RESUMEN
SET and RING-finger-associated (SRA) domain is involved in establishment and maintenance of DNA methylation in eukaryotes. Proteins containing SRA domains exist in mammals, plants, even microorganisms. It has been established that mammalian SRA domain recognizes 5-methylcytosine (5mC) through a base-flipping mechanism. Here, we identified and characterized two SRA domain-containing proteins with the common domain architecture of N-terminal SRA domain and C-terminal HNH nuclease domain, Sco5333 from Streptomyces coelicolor and Tbis1 from Thermobispora bispora. Both sco5333 and tbis1 cannot establish in methylated Escherichia coli hosts (dcm(+)), and this in vivo toxicity requires both SRA and HNH domain. Purified Sco5333 and Tbis1 displayed weak DNA cleavage activity in the presence of Mg(2+), Mn(2+) and Co(2+) and the cleavage activity was suppressed by Zn(2+). Both Sco5333 and Tbis1 bind to 5mC-containing DNA in all sequence contexts and have at least a preference of 100 folds in binding affinity for methylated DNA over non-methylated one. We suggest that linkage of methyl-specific SRA domain and weakly active HNH domain may represent a universal mechanism in competing alien methylated DNA but to maximum extent minimizing damage to its own chromosome.
Asunto(s)
5-Metilcitosina/metabolismo , Proteínas Bacterianas/metabolismo , División del ADN , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Actinomycetales , Proteínas Bacterianas/química , Cationes Bivalentes , Proteínas de Unión al ADN/química , Endodesoxirribonucleasas/química , Unión Proteica , Estructura Terciaria de Proteína , Streptomyces coelicolorRESUMEN
MspJI is a novel modification-dependent restriction endonuclease that cleaves at a fixed distance away from the modification site. Here, we present the biochemical characterization of several MspJI homologs, including FspEI, LpnPI, AspBHI, RlaI, and SgrTI. All of the enzymes specifically recognize cytosine C5 modification (methylation or hydroxymethylation) in DNA and cleave at a constant distance (N(12)/N(16)) away from the modified cytosine. Each displays its own sequence context preference, favoring different nucleotides flanking the modified cytosine. By cleaving on both sides of fully modified CpG sites, they allow the extraction of 32-base long fragments around the modified sites from the genomic DNA. These enzymes provide powerful tools for direct interrogation of the epigenome. For example, we show that RlaI, an enzyme that prefers (m)CWG but not (m)CpG sites, generates digestion patterns that differ between plant and mammalian genomic DNA, highlighting the difference between their epigenomic patterns. In addition, we demonstrate that deep sequencing of the digested DNA fragments generated from these enzymes provides a feasible method to map the modified sites in the genome. Altogether, the MspJI family of enzymes represent appealing tools of choice for method development in DNA epigenetic studies.
Asunto(s)
Enzimas de Restricción del ADN , Epigénesis Genética , Epigenómica/métodos , Técnicas Genéticas , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Mapeo Cromosómico/métodos , Biología Computacional , ADN/química , ADN/genética , ADN/aislamiento & purificación , Metilación de ADN , Enzimas de Restricción del ADN/genética , Enzimas de Restricción del ADN/metabolismo , Biblioteca de Genes , Células HeLa , Humanos , Células Jurkat , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Properly regulated apoptosis in the developing central nervous system is crucial for normal morphogenesis and homeostasis. In Drosophila, a subset of neural stem cells, or neuroblasts, undergo apoptosis during embryogenesis. Of the 30 neuroblasts initially present in each abdominal hemisegment of the embryonic ventral nerve cord, only three survive into larval life, and these undergo apoptosis in the larvae. Here, we use loss-of-function analysis to demonstrate that neuroblast apoptosis during embryogenesis requires the coordinated expression of the cell death genes grim and reaper, and possibly sickle. These genes are clustered in a 140 kb region of the third chromosome and show overlapping patterns of expression. We show that expression of grim, reaper and sickle in embryonic neuroblasts is controlled by a common regulatory region located between reaper and grim. In the absence of grim and reaper, many neuroblasts survive the embryonic period of cell death and the ventral nerve cord becomes massively hypertrophic. Deletion of grim alone blocks the death of neuroblasts in the larvae. The overlapping activity of these multiple cell death genes suggests that the coordinated regulation of their expression provides flexibility in this crucial developmental process.
Asunto(s)
Apoptosis/genética , Proteínas de Drosophila/genética , Drosophila/embriología , Regulación del Desarrollo de la Expresión Génica , Células-Madre Neurales/fisiología , Neuropéptidos/genética , Animales , Supervivencia Celular , Sistema Nervioso Central/embriología , Proteínas de Drosophila/biosíntesis , Técnicas de Inactivación de Genes , Hibridación Fluorescente in Situ , Células-Madre Neurales/citología , Neuropéptidos/biosíntesis , Reacción en Cadena de la Polimerasa , Regiones Promotoras GenéticasRESUMEN
TSC22D1, which encodes transforming growth factor beta-stimulated clone 22 (TSC-22), is thought to be a tumor suppressor because its expression is lost in many glioblastoma, salivary gland, and prostate cancers. TSC-22 is the founding member of the TSC-22/DIP/Bun family of leucine zipper transcription factors; its functions have not been investigated in a multicellular environment. Genetic studies in the model organism Drosophila melanogaster often provide fundamental insights into mechanisms disrupted in carcinogenesis, because of the strong evolutionary conservation of molecular mechanisms between flies and humans. Whereas humans and mice have four TSC-22 domain genes with numerous isoforms, Drosophila has only one TSC-22 domain gene, bunched (bun), which encodes both large and small protein isoforms. Surprisingly, Drosophila Bun proteins promote cellular growth and proliferation in ovarian follicle cells. Loss of both large isoforms has the strongest phenotypes, including increased apoptosis. Cultured S2 cells depleted for large Bun isoforms show increased apoptosis and less frequent cell division, with decreased cell size. Altogether, these data indicate that Drosophila TSC-22/DIP/Bun proteins are necessary for cellular growth, proliferation, and survival both in culture and in an epithelial context. Previous work demonstrated that bun prevents recruitment of epithelial cells to a migratory fate and, thus, maintains epithelial organization. We speculate that reduced TSC22D1 expression generally reduces cellular fitness and only contributes to carcinogenesis in specific tissue environments.