RESUMO
The LAT gene encodes an adaptor molecule that links receptor engagement to critical downstream signaling events. Previously, we identified the proximal promoter for the human LAT gene and found that it contains binding sites for members of the Ets and Runx transcription factor families. In the present study, we show that the promoter also contains 5 GC-rich elements that contribute to promoter activity and that are capable of binding the transcription factors Sp1 and Sp3. Overexpression of either Sp1 or full-length Sp3 was shown to augment LAT promoter activity, while siRNA-mediated knockdown of each transcription factor was demonstrated to have an inhibitory effect. We also discovered a cell-type specific DNase hypersensitive site that maps to the Sp1/Sp3 and adjacent Ets and Runx binding sites. Collectively, these results provide compelling data that implicates Sp1 and Sp3 in the transcriptional regulation of the human LAT gene.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Membrana/genética , Elementos Reguladores de Transcrição , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , DNA/genética , DNA/metabolismo , Drosophila , Sequência Rica em GC , Células HeLa , Humanos , Células Jurkat , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , Fator de Transcrição Sp1/antagonistas & inibidores , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3/antagonistas & inibidores , Fator de Transcrição Sp3/genética , Transcrição GênicaRESUMO
BACKGROUND: The LAT gene encodes an intracellular adaptor protein that links cell-surface receptor engagement to numerous downstream signalling events, and thereby plays an integral role in the function of cell types that express the gene, including T cells, mast cells, natural killer cells, and platelets. To date, the mechanisms responsible for the transcriptional regulation of this gene have not been investigated. RESULTS: In this study we have mapped the transcriptional start sites for the human LAT gene and localized the 5' and 3' boundaries of the proximal promoter. We find that the promoter contains both positive and negative regulatory regions, and that two binding sites for the Ets family of transcription factors have a strong, positive effect on gene expression. Each site binds the Ets family member Elf-1, and overexpression of Elf-1 augments LAT promoter activity. The promoter also contains a Runx binding site adjacent to one of the Ets sites. This site, which is shown to bind Runx-1, has an inhibitory effect on gene expression. Finally, data is also presented indicating that the identified promoter may regulate cell-type specific expression. CONCLUSION: Collectively, these results provide the first insights into the transcriptional regulation of the LAT gene, including the discovery that the Ets transcription factor Elf-1 may play a central role in its expression.