RESUMEN
The chromatin-remodeling enzyme helicase lymphoid-specific (HELLS) interacts with cell division cycle-associated 7 (CDCA7) on nucleosomes and is involved in the regulation of DNA methylation in higher organisms. Mutations in these genes cause immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, which also results in DNA hypomethylation of satellite repeat regions. We investigated the functional domains of human CDCA7 in HELLS using several mutant CDCA7 proteins. The central region is critical for binding to HELLS, activation of ATPase, and nucleosome sliding activities of HELLS-CDCA7. The N-terminal region tends to inhibit ATPase activity. The C-terminal 4CXXC-type zinc finger domain contributes to CpG and hemimethylated CpG DNA preference for DNA-dependent HELLS-CDCA7 ATPase activity. Furthermore, CDCA7 showed a binding preference to DNA containing hemimethylated CpG, and replication-dependent pericentromeric heterochromatin foci formation of CDCA7 with HELLS was observed in mouse embryonic stem cells; however, all these phenotypes were lost in the case of an ICF syndrome mutant of CDCA7 mutated in the zinc finger domain. Thus, CDCA7 most likely plays a role in the recruitment of HELLS, activates its chromatin remodeling function, and efficiently induces DNA methylation, especially at hemimethylated replication sites.
RESUMEN
In higher eukaryotes, a single DOT1 histone H3 lysine 79 (H3K79) methyltransferase processively produces H3K79me2/me3 through histone H2B mono-ubiquitin interaction, while the kinetoplastid Trypanosoma brucei di-methyltransferase DOT1A and tri-methyltransferase DOT1B efficiently methylate the homologous H3K76 without H2B mono-ubiquitination. Based on structural and biochemical analyses of DOT1A, we identify key residues in the methyltransferase motifs VI and X for efficient ubiquitin-independent H3K76 methylation in kinetoplastids. Substitution of a basic to an acidic residue within motif VI (Gx6K) is essential to stabilize the DOT1A enzyme-substrate complex, while substitution of the motif X sequence VYGE by CAKS renders a rigid active-site loop flexible, implying a distinct mechanism of substrate recognition. We further reveal distinct methylation kinetics and substrate preferences of DOT1A (H3K76me0) and DOT1B (DOT1A products H3K76me1/me2) in vitro, determined by a Ser and Ala residue within motif IV, respectively, enabling DOT1A and DOT1B to mediate efficient H3K76 tri-methylation non-processively but cooperatively, and suggesting why kinetoplastids have evolved two DOT1 enzymes.
Asunto(s)
Histonas , Ubiquitina , Histonas/metabolismo , Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , MetilaciónRESUMEN
In Saccharomyces cerevisiae, the pre-mRNA leakage 39-kDa protein (ScPml39) was reported to retain unspliced pre-mRNA prior to export through nuclear pore complexes (NPCs). Pml39 homologs outside the Saccharomycetaceae family are currently unknown, and mechanistic insight into Pml39 function is lacking. Here we determined the crystal structure of ScPml39 at 2.5 Å resolution to facilitate the discovery of orthologs beyond Saccharomycetaceae, e.g. in Schizosaccharomyces pombe or human. The crystal structure revealed integrated zf-C3HC and Rsm1 modules, which are tightly associated through a hydrophobic interface to form a single domain. Both zf-C3HC and Rsm1 modules belong to the Zn-containing BIR (Baculovirus IAP repeat)-like super family, with key residues of the canonical BIR domain being conserved. Features unique to the Pml39 modules refer to the spacing between the Zn-coordinating residues, giving rise to a substantially tilted helix αC in the zf-C3HC and Rsm1 modules, and an extra helix αAB' in the Rsm1 module. Conservation of key residues responsible for its distinct features identifies S. pombe Rsm1 and Homo sapiens NIPA/ZC3HC1 as structural orthologs of ScPml39. Based on the recent functional characterization of NIPA/ZC3HC1 as a scaffold protein that stabilizes the nuclear basket of the NPC, our data suggest an analogous function of ScPml39 in S. cerevisiae.
Asunto(s)
Proteínas Nucleares , Proteínas de Saccharomyces cerevisiae , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Poro Nuclear/metabolismo , Proteínas Nucleares/química , Precursores del ARN/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMEN
The tumor suppressor p53 integrates stress response pathways by selectively engaging one of several potential transcriptomes, thereby triggering cell fate decisions (e.g., cell cycle arrest, apoptosis). Foundational to this process is the binding of tetrameric p53 to 20-bp response elements (REs) in the genome (RRRCWWGYYYN0-13RRRCWWGYYY). In general, REs at cell cycle arrest targets (e.g. p21) are of higher affinity than those at apoptosis targets (e.g., BAX). However, the RE sequence code underlying selectivity remains undeciphered. Here, we identify molecular mechanisms mediating p53 binding to high- and low-affinity REs by showing that key determinants of the code are embedded in the DNA shape. We further demonstrate that differences in minor/major groove widths, encoded by G/C or A/T bp content at positions 3, 8, 13, and 18 in the RE, determine distinct p53 DNA-binding modes by inducing different Arg248 and Lys120 conformations and interactions. The predictive capacity of this code was confirmed in vivo using genome editing at the BAX RE to interconvert the DNA-binding modes, transcription pattern, and cell fate outcome.
Asunto(s)
Diferenciación Celular/genética , Diferenciación Celular/fisiología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/genética , Ciclo Celular , Puntos de Control del Ciclo Celular , Línea Celular , ADN/química , Proteínas de Unión al ADN , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Modelos Moleculares , Conformación Molecular , Unión Proteica/genética , Elementos de RespuestaRESUMEN
Prozymes are pseudoenzymes that stimulate the function of weakly active enzymes through complex formation. The major Trypanosoma brucei protein arginine methyltransferase, TbPRMT1 enzyme (ENZ), requires TbPRMT1 prozyme (PRO) to form an active heterotetrameric complex. Here, we present the X-ray crystal structure of the TbPRMT1 ENZ-Δ52PRO tetrameric complex with the cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.4 Å resolution. The individual ENZ and PRO units adopt the highly-conserved PRMT domain architecture and form an antiparallel heterodimer that corresponds to the canonical homodimer observed in all previously reported PRMTs. In turn, two such heterodimers assemble into a tetramer both in the crystal and in solution with twofold rotational symmetry. ENZ is unstable in absence of PRO and incapable of forming a homodimer due to a steric clash of an ENZ-specific tyrosine within the dimerization arm, rationalizing why PRO is required to complement ENZ to form a PRMT dimer that is necessary, but not sufficient for PRMT activity. The PRO structure deviates from other, active PRMTs in that it lacks the conserved η2 310-helix within the Rossmann fold, abolishing cofactor binding. In addition to its chaperone function for ENZ, PRO substantially contributes to substrate binding. Heterotetramerization is required for catalysis, as heterodimeric ENZ-PRO mutants lack binding affinity and methyltransferase activity toward the substrate protein TbRGG1. Together, we provide a structural basis for TbPRMT1 ENZ activation by PRO heterotetramer formation, which is conserved across all kinetoplastids, and describe a chaperone function of the TbPRMT1 prozyme, which represents a novel mode of PRMT regulation.
Asunto(s)
Complejos Multiproteicos/ultraestructura , Conformación Proteica , Proteína-Arginina N-Metiltransferasas/ultraestructura , S-Adenosilhomocisteína/química , Trypanosoma brucei brucei/ultraestructura , Secuencia de Aminoácidos/genética , Catálisis , Cristalografía por Rayos X , Dimerización , Metilación , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/genética , Especificidad por Sustrato/genética , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/genéticaRESUMEN
Bromodomains recognize a wide range of acetylated lysines in histones and other nuclear proteins. Substrate specificity is critical for their biological function and arises from unique acetyl-lysine binding sites formed by variable loop regions. Here, we analyzed substrate affinity and specificity of the yeast ScSth1p bromodomain, an essential component of the "Remodels the Structure of Chromatin" complex, and found that the wild-type bromodomain preferentially recognizes H3K14ac and H4K20ac peptides. Mutagenesis studies-guided by our crystal structure determined at 2.7-Å resolution-revealed loop residues Ser1276 and Trp1338 as key determinants for such interactions. Strikingly, point mutations of each of these residues substantially increased peptide binding affinity and selectivity, respectively. Our data demonstrate that the ScSth1p bromodomain is not optimized for binding to an individual acetylation mark, but fine-tuned for interactions with several such modifications, consistent with the versatile and multivalent nature of histone recognition by reader modules such as bromodomains.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Mutación Puntual , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilación , Proteínas de Ciclo Celular/genética , Cristalografía por Rayos X , Histonas/metabolismo , Humanos , Modelos Moleculares , Proteínas Nucleares/genética , Conformación Proteica , Dominios Proteicos , Proteínas de Saccharomyces cerevisiae/genética , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/genética , Cara/anomalías , Genoma , Mutación Missense , Proteínas Nucleares/genética , Enfermedades de Inmunodeficiencia Primaria/genética , Proteínas Represoras/química , Factores de Transcripción/química , Dedos de Zinc/genética , Animales , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Cara/patología , Expresión Génica , Sitios Genéticos , Vectores Genéticos , Humanos , Ratones , Modelos Moleculares , Proteínas Nucleares/metabolismo , Motivos de Nucleótidos , Enfermedades de Inmunodeficiencia Primaria/metabolismo , Enfermedades de Inmunodeficiencia Primaria/patología , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Metilación de ADN , ADN/metabolismo , Proteínas Represoras/metabolismo , 5-Metilcitosina/metabolismo , Sitios de Unión , Factor de Unión a CCCTC , Clonación Molecular , Cristalografía por Rayos X , ADN/química , ADN/genética , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Estabilidad Proteica , Proteínas Represoras/química , Proteínas Represoras/genética , Relación Estructura-Actividad , Repeticiones de Trinucleótidos , Dedos de ZincRESUMEN
The oncogenic transcription factor MYC and its binding partner MAX regulate gene expression by binding to DNA at enhancer-box (E-box) elements 5Î-CACGTG-3Î. In mammalian genomes, the central E-box CpG has the potential to be methylated at the 5-position of cytosine (5mC), or to undergo further oxidation to the 5-hydroxymethyl (5hmC), 5-formyl (5fC), or 5-carboxyl (5caC) forms. We find that MAX exhibits the greatest affinity for a 5caC or unmodified C-containing E-box, and much reduced affinities for the corresponding 5mC, 5hmC or 5fC forms. Crystallization of MAX with a 5caC modified E-box oligonucleotide revealed that MAX Arg36 recognizes 5caC using a 5caC-Arg-Guanine triad, with the next nearest residue to the carboxylate group being Arg60. In an analysis of >800 primary multiple myelomas, MAX alterations occurred at a frequency of â¼3%, more than half of which were single nucleotide substitutions affecting a basic clamp-like interface important for DNA interaction. Among these, arginines 35, 36 and 60 were the most frequently altered. In vitro binding studies showed that whereas mutation of Arg36 (R36W) or Arg35 (R35H/L) completely abolished DNA binding, mutation of Arg60 (R60Q) significantly reduced DNA binding, but retained a preference for the 5caC modified E-box. Interestingly, MAX alterations define a subset of myeloma patients with lower MYC expression and a better overall prognosis. Together these data indicate that MAX can act as a direct epigenetic sensor of E-box cytosine modification states and that local CpG modification and MAX variants converge to modulate the MAX-MYC transcriptional network.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/química , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Citosina/análogos & derivados , Elementos E-Box , Mieloma Múltiple/genética , Proteínas Represoras/química , Proteínas Represoras/genética , Arginina/química , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Islas de CpG , Citosina/química , Citosina/metabolismo , ADN/química , ADN/metabolismo , Epigénesis Genética , Mutación , Unión Proteica , Conformación Proteica , Proteínas Represoras/metabolismoRESUMEN
Reprogramming of mammalian genome methylation is critically important but poorly understood. Klf4, a transcription factor directing reprogramming, contains a DNA binding domain with three consecutive C2H2 zinc fingers. Klf4 recognizes CpG or TpG within a specific sequence. Mouse Klf4 DNA binding domain has roughly equal affinity for methylated CpG or TpG, and slightly lower affinity for unmodified CpG. The structural basis for this key preference is unclear, though the side chain of Glu446 is known to contact the methyl group of 5-methylcytosine (5mC) or thymine (5-methyluracil). We examined the role of Glu446 by mutagenesis. Substituting Glu446 with aspartate (E446D) resulted in preference for unmodified cytosine, due to decreased affinity for 5mC. In contrast, substituting Glu446 with proline (E446P) increased affinity for 5mC by two orders of magnitude. Structural analysis revealed hydrophobic interaction between the proline's aliphatic cyclic structure and the 5-methyl group of the pyrimidine (5mC or T). As in wild-type Klf4 (E446), the proline at position 446 does not interact directly with either the 5mC N4 nitrogen or the thymine O4 oxygen. In contrast, the unmethylated cytosine's exocyclic N4 amino group (NH2) and its ring carbon C5 atom hydrogen bond directly with the aspartate carboxylate of the E446D variant. Both of these interactions would provide a preference for cytosine over thymine, and the latter one could explain the E446D preference for unmethylated cytosine. Finally, we evaluated the ability of these Klf4 mutants to regulate transcription of methylated and unmethylated promoters in a luciferase reporter assay.
Asunto(s)
Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Mutación , 5-Metilcitosina/metabolismo , Animales , Islas de CpG , Cristalografía por Rayos X , ADN/metabolismo , Metilación de ADN , Polarización de Fluorescencia , Células HEK293 , Humanos , Enlace de Hidrógeno , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/química , Ratones , Prolina/metabolismo , Conformación Proteica , Timina/metabolismoRESUMEN
Mutations in human zinc-finger transcription factor WT1 result in abnormal development of the kidneys and genitalia and an array of pediatric problems including nephropathy, blastoma, gonadal dysgenesis and genital discordance. Several overlapping phenotypes are associated with WT1 mutations, including Wilms tumors, Denys-Drash syndrome (DDS), Frasier syndrome (FS) and WAGR syndrome (Wilms tumor, aniridia, genitourinary malformations, and mental retardation). These conditions vary in severity from individual to individual; they can be fatal in early childhood, or relatively benign into adulthood. DDS mutations cluster predominantly in zinc fingers (ZF) 2 and 3 at the C-terminus of WT1, which together with ZF4 determine the sequence-specificity of DNA binding. We examined three DDS associated mutations in ZF2 of human WT1 where the normal glutamine at position 369 is replaced by arginine (Q369R), lysine (Q369K) or histidine (Q369H). These mutations alter the sequence-specificity of ZF2, we find, changing its affinity for certain bases and certain epigenetic forms of cytosine. X-ray crystallography of the DNA binding domains of normal WT1, Q369R and Q369H in complex with preferred sequences revealed the molecular interactions responsible for these affinity changes. DDS is inherited in an autosomal dominant fashion, implying a gain of function by mutant WT1 proteins. This gain, we speculate, might derive from the ability of the mutant proteins to sequester WT1 into unproductive oligomers, or to erroneously bind to variant target sequences.
Asunto(s)
Síndrome de Denys-Drash/genética , Epigénesis Genética , Mutación , Proteínas WT1/genética , Proteínas WT1/metabolismo , Adenina/metabolismo , Sustitución de Aminoácidos , Cristalografía por Rayos X , Citosina/química , Citosina/metabolismo , ADN/química , ADN/metabolismo , Glutamina/genética , Guanina/metabolismo , Humanos , Proteínas WT1/químicaRESUMEN
The family of ten-eleven translocation (Tet) dioxygenases is widely distributed across the eukaryotic tree of life, from mammals to the amoeboflagellate Naegleria gruberi. Like mammalian Tet proteins, the Naegleria Tet-like protein, NgTet1, acts on 5-methylcytosine (5mC) and generates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in three consecutive, Fe(II)- and α-ketoglutarate-dependent oxidation reactions. The two intermediates, 5hmC and 5fC, could be considered either as the reaction product of the previous enzymatic cycle or the substrate for the next cycle. Here we present a new crystal structure of NgTet1 in complex with DNA containing a 5hmC. Along with the previously solved NgTet1-5mC structure, the two complexes offer a detailed picture of the active site at individual stages of the reaction cycle. In the crystal, the hydroxymethyl (OH-CH2-) moiety of 5hmC points to the metal center, representing the reaction product of 5mC hydroxylation. The hydroxyl oxygen atom could be rotated away from the metal center, to a hydrophobic pocket formed by Ala212, Val293 and Phe295. Such rotation turns the hydroxyl oxygen atom away from the product conformation, and exposes the target CH2 towards the metal-ligand water molecule, where a dioxygen O2 molecule would occupy to initiate the next round of reaction by abstracting a hydrogen atom from the substrate. The Ala212-to-Val (A212V) mutant profoundly limits the product to 5hmC, probably because the reduced hydrophobic pocket size restricts the binding of 5hmC as a substrate.
Asunto(s)
Citosina/análogos & derivados , ADN/química , Dioxigenasas/química , Naegleria/enzimología , 5-Metilcitosina/química , Sustitución de Aminoácidos , Dominio Catalítico , Citosina/química , Dioxigenasas/genética , Modelos Moleculares , Timina/químicaRESUMEN
One of the most fundamental questions in the control of gene expression in mammals is how the patterns of epigenetic modifications of DNA are generated, recognized, and erased. This includes covalent cytosine methylation of DNA and its associated oxidation states. An array of AdoMet-dependent methyltransferases, Fe(II)- and α-ketoglutarate-dependent dioxygenases, base excision glycosylases, and sequence-specific transcription factors is responsible for changing, maintaining, and 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 thymine (5-methyluracil).
Asunto(s)
5-Metilcitosina/metabolismo , ADN Glicosilasas/metabolismo , Dioxigenasas/metabolismo , Metiltransferasas/metabolismo , Procesamiento Proteico-Postraduccional , Timina/metabolismo , Factores de Transcripción/metabolismo , ADN Glicosilasas/genética , Metilación de ADN , Reparación del ADN , Dioxigenasas/química , Dioxigenasas/genética , Epigénesis Genética , Expresión Génica , Humanos , Hierro/metabolismo , Ácidos Cetoglutáricos/metabolismo , Metiltransferasas/genética , Modelos Moleculares , Oxidación-Reducción , Factores de Transcripción/genéticaRESUMEN
Fragile X syndrome (FXS) results in intellectual disability (ID) most often caused by silencing of the fragile X mental retardation 1 (FMR1) gene. The resulting absence of fragile X mental retardation protein 1 (FMRP) leads to both pre- and postsynaptic defects, yet whether the pre- and postsynaptic functions of FMRP are independent and have distinct roles in FXS neuropathology remain poorly understood. Here, we demonstrate an independent presynaptic function for FMRP through the study of an ID patient with an FMR1 missense mutation. This mutation, c.413G > A (R138Q), preserves FMRP's canonical functions in RNA binding and translational regulation, which are traditionally associated with postsynaptic compartments. However, neuronally driven expression of the mutant FMRP is unable to rescue structural defects at the neuromuscular junction in fragile x mental retardation 1 (dfmr1)-deficient Drosophila, suggesting a presynaptic-specific impairment. Furthermore, mutant FMRP loses the ability to rescue presynaptic action potential (AP) broadening in Fmr1 KO mice. The R138Q mutation also disrupts FMRP's interaction with the large-conductance calcium-activated potassium (BK) channels that modulate AP width. These results reveal a presynaptic- and translation-independent function of FMRP that is linked to a specific subset of FXS phenotypes.
Asunto(s)
Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil , Mutación Missense , Convulsiones , Potenciales de Acción/genética , Sustitución de Aminoácidos , Animales , Niño , Preescolar , Drosophila , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/patología , Síndrome del Cromosoma X Frágil/fisiopatología , Regulación de la Expresión Génica/genética , Humanos , Masculino , Ratones , Convulsiones/genética , Convulsiones/metabolismo , Convulsiones/patología , Convulsiones/fisiopatologíaRESUMEN
Fragile X syndrome, a common cause of intellectual disability and autism, is due to mutational silencing of the FMR1 gene leading to the absence of its gene product, fragile X mental retardation protein (FMRP). FMRP is a selective RNA binding protein owing to two central K-homology domains and a C-terminal arginine-glycine-glycine (RGG) box. However, several properties of the FMRP amino terminus are unresolved. It has been documented for over a decade that the amino terminus has the ability to bind RNA despite having no recognizable functional motifs. Moreover, the amino terminus has recently been shown to bind chromatin and influence the DNA damage response as well as function in the presynaptic space, modulating action potential duration. We report here the amino terminal crystal structures of wild-type FMRP, and a mutant (R138Q) that disrupts the amino terminus function, containing an integral tandem Agenet and discover a novel KH motif.
Asunto(s)
Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/química , Secuencias de Aminoácidos , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Humanos , Mutación Missense , Estructura Terciaria de ProteínaRESUMEN
Arabidopsis thaliana repressor of silencing 1 (ROS1) is a multi-domain bifunctional DNA glycosylase/lyase, which excises 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) as well as thymine and 5-hydroxymethyluracil (i.e., the deamination products of 5mC and 5hmC) when paired with a guanine, leaving an apyrimidinic (AP) site that is subsequently incised by the lyase activity. ROS1 is slow in base excision and fast in AP lyase activity, indicating that the recognition of pyrimidine modifications might be a rate-limiting step. In the C-terminal half, the enzyme harbors a helix-hairpin-helix DNA glycosylase domain followed by a unique C-terminal domain. We show that the isolated glycosylase domain is inactive for base excision but retains partial AP lyase activity. Addition of the C-terminal domain restores the base excision activity and increases the AP lyase activity as well. Furthermore, the two domains remain tightly associated and can be co-purified by chromatography. We suggest that the C-terminal domain of ROS1 is indispensable for the 5mC DNA glycosylase activity of ROS1.
Asunto(s)
5-Metilcitosina/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , ADN Glicosilasas/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Proteínas Nucleares/metabolismo , Animales , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , ADN Glicosilasas/genética , Ratones , Mutación/genética , Proteínas Nucleares/genética , Estructura Terciaria de ProteínaRESUMEN
In mammalian DNA, cytosine occurs in several chemical forms, including unmodified cytosine (C), 5-methylcytosine (5 mC), 5-hydroxymethylcytosine (5 hmC), 5-formylcytosine (5 fC), and 5-carboxylcytosine (5 caC). 5 mC is a major epigenetic signal that acts to regulate gene expression. 5 hmC, 5 fC, and 5 caC are oxidized derivatives that might also act as distinct epigenetic signals. We investigated the response of the zinc finger DNA-binding domains of transcription factors early growth response protein 1 (Egr1) and Wilms tumor protein 1 (WT1) to different forms of modified cytosine within their recognition sequence, 5'-GCG(T/G)GGGCG-3'. Both displayed high affinity for the sequence when C or 5 mC was present and much reduced affinity when 5 hmC or 5 fC was present, indicating that they differentiate primarily oxidized C from unoxidized C, rather than methylated C from unmethylated C. 5 caC affected the two proteins differently, abolishing binding by Egr1 but not by WT1. We ascribe this difference to electrostatic interactions in the binding sites. In Egr1, a negatively charged glutamate conflicts with the negatively charged carboxylate of 5 caC, whereas the corresponding glutamine of WT1 interacts with this group favorably. Our analyses shows that zinc finger proteins (and their splice variants) can respond in modulated ways to alternative modifications within their binding sequence.
Asunto(s)
Citosina/análogos & derivados , Proteínas WT1/química , Proteínas WT1/genética , Proteínas WT1/metabolismo , Cristalización , Citosina/metabolismo , Metilación de ADN , Proteína 1 de la Respuesta de Crecimiento Precoz/química , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Humanos , Modelos Moleculares , Mutación , Oxidación-Reducción , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Transcription factor Krüppel-like factor 4 (Klf4), one of the factors directing cellular reprogramming, recognizes the CpG dinucleotide (whether methylated or unmodified) within a specific G/C-rich sequence. The binding affinity of the mouse Klf4 DNA-binding domain for methylated DNA is only slightly stronger than that for an unmodified oligonucleotide. The structure of the C-terminal three Krüppel-like zinc fingers (ZnFs) of mouse Klf4, in complex with fully methylated DNA, was determined at 1.85 Å resolution. An arginine and a glutamate interact with the methyl group. By comparison with two other recently characterized structures of ZnF protein complexes with methylated DNA, we propose a common principle of recognition of methylated CpG by C2H2 ZnF proteins, which involves a spatially conserved Arg-Glu pair.
Asunto(s)
Metilación de ADN , Factores de Transcripción de Tipo Kruppel/química , Secuencia de Aminoácidos , Animales , Arginina/química , Islas de CpG , Ácido Glutámico/química , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Datos de Secuencia Molecular , Unión Proteica , Alineación de Secuencia , Factores de Transcripción/químicaRESUMEN
Cytosine residues in mammalian DNA occur in five forms: cytosine (C), 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The ten-eleven translocation (Tet) dioxygenases convert 5mC to 5hmC, 5fC and 5caC in three consecutive, Fe(II)- and α-ketoglutarate-dependent oxidation reactions. The Tet family of dioxygenases is widely distributed across the tree of life, including in the heterolobosean amoeboflagellate Naegleria gruberi. The genome of Naegleria encodes homologues of mammalian DNA methyltransferase and Tet proteins. Here we study biochemically and structurally one of the Naegleria Tet-like proteins (NgTet1), which shares significant sequence conservation (approximately 14% identity or 39% similarity) with mammalian Tet1. Like mammalian Tet proteins, NgTet1 acts on 5mC and generates 5hmC, 5fC and 5caC. The crystal structure of NgTet1 in complex with DNA containing a 5mCpG site revealed that NgTet1 uses a base-flipping mechanism to access 5mC. The DNA is contacted from the minor groove and bent towards the major groove. The flipped 5mC is positioned in the active-site pocket with planar stacking contacts, Watson-Crick polar hydrogen bonds and van der Waals interactions specific for 5mC. The sequence conservation between NgTet1 and mammalian Tet1, including residues involved in structural integrity and functional significance, suggests structural conservation across phyla.
Asunto(s)
5-Metilcitosina/metabolismo , ADN/química , ADN/metabolismo , Dioxigenasas/química , Dioxigenasas/metabolismo , Naegleria/enzimología , 5-Metilcitosina/química , Secuencia de Aminoácidos , Animales , Dominio Catalítico/genética , Secuencia Conservada , Cristalografía por Rayos X , Citosina/análogos & derivados , Citosina/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/química , Células HEK293 , Humanos , Enlace de Hidrógeno , Ratones , Oxigenasas de Función Mixta/química , Modelos Moleculares , Datos de Secuencia Molecular , Naegleria/genética , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Homología Estructural de Proteína , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
DNA methyltransferases (DNMTs) are important enzymes involved in epigenetic control of gene expression and represent valuable targets in cancer chemotherapy. A number of nucleoside DNMT inhibitors (DNMTi) have been studied in cancer, including in cancer stem cells, and two of them (azacytidine and decitabine) have been approved for treatment of myelodysplastic syndromes. However, only a few non-nucleoside DNMTi have been identified so far, and even fewer have been validated in cancer. Through a process of hit-to-lead optimization, we report here the discovery of compound 5 as a potent non-nucleoside DNMTi that is also selective toward other AdoMet-dependent protein methyltransferases. Compound 5 was potent at single-digit micromolar concentrations against a panel of cancer cells and was less toxic in peripheral blood mononuclear cells than two other compounds tested. In mouse medulloblastoma stem cells, 5 inhibited cell growth, whereas related compound 2 showed high cell differentiation. To the best of our knowledge, 2 and 5 are the first non-nucleoside DNMTi tested in a cancer stem cell line.