RESUMEN
T-cell acute lymphoblastic leukemia (T-ALL) protein 1 (TAL1) is a central transcription factor in hematopoiesis. The timing and level of TAL1 expression orchestrate the differentiation to specialized blood cells and its overexpression is a common cause of T-ALL. Here, we studied the 2 protein isoforms of TAL1, short and long, which are generated by the use of alternative promoters as well as by alternative splicing. We analyzed the expression of each isoform by deleting an enhancer or insulator, or by opening chromatin at the enhancer location. Our results show that each enhancer promotes expression from a specific TAL1 promoter. Expression from a specific promoter gives rise to a unique 5' UTR with differential regulation of translation. Moreover, our study suggests that the enhancers regulate TAL1 exon 3 alternative splicing by inducing changes in the chromatin at the splice site, which we demonstrate is mediated by KMT2B. Furthermore, our results indicate that TAL1-short binds more strongly to TAL1 E-protein partners and functions as a stronger transcription factor than TAL1-long. Specifically TAL1-short has a unique transcription signature promoting apoptosis. Finally, when we expressed both isoforms in mice bone marrow, we found that while overexpression of both isoforms prevents lymphoid differentiation, expression of TAL1-short alone leads to hematopoietic stem cell exhaustion. Furthermore, we found that TAL1-short promoted erythropoiesis and reduced cell survival in the CML cell line K562. While TAL1 and its partners are considered promising therapeutic targets in the treatment of T-ALL, our results show that TAL1-short could act as a tumor suppressor and suggest that altering TAL1 isoform's ratio could be a preferred therapeutic approach.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Animales , Ratones , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cromatina , Hematopoyesis/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Isoformas de Proteínas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteína 1 de la Leucemia Linfocítica T Aguda/genética , Factores de Transcripción/metabolismoRESUMEN
Splicing of precursor mRNA (pre-mRNA) is an important regulatory step in gene expression. Recent evidence points to a regulatory role of chromatin-related proteins in alternative splicing regulation. Using an unbiased approach, we have identified the acetyltransferase p300 as a key chromatin-related regulator of alternative splicing. p300 promotes genome-wide exon inclusion in both a transcription-dependent and -independent manner. Using CD44 as a paradigm, we found that p300 regulates alternative splicing by modulating the binding of splicing factors to pre-mRNA. Using a tethering strategy, we found that binding of p300 to the CD44 promoter region promotes CD44v exon inclusion independently of RNAPII transcriptional elongation rate. Promoter-bound p300 regulates alternative splicing by acetylating splicing factors, leading to exclusion of hnRNP M from CD44 pre-mRNA and activation of Sam68. p300-mediated CD44 alternative splicing reduces cell motility and promotes epithelial features. Our findings reveal a chromatin-related mechanism of alternative splicing regulation and demonstrate its impact on cellular function.
Asunto(s)
Empalme Alternativo , Neoplasias de la Mama/genética , Cromatina/metabolismo , Proteína p300 Asociada a E1A/metabolismo , Regulación Neoplásica de la Expresión Génica , Receptores de Hialuranos/genética , Factores de Empalme de ARN/química , Acetilación , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cromatina/genética , Proteína p300 Asociada a E1A/genética , Exones , Femenino , Humanos , Receptores de Hialuranos/metabolismo , Regiones Promotoras Genéticas , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Enhancer demethylation in leukemia has been shown to lead to overexpression of genes which promote cancer characteristics. The vascular endothelial growth factor A (VEGFA) enhancer, located 157 Kb downstream of its promoter, is demethylated in chronic myeloid leukemia (CML). VEGFA has several alternative splicing isoforms with different roles in cancer progression. Since transcription and splicing are coupled, we wondered whether VEGFA enhancer activity can also regulate the gene's alternative splicing to contribute to the pathology of CML. Our results show that mutating the VEGFA +157 enhancer promotes exclusion of exons 6a and 7 and activating the enhancer by tethering a chromatin activator has the opposite effect. In line with these results, CML patients present with high expression of +157 eRNA and inclusion of VEGFA exons 6a and 7. In addition, our results show that the positive regulator of RNAPII transcription elongation, CCNT2, binds VEGFA's promoter and enhancer, and its silencing promotes exclusion of exons 6a and 7 as it slows down RNAPII elongation rate. Thus our results suggest that VEGFA's +157 enhancer regulates its alternative splicing by increasing RNAPII elongation rate via CCNT2. Our work demonstrates for the first time a connection between an endogenous enhancer and alternative splicing regulation of its target gene.