RESUMO
Multiple transcription factors guide the development of mature functional natural killer (NK) cells, yet little is known about their function. We used global gene expression and genome-wide binding analyses combined with developmental and functional studies to unveil three roles for the ETS1 transcription factor in NK cells. ETS1 functions at the earliest stages of NK cell development to promote expression of critical transcriptional regulators including T-BET and ID2, NK cell receptors (NKRs) including NKp46, Ly49H, and Ly49D, and signaling molecules essential for NKR function. As a consequence, Ets1(-/-) NK cells fail to degranulate after stimulation through activating NKRs. Nonetheless, these cells are hyperresponsive to cytokines and have characteristics of chronic stimulation including increased expression of inhibitory NKRs and multiple activation-associated genes. Therefore, ETS1 regulates a broad gene expression program in NK cells that promotes target cell recognition while limiting cytokine-driven activation.
Assuntos
Células Matadoras Naturais/imunologia , Proteína Proto-Oncogênica c-ets-1/deficiência , Motivos de Aminoácidos , Animais , Sítios de Ligação , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Proteína 2 Inibidora de Diferenciação/biossíntese , Proteína 2 Inibidora de Diferenciação/genética , Interleucina-15/farmacologia , Interleucina-15/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Proteína Proto-Oncogênica c-ets-1/genética , Proteína Proto-Oncogênica c-ets-1/fisiologia , Quimera por Radiação , Receptores de Células Matadoras Naturais/biossíntese , Receptores de Células Matadoras Naturais/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Proteínas com Domínio T/biossíntese , Proteínas com Domínio T/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/imunologiaRESUMO
RNA polymerase (Pol) III transcribes many noncoding RNAs (for example, transfer RNAs) important for translational capacity and other functions. We localized Pol III, alternative TFIIIB complexes (BRF1 or BRF2) and TFIIIC in HeLa cells to determine the Pol III transcriptome, define gene classes and reveal 'TFIIIC-only' sites. Pol III localization in other transformed and primary cell lines reveals previously uncharacterized and cell type-specific Pol III loci as well as one microRNA. Notably, only a fraction of the in silico-predicted Pol III loci are occupied. Many occupied Pol III genes reside within an annotated Pol II promoter. Outside of Pol II promoters, occupied Pol III genes overlap with enhancer-like chromatin and enhancer-binding proteins such as ETS1 and STAT1. Moreover, Pol III occupancy scales with the levels of nearby Pol II, active chromatin and CpG content. These results suggest that active chromatin gates Pol III accessibility to the genome.
Assuntos
DNA Polimerase II/genética , Perfilação da Expressão Gênica , RNA Polimerase III/genética , Linhagem Celular , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Genes , Loci Gênicos , Genômica , Células HeLa , Humanos , Células Jurkat , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-ets-1/metabolismo , RNA Polimerase III/análise , RNA de Transferência/genética , Fator de Transcrição STAT1/metabolismoRESUMO
To elucidate how genomic sequences build transcriptional control networks, we need to understand the connection between DNA sequence and transcription factor binding and function. Binding predictions based solely on consensus predictions are limited, because a single factor can use degenerate sequence motifs and because related transcription factors often prefer identical sequences. The ETS family transcription factor, ETS1, exemplifies these challenges. Unexpected, redundant occupancy of ETS1 and other ETS proteins is observed at promoters of housekeeping genes in T cells due to common sequence preferences and the presence of strong consensus motifs. However, ETS1 exhibits a specific function in T cell activation; thus, unique transcriptional targets are predicted. To uncover the sequence motifs that mediate specific functions of ETS1, a genome-wide approach, chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), identified both promoter and enhancer binding events in Jurkat T cells. A comparison with DNase I sensitivity both validated the dataset and also improved accuracy. Redundant occupancy of ETS1 with the ETS protein GABPA occurred primarily in promoters of housekeeping genes, whereas ETS1 specific occupancy occurred in the enhancers of T cell-specific genes. Two routes to ETS1 specificity were identified: an intrinsic preference of ETS1 for a variant of the ETS family consensus sequence and the presence of a composite sequence that can support cooperative binding with a RUNX transcription factor. Genome-wide occupancy of RUNX factors corroborated the importance of this partnership. Furthermore, genome-wide occupancy of co-activator CBP indicated tight co-localization with ETS1 at specific enhancers, but not redundant promoters. The distinct sequences associated with redundant versus specific ETS1 occupancy were predictive of promoter or enhancer location and the ontology of nearby genes. These findings demonstrate that diversity of DNA binding motifs may enable variable transcription factor function at different genomic sites.