RESUMO
Methylation of lysine 4 on histone H3 (H3K4) at promoters is tightly linked to transcriptional regulation in human cells. At least six different COMPASS-like multisubunit (SET1/MLL) complexes that contain methyltransferase activity for H3K4 have been described, but a comprehensive and quantitative analysis of these SET1/MLL complexes is lacking. We applied label-free quantitative mass spectrometry to determine the subunit composition and stoichiometry of the human SET1/MLL complexes. We identified both known and novel, unique and shared interactors and determined their distribution and stoichiometry over the different SET1/MLL complexes. In addition to being a core COMPASS subunit, the Dpy30 protein is a genuine subunit of the NURF chromatin remodeling complex. Furthermore, we identified the Bod1 protein as a discriminator between the SET1B and SET1A complexes, and we show that the H3K36me-interactor Psip1 preferentially binds to the MLL2 complex. Finally, absolute protein quantification in crude lysates mirrors many of the observed SET1/MLL complex stoichiometries. Our findings provide a molecular framework for understanding the diversity and abundance of the different SET1/MLL complexes, which together establish the H3K4 methylation landscape in human cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas de Neoplasias/metabolismo , Subunidades Proteicas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/isolamento & purificação , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Cromatografia de Afinidade , Proteínas de Ligação a DNA/isolamento & purificação , Células HeLa , Histona-Lisina N-Metiltransferase/isolamento & purificação , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteína de Leucina Linfoide-Mieloide/isolamento & purificação , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Neoplasias/isolamento & purificação , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Mapeamento de Interação de Proteínas , Subunidades Proteicas/isolamento & purificação , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismoRESUMO
Expression of the AF4-MLL fusion protein in murine hematopoietic progenitor/stem cells results in the development of proB acute lymphoblastic leukemia. In this study, we affinity purified the AF4-MLL and AF4 protein complexes to elucidate their function. We observed that the AF4 complex consists of 11 binding partners and exhibits positive transcription elongation factor b (P-TEFb)-mediated activation of promoter-arrested RNA polymerase (pol) II in conjunction with several chromatin-modifying activities. In contrast, the AF4-MLL complex consists of at least 16 constituents including P-TEFb kinase, H3K4(me3) and H3K79(me3) histone methyltransferases (HMT), a protein arginine N-methyltransferase and a histone acetyltransferase. These findings suggest that the AF4-MLL protein disturbs the fine-tuned activation cycle of promoter-arrested RNA Pol II and causes altered histone methylation signatures. Thus, we propose that these two processes are key to trigger cellular reprogramming that leads to the onset of acute leukemia.
