RESUMO
SKI is a transcriptional co-regulator and overexpressed in various human tumors, for example in acute myeloid leukemia (AML). SKI contributes to the origin and maintenance of the leukemic phenotype. Here, we use ChIP-seq and RNA-seq analysis to identify the epigenetic alterations induced by SKI overexpression in AML cells. We show that approximately two thirds of differentially expressed genes are up-regulated upon SKI deletion, of which >40% harbor SKI binding sites in their proximity, primarily in enhancer regions. Gene ontology analysis reveals that many of the differentially expressed genes are annotated to hematopoietic cell differentiation and inflammatory response, corroborating our finding that SKI contributes to a myeloid differentiation block in HL60 cells. We find that SKI peaks are enriched for RUNX1 consensus motifs, particularly in up-regulated SKI targets upon SKI deletion. RUNX1 ChIP-seq displays that nearly 70% of RUNX1 binding sites overlap with SKI peaks, mainly at enhancer regions. SKI and RUNX1 occupy the same genomic sites and cooperate in gene silencing. Our work demonstrates for the first time the predominant co-repressive function of SKI in AML cells on a genome-wide scale and uncovers the transcription factor RUNX1 as an important mediator of SKI-dependent transcriptional repression.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Proteínas de Ligação a DNA/metabolismo , Leucemia Mieloide Aguda/genética , Proteínas Proto-Oncogênicas/metabolismo , Elementos Reguladores de Transcrição/genética , Sítios de Ligação/genética , Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Inativação Gênica , Genes/genética , Células HL-60 , Humanos , Leucemia Mieloide Aguda/patologia , Proteínas Proto-Oncogênicas/genética , Ativação Transcricional/genética , Transcriptoma/genéticaRESUMO
Protein arginine methyltransferase 6 (PRMT6) catalyzes asymmetric dimethylation of histone H3 at arginine 2 (H3R2me2a). This mark has been reported to associate with silent genes. Here, we use a cell model of neural differentiation, which upon PRMT6 knockout exhibits proliferation and differentiation defects. Strikingly, we detect PRMT6-dependent H3R2me2a at active genes, both at promoter and enhancer sites. Loss of H3R2me2a from promoter sites leads to enhanced KMT2A binding and H3K4me3 deposition together with increased target gene transcription, supporting a repressive nature of H3R2me2a. At enhancers, H3R2me2a peaks co-localize with the active enhancer marks H3K4me1 and H3K27ac. Here, loss of H3R2me2a results in reduced KMT2D binding and H3K4me1/H3K27ac deposition together with decreased transcription of associated genes, indicating that H3R2me2a also exerts activation functions. Our work suggests that PRMT6 via H3R2me2a interferes with the deposition of adjacent histone marks and modulates the activity of important differentiation-associated genes by opposing transcriptional effects.
Assuntos
Código das Histonas , Histonas/metabolismo , Proteínas Nucleares/genética , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/genética , Ativação Transcricional , Animais , Elementos Facilitadores Genéticos , Células HEK293 , Células HeLa , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Humanos , Metilação , Camundongos , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Neurogênese/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteína-Arginina N-Metiltransferases/metabolismoRESUMO
Protein arginine methyltransferase 4 (PRMT4) is an essential epigenetic regulator of fundamental and conserved processes during vertebrate development, such as pluripotency and differentiation. Surprisingly, PRMT4 homologs have been identified in nearly all vertebrate classes except the avian genome. This raises the possibility that in birds PRMT4 functions are taken over by other PRMT family members. Here, we reveal the existence of a bona fide PRMT4 homolog in the chicken, Gallus gallus. Using a biochemical approach, we initially purified a putative chicken PRMT4 protein and thus provided the first evidence for the presence of an endogenous PRMT4-specific enzymatic activity toward histone H3 arginine 17 (H3R17) in avian cells. We then isolated a G. gallus PRMT4 (ggPRMT4) transcript encompassing the complete open reading frame. Recombinant ggPRMT4 possesses intrinsic methyltransferase activity toward H3R17. CRISPR/Cas9-mediated deletion of ggPRMT4 demonstrated that the transcript identified here encodes avian PRMT4. Combining protein-protein docking and homology modeling based on published crystal structures of murine PRMT4, we found a strong structural similarity of the catalytic core domain between chicken and mammalian PRMT4. Strikingly, in silico structural comparison of the N-terminal Pleckstrin homology (PH) domain of avian and murine PRMT4 identified strictly conserved amino acids that are involved in an interaction interface toward the catalytic core domain, facilitating for the first time a prediction of the relative spatial arrangement of these two domains. Our novel findings are particularly exciting in light of the essential function of the PH domain in substrate recognition and methylation by PRMT4.