RESUMO
The coordinated recruitment of epigenetic regulators of gene expression by transcription factors such as RUNX1 (AML1, acute myeloid leukemia 1) is crucial for hematopoietic differentiation. Here, we identify protein arginine methyltransferase 6 (PRMT6) as a central functional component of a RUNX1 corepressor complex containing Sin3a and HDAC1 in human hematopoietic progenitor cells. PRMT6 is recruited by RUNX1 and mediates asymmetric histone H3 arginine-2 dimethylation (H3R2me2a) at megakaryocytic genes in progenitor cells. H3R2me2a keeps RUNX1 target genes in an intermediate state with concomitant H3K27me3 and H3K4me2 but not H3K4me3. Upon megakaryocytic differentiation PRMT6 binding is lost, the H3R2me2a mark decreases and a coactivator complex containing WDR5/MLL and p300/pCAF is recruited. This leads to an increase of H3K4me3 and H3K9ac, which result in augmented gene expression. Our results provide novel mechanistic insight into how RUNX1 activity in hematopoietic progenitor cells maintains differentiation genes in a suppressed state but poised for rapid transcriptional activation.
Assuntos
Arginina/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Diferenciação Celular , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células K562 , Células Progenitoras de Megacariócitos/fisiologia , Megacariócitos/metabolismo , Metilação , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Proteína-Arginina N-Metiltransferases/genética , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Bacterial toxins are known to be effective for cancer therapy. Clostridium perfringens enterotoxin (CPE) is produced by the bacterial Clostridium type A strain. The transmembrane proteins claudin-3 and -4, often overexpressed in numerous human epithelial tumors (for example, colon, breast, pancreas, prostate and ovarian), are the targeted receptors for CPE. CPE binding to them triggers formation of membrane pore complexes leading to rapid cell death. In this study, we aimed at selective tumor cell killing by CPE gene transfer. We generated expression vectors bearing the bacterial wild-type CPE cDNA (wtCPE) or translation-optimized CPE (optCPE) cDNA for in vitro and in vivo gene therapy of claudin-3- and -4-overexpressing tumors. The CPE expression analysis at messenger RNA and protein level revealed more efficient expression of optCPE compared with wtCPE. Expression of optCPE showed rapid cytotoxic activity, hightened by CPE release as bystander effect. Cytotoxicity of up to 100% was observed 72 h after gene transfer and is restricted to claudin-3-and -4-expressing tumor lines. MCF-7 and HCT116 cells with high claudin-4 expression showed dramatic sensitivity toward CPE toxicity. The claudin-negative melanoma line SKMel-5, however, was insensitive toward CPE gene transfer. The non-viral intratumoral in vivo gene transfer of optCPE led to reduced tumor growth in MCF-7 and HCT116 tumor-bearing mice compared with the vector-transfected control groups. This novel approach demonstrates that CPE gene transfer can be employed for a targeted suicide gene therapy of claudin-3- and -4-overexpressing tumors, leading to the rapid and efficient tumor cell killing in vitro and in vivo.
