The elongation domain of ELL is dispensable but its ELL-associated factor 1 interaction domain is essential for MLL-ELL-induced leukemogenesis - PubMed (original) (raw)

The elongation domain of ELL is dispensable but its ELL-associated factor 1 interaction domain is essential for MLL-ELL-induced leukemogenesis

R T Luo et al. Mol Cell Biol. 2001 Aug.

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.

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Figures

FIG. 1

FIG. 1

Determination of the sequences of ELL essential to the immortalization of hematopoietic progenitor cells by MLL-ELL in a myeloid clonogenic assay. The bars in the right column represent the number of tertiary colonies generated by the respective mutants depicted in the left column.

FIG. 2

FIG. 2

Western blot and RT-PCR analysis of the expression of the various constructs. The upper panels show expression of the various MLL fusion protein constructs in transiently transfected BOSC cells using antibodies to MLL, ELL, or EAF1. The lower panels show RT-PCR expression analysis in the transduced myeloid clonogenic cells harvested from primary methylcellulose cultures. Amplification from reverse transcribed cDNA is indicated by a plus symbol (+). To exclude amplification of integrated retroviral genomic DNA, a no-RT control is indicated by a minus symbol (−).

FIG. 3

FIG. 3

Mapping of the EAF1 interaction domain within ELL. (A) The human 293 cell line was transfected with FLAG-tagged constructs containing different regions of ELL. Western blot analysis of cell lysates confirmed the expression of each of the constructs. (B) Immunoprecipitation of cell lysates was performed with the FLAG antibody, followed by stringent washes of the immunoprecipitated complexes. The minimal domain that coprecipitated with endogenous EAF1 mapped to amino acids 508 to 621 within ELL. Endogenous EAF1 migrates at approximately 43 kDa.

FIG. 4

FIG. 4

Survival curves of the mice transplanted with BM progenitors transduced by the MLL-ELL, MLL-ELL508–621, and MLL-EAF1 encoding vectors.

FIG. 5

FIG. 5

Morphology of the neoplastic cells in MLL-ELL (left column) and MLL-EAF1 leukemic mice (right column). Wright-Giemsa staining of BM cytospin preparation (A, B, C, and D). (A) MLL-ELL mouse with acute monoblastic leukemia (poorly differentiated). (B) MLL-EAF1 mouse with acute monocytic leukemia (differentiated). (C and D) MLL-ELL and MLL-EAF1 mice, respectively, with acute myelomonocytic leukemia. (E and F) PB smears showing circulating blast cells. (G and H) Histological analysis of the liver showing infiltration by leukemia cells. Bar, 300 μm.

FIG. 6

FIG. 6

(A) Immunophenotype of the leukemic BM of MLL-ELL (left) and MLL-EAF1 (right) mice. The histograms show the expression of the EGFP marker, and the region used to gate the EGFP-positive population is indicated, along with the percentage of cells it comprises. The dot plots represent the Mac-1 versus Gr-1 or cKit staining of the EGFP-expressing cells. Percentage values correspond to the content of the adjacent quadrants. (B) Southern blot analysis of spleen DNA obtained from leukemic mice, digested with _Bam_HI, and probed with an MLL cDNA fragment. As indicated by the arrows, one to three integration sites could be detected, indicating that the leukemias were mono- or pauciclonal in the MLL-ELL and the MLL-EAF1 mice. A 9-kb band is also detected, corresponding to the endogenous murine Mll gene.

FIG. 7

FIG. 7

Model of 11q23 leukemogenesis involving AF4 family members and ELL. A subset of 11q23 translocations involves a fusion of MLL to the AF4, LAF4 and AF5q31 genes that contain transcriptional activation domain rich in serine (S), aspartic acid (D), and glutamic acid (E) residues. In the MLL-ELL fusion that results from the t(11;19)(q23;p13.1), ELL retains its interaction domain with EAF1, a transcriptional activator with homology to the serine (S)-, aspartic acid (D)-, and glutamic acid (E)-rich activation domains of AF4, LAF4, and AF5q31.

FIG. 8

FIG. 8

CLUSTALW alignment of EAF1 with LAF4, AF5q31, and AF4. Amino acid identity is indicated by dark gray boxes, and amino acid similarity is indicated by light gray boxes.

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