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The MLL-ELL chimeric gene is the product of the (11;19)(q23p13.1) translocation associated with de novo and therapy-related acute myeloid leukemias (AML). ELL is an RNA polymerase II elongation factor that interacts with the recently identified EAF1 (ELL associated factor 1) protein. EAF1 contains a limited region of homology with the transcriptional activation domains of three other genes fused to MLL in leukemias, AF4, LAF4, and AF5q31. Using an in vitro transformation assay of retrovirally transduced myeloid progenitors, we conducted a structure-function analysis of MLL-ELL. Whereas the elongation domain of ELL was dispensable, the EAF1 interaction domain of ELL was critical to the immortalizing properties of MLL-ELL in vitro. To confirm these results in vivo, we transplanted mice with bone marrow transduced with MLL fused to the minimal EAF1 interaction domain of ELL. These mice all developed AML, with a longer latency than mice transplanted with the wild-type MLL-ELL fusion. Based on these results, we generated a heterologous MLL-EAF1 fusion gene and analyzed its transforming potential. Strikingly, we found that MLL-EAF1 immortalized myeloid progenitors in the same manner as that of MLL-ELL. Furthermore, transplantation of bone marrow transduced with MLL-EAF1 induced AML with a shorter latency than mice transplanted with the MLL-ELL fusion. Taken together, these results indicate that the leukemic activity of MLL-ELL requires the EAF1 interaction domain of ELL, suggesting that the recruitment by MLL of a transactivation domain similar to that in EAF1 or the AF4/LAF4/AF5q31 family may be a critical common feature of multiple 11q23 translocations. In addition, these studies support a critical role for MLL partner genes and their protein-protein interactions in 11q23 leukemogenesis.11q23 translocations occur frequently in hematologic malignancies. The MLL gene spans the 11q23 chromosomal translocation breakpoint and contains significant homology with the Drosophila trithorax gene (9,26,29,32). More than 30 different recurring cytogenetic aberrations that affect the MLL gene have been described (5). The critical feature of these chromosomal rearrangements is the generation of a chimeric transcript consisting of 5Ј MLL and 3Ј sequences of the gene on the partner chromosome. At present, more than 20 MLL partner genes at 11q23 partner chromosomal breakpoints have been cloned (3, 5). The functions of most MLL partner genes are not yet known. Although no consistent homologies or motifs among the partner gene sequences have been identified that might explain how their fusion to MLL results in leukemia, certain groups of partner genes have similar features. These include ENL and AF9, which are serine-and proline-rich and share extensive amino acid homology (15,29). AF4, LAF4, and AF5q31 are also rich in serines and prolines and exhibit homology with ENL and AF9 (8,14,25). AF4, ENL, and AF9 contain transcriptional activation domains with similar properties in reporter gene assays (17,20).The (11;19)(q23;p13.1) transloca...

BCL6, a gene on chromosome 3 band q27, encodes a Kruppel-type zinc finger transcriptional repressor. Rearrangements of this gene are frequent in various kinds of lymphomas, particularly of the large-cell B-cell type. The BCL6 nuclear phosphoprotein is expressed in a variety of tissues and is up-regulated particularly in lymph node germinal centers. The zinc fingers of BCL6 bind DNA in a sequence-specific manner. To identify targets of the BCL6 repressive effects, we used a VP16-BCL6 fusion protein containing the zinc fingers but devoid of the repressor domains to compete with the binding of endogenous BCL6 in a transiently transfected B-cell line and then performed subtractive hybridization by using a method to selectively amplify sequences that are differentially expressed. We found that the programmed cell death-2 (PDCD2) gene is a target of BCL6 repression. This gene is the human homolog of Rp8, a rat gene associated with programmed cell death in thymocytes. Immunohistochemistry reveals the anticipated inverse relationship between BCL6 and PDCD2 expression in human tonsil. PDCD2 is detectable in cells of the germinal center in areas where there is less BCL6 expression as well as in the mantle zone, where there is little or no BCL6 expression. These results raise the possibility that BCL6 may regulate apoptosis by means of its repressive effects on PDCD2. BCL6 deregulation may lead to persistent down-regulation of PDCD2, reduced apoptosis, and, as a consequence, accumulation of BCL6-containing lymphoma cells.B CL6 is a gene on chromosome 3 band q27 that we and others identified (1-4) in association with chromosomal rearrangements that accompany a variety of lymphoid malignancies, particularly diffuse large-cell B-cell lymphomas (5). Although the most common translocations involving BCL6 are the t(3;14)(q27;q32) and the t(3;22)(q27;q11), many other chromosomal partners have been identified in association with BCL6 rearrangements (6, 7). The breakpoints cluster around the noncoding first exon of BCL6 (2-4), and it is believed likely that BCL6 expression becomes deregulated by translocations in which heterologous promoters͞enhancers are substituted for normal BCL6 regulatory sequences (8).BCL6 encodes a transcription factor containing six C-terminal Cys 2 -His 2 zinc fingers (3, 4). Two domains upstream of the zinc finger domain (ZF) independently convey transcriptional repressor activity (9-12)-an N-terminal evolutionarily conserved POZ domain and a more central domain. BCL6 is a nuclear phosphoprotein whose expression is up-regulated 3-34-fold in lymph node germinal center B cells (13-15). The BCL6 ZF binds DNA readily in a sequence-specific manner (9-11, 16, 17).We sought to identify genes that are the targets of the BCL6 repressive effects. We used a construct expressing the BCL6 ZF fused to the VP16 activating domain of herpes simplex virus (VP16-BCL6ZF) to compete with the binding of endogenous BCL6 in BJAB cells, an Epstein-Barr virus-negative Burkitt lymphoma cell line expressing high levels of BCL6 (15)....

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 dif-