RESUMEN

The MLL gene is a common target of chromosomal translocations found in human leukemia. MLL-fusion leukemia has a consistently poor outcome. One of the most common translocation partners is AF9 (MLLT3). MLL-AF9 recruits DOT1L, a histone 3 lysine 79 methyltransferase (H3K79me1/me2/me3), leading to aberrant gene transcription. We show that DOT1L has three AF9 binding sites and present the nuclear magnetic resonance (NMR) solution structure of a DOT1L-AF9 complex. We generate structure-guided point mutations and find that they have graded effects on recruitment of DOT1L to MLL-AF9. Chromatin immunoprecipitation sequencing (ChIP-seq) analyses of H3K79me2 and H3K79me3 show that graded reduction of the DOT1L interaction with MLL-AF9 results in differential loss of H3K79me2 and me3 at MLL-AF9 target genes. Furthermore, the degree of DOT1L recruitment is linked to the level of MLL-AF9 hematopoietic transformation.

Asunto(s)

Histonas/metabolismo , Metiltransferasas/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Inmunoprecipitación de Cromatina , N-Metiltransferasa de Histona-Lisina , Humanos , Espectroscopía de Resonancia Magnética , Metilación , Metiltransferasas/química , Metiltransferasas/genética , Datos de Secuencia Molecular , Proteína de la Leucemia Mieloide-Linfoide/química , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/genética , Mutación Puntual , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Análisis de Secuencia de ADN

RESUMEN

Acute leukemias caused by translocations of the MLL gene at chromosome 11 band q23 (11q23) are characterized by a unique gene expression profile. More recently, data from several laboratories indicate that the most commonly encountered MLL fusion proteins, MLLT1, MLLT3, and AFF1 are found within a molecular complex that facilitates the elongation phase of mRNA transcription. Mutational analyses suggest that interaction between the MLLT1/3 proteins and AFF family proteins are required for experimental transformation of hematopoietic progenitor cells (HPCs). Here, we define a specific pairing of two amino acids that creates a salt bridge between MLLT1/3 and AFF proteins that is critically important for MLL-mediated transformation of HPCs. Our findings, coupled with the newly defined structure of MLLT3 in complex with AFF1, should facilitate the development of small molecules that block this amino acid interaction and interfere with the activity of the most common MLL oncoproteins.

Asunto(s)

Aminoácidos/genética , Proteínas de Unión al ADN/genética , Leucemia Experimental/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Proteínas de Unión al ADN/química , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/química , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Factores de Elongación Transcripcional

RESUMEN

The MLL CXXC domain binds nonmethylated CpG-containing DNA and is essential for the oncogenic properties of MLL fusion proteins. To determine potential functional promiscuity of similar DNA binding domains, we replaced the MLL CXXC domain in the context of the leukemogenic MLL-AF9 fusion with CXXC domains from DNMT1, CGBP (CFP1), and MBD1, or with a methyl-CpG-binding domain (MBD) from MBD1. MLL(DNMT1 CXXC)-AF9 shows robust in vitro colony forming activity and in vivo leukemogenesis, similar to MLL-AF9. However, colony forming ability and leukemogenicity are abrogated in MLL-AF9 containing either the CGBP or MBD1 CXXC domains or the MBD1 MBD domain. Direct comparison of in vitro DNA binding affinity of the isolated CXXC or MBD domains demonstrated that MLL, DNMT1, and CGBP CXXC domains could each bind to unmethylated DNA but with differing affinity. In contrast, the isolated MBD1 CXXC and MBD1 MBD domains were unable to bind to the same DNA. However, all substituted domains still allowed targeting of the MLL fusions to the functionally important Hoxa9 locus in primary bone marrow progenitor cells. In addition to DNA binding activity, it was critical that specific CpG residues in the Hoxa9 locus were protected from methylation for leukemia development. This ultimately prevented histone 3 lysine 9 trimethylation (H3K9me3) of the locus and enabled Hoxa9 expression. These were properties shared by MLL and DNMT1 CXXC domains but not by CGBP CXXC or the other swapped fusions tested. We demonstrate that similar CXXC domains can be mechanistically distinguished by specificity of CpG nucleotides preferentially protected from DNA methylation.

Asunto(s)

Islas de CpG , ADN/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Línea Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , ADN/genética , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , N-Metiltransferasa de Histona-Lisina , Histonas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Leucemia Experimental/genética , Leucemia Experimental/metabolismo , Leucemia Experimental/patología , Lisina/metabolismo , Metilación , Ratones , Datos de Secuencia Molecular , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Unión Proteica , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

KDM1A/LSD1, a histone H3K4/K9 demethylase and epigenetic regulator with roles in both gene activation and repression, has increased expression in multiple cancer types. Harris et al., in this issue of Cancer Cell, and Schenk et al. show that KDM1A may be a viable therapeutic target in treating AML.