RESUMEN
During class switch recombination (CSR), antigen-stimulated B-cells rearrange their immunoglobulin constant heavy chain (CH) loci to generate antibodies with different effector functions. CSR is initiated by activation-induced deaminase (AID), which converts cytosines in switch (S) regions, repetitive sequences flanking the CH loci, to uracils. Although U/G mispairs arising in this way are generally efficiently repaired to C/Gs by uracil DNA glycosylase (UNG)-initiated base excision repair (BER), uracil processing in S-regions of activated B-cells occasionally gives rise to double strand breaks (DSBs), which trigger CSR. Surprisingly, genetic experiments revealed that CSR is dependent not only on AID and UNG, but also on mismatch repair (MMR). To elucidate the role of MMR in CSR, we studied the processing of uracil-containing DNA substrates in extracts of MMR-proficient and -deficient human cells, as well as in a system reconstituted from recombinant BER and MMR proteins. Here, we show that the interplay of these repair systems gives rise to DSBs in vitro and to genomic deletions and mutations in vivo, particularly in an S-region sequence. Our findings further suggest that MMR affects pathway choice in DSB repair. Given its amenability to manipulation, our system represents a powerful tool for the molecular dissection of CSR.
Asunto(s)
Linfocitos B/metabolismo , Reparación de la Incompatibilidad de ADN/inmunología , ADN/genética , Cambio de Clase de Inmunoglobulina/genética , Regiones Constantes de Inmunoglobulina/genética , Uracilo/metabolismo , Linfocitos B/citología , Linfocitos B/inmunología , Línea Celular Tumoral , Citidina Desaminasa/genética , Citidina Desaminasa/inmunología , Citosina/inmunología , Citosina/metabolismo , ADN/inmunología , Roturas del ADN de Doble Cadena , Regulación de la Expresión Génica , Células HEK293 , Humanos , Transducción de Señal , Uracilo/inmunología , Uracil-ADN Glicosidasa/genética , Uracil-ADN Glicosidasa/inmunologíaRESUMEN
The enhancer/promoter of the vitellogenin II gene (VTG) has been extensively studied as a model system of vertebrate transcriptional control. While deletion mutagenesis and in vivo footprinting identified the transcription factor (TF) binding sites governing its tissue specificity, DNase hypersensitivity and DNA methylation studies revealed the epigenetic changes accompanying its hormone-dependent activation. Moreover, upon induction with estrogen (E2), the region flanking the estrogen-responsive element (ERE) was reported to undergo active DNA demethylation. We now show that although the VTG ERE is methylated in embryonic chicken liver and in LMH/2A hepatocytes, its induction by E2 was not accompanied by extensive demethylation. In contrast, E2 failed to activate a VTG enhancer/promoter-controlled luciferase reporter gene methylated by SssI. Surprisingly, this inducibility difference could be traced not to the ERE but rather to a single CpG in an E-box (CACGTG) sequence upstream of the VTG TATA box, which is unmethylated in vivo but methylated by SssI. We demonstrate that this E-box binds the upstream stimulating factor USF1/2. Selective methylation of the CpG within this binding site with an E-box-specific DNA methyltransferase, Eco72IM, was sufficient to attenuate USF1/2 binding in vitro and abolish the hormone-induced transcription of the VTG gene in the reporter system.
Asunto(s)
Expresión Génica Ectópica/genética , Receptor alfa de Estrógeno/genética , Vitelogeninas/genética , Animales , Sitios de Unión , Línea Celular , Embrión de Pollo , Islas de CpG/genética , Metilación de ADN/genética , Proteínas de Unión al ADN/metabolismo , ADN-Citosina Metilasas/metabolismo , Expresión Génica Ectópica/efectos de los fármacos , Receptor alfa de Estrógeno/metabolismo , Estrógenos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/metabolismo , Vitelogeninas/metabolismoRESUMEN
The cytotoxicity of SN1-type alkylating agents such as N-methyl-N'-nitrosourea (MNU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), or the cancer chemotherapeutics temozolomide, dacarbazine and streptozotocin has been ascribed to the persistence of O(6)-methylguanine ((me)G) in genomic DNA. One hypothesis posits that (me)G toxicity is caused by futile attempts of the mismatch repair (MMR) system to process (me)G/C or (me)G/T mispairs arising during replication, while an alternative proposal suggests that the latter lesions activate DNA damage signaling, cell cycle arrest and apoptosis directly. Attempts to elucidate the molecular mechanism of (me)G-induced cell killing in vivo have been hampered by the fact that the above reagents induce several types of modifications in genomic DNA, which are processed by different repair pathways. In contrast, defined substrates studied in vitro did not undergo replication. We set out to re-examine this phenomenon in replication-competent Xenopus laevis egg extracts, using either phagemid substrates containing a single (me)G residue, or methylated sperm chromatin. Our findings provide further support for the futile cycling hypothesis.