RESUMEN
The murine complement receptor type 2 gene (Cr2/CD21) is expressed by murine B and follicular dendritic cells, but not murine T cells. We have previously shown that appropriate transcriptional control of the CD21 gene requires the CD21 promoter as well as intronic sequences. We have also demonstrated that altering chromatin structure by inhibiting histone deacetylases induces CD21 expression in murine T cells by increasing the accessibility of promoter and intronic regulatory elements. In this report, we identify seven distinct regulatory areas within the first intron of the murine CD21 gene that are conserved between mouse and human CD21 intronic sequences. EMSA competition and supershift analyses reveal the formation of multiple DNA-protein complexes at these sites that include Yin Yang 1, Oct1, and NFAT-4. NFAT-containing complexes were altered in B cells treated with the NFAT inhibitor cyclosporin A and correlated with a repression of CD21 gene transcription implicating NFAT transcriptional control. Functional data revealed that no single region conferred cell-specific reporter gene expression, but rather the entire CD21 regulatory element was required to confer cell-specific gene expression. Taken together, these data demonstrate the formation of repeating, overlapping regulatory modules, all of which are required to coordinately control the cell-specific expression of the murine CD21 gene. We propose a model in which Yin Yang 1 and Oct1 may recruit histone deacetylase to multiple sites in the CD21 intronic regulatory element in nonexpressing cells and NFAT either displaces this histone deacetylase or recruits a histone acetylase to allow the formation of a functional transcriptional complex in expressing cells.
Asunto(s)
Ciclosporina/farmacología , Proteínas de Unión al ADN/genética , Intrones/inmunología , Región de Control de Posición/inmunología , Transportador 1 de Catión Orgánico/genética , Receptores de Complemento 3d/genética , Secuencias Repetitivas de Ácidos Nucleicos/efectos de los fármacos , Factores de Transcripción/genética , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Secuencia de Bases , Sitios de Unión/efectos de los fármacos , Sitios de Unión/genética , Sitios de Unión/inmunología , Proteínas de Unión al ADN/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Humanos , Intrones/efectos de los fármacos , Región de Control de Posición/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Factores de Transcripción NFATC , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transportador 1 de Catión Orgánico/metabolismo , Receptores de Complemento 3d/antagonistas & inhibidores , Receptores de Complemento 3d/biosíntesis , Homología de Secuencia de Ácido Nucleico , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Factor de Transcripción YY1RESUMEN
In a high proportion of Burkitt lymphomas, transcription of the c-myc gene is initiated from a cryptic promoter in the first intron, creating abnormal messenger RNA molecules in which intron sequences, normally spliced out of the nascent transcripts, persist. An antisense oligodeoxynucleotide directed against these intron sequences greatly inhibited the proliferation of Burkitt lymphoma cell lines containing the abnormal transcripts (ST486 and JD38), but not that of cell lines containing normal c-myc transcripts (KK124). Flow cytometry showed a pronounced reduction in intracellular c-myc protein levels in cell lines containing aberrant myc transcripts, but no change in other cellular proteins. Control oligonucleotide did not inhibit c-myc protein expression or growth. These experiments provide evidence that antisense oligonucleotides targeted against tumour-specific, aberrant RNA species could be effective in controlling the proliferation of tumour cells without affecting normal cells.