RESUMO
DNA fragmentation in apoptosis, especially in lymphocytic cells, is initiated at scaffold/matrix attachment regions (S/MARs) and is preceded by the degradation of nuclear proteins. The present study was performed to establish whether the same mechanism occurred in human NT2 cells subjected to oxygen and glucose deprivation (OGD). We analyzed the integrity of c-myc S/MAR containing a base-unpairing region (BUR)-like element, which we established to be a binding site of the transcription factor Sox2. An accumulation of DNA breaks in close proximity to this element and a degradation of Sox2 were observed early in the OGD-induced apoptotic response. Identification of Sox2 as a novel c-myc BUR-binding protein was achieved through yeast one-hybrid screening and the Sox2/DNA interaction was confirmed by electrophoretic mobility shift assay and immunoprecipitation with Sox2 antibody. Our data support the notion that early proteolysis of unique BUR-binding proteins might represent a universal mechanism that renders these DNA sites vulnerable to endonucleolysis.
Assuntos
Apoptose/fisiologia , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genes myc/fisiologia , Proteínas HMGB/metabolismo , Regiões de Interação com a Matriz/fisiologia , Neurônios/citologia , Fatores de Transcrição/metabolismo , Apoptose/genética , Sequência de Bases , Carcinoma Embrionário/genética , Carcinoma Embrionário/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/genética , Genes myc/genética , Proteínas HMGB/genética , Humanos , Células Jurkat , Regiões de Interação com a Matriz/genética , Neurônios/metabolismo , Reação em Cadeia da Polimerase , Fatores de Transcrição SOXB1 , Fatores de Transcrição/genéticaRESUMO
Apoptotic DNA degradation could be initiated by the accumulation of single-strand (ss) breaks in vulnerable chromatin regions, such as base unpairing regions (BURs), which might be preferentially targeted for degradation by both proteases and nucleases. We tested this hypothesis in anti-Fas-treated apoptotic Jurkat cells. Several nuclear proteins known for their association with both MARs and the nuclear matrix, that is, PARP, NuMA, lamin B and SATB1, were degraded, but the morphological rearrangement of the BUR-binding SATB1 protein was one of the earliest detected changes. Subsequently, we have identified several genes containing sequences homologous to the 25 bp BUR element of the IgH gene, a known SATB1-binding site, and examined the integrity of genomic DNA in their vicinity. Multiple ss breaks were found in close proximity to these sites relative to adjacent regions of DNA. Consistent with our prediction, the results indicated that the initiation of DNA cleavage in anti-Fas-treated Jurkat cells occurred within the BUR sites, which likely became accessible to endonucleases due to the degradation of BUR-binding proteins.
