites have the expanded consensus binding specificity 5'-C(A/G)-CANNTG-3', with an added outer Cp(A/G) dinucleotide; for example in the promoter for CNIH3, a gene involved in opioid dependence. In mammalian genomes, particularly brain, the CpG and CpA dinucleotides can be methylated at the 5-position of cytosine (5mC), and then may undergo successive oxidations to the 5-hydroxymethyl (5hmC), 5-formyl (5fC), and 5-carboxyl (5caC) forms. We find that, in the context of 5'-0CG-1CA-2CG-3TG-3'(where the numbers indicate successive dinucleotides), modification of the central E-box 2CG has very little effect on TCF4 binding, E-box 1CA modification has a negative influence on binding, while modification of the flanking 0CG, particularly carboxylation, has a strong positive impact on TCF4 binding to DNA. Crystallization of TCF4 in complex with unmodified or 5caC-modified oligonucleotides revealed that the basic region of bHLH domain adopts multiple conformations, including an extended loop going through the DNA minor groove, or the N-terminal portion of a long helix binding in the DNA major groove. The different protein conformations enable arginine 576 (R576) to interact, respectively, with a thymine in the minor groove, a phosphate group of DNA backbone, or 5caC in the major groove. The Pitt-Hopkins syndrome mutations affect five arginine residues in the basic region, two of them (R569 and R576) involved in 5caC recognition. Our analyses indicate, and suggest a structural basis for, the preferential recognition of 5caC by a transcription factor centrally important in brain development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkz381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895265PMC
September 2019

Discovery of a Novel Chemotype of Histone Lysine Methyltransferase EHMT1/2 (GLP/G9a) Inhibitors: Rational Design, Synthesis, Biological Evaluation, and Co-crystal Structure.

J Med Chem 2019 03 26;62(5):2666-2689. Epub 2019 Feb 26.

Department of Molecular and Cellular Oncology , The University of Texas MD Anderson Cancer Center , Houston , Texas 77030 , United States.

Since the discovery of compound BIX01294 over 10 years ago, only a very limited number of nonquinazoline inhibitors of H3K9-specific methyltransferases G9a and G9a-like protein (GLP) have been reported. Herein, we report the identification of a novel chemotype for G9a/GLP inhibitors, based on the underinvestigated 2-alkyl-5-amino- and 2-aryl-5-amino-substituted 3 H-benzo[ e][1,4]diazepine scaffold. Our research efforts resulted in the identification 12a (EML741), which not only maintained the high in vitro and cellular potency of its quinazoline counterpart, but also displayed improved inhibitory potency against DNA methyltransferase 1, improved selectivity against other methyltransferases, low cell toxicity, and improved apparent permeability values in both parallel artificial membrane permeability assay (PAMPA) and blood-brain barrier-specific PAMPA, and therefore might potentially be a better candidate for animal studies. Finally, the co-crystal structure of GLP in complex with 12a provides the basis for the further development of benzodiazepine-based G9a/GLP inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.8b02008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430049PMC
March 2019

SETD3 is an actin histidine methyltransferase that prevents primary dystocia.

Nature 2019 01 10;565(7739):372-376. Epub 2018 Dec 10.

Department of Biology, Stanford University, Stanford, CA, USA.

For more than 50 years, the methylation of mammalian actin at histidine 73 has been known to occur. 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 the physiological actin His73 methyltransferase. Structural studies reveal that an extensive network of interactions clamps the actin peptide onto the surface of SETD3 to orient His73 correctly within the catalytic pocket and to facilitate methyl transfer. His73 methylation reduces the nucleotide-exchange rate on actin monomers and modestly accelerates the assembly of actin filaments. Mice that lack SETD3 show complete loss of actin His73 methylation in several tissues, and quantitative proteomics analysis shows that actin His73 methylation is the only detectable physiological substrate of SETD3. SETD3-deficient female mice have severely decreased litter sizes owing to primary maternal dystocia that is refractory to ecbolic induction agents. Furthermore, depletion of SETD3 impairs signal-induced contraction in primary human uterine smooth muscle cells. Together, our results identify a mammalian histidine methyltransferase and uncover a pivotal role for SETD3 and actin His73 methylation in the regulation of smooth muscle contractility. Our data also support the broader hypothesis that protein histidine methylation acts as a common regulatory mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-018-0821-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511263PMC
January 2019

Structural basis for effects of CpA modifications on C/EBPβ binding of DNA.

Nucleic Acids Res 2019 02;47(4):1774-1785

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

CCAAT/enhancer binding proteins (C/EBPs) regulate gene expression in a variety of cells/tissues/organs, during a range of developmental stages, under both physiological and pathological conditions. C/EBP-related transcription factors have a consensus binding specificity of 5'-TTG-CG-CAA-3', with a central CpG/CpG and two outer CpA/TpG dinucleotides. Methylation of the CpG and CpA sites generates a DNA element with every pyrimidine having a methyl group in the 5-carbon position (thymine or 5-methylcytosine (5mC)). To understand the effects of both CpG and CpA modification on a centrally-important transcription factor, we show that C/EBPβ binds the methylated 8-bp element with modestly-increased (2.4-fold) binding affinity relative to the unmodified cognate sequence, while cytosine hydroxymethylation (particularly at the CpA sites) substantially decreased binding affinity (36-fold). The structure of C/EBPβ DNA binding domain in complex with methylated DNA revealed that the methyl groups of the 5mCpA/TpG make van der Waals contacts with Val285 in C/EBPβ. Arg289 recognizes the central 5mCpG by forming a methyl-Arg-G triad, and its conformation is constrained by Val285 and the 5mCpG methyl group. We substituted Val285 with Ala (V285A) in an Ala-Val dipeptide, to mimic the conserved Ala-Ala in many members of the basic leucine-zipper family of transcription factors, important in gene regulation, cell proliferation and oncogenesis. The V285A variant demonstrated a 90-fold binding preference for methylated DNA (particularly 5mCpA methylation) over the unmodified sequence. The smaller side chain of Ala285 permits Arg289 to adopt two alternative conformations, to interact in a similar fashion with either the central 5mCpG or the TpG of the opposite strand. Significantly, the best-studied cis-regulatory elements in RNA polymerase II promoters and enhancers have variable sequences corresponding to the central CpG or reduced to a single G:C base pair, but retain a conserved outer CpA sequence. Our analyses suggest an important modification-dependent CpA recognition by basic leucine-zipper transcription factors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gky1264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393304PMC
February 2019

Structure-Based Engineering of Irreversible Inhibitors against Histone Lysine Demethylase KDM5A.

J Med Chem 2018 12 15;61(23):10588-10601. Epub 2018 Nov 15.

Department of Molecular and Cellular Oncology , The University of Texas MD Anderson Cancer Center , Houston , Texas 77030 , United States.

The active sites of hundreds of human α-ketoglutarate (αKG) and Fe(II)-dependent dioxygenases are exceedingly well preserved, which challenges the design of selective inhibitors. We identified a noncatalytic cysteine (Cys481 in KDM5A) near the active sites of KDM5 histone H3 lysine 4 demethylases, which is absent in other histone demethylase families, that could be explored for interaction with the cysteine-reactive electrophile acrylamide. We synthesized analogs of a thienopyridine-based inhibitor chemotype, namely, 2-((3-aminophenyl)(2-(piperidin-1-yl)ethoxy)methyl)thieno[3,2- b]pyridine-7-carboxylic acid (N70) and a derivative containing a (dimethylamino)but-2-enamido)phenyl moiety (N71) designed to form a covalent interaction with Cys481. We characterized the inhibitory and binding activities against KDM5A and determined the cocrystal structures of the catalytic domain of KDM5A in complex with N70 and N71. Whereas the noncovalent inhibitor N70 displayed αKG-competitive inhibition that could be reversed after dialysis, inhibition by N71 was dependent on enzyme concentration and persisted even after dialysis, consistent with covalent modification.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.8b01219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467790PMC
December 2018

KDM5 histone demethylases repress immune response via suppression of STING.

PLoS Biol 2018 08 6;16(8):e2006134. Epub 2018 Aug 6.

Department of Pathology, Yale School of Medicine, New Haven, Connecticut, United States of America.

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 clinical trials. However, STING is commonly silenced in cancer cells via unclear mechanisms, limiting the application of these agonists. Here, we report that the expression of STING is epigenetically suppressed by the histone H3K4 lysine demethylases KDM5B and KDM5C and is activated by the opposing H3K4 methyltransferases. The induction of STING expression by KDM5 blockade triggered a robust interferon response in a cytosolic DNA-dependent manner in breast cancer cells. This response resulted in resistance to infection by DNA and RNA viruses. In human tumors, KDM5B expression is inversely associated with STING expression in multiple cancer types, with the level of intratumoral CD8+ T cells, and with patient survival in cancers with a high level of cytosolic DNA, such as human papilloma virus (HPV)-positive head and neck cancer. These results demonstrate a novel epigenetic regulatory pathway of immune response and suggest that KDM5 demethylases are potential targets for antipathogen treatment and anticancer immunotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.2006134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6095604PMC
August 2018

Detecting and interpreting DNA methylation marks.

Curr Opin Struct Biol 2018 12 19;53:88-99. Epub 2018 Jul 19.

Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. Electronic address:

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. Sequence-specific transcription factors are responsible for interpreting the modification status of specific regions of chromatin. This review focuses on recent developments in characterizing the functional and structural links between the modification status of two DNA bases: 5-methylcytosine and 5-methyluracil (thymine).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.sbi.2018.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322410PMC
December 2018

DNA Conformation Induces Adaptable Binding by Tandem Zinc Finger Proteins.

Cell 2018 03 15;173(1):221-233.e12. Epub 2018 Mar 15.

Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA; Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. Electronic address:

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 protein conformations deviate from the conventional one finger-three bases recognition, with individual ZFs contacting 2, 3, or 4 bases and recognizing thymine on the opposite strand. These interactions arise from a shortened minor groove caused by an AT-rich stretch, suggesting adaptability of ZF arrays to sequence variations. Despite conservation in mammals, mutations at Igf2 and ZFP568 reduce their binding affinity in chimpanzee and humans. Our studies provide important insights into the evolutionary and structural dynamics of ZF-DNA interactions that play a key role in mammalian development and evolution.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2018.02.058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5877318PMC
March 2018

Insights into the Action of Inhibitor Enantiomers against Histone Lysine Demethylase 5A.

J Med Chem 2018 04 23;61(7):3193-3208. Epub 2018 Mar 23.

Department of Molecular and Cellular Oncology , The University of Texas MD Anderson Cancer Center , Houston , Texas 77030 , United States.

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)-1 H-pyrrolo[3,2- b]pyridine-7-carboxylic acid (compounds N51 and N52) and ( R) - and ( S) -N-(1-(3-isopropyl-1 H-pyrazole-5-carbonyl)pyrrolidin-3-yl)cyclopropanecarboxamide (compounds N54 and N55). In vitro, the S enantiomer of the N51/N52 pair (N52) and the R enantiomer of the N54/N55 pair (N54) exhibited about 4- to 5-fold greater binding affinity. The more potent enzyme inhibition of KDM5A by the R-isoform for the cell-permeable N54/N55 pair translated to differences in growth inhibitory activity. We determined structures of the KDM5A catalytic domain in complex with all 12 inhibitors, which revealed the interactions (or lack thereof) responsible for the differences in binding affinity. These results provide insights to guide improvements in binding potency and avenues for development of cell permeable inhibitors of the KDM5 family.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.8b00261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322411PMC
April 2018

Role for first zinc finger of WT1 in DNA sequence specificity: Denys-Drash syndrome-associated WT1 mutant in ZF1 enhances affinity for a subset of WT1 binding sites.

Nucleic Acids Res 2018 05;46(8):3864-3877

Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.

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 and ZF4. In the -KTS isoform, ZF2-4 determine the sequence-specificity of DNA binding, whereas the function of ZF1 remains elusive. Three X-ray structures are described here for wild-type -KTS isoform ZF1-4 in complex with its cognate DNA sequence. We observed four unique ZF1 conformations. First, like ZF2-4, ZF1 can be positioned continuously in the DNA major groove forming a 'near-cognate' complex. Second, while ZF2-4 make base-specific interactions with one DNA molecule, ZF1 can interact with a second DNA molecule (or, presumably, two regions of the same DNA molecule). Third, ZF1 can intercalate at the joint of two tail-to-head DNA molecules. If such intercalation occurs on a continuous DNA molecule, it would kink the DNA at the ZF1 binding site. Fourth, two ZF1 units can dimerize. Furthermore, we examined a Denys-Drash syndrome-associated ZF1 mutation (methionine at position 342 is replaced by arginine). This mutation enhances WT1 affinity for a guanine base. X-ray crystallography of the mutant in complex with its preferred sequence revealed the interactions responsible for this affinity change. These results provide insight into the mechanisms of action of WT1, and clarify the fact that ZF1 plays a role in determining sequence specificity of this critical transcription factor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkx1274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5934627PMC
May 2018

Structural Basis for the Versatile and Methylation-Dependent Binding of CTCF to DNA.

Mol Cell 2017 Jun 18;66(5):711-720.e3. Epub 2017 May 18.

Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road NE, Atlanta, GA 30322, USA; Department of Molecular and Cellular Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. Electronic address:

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 15-bp consensus sequence. Each conserved nucleotide makes base-specific hydrogen bonds with a particular residue. Most of the variable base pairs within the core sequence also engage in interactions with the protein. These interactions compensate for deviations from the consensus sequence, allowing CTCF to adapt to sequence variations. CTCF is sensitive to cytosine methylation at position 2, but insensitive at position 12 of the 15-bp core sequence. These differences can be rationalized structurally. Although included in crystallizations, ZF10 and ZF11 are not visible, while ZF8 and ZF9 span the backbone of the DNA duplex, conferring no sequence specificity but adding to overall binding stability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2017.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5542067PMC
June 2017

Methyl-dependent and spatial-specific DNA recognition by the orthologous transcription factors human AP-1 and Epstein-Barr virus Zta.

Nucleic Acids Res 2017 03;45(5):2503-2515

Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA.

T: Activator protein 1 (AP-1) is a transcription factor that recognizes two versions of a 7-base pair response element, either 5΄- GAG CA-3΄ or 5΄- GAG CA-3΄ (where M = 5-methylcytosine). These two elements share the feature that 5-methylcytosine and thymine both have a methyl group in the same position, 5-carbon of the pyrimidine, so each of them has two methyl groups at nucleotide positions 1 and 5 from the 5΄ end, resulting in four methyl groups symmetrically positioned in duplex DNA. Epstein-Barr Virus Zta is a key transcriptional regulator of the viral lytic cycle that is homologous to AP-1. Zta recognizes several methylated Zta-response elements, including meZRE1 (5΄- GAG C A-3΄) and meZRE2 (5΄- GAG G A-3΄), where a methylated cytosine occupies one of the inner thymine residues corresponding to the AP-1 element, resulting in the four spatially equivalent methyl groups. Here, we study how AP-1 and Zta recognize these methyl groups within their cognate response elements. These methyl groups are in van der Waals contact with a conserved di-alanine in AP-1 dimer (Ala265 and Ala266 in Jun), or with the corresponding Zta residues Ala185 and Ser186 (via its side chain carbon Cβ atom). Furthermore, the two ZRE elements differ at base pair 6 (C:G versus G:C), forming a pseudo-symmetric sequence (meZRE1) or an asymmetric sequence (meZRE2). In vitro DNA binding assays suggest that Zta has high affinity for all four sequences examined, whereas AP-1 has considerably reduced affinity for the asymmetric sequence (meZRE2). We ascribe this difference to Zta Ser186 (a unique residue for Zta) whose side chain hydroxyl oxygen atom interacts with the two half sites differently, whereas the corresponding Ala266 of AP-1 Jun protein lacks such flexibility. Our analyses demonstrate a novel mechanism of 5mC/T recognition in a methylation-dependent, spatial and sequence-specific approach by basic leucine-zipper transcriptional factors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkx057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389525PMC
March 2017

Structural Basis for KDM5A Histone Lysine Demethylase Inhibition by Diverse Compounds.

Cell Chem Biol 2016 07 14;23(7):769-781. Epub 2016 Jul 14.

Department of Biochemistry, Emory University, Atlanta, GA 30322, USA; The Winship Cancer Institute, Emory University, Atlanta, GA 30322, USA. Electronic address:

The KDM5/JARID1 family of Fe(II)- and α-ketoglutarate-dependent demethylases removes methyl groups from methylated lysine 4 of histone H3. Accumulating evidence supports a role for KDM5 family members as oncogenic drivers. We compare the in vitro inhibitory properties and binding affinity of ten diverse compounds with all four family members, and present the crystal structures of the KDM5A-linked Jumonji domain in complex with eight of these inhibitors in the presence of Mn(II). All eight inhibitors structurally examined occupy the binding site of α-ketoglutarate, but differ in their specific binding interactions, including the number of ligands involved in metal coordination. We also observed inhibitor-induced conformational changes in KDM5A, particularly those residues involved in the binding of α-ketoglutarate, the anticipated peptide substrate, and intramolecular interactions. We discuss how particular chemical moieties contribute to inhibitor potency and suggest strategies that might be utilized in the successful design of selective and potent epigenetic inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2016.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4958579PMC
July 2016

Structural basis for human PRDM9 action at recombination hot spots.

Genes Dev 2016 Feb;30(3):257-65

Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA;

The multidomain zinc finger (ZnF) protein PRDM9 (PRD1-BF1-RIZ1 homologous domain-containing 9) is thought to influence the locations of recombination hot spots during meiosis by sequence-specific DNA binding and trimethylation of histone H3 Lys4. The most common variant of human PRDM9, allele A (hPRDM9A), recognizes the consensus sequence 5'-NCCNCCNTNNCCNCN-3'. We cocrystallized ZnF8-12 of hPRDM9A with an oligonucleotide representing a known hot spot sequence and report the structure here. ZnF12 was not visible, but ZnF8-11, like other ZnF arrays, follows the right-handed twist of the DNA, with the α helices occupying the major groove. Each α helix makes hydrogen-bond (H-bond) contacts with up to four adjacent bases, most of which are purines of the complementary DNA strand. The consensus C:G base pairs H-bond with conserved His or Arg residues in ZnF8, ZnF9, and ZnF11, and the consensus T:A base pair H-bonds with an Asn that replaces His in ZnF10. Most of the variable base pairs (N) also engage in H bonds with the protein. These interactions appear to compensate to some extent for changes from the consensus sequence, implying an adaptability of PRDM9 to sequence variations. We investigated the binding of various alleles of hPRDM9 to different hot spot sequences. Allele C was found to bind a C-specific hot spot with higher affinity than allele A bound A-specific hot spots, perhaps explaining why the former is dominant in A/C heterozygotes. Allele L13 displayed higher affinity for several A-specific sequences, allele L9/L24 displayed lower affinity, and allele L20 displayed an altered sequence preference. These differences can be rationalized structurally and might contribute to the variation observed in the locations and activities of meiotic recombination hot spots.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.274928.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4743056PMC
February 2016

Characterization of a Linked Jumonji Domain of the KDM5/JARID1 Family of Histone H3 Lysine 4 Demethylases.

J Biol Chem 2016 Feb 8;291(6):2631-46. Epub 2015 Dec 8.

From the Departments of Biochemistry, the Winship Cancer Institute, Emory University, Atlanta, Georgia 30322

The KDM5/JARID1 family of Fe(II)- and α-ketoglutarate-dependent demethylases remove methyl groups from tri- and dimethylated lysine 4 of histone H3. Accumulating evidence from primary tumors and model systems supports a role for KDM5A (JARID1A/RBP2) and KDM5B (JARID1B/PLU1) as oncogenic drivers. The KDM5 family is unique among the Jumonji domain-containing histone demethylases in that there is an atypical insertion of a DNA-binding ARID domain and a histone-binding PHD domain into the Jumonji domain, which separates the catalytic domain into two fragments (JmjN and JmjC). Here we demonstrate that internal deletion of the ARID and PHD1 domains has a negligible effect on in vitro enzymatic kinetics of the KDM5 family of enzymes. We present a crystal structure of the linked JmjN-JmjC domain from KDM5A, which reveals that the linked domain fully reconstitutes the cofactor (metal ion and α-ketoglutarate) binding characteristics of other structurally characterized Jumonji domain demethylases. Docking studies with GSK-J1, a selective inhibitor of the KDM6/KDM5 subfamilies, identify critical residues for binding of the inhibitor to the reconstituted KDM5 Jumonji domain. Further, we found that GSK-J1 inhibited the demethylase activity of KDM5C with 8.5-fold increased potency compared with that of KDM5B at 1 mm α-ketoglutarate. In contrast, JIB-04 (a pan-inhibitor of the Jumonji demethylase superfamily) had the opposite effect and was ~8-fold more potent against KDM5B than against KDM5C. Interestingly, the relative selectivity of JIB-04 toward KDM5B over KDM5C in vitro translates to a ~10-50-fold greater growth-inhibitory activity against breast cancer cell lines. These data define the minimal requirements for enzymatic activity of the KDM5 family to be the linked JmjN-JmjC domain coupled with the immediate C-terminal helical zinc-binding domain and provide structural characterization of the linked JmjN-JmjC domain for the KDM5 family, which should prove useful in the design of KDM5 demethylase inhibitors with improved potency and selectivity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M115.698449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4742734PMC
February 2016

Structures of Escherichia coli DNA adenine methyltransferase (Dam) in complex with a non-GATC sequence: potential implications for methylation-independent transcriptional repression.

Nucleic Acids Res 2015 Apr 6;43(8):4296-308. Epub 2015 Apr 6.

Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA

DNA adenine methyltransferase (Dam) is widespread and conserved among the γ-proteobacteria. Methylation of the Ade in GATC sequences regulates diverse bacterial cell functions, including gene expression, mismatch repair and chromosome replication. Dam also controls virulence in many pathogenic Gram-negative bacteria. An unexplained and perplexing observation about Escherichia coli Dam (EcoDam) is that there is no obvious relationship between the genes that are transcriptionally responsive to Dam and the promoter-proximal presence of GATC sequences. Here, we demonstrate that EcoDam interacts with a 5-base pair non-cognate sequence distinct from GATC. The crystal structure of a non-cognate complex allowed us to identify a DNA binding element, GTYTA/TARAC (where Y = C/T and R = A/G). This element immediately flanks GATC sites in some Dam-regulated promoters, including the Pap operon which specifies pyelonephritis-associated pili. In addition, Dam interacts with near-cognate GATC sequences (i.e. 3/4-site ATC and GAT). Taken together, these results imply that Dam, in addition to being responsible for GATC methylation, could also function as a methylation-independent transcriptional repressor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkv251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4417163PMC
April 2015

Nonsynonymous single nucleotide polymorphisms of NHE3 differentially decrease NHE3 transporter activity.

Am J Physiol Cell Physiol 2015 May 25;308(9):C758-66. Epub 2015 Feb 25.

Departments of Physiology and Medicine, Division of Gastroenterology, Johns Hopkins University School of Medicine, Baltimore, Maryland; and

Genetic determinants appear to play a role in susceptibility to chronic diarrhea, but the genetic abnormalities involved have only been identified in a few conditions. The Na⁺/H⁺ exchanger 3 (NHE3) accounts for a large fraction of physiologic intestinal Na⁺ absorption. It is highly regulated through effects on its intracellular COOH-terminal regulatory domain. The impact of genetic variation in the NHE3 gene, such as single nucleotide polymorphisms (SNPs), on transporter activity remains unexplored. From a total of 458 SNPs identified in the entire NHE3 gene, we identified three nonsynonymous mutations (R474Q, V567M, and R799C), which were all in the protein's intracellular COOH-terminal domain. Here we evaluated whether these SNPs affect NHE3 activity by expressing them in a mammalian cell line that is null for all plasma membrane NHEs. These variants significantly reduced basal NHE3 transporter activity through a reduction in intrinsic NHE3 function in variant R474Q, abnormal trafficking in variant V567M, or defects in both intrinsic NHE3 function and trafficking in variant R799C. In addition, variants NHE3 R474Q and R799C failed to respond to acute dexamethasone stimulation, suggesting cells with these mutant proteins might be defective in NHE3 function during postprandial stimulation and perhaps under stressful conditions. Finally, variant R474Q was shown to exhibit an aberrant interaction with calcineurin B homologous protein (CHP), an NHE3 regulatory protein required for basal NHE3 activity. Taken together, these results demonstrate decreased transport activity in three SNPs of NHE3 and provide mechanistic insight into how these SNPs impact NHE3 function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajpcell.00421.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4420795PMC
May 2015