ABSTRACT
The (11;19)(q23;p13.1) translocation in acute leukemia leads to the generation of a chimeric protein that fuses MLL to the transcriptional elongation factor ELL. A novel protein was isolated from a yeast 2-hybrid screen with ELL that was named EAF1 for ELL-associated factor 1. Using specific antibodies, the endogenous EAF1 and ELL proteins were coimmunoprecipitated from multiple cell lines. In addition, endogenous EAF1 also exhibited the capacity to interact with ELL2. Database comparisons with EAF1 identified a region with a high content of serine, aspartic acid, and glutamic acid residues that exhibited homology with the transcriptional activation domains of several translocation partner proteins of MLL, including AF4, LAF4, and AF5q31. A similar transcriptional activation domain has been identified in this region of EAF1. By confocal microscopy, endogenous EAF1 and ELL colocalized in a distinct nuclear speckled pattern. Transfection of the MLL-ELL fusion gene delocalized EAF1 from its nuclear speckled distribution to a diffuse nucleoplasmic pattern. In leukemic cell lines derived from mice transplanted with MLL-ELL-transduced bone marrow, EAF1 speckles were not detected. Taken together, these data suggest that expression of the MLL-ELL fusion protein may have a dominant effect on the normal protein-protein interactions of ELL.
Subject(s)
DNA-Binding Proteins/drug effects , DNA-Binding Proteins/metabolism , Oncogene Proteins, Fusion/pharmacology , Peptide Elongation Factors , Proto-Oncogenes , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Histone-Lysine N-Methyltransferase , Humans , Mice , Molecular Sequence Data , Myeloid-Lymphoid Leukemia Protein , Neoplasm Proteins , Precipitin Tests , Protein Binding , Sequence Alignment , Transcription Factors/isolation & purification , Transcriptional Activation , Transcriptional Elongation Factors , Transfection , Tumor Cells, CulturedABSTRACT
To characterize the functions of MLL fusion transcripts, we cloned the gene that fuses to MLL in the translocation t(11;19)(q23;p13.1). This translocation is distinct from another type of 11;19 translocation with a 19p13.3 breakpoint that results in the fusion of MLL to the ENL gene. By PCR screening of a cDNA library prepared from a patient's leukemia cells with this translocation, we obtained a fusion transcript containing exon 7 of MLL and sequence of an unknown gene. The sequence of this gene was amplified and used as a probe to screen a fetal brain cDNA library. On Northern blot analysis, this cDNA detected a 4.4-kb transcript that was abundant in peripheral blood leukocytes, skeletal muscle, placenta, and testis and expressed at lower levels in spleen, thymus, heart, brain, lung, kidney, liver, and ovary. In addition, a 2.8-kb transcript was present in peripheral blood, testis, and placenta. On "zoo blots," this gene was shown to be evolutionarily conserved in 10 mammalian species as well as in chicken, frog, and fish. We have named this gene ELL (for eleven-nineteen lysine-rich leukemia gene). A highly basic, lysine-rich motif of the predicted ELL protein is homologous to similar regions of several proteins, including the DNA-binding domain of poly(ADP-ribose) polymerase. The characterization of the normal functions of ELL as well as its altered function when fused to MLL will be critical to further our understanding of the mechanisms of leukemogenesis.
