RESUMO
The human transcription factor Sp3 has been widely studied at the translational level and has been described as a regulatory factor for a number of genes. Sp3 is currently characterized as a bifunctional transcription factor having the ability to behave as both an activator and/or a repressor in various promoter regions. Previous translational studies have attempted to determine the basis for these diverse functions with mostly contradictory evidence to date. Little data are available, however, concerning genomic structure, full-length cDNA, potential transcript variants, or location of translation initiation sites for the large isoform of the Sp3 gene. In this study, bacterial artificial chromosome (BAC) sequencing, reverse transcription-polymerase chain reaction (RT-PCR), genomic PCR, and database mining indicate that the Sp3 gene encompasses seven exons spanning more than 55 kb of genomic DNA on Chromosome 2. The 5' end of this sequence contains a large CpG island. This work also detected a processed pseudogene, psiSp3, located on Chromosome 13, spanning approximately 4.0 kb. Northern blot analysis detected three predominant transcripts at 4.0, 6.0 and 2.5 kb. Sequence analysis indicated that alternative splicing of exon 3 allows for multiple transcripts of Sp3. Each sequenced transcript possesses three to five potential translation initiation sites. This diversity at the level of gene expression will likely be key to understanding the diverse functions of Sp3.
Assuntos
Processamento Alternativo , Proteínas de Ligação a DNA/genética , Pseudogenes/genética , Fatores de Transcrição/genética , Sequência de Bases , Northern Blotting , Linhagem Celular Tumoral , Cromossomos Humanos Par 13/genética , Cromossomos Humanos Par 2/genética , Clonagem Molecular , DNA/química , DNA/genética , DNA Complementar/química , DNA Complementar/genética , Éxons , Perfilação da Expressão Gênica , Genes/genética , Células HeLa , Humanos , Íntrons , Células Jurkat , Masculino , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Isoformas de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Fator de Transcrição Sp3 , Transcrição Gênica/genéticaRESUMO
We previously reported the lack of expression of the bifunctional transcription factor Sp3 in peripheral blood mononuclear cells from most patients with multiple sclerosis (MS) (Grekova et al, 1996). An RT-PCR technique was developed to evaluate Sp3 mRNA levels in peripheral blood mononuclear cell subsets. Semi-quantitative and quantitative competitive RT-PCR assays were used to compare the level of Sp3 expression among subjects and among immune cell subsets. The competitor DNA fragment contained a deletion from the normal Sp3 cDNA sequence. The wild-type Sp3 cDNA and the competitor DNA fragment amplified with equal efficiency, and the two PCR products were distinguished by size. These studies demonstrated that normal CD4(+) and CD8(+) T cells, B cells, and macrophages expressed comparable amounts of Sp3 mRNA. No Sp3 expression could be detected in normal natural killer cells nor in any of these cell types from Sp3-negative MS patients. We propose that transcription of the Sp3 gene is blocked in immune cells from most patients with MS and that this contributes to the development of central nervous system inflammation in the disease.