RESUMEN
Given the intimate link between inflammation and dysregulated cell proliferation in cancer, we investigated cytokine-triggered gene expression in different cell cycle stages. Transcriptome analysis revealed that G1 release through cyclin-dependent kinase 6 (CDK6) and CDK4 primes and cooperates with the cytokine-driven gene response. CDK6 physically and functionally interacts with the NF-κB subunit p65 in the nucleus and is found at promoters of many transcriptionally active NF-κB target genes. CDK6 recruitment to distinct chromatin regions of inflammatory genes was essential for proper loading of p65 to its cognate binding sites and for the function of p65 coactivators, such as TRIP6. Furthermore, cytokine-inducible nuclear translocation and chromatin association of CDK6 depends on the kinase activity of TAK1 and p38. These results have widespread biological implications, as aberrant CDK6 expression or activation that is frequently observed in human tumors modulates NF-κB to shape the cytokine and chemokine repertoires in chronic inflammation and cancer.
Asunto(s)
Cromatina/metabolismo , Quinasa 6 Dependiente de la Ciclina/fisiología , FN-kappa B/genética , Ciclo Celular/genética , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/fisiología , Quinasa 6 Dependiente de la Ciclina/análisis , Quinasa 6 Dependiente de la Ciclina/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Interleucina-1/metabolismo , Interleucina-1/fisiología , Interleucina-8/genética , Interleucina-8/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/fisiología , Regiones Promotoras Genéticas , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Factor de Transcripción ReIA/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
The inflammatory gene response requires activation of the protein kinase TAK1, but it is currently unknown how TAK1-derived signals coordinate transcriptional programs in the genome. We determined the genome-wide binding of the TAK1-controlled NF-κB subunit p65 in relation to active enhancers and promoters of transcribed genes by chromatin immunoprecipitation sequencing (ChIP-seq) experiments. Out of 35,000 active enhancer regions, 410 H3K4me1-positive enhancers show interleukin 1 (IL-1)-induced H3K27ac and p65 binding. Inhibition of TAK1 or IKK2 or depletion of p65 blocked inducible enhancer activation and gene expression. As exemplified by the CXC chemokine cluster located on chromosome 4, the TAK1-p65 pathway also regulates the recruitment kinetics of the histone acetyltransferase CBP, of NF-κB p50, and of AP-1 transcription factors to both promoters and enhancers. This study provides a high-resolution view of epigenetic changes occurring during the IL-1 response and allows the genome-wide identification of a distinct class of inducible p65 NF-κB-dependent enhancers in epithelial cells.
RESUMEN
Nuclear factor kappa-B (NF-κB) activates multiple genes with overlapping roles in cell proliferation, inflammation and cancer. Using an unbiased approach we identified human CDK6 as a novel kinase phosphorylating NF-κB p65 at serine 536. Purified and reconstituted CDK6/cyclin complexes phosphorylated p65 in vitro and in transfected cells. The physiological role of CDK6 for basal as well as cytokine-induced p65 phosphorylation or NF-κB activation was revealed upon RNAi-mediated suppression of CDK6. Inhibition of CDK6 catalytic activity by PD332991 suppressed activation of NF-κB and TNF-induced gene expression. In complex with a constitutively active viral cyclin CDK6 stimulated NF-κB p65-mediated transcription in a target gene specific manner and this effect was partially dependent on its ability to phosphorylate p65 at serine 536. Tumor formation in thymi and spleens of v-cyclin transgenic mice correlated with increased levels of p65 Ser536 phosphorylation, increased expression of CDK6 and upregulaton of the NF-κB target cyclin D3. These results suggest that aberrant CDK6 expression or activation that is frequently observed in human tumors can contribute through NF-κB to chronic inflammation and neoplasia.