RESUMO
Transcription factors regulate gene networks controlling normal hematopoiesis and are frequently deregulated in acute myeloid leukemia (AML). Critical to our understanding of the mechanism of cellular transformation by oncogenic transcription factors is the ability to define their direct gene targets. However, gene network cascades can change within minutes to hours, making it difficult to distinguish direct from secondary or compensatory transcriptional changes by traditional methodologies. To overcome this limitation, we devised cell models in which the AML1-ETO protein could be quickly degraded upon addition of a small molecule. The rapid kinetics of AML1-ETO removal, when combined with analysis of transcriptional output by nascent transcript analysis and genome-wide AML1-ETO binding by CUT&RUN, enabled the identification of direct gene targets that constitute a core AML1-ETO regulatory network. Moreover, derepression of this gene network was associated with RUNX1 DNA binding and triggered a transcription cascade ultimately resulting in myeloid differentiation.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , RNA Neoplásico/biossíntese , Proteína 1 Parceira de Translocação de RUNX1/metabolismo , Transcrição Gênica , Acetilação , Sítios de Ligação , Ligação Competitiva , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Autorrenovação Celular , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Sangue Fetal/citologia , Regulação Leucêmica da Expressão Gênica , Redes Reguladoras de Genes , Células HEK293 , Células-Tronco Hematopoéticas/patologia , Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/patologia , Proteínas de Fusão Oncogênica/genética , Ligação Proteica , Proteólise , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Neoplásico/genética , Proteína 1 Parceira de Translocação de RUNX1/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Tempo , TranscriptomaRESUMO
The Male-Specific Lethal (MSL) complex regulates dosage compensation of the male X chromosome in Drosophila. Here, we report the crystal structure of its MSL1/MSL2 core, where two MSL2 subunits bind to a dimer formed by two molecules of MSL1. Analysis of structure-based mutants revealed that MSL2 can only interact with the MSL1 dimer, but MSL1 dimerization is MSL2 independent. We show that Msl1 is a substrate for Msl2 E3 ubiquitin ligase activity. ChIP experiments revealed that Msl1 dimerization is essential for targeting and spreading of the MSL complex on X-linked genes; however, Msl1 binding to promoters of male and female cells is independent of the dimer status and other MSL proteins. Finally, we show that loss of Msl1 dimerization leads to male-specific lethality. We propose that Msl1-mediated dimerization of the entire MSL complex is required for Msl2 binding, X chromosome recognition, and spreading along the X chromosome.