RESUMEN
We investigated in different human cell types nuclear positioning and transcriptional regulation of the functionally unrelated genes GASZ, CFTR, and CORTBP2, mapping to adjacent loci on human chromosome 7q31. When inactive, GASZ, CFTR, and CORTBP2 preferentially associated with the nuclear periphery and with perinuclear heterochromatin, whereas in their actively transcribed states the gene loci preferentially associated with euchromatin in the nuclear interior. Adjacent genes associated simultaneously with these distinct chromatin fractions localizing at different nuclear regions, in accordance with their individual transcriptional regulation. Although the nuclear localization of CFTR changed after altering its transcription levels, the transcriptional status of CFTR was not changed by driving this gene into a different nuclear environment. This implied that the transcriptional activity affected the nuclear positioning, and not vice versa. Together, the results show that small chromosomal subregions can display highly flexible nuclear organizations that are regulated at the level of individual genes in a transcription-dependent manner.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Proteínas del Tejido Nervioso/genética , Transcripción Genética , Proteínas Portadoras/metabolismo , Línea Celular , Cromatina/metabolismo , Cromosomas Humanos Par 7 , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Eucromatina/metabolismo , Regulación de la Expresión Génica , Heterocromatina/metabolismo , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Proteínas de Transporte de Membrana , Microscopía Confocal , Proteínas del Tejido Nervioso/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Classical gene therapy for cystic fibrosis has had limited success because of immune response against viral vectors and short-term expression of cDNA-based transgenes. These limitations could be overcome by delivering the complete genomic CFTR gene on nonintegrating human artificial chromosomes (HACs). Here, we report reconstruction of the genomic CFTR locus and analyze incorporation into HACs of three P1 phage-based and F factor bacteria-based artificial chromosomes (PACs/BACs) of various sizes: (1) 5A, a large, nonselectable BAC containing the entire wild-type CFTR locus extending into both adjacent genes (296.8-kb insert, from kb -58.4 to +51.4) containing all regulators; (2) CGT21, a small, selectable, telomerized PAC (134.7 kb, from kb -60.7 to + 2) containing a synthetic last exon joining exon 10, EGFP, exon 24, and the 3' untranslated region; and (3) CF225, a midsized, nonselectable PAC (225.3 kb, from kb -60.7 to +9.8) ligated from two PACs with optimized codons and a silent XmaI restriction variant to discriminate transgene from endogenous expression. Cotransfection with telomerized, blasticidin-S-selectable, centromere-proficient α-satellite constructs into HT1080 cells revealed a workable HAC formation rate of 1 per â¼25 lines when using CGT21 or 5A. CF225 was not incorporated into a de novo HAC in 122 lines analyzed, but integrants were expressed. Stability analyses suggest the feasibility of prefabricating a large, tagged CFTR transgene that stably replicates in the proximity of a functional centromere. Although definite conclusions about HAC-proficient construct configurations cannot be drawn at this stage, important transfer resources were generated and characterized, demonstrating the promise of de novo HACs as potentially ideal gene therapy vector systems.