RESUMEN
Non-coding RNAs are much more common than previously thought. However, for the vast majority of non-coding RNAs, the cellular function remains enigmatic. The two long non-coding RNA (lncRNA) genes DLEU1 and DLEU2 map to a critical region at chromosomal band 13q14.3 that is recurrently deleted in solid tumors and hematopoietic malignancies like chronic lymphocytic leukemia (CLL). While no point mutations have been found in the protein coding candidate genes at 13q14.3, they are deregulated in malignant cells, suggesting an epigenetic tumor suppressor mechanism. We therefore characterized the epigenetic makeup of 13q14.3 in CLL cells and found histone modifications by chromatin-immunoprecipitation (ChIP) that are associated with activated transcription and significant DNA-demethylation at the transcriptional start sites of DLEU1 and DLEU2 using 5 different semi-quantitative and quantitative methods (aPRIMES, BioCOBRA, MCIp, MassARRAY, and bisulfite sequencing). These epigenetic aberrations were correlated with transcriptional deregulation of the neighboring candidate tumor suppressor genes, suggesting a coregulation in cis of this gene cluster. We found that the 13q14.3 genes in addition to their previously known functions regulate NF-kB activity, which we could show after overexpression, siRNA-mediated knockdown, and dominant-negative mutant genes by using Western blots with previously undescribed antibodies, by a customized ELISA as well as by reporter assays. In addition, we performed an unbiased screen of 810 human miRNAs and identified the miR-15/16 family of genes at 13q14.3 as the strongest inducers of NF-kB activity. In summary, the tumor suppressor mechanism at 13q14.3 is a cluster of genes controlled by two lncRNA genes that are regulated by DNA-methylation and histone modifications and whose members all regulate NF-kB. Therefore, the tumor suppressor mechanism in 13q14.3 underlines the role both of epigenetic aberrations and of lncRNA genes in human tumorigenesis and is an example of colocalization of a functionally related gene cluster.
Asunto(s)
Metilación de ADN/genética , Leucemia , ARN Largo no Codificante , Proteínas Supresoras de Tumor , Adulto , Anciano , Anciano de 80 o más Años , Transformación Celular Neoplásica , Cromatina/genética , Cromosomas Humanos Par 13/genética , Regulación hacia Abajo , Epigénesis Genética/genética , Femenino , Células HEK293 , Humanos , Leucemia/sangre , Leucemia/genética , Leucemia/fisiopatología , Masculino , Persona de Mediana Edad , Mutación , FN-kappa B/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Sitio de Iniciación de la Transcripción , Transferasas , Proteínas Supresoras de Tumor/sangre , Proteínas Supresoras de Tumor/genética , Regulación hacia ArribaRESUMEN
Loss of a critical region in 13q14.3 [del(13q)] is the most common genomic aberration in chronic lymphocytic leukemia (CLL), occurring in more than 50% of patients (Stilgenbauer et al., Oncogene 1998;16:1891 - 1897, Dohner et al., N Engl J Med 2000;343:1910 - 1916). Despite extensive investigations, no point mutations have been found in the remaining allele that would inactivate one of the candidate tumor suppressor genes and explain the pathomechanism postulated for this region. However, the genes in the region are significantly down-regulated in CLL cells, more than would be expected by gene dosage, and recently a complex epigenetic regulatory mechanism was identified for 13q14.3 in non-malignant cells that involves asynchronous replication timing and monoallelic expression of candidate tumor suppressor genes. Here, we propose a model of a multigenic pathomechanism in 13q14.3, where several tumor suppressor genes, including the miRNA genes miR-16-1 and miR-15a, are co-regulated by the two long non-coding RNA genes DLEU1 and DLEU2 that span the critical region. Furthermore, we propose these co-regulated genes to be involved in the same molecular pathways, thereby also forming a functional gene cluster. Elucidating the molecular and cellular function of the 13q14.3 candidate genes will shed light on the underlying pathomechanism of CLL.