The Elongation Domain of ELL Is Dispensable but Its ELL-Associated Factor 1 Interaction Domain Is Essential for MLL-ELL-Induced Leukemogenesis

Abstract

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...

The MYO1F, unconventional myosin type 1F, gene is fused to MLL in infant acute monocytic leukemia with a complex translocation involving chromosomes 7, 11, 19 and 22

“…(Hsu and Look, 2003;Daser and Rabbitts, 2004). One mechanism leads to the aberrant activation of target genes including HOX by the fusion of MLL with transcriptional activation domains within translocation partners that are transcriptional factors and located in the nucleus, such as ENL, ELL, AF10, and AFX (Lavau et al, 1997;Luo et al, 2001;DiMartino et al, 2002;So and Cleary, 2002). The other mechanism leads to the similar aberrant activation of target genes by the dimerization of MLL fusion proteins, through oligomerization domains within various translocation partners that are located in the cytoplasm, such as GAS7, AF1p and Gephyrin (Martin et al, 2003;So et al, 2003;Eguchi et al, 2004).…”

U19/Eaf2 knockout causes lung adenocarcinoma, B-cell lymphoma, hepatocellular carcinoma and prostatic intraepithelial neoplasia

“…In addition, ELL and Eaf1 colocalize to nuclear suborganelles called Cajal bodies thought to be involved in nucleoli formation, RNA processing and transport of small ribonucleoproteins (Gall, 2003;Cioce and Lamond, 2005). Studies by Luo et al (2001) argue that the Eaf proteins are important in MLL-ELL leukemogenesis. Our previous studies showed that U19/Eaf2 inhibits xenograft prostate tumor growth and is downregulated in prostate cancer cell lines (Xiao et al, 2003).…”

Transcriptional activation is a key function encoded by MLL fusion partners

“…It has been previously shown that this test is also a good predictor of the in vivo transforming capacity of MLL fusion proteins. 15,16,29 To assess the oncogenic properties of the artificial MLL fusion proteins bone marrow cells from 5-fluorouracil conditioned Balb/C mice were retrovirally transduced with the respective constructs and plated in methocel. Selection for neomycin resistance during the first round of plating ensured that only transduced cells survived.…”

“…[12][13][14] Moreover, several structurefunction studies testing the transforming capacity of MLL fusions in a bone marrow transformation assay found a critical contribution of the fusion partner. [15][16][17] Whereas all studies agreed on the necessity of two critical DNA binding motifs in the MLL moiety, namely the AT hooks and the methyltransferase homology (MT) domain, the crucial function of the fusion partner is still unknown. In a knock-in experiment a fusion of Mll with the bacterial LacZ protein was sufficient to elicit leukemias in mice, albeit only after an extended latency period and with low penetrance.…”

“…17 The crucial domain of another MLL partner, ELL, is a protein-protein interaction domain that binds to EAF1 (ELL associated factor). 16,19 EAF1, in turn, comprises a stretch of amino acids with homology to the transactivator domain of AF4. 20 Finally, the MLL partners CBP and p300 are general cofactors in transcriptional activation.…”