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NEOPLASIA
From the Imperial Cancer Research Fund, Department of
Medical Oncology, St Bartholomew's Hospital Medical College,
London, United Kingdom; Department of Human Genetics, University
Hospital Nijmegen, The Netherlands; and the Department of Human
Genetics, Medical School, University of Saar, Homburg/ Saar, Germany.
The AF10 gene encodes a putative transcription factor
containing an N-terminal LAP/PHD zinc finger motif, a functional
nuclear localization signal, an AT-hook domain, and a leucine zipper
toward the C-terminus. AF10 is involved in 2 distinct
chromosomal translocations associated with hematologic malignancy. The
chimeric fusion proteins MLL/AF10 and CALM/AF10, resulting from the
t(10;11)(p12;q23) and the t(10;11)(p12;q14), respectively, consistently
retain the leucine zipper motif of AF10. This part of the
C-terminal region was used as bait in a yeast 2 hybrid screening of a
testis complementary DNA library. The leucine zipper interacted with
GAS41, a protein previously identified as the product of an amplified
gene in a glioblastoma. GAS41 shows significant homology to the
Saccharomyces cerevisiae protein ANC1 and to the human MLL
fusion partners AF9 and ENL. The interaction was confirmed in vivo.
Furthermore, the study showed by coimmunoprecipitation that GAS41
interacts with INI1 (Integrase Interactor 1) and that INI1 was present
in the AF10 immunoprecipitate. INI1 is the human homologue of the yeast SNF5 protein, a component of the SWI/SNF complex, which acts to remodel
chromatin and to modulate transcription. The retention of the leucine
zipper in the MLL and CALM fusions suggests that a key feature of these
chimeric proteins may be their ability to interfere in normal
gene regulation through interaction with the adenosine
triphosphate-dependent chromatinremodeling complexes.
(Blood. 2002;99:275-281) The disruption of the human homologue of the
Drosophila Trithorax (trx) gene, MLL on
11q231-4 by chromosomal translocations is a frequent event
in human acute leukemia. These translocations, leading to the
juxtaposition of genetic elements and formation of MLL
fusion genes, occur in approximately 5% to 10% of human acute
leukemias, but with a higher frequency in infant
leukemias5 and secondary leukemias.6 Although
the full biological function of MLL is uncertain, it is
known to act as a positive regulator of HOX gene expression
in development.7,8 Currently, 20 different translocations
affecting the MLL gene have been molecularly cloned and the
partner genes identified.9 Significantly, all such translocations result in in-frame fusions at the messenger RNA level,
and, therefore, the rearrangements result in the production of chimeric
proteins in which the N-terminus of MLL is consistently fused to the C-terminus encoded by the partner gene.10-12
Because such a diverse group of proteins can be fused to MLL, the role of the fusion proteins in leukemogenesis remains
obscure.13 Although most of the fusion partners are
structurally and functionally unrelated to each other,14 a
number are involved in transcriptional regulation. For example, ENL,
AF9, and AF4 activate transcription from synthetic reporter genes in
vivo.15-20 The AF10 gene is one of the few
MLL partner genes to be independently rearranged with a
third gene in leukemia, the CALM gene in the
t(10;11)(p12;q14) translocation.21 AF10
complementary DNA (cDNA) encodes a 1084-aa protein, a member of a
family of proteins including MLL, all carrying a conserved LAP/PHD
finger domain.22,23 There is also a putative AT-hook
motif,24 a bipartite nuclear localization signal, a leucine zipper domain, and a glutamine-rich region at the C-terminus. The latter is not present in all isoforms, as a result of alternative splicing.25,26 Although different breakpoints have been
described for AF10, the resultant fusion protein in both
products consistently loses the LAP/PHD finger but retains the putative
leucine zipper region. The leucine zipper motif of AF10 along with its
immediate upstream region was found to be homologous to the equivalent
region in AF1727,28 and to be conserved among other
species (Figure 1A). To gain an insight
into the potential role of this motif in leukemogenesis, we have
investigated its potential protein interactions. We have established
that the leucine zipper interacts both in vitro and in vivo with GAS41,
previously identified as the product of an amplified gene in a
glioblastoma.29 GAS41 shows significant homology to the
human AF9 and ENL proteins and to the ANC1 protein in
yeast.30 Furthermore, we have shown that GAS41
interacts with INI1 (Integrase Interactor 1) the human homologue of the
yeast SNF5, a component of the SWI/SNF complex,31-33 and INI1 was detected in the AF10 immunoprecipitate. The evolutionarily conserved SWI/SNF complex is one of several multiprotein complexes that
modulate transcription by remodeling chromatin in an adenosine triphosphate-dependent manner.
Yeast 2 hybrid screening
In vitro glutathione-S-transferase pull-down
assay
Cell culture KG1a cells35 were grown in RPMI 1640 medium containing 1% penicillin/streptomycin and 1% glutamine and were supplemented with 10% heat-inactivated fetal calf serum. Cells were grown at 37°C in a humidified incubator (MK11 Leec, Nottingham, United Kingdom) supplemented with 5% carbon dioxide.Protein extraction Approximately 5 million cells were pelleted at 1400 rpm for 10 minutes and washed 3 times in ice-cold 1× PBS. The proteins were extracted with ice-cold lysis buffer (60 mM Tris-HCl pH 7.5, 5 mM EDTA pH 8.0, 150 mM NaCl, 1.0% Triton X-100, 10% glycerol, 5 µg/mL aprotinin, 5 µg/mL leupeptin, 1 mM Na3VO4, 1 µg/mL pepstatin, and 1 mM PMSF). The lysate was kept on ice for 20 minutes, then microfuged at 14 000 rpm for 5 minutes. Protein concentrations were measured against a calibration curve by using bovine serum albumin (BSA) as a reference in a Bradford assay (Bio-Rad, Hercules, CA). Aliquots containing 30 µg protein lysate were mixed with an equal volume of 2× SDS sample buffer, boiled for 4 minutes, and stored at 20°C.
Immunoprecipitation Immunoprecipitation assays were performed with KG1a cells. Thirty million cells were lysed in 1.3 mL lysis buffer (60 mM Tris-HCl pH 7.5, 5 mM EDTA pH 8.0, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 5 µg/mL aprotinin, 5 µg/mL leupeptin, 1 mM Na3VO4, 1 µg/mL pepstatin, and 1 mM PMSF) for 20 minutes in ice. Whole cell extracts were clarified by centrifugation and quantified. For each immunoprecipitation, 1 mg total lysate was diluted in 1 mL lysis buffer and incubated for 2 hours at 4°C with 50 µL precleared Protein A Sepharose CL-4B (Pharmacia). The supernatant was then incubated at 4°C overnight either with 10 µg primary antibody or preimmune serum and further incubated with 50 µL fresh Protein A for 1 hour at 4°C with agitation. The precipitate was washed 3 times in buffer A (1× PBS, 1% NP-40, 100 µM Na3VO4), twice in buffer B (100 mM Tris-HCl pH 7.4, 5 mM LiCl, 100 µM Na3VO4), and twice in buffer C (100 mM Tris-HCl pH 7.4, 5 mM EDTA pH 8.0, 5 mM NaCl, 100 µM Na3VO4). The pellet was resuspended in 2× SDS sample buffer, boiled, and analyzed on SDS polyacrylamide gel for coimmunoprecipitated proteins.Immunoblot analysis Samples were electrophoresed on an SDS polyacrylamide gel in a Mighty Small miniature slab gel unit (Hoefer, Uppsala, Sweden) in running buffer (25 mM Tris-HCl, 192 mM glycine, and 0.1% SDS) and blotted onto a polyvinylidene diflouride membrane (Immobilon-P; Millipore, Bedford, MA) at 300 mA for 2.5 hours at 4°C by using a Bio-Rad Mini Trans-Blot Cell, containing transfer buffer (25 mM Tris/HCl, 192 mM glycine, and 20% methanol). The filters were blocked (3% nonfat dried milk, 0.1% Tween 20 in 1× Tris-buffered saline: 20 mM Tris-HCl pH 7.6, 137 mM NaCl) for 1 hour at room temperature and then incubated with the primary antibodies diluted in blocking solution, with gentle agitation for 2 hours at room temperature. Membranes were washed 5 times with 0.1% Tween 20 in 1× Tris-buffered saline and then incubated with the diluted horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. The blots were washed and proteins detected by using the Super Signal West Dura Substrate Working Solution (Pierce, Rockford, IL) according to the manufacturers' instructions.Immunofluorescence KG1a cells were washed twice in 1× PBS and finally resuspended at a concentration of 106 cells/mL. A total of 100 mL of this suspension was pipetted onto a cytospin column and spun at 400 rpm for 5 minutes (Cytospin3; Thermo-Shandon, Runcorn, United Kingdom) on poly-L-lysine-coated glass slides. Cells were immediately fixed in 2% paraformaldehyde for 20 minutes and then permeabilized in 0.1% saponin for 10 minutes. For AF10/GAS41 colocalization cells were rinsed in 1× PBS and covered for 1 hour with 200 µL chicken anti-AF10 antibody diluted 100 times in 1× PBS, 1% BSA, and 0.02 M sodium azide. Slides were washed 3 times in 1× PBS and 200 µL rabbit anti-GAS41 antibody, diluted 50 times, and was applied at room temperature for 1 hour. After 3 washes, cells were first incubated with fluorescein isothiocyanate (FITC)-conjugated rabbit antichicken secondary antibody for 45 minutes at room temperature and then with the Cy3-labeled goat antirabbit antibody again for 45 minutes. Slides were washed 3 times and then mounted in 70% glycerol in PBS containing antifade. Staining was visualized by using the Bio-Rad Laser sharp MRC-600 confocal imaging system.Antibodies Primary antibodies for immunoblot and immunoprecipitation of AF10, a rabbit polyclonal antibody obtained from rabbit immunized with the first 331 amino acids produced as GST fusion protein (Clare Hall, ICRF)25 was used. The rabbit polyclonal antibody, GAS41-N, against GAS41 was a kind gift of Dr A. Munnia (Department of Human Genetics, University Hospital, Homburg, Germany). Anti INI1, C-18, is a goat polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA). For the immunofluorescence of AF10, a chicken antibody was used (BMA, Biomedicals, AG, Augst, Switzerland). Secondary antibodies for immunoblot were donkey antirabbit immunoglobulin g (IgG) HRP-conjugated (Amersham, Uppsala, Sweden) and antigoat IgG HRP-conjugated (Santa Cruz, Santa Cruz, CA). Secondary antibodies for immunofluorescence were FITC-conjugated rabbit antichicken IgG (Sigma, St Louis, MO) and fluoroLinkTM CyTM3-labeled goat antirabbit IgG (Amersham).
AF10 interacts with GAS41 in a yeast 2 hybrid screening A C-terminal region of AF10 containing the leucine zipper motif (aa 683-972) was used as bait for yeast 2 hybrid screening. The screening of a human testis cDNA library led to the identification of 17 positive clones. Sequence analysis identified 4 independent clones containing the complete open-reading frame (227 amino acids) of the GAS41 gene, linked in-frame to the Gal4 transactivation domain. The GAS41 gene was previously cloned (Glioma Amplified Sequence) from a glioblastoma cell line.29 The first N-terminal 100 amino acids of GAS41 are homologous to AF9 and ENL in humans and to ANC1 and YNK7 in Saccharomyces cerevisiae, YD67 and SPAC22H12.02 in Schizosaccharomyces pombe, and M04B2.3 in Caenorhabditis elegans (Figure 1B). The remainder of GAS41 consists of a C-terminal coiled-coil domain. To determine the specificity of the AF10/GAS41 interaction, we generated a series of deletions encoding portions of AF10, in-frame with the binding domain of Gal4. Yeast plated on selective media was able to grow only when the leucine zipper region was present in the expressed proteins, following cotransformation with the AF10 constructs and the pAD-Gal4 vector expressing GAS41. The shortest active segment identified contained the leucine zipper motif alone (aa 733-806) (Figure 2).
The leucine zipper motif of AF10 interacts with GAS41 in a GST pull-down experiment Four AF10 constructs, one containing the cysteine-rich region (aa 1-331), 2 containing the leucine zipper motif (aa 733-806 and aa 683-972), and the fourth containing the C-terminal portion of AF10 (aa 819-972), were expressed in bacteria as GST fusion products and bound to Glutathione-Sepharose beads. Binding of radiolabeled GAS41 protein was examined against the 4 GST chimeric polypeptides and GST alone. Only the constructs containing the leucine zipper motif were capable of binding to GAS41 protein (Figure 3A, lanes 3 and 4). This result demonstrated that the leucine zipper motif of AF10 was essential for the in vitro binding of AF10 to GAS41. Two deleted constructs of GAS41 (aa 1-95 and aa 163-227) were fused to GST and used to map the region of interaction with AF10. In vitro-translated and -radiolabeled AF10 bound the C-terminal coiled-coil region of GAS41 fused to GST (Figure 3B, lane 3) but not the N-terminal ENL/AF9-like domain (Figure 3B, lane 2). The leucine zipper of AF17 (aa 696-769) was expressed as a GST fusion protein and incubated with the radiolabeled in vitro-translated GAS41 protein. AF17 leucine zipper was unable to mediate the binding to GAS41 (Figure 3C, lane 2), suggesting that GAS41 may have specificity for AF10.
AF10 and GAS41 interact in vivo: coimmunoprecipitation of the endogenous proteins To establish whether endogenous AF10 and GAS41 interacted in vivo, the 2 proteins were independently immunoprecipitated from KG1a cell line with polyclonal antibodies to AF10 and to GAS41 proteins, respectively. Both immunoprecipitates were loaded on separate denaturing polyacrylamide gels and immunoblotted with the 2 antibodies (rabbit polyclonal anti-AF10 and rabbit polyclonal anti-GAS41). An immunoblot on total lysate was performed to test the antibodies (Figure 4A, lane 3, and 4B, lane 3). In the AF10 immunoprecipitate, the rabbit antibody to GAS41 identified a single band of the size expected for the endogenous GAS41 (26.5 KDa) (Figure 4B, lane 1), whereas the rabbit antibody to AF10 detected, in the GAS41 immunoprecipitate, a band of about 120 KDa, the size predicted for the AF10 protein (Figure 4A, lane 2). These experiments suggest a direct physical interaction in vivo between GAS41 and AF10.
Cytoplasmic and nuclear colocalization of AF10 and GAS41 For a further validation of the AF10/GAS41 interaction, we determined the subcellular localization of both proteins by using immunofluorescence assays in KG1a cells. The cells were first alternatively incubated with the 2 primary antibodies and then with a FITC-conjugated secondary antibody to detect AF10 (Figure 5A) and with a Cy3-labeled secondary antibody for GAS41 (Figure 5B). The images obtained at the confocal microscope showed for both the proteins a cytoplasmic localization, although a lighter signal was detected in the nucleus. After overlay, a mixed (yellow) color was observed for all signals (Figure 5C), which is indicative of colocalization. A phase contrast image of the same slides (Figure 5D) indicated the subcellular compartments. Debris of cells lightly stained was visible in the background.
GAS41 interacts with INI1 both in vivo and in vitro It has been previously shown in S cerevisiae that the ANC1 protein interacted with SNF5, a protein component of the SWI/SNF complex.30 Because GAS41 appears to be the human homologue of ANC1, we investigated the possible interaction with INI1, the human homologue of SNF5. The GAS41 immunoprecipitate was immunoblotted with a goat anti-INI1 antibody that detected a 44-KDa band corresponding to the expected size of INI1, suggesting an association of GAS41 with INI1 in vivo (Figure 6A, lane 1). The interaction was mapped by using 2 GST-deleted constructs of GAS41 (aa 1-95 and aa 163-227) expressed in bacteria, both of which pulled down in vitro-translated INI1 (Figure 6B, lanes 2 and 3). The results of the experiments shown above prompted us to investigate whether a similar association could be shown between AF10 and INI1. An AF10 immunoprecipitate was immunoblotted with the anti-INI1 antibody. The antibody identified a protein of 44 KDa, the expected size for INI1 (Figure 6C, lane 1). Further experiments were performed to investigate whether this interaction was direct. In a yeast 2 hybrid experiment, yeast plated on selective media was not able to grow when cotransformed with the leucine zipper region (aa 683-972) of AF10 fused in-frame with the DNA-binding domain of Gal4 and the pAD-Gal4 vector expressing INI1 (data not shown). We therefore concluded that AF10, GAS41, and INI1 may be present in the same complex but without a direct interaction between AF10 and INI1.
The AF10 gene was first identified as a translocated partner of MLL.34 It has been subsequently found that the AF10 gene is one of the few MLL partner genes to be independently rearranged, with a third gene, CALM, encoding a clathrin assembly protein.21 Although the MLL/AF10 fusions were initially thought to be restricted to the AML M4/M5 subtype, recent evidence36 suggests that they are more widely distributed among the AML subtypes. The fusion of AF10 with CALM occurs in a wider spectrum of hematologic malignancies, including acute myeloid and lymphoid leukemias and lymphomas.37-39 A consistent feature of both the MLL/AF10 and CALM/AF10 fusion proteins appears to be the juxtaposition of the leucine zipper motif of AF10 onto the N-terminal region of MLL or CALM.27,40 The fact that in all these cases the leucine zipper region is retained underlines its relevance in leukemic transformation, as has been already demonstrated in murine stem cells retrovirally transduced with MLL/AF10 fusion cDNAs. Constructs containing the leucine zipper were capable of mediating transformation, whereas those lacking this motif were not.41 Because of this observation, a C-terminal fragment of AF10 containing the leucine zipper motif was used as bait in a yeast 2 hybrid screening to identify candidate interacting proteins. Four independent clones were isolated, each containing the full open-reading frame of GAS41, a previously known protein that was identified as the product of a gene amplified in a glioblastoma.29 Deletion constructs of AF10 indicated that the region of interaction with GAS41 in yeast was confined to the leucine zipper motif. We have confirmed this interaction by using in vitro GST pull-down experiments and have furthermore demonstrated that the endogenous AF10 and GAS41 proteins can be coimmunoprecipitated from cell lines. AF17, together with AF10 and MLL, is a member of the LAP/PHD zinc finger-containing family and is itself involved in a t(11;17)(q23;q21) translocation with MLL.28 The leucine zipper of AF17 has 77% (27 of 35 aa) identity with the leucine zipper motif of AF10 (Figure 1A). The published data suggest that, as in the case of AF10, the leucine zipper of AF17 is consistently retained in the fusion product of the translocation. Thus, there has been selective pressure to maintain the motif, suggesting that it has an important functional role. However, the leucine zipper motif of AF17, when tested in vitro, did not pull down GAS41. GAS41 appears to be a human homologue of ANC1, a 244-amino acid protein in S cerevisiae known to be an integral member of 2 basal transcription factor complexes, TFIID and TFIIF, and to be an interacting component of the SWI/SNF chromatin-remodeling complex.30 GAS41, ANC1, and the other yeast homologues have a significant similarity (Figure 1B) in their N-terminal region to 2 human proteins involved in fusion with MLL, AF942 and ENL.43 Both AF9 and ENL, when fused to MLL, are known to mediate transformation in mice.15,17,18,44 The biochemical role ANC1 plays in the transcription complexes is not yet clear, but ANC1 is known to bind to the SWI/SNF complex through its interaction with SNF5. Homologues of SNF5 have been isolated in both humans and Drosophila, INI1, and Snr1, respectively. They have been shown to be associated in large complexes equivalent to the yeast SWI/SNF45,46 and to bind the SET domains of MLL and Trx genes.47 To extend these findings and investigate a putative relation of AF10 with the chromatin-remodeling complexes, we have further shown that GAS41 interacts in vitro and in vivo with INI1. Although a yeast 2 hybrid experiment excluded the direct interaction between AF10 and INI1, the presence of the latter in an AF10 immunoprecipitated, suggested the possibility that the 3 proteins exist in a protein complex. It has been shown that the AT-hook motif of AF10 mediated the binding to synthetic cruciform DNA.26 The published data suggested that proteins containing AT-hooks, like HMG-I (Y), play an important role in chromatin structure and transcriptional regulation by acting as accessory factors that influence the association of transcription factors with chromatin.48,49 It is plausible that AF10 and GAS41 when bound together recruit the SWI/SNF complex through the interaction with INI1. The AT-hook motif of AF10 could use AT-rich tracks to target specific regions on chromatin. When AF10 is fused to MLL, the AT-hook region of AF10 will not be consistently present in the fusion proteins, whereas the N-terminal AT-hooks of MLL will be retained. Fused MLL lacks the C-terminal SET domain that mediates the interaction with INI1.47 The MLL/AF10 fusion would create a protein, which could still interact with GAS41 and which could still bind DNA, but the interaction with INI1 would be mediated by GAS41, if still possible, leading to the loss of regulation of MLL target genes, thus compromising its role in development and affecting the expression of AF10 target genes. The interaction described here may therefore play an important role in neoplastic transformation, and further work is required to determine the role of the AF10 fusion proteins in leukemogenesis.
We thank Professor Ad Geurts van Kessel (Department of Human Genetics, University Medical Center St Radbound, Nijmegen, The Netherlands) for gifts of the human testis cDNA library, all the expressing vectors, the oligonucleotides, as well as the yeast strain used in the yeast 2 hybrid screening. We also thank Dr A. Munnia (Department of Human Genetics, Medical School, University of Saar, Homburg/Saar, Germany) for the gift of the rabbit polyclonal antibody GAS41-N. Finally, we thank Dr V. Saha for proofreading the paper.
Submitted February 16, 2001; accepted August 22, 2001.
Supported by the Kay Kendall Leukaemia Fund (S.D.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Bryan D. Young, ICRF Med Oncology Unit, St Bartholomew's Hospital Medical School, Charterhouse Square, London EC1M 6BQ, United Kingdom; e-mail: b.young{at}icrf.icnet.uk.
1.
Ziemin-van der Poel S, McCabe NR, Gill HJ, et al.
Identification of a gene, MLL, that spans the breakpoint in 11q23 translocations associated with human leukemias [published erratum appears in Proc Natl Acad Sci U S A. 1992 May 1;89(9):4220].
Proc Natl Acad Sci U S A.
1991;88:10735-10739 2. Gu Y, Nakamura T, Alder H, et al. The t(4;11) chromosome translocation of human acute leukemias fuses the ALL-1 gene, related to Drosophila trithorax, to the AF-4 gene. Cell. 1992;71:701-708[CrossRef][Medline] [Order article via Infotrieve]. 3. Tkachuk DC, Kohler S, Cleary ML. Involvement of a homolog of Drosophila trithorax by 11q23 chromosomal translocations in acute leukemias. Cell. 1992;71:691-700[CrossRef][Medline] [Order article via Infotrieve]. 4. Djabali M, Selleri L, Parry P, Bower M, Young BD, Evans GA. A trithorax-like gene is interrupted by chromosome 11q23 translocations in acute leukaemias. Nat Genet. 1992;2:113-118[CrossRef][Medline] [Order article via Infotrieve]. 5. Satake N, Maseki N, Nishiyama M, et al. Chromosome abnormalities and MLL rearrangements in acute myeloid leukemia of infants. Leukemia. 1999;13:1013-1017[CrossRef][Medline] [Order article via Infotrieve]. 6. Felix CA. Secondary leukemias induced by topoisomerase-targeted drugs. Biochim Biophys Acta. 1998;1400:233-255[Medline] [Order article via Infotrieve]. 7. Yu BD, Hess JL, Horning SE, Brown GA, Korsmeyer SJ. Altered Hox expression and segmental identity in Mll-mutant mice. Nature. 1995;378:505-508[CrossRef][Medline] [Order article via Infotrieve].
8.
Hanson RD, Hess JL, Yu BD, et al.
Mammalian Trithorax and polycomb-group homologues are antagonistic regulators of homeotic development.
Proc Natl Acad Sci U S A.
1999;96:14372-14377 9. Dimartino JF, Cleary ML. Mll rearrangements in haematological malignancies: lessons from clinical and biological studies. Br J Haematol. 1999;106:614-626[CrossRef][Medline] [Order article via Infotrieve]. 10. Young BD, Saha V. Chromosome abnormalities in leukaemia: the 11q23 paradigm. Cancer Surv. 1996;28:225-245[Medline] [Order article via Infotrieve]. 11. Saha V, Young BD, Freemont PS. Translocations, fusion genes, and acute leukemia. J Cell Biochem Suppl. 1998;30-31:264-276. 12. Rowley JD. Molecular genetics in acute leukemia. Leukemia. 2000;14:513-517[CrossRef][Medline] [Order article via Infotrieve]. 13. Rowley JD. The role of chromosome translocations in leukemogenesis. Semin Hematol. 1999;36:59-72[Medline] [Order article via Infotrieve]. 14. Bernard OA, Mauchauffe M, Mecucci C, Van den Berghe H, Berger R. A novel gene, AF-1p, fused to HRX in t(1;11)(p32;q23), is not related to AF-4, AF-9 nor ENL. Oncogene. 1994;9:1039-1045[Medline] [Order article via Infotrieve]. 15. Lavau C, Szilvassy SJ, Slany R, Cleary ML. Immortalization and leukemic transformation of a myelomonocytic precursor by retrovirally transduced HRX-ENL. EMBO J. 1997;16:4226-4237[CrossRef][Medline] [Order article via Infotrieve]. 16. Schreiner SA, Garcia-Cuellar MP, Fey GH, Slany RK. The leukemogenic fusion of MLL with ENL creates a novel transcriptional transactivator. Leukemia. 1999;13:1525-1533[CrossRef][Medline] [Order article via Infotrieve].
17.
Slany RK, Lavau C, Cleary ML.
The oncogenic capacity of HRX-ENL requires the transcriptional transactivation activity of ENL and the DNA binding motifs of HRX.
Mol Cell Biol.
1998;18:122-129 18. Dobson CL, Warren AJ, Pannell R, et al. The mll-AF9 gene fusion in mice controls myeloproliferation and specifies acute myeloid leukaemogenesis. EMBO J. 1999;18:3564-3574[CrossRef][Medline] [Order article via Infotrieve].
19.
Domer PH, Fakharzadeh SS, Chen CS, et al.
Acute mixed-lineage leukemia t(4;11)(q21;q23) generates an MLL-AF4 fusion product.
Proc Natl Acad Sci U S A.
1993;90:7884-7888
20.
Isnard P, Core N, Naquet P, Djabali M.
Altered lymphoid development in mice deficient for the mAF4 proto-oncogene.
Blood.
2000;96:705-710
21.
Dreyling MH, Martinez-Climent JA, Zheng M, Mao J, Rowley JD, Bohlander SK.
The t(10;11)(p13;q14) in the U937 cell line results in the fusion of the AF10 gene and CALM, encoding a new member of the AP-3 clathrin assembly protein family.
Proc Natl Acad Sci U S A.
1996;93:4804-4809
22.
Saha V, Chaplin T, Gregorini A, Ayton P, Young BD.
The leukemia-associated-protein (LAP) domain, a cysteine-rich motif, is present in a wide range of proteins, including MLL, AF10, and MLLT6 proteins.
Proc Natl Acad Sci U S A.
1995;92:9737-9741 23. Aasland R, Gibson TJ, Stewart AF. The PHD finger: implications for chromatin-mediated transcriptional regulation. Trends Biochem Sci. 1995;20:56-59[CrossRef][Medline] [Order article via Infotrieve].
24.
Aravind L, Landsman D.
AT-hook motifs identified in a wide variety of DNA-binding proteins.
Nucleic Acids Res.
1998;26:4413-4421 25. Linder B, Jones LK, Chaplin T, et al. Expression pattern and cellular distribution of the murine homologue of AF10. Biochim Biophys Acta. 1998;1443:285-296[Medline] [Order article via Infotrieve]. 26. Linder B, Newman R, Jones LK, et al. Biochemical analyses of the AF10 protein: the extended LAP/PHD-finger mediates oligomerisation. J Mol Biol. 2000;299:369-378[CrossRef][Medline] [Order article via Infotrieve].
27.
Chaplin T, Bernard O, Beverloo HB, et al.
The t(10;11) translocation in acute myeloid leukemia (M5) consistently fuses the leucine zipper motif of AF10 onto the HRX gene.
Blood.
1995;86:2073-2076
28.
Prasad R, Leshkowitz D, Gu Y, et al.
Leucine-zipper dimerization motif encoded by the AF17 gene fused to ALL-1 (MLL) in acute leukemia.
Proc Natl Acad Sci U S A.
1994;91:8107-8111
29.
Fischer U, Heckel D, Michel A, Janka M, Hulsebos T, Meese E.
Cloning of a novel transcription factor-like gene amplified in human glioma including astrocytoma grade I.
Hum Mol Genet.
1997;6:1817-1822 30. Cairns BR, Henry NL, Kornberg RD. TFG/TAF30/ANC1, a component of the yeast SWI/SNF complex that is similar to the leukemogenic proteins ENL and AF-9. Mol Cell Biol. 1996;16:3308-3316[Abstract]. 31. Phelan ML, Sif S, Narlikar GJ, Kingston RE. Reconstitution of a core chromatin remodeling complex from SWI/SNF subunits. Mol Cell. 1999;3:247-253[CrossRef][Medline] [Order article via Infotrieve].
32.
Morozov A, Yung E, Kalpana GV.
Structure-function analysis of integrase interactor 1/hSNF5L1 reveals differential properties of two repeat motifs present in the highly conserved region.
Proc Natl Acad Sci U S A.
1998;95:1120-1125 33. Muchardt C, Yaniv M. The mammalian SWI/SNF complex and the control of cell growth. Semin Cell Dev Biol. 1999;10:189-195[CrossRef][Medline] [Order article via Infotrieve].
34.
Chaplin T, Ayton P, Bernard OA, et al.
A novel class of zinc finger/leucine zipper genes identified from the molecular cloning of the t(10;11) translocation in acute leukemia.
Blood.
1995;85:1435-1441
35.
Furley AJ, Reeves BR, Mizutani S, et al.
Divergent molecular phenotypes of KG1 and KG1a myeloid cell lines.
Blood.
1986;68:1101-1107 36. Gore L, Ess J, Bitter MA, et al. Protean clinical manifestations in children with leukemias containing MLL-AF10 fusion. Leukemia. 2000;14:2070-2075[CrossRef][Medline] [Order article via Infotrieve]. 37. Kobayashi H, Hosoda F, Maseki N, et al. Hematologic malignancies with the t(10;11) (p13;q21) have the same molecular event and a variety of morphologic or immunologic phenotypes. Genes Chromosomes Cancer. 1997;20:253-259[CrossRef][Medline] [Order article via Infotrieve]. 38. Carlson KM, Vignon C, Bohlander S, et al. Identification and molecular characterisation of CALM/AF10 fusion products in T cell acute lymphoblastic leukemia and acute myeloid leukemia. Leukemia. 2000;14:100-104[CrossRef][Medline] [Order article via Infotrieve]. 39. Bohlander SK, Muschinsky V, Schrader K, et al. Molecular analysis of the CALM/AF10 fusion: identical rearrangements in acute myeloid leukemia, acute lymphoblastic leukemia and malignant lymphoma. Leukemia. 2000;14:93-99[CrossRef][Medline] [Order article via Infotrieve]. 40. Bohlander SK, Muschinsky V, Schrader K, et al. Molecular analysis of the CALM/AF10 fusion: identical rearrangements in acute myeloid leukemia, acute lymphoblastic leukemia and malignant lymphoma patients. Leukemia. 2000;14:93-99. 41. DiMartino JF, Ayton P, Young BD, Shilatifard A, Cleary ML. Structure-function analysis of MLL-AF10 and MLL-ELL fusion proteins. Blood. 1999;94:56a. 42. Super HG, Strissel PL, Sobulo OM, et al. Identification of complex genomic breakpoint junctions in the t(9;11) MLL-AF9 fusion gene in acute leukemia. Genes Chromosomes Cancer. 1997;20:185-195[CrossRef][Medline] [Order article via Infotrieve].
43.
Rubnitz JE, Behm FG, Curcio-Brint AM, et al.
Molecular analysis of t(11;19) breakpoints in childhood acute leukemias.
Blood.
1996;87:4804-4808 44. Corral J, Lavenir I, Impey H, et al. An Mll-AF9 fusion gene made by homologous recombination causes acute leukemia in chimeric mice: a method to create fusion oncogenes. Cell. 1996;85:853-861[CrossRef][Medline] [Order article via Infotrieve].
45.
Wang W, Xue Y, Zhou S, Kuo A, Cairns BR, Crabtree GR.
Diversity and specialization of mammalian SWI/SNF complexes.
Genes Dev.
1996;10:2117-2130 46. Dingwall AK, Beek SJ, McCallum CM, et al. The Drosophila snr1 and brm proteins are related to yeast SWI/SNF proteins and are components of a large protein complex. Mol Biol Cell. 1995;6:777-791[Abstract].
47.
Rozenblatt-Rosen O, Rozovskaia T, Burakov D, et al.
The C-terminal SET domains of ALL-1 and TRITHORAX interact with the INI1 and SNR1 proteins, components of the SWI/SNF complex.
Proc Natl Acad Sci U S A.
1998;95:4152-4157 48. Falvo JV, Thanos D, Maniatis T. Reversal of intrinsic DNA bends in the IFN beta gene enhancer by transcription factors and the architectural protein HMG I(Y). Cell. 1995;83:1101-1111[CrossRef][Medline] [Order article via Infotrieve]. 49. Strick R, Laemmli UK. SARs are cis DNA elements of chromosome dynamics: synthesis of a SAR repressor protein. Cell. 1995;83:1137-1148[CrossRef][Medline] [Order article via Infotrieve].
© 2002 by The American Society of Hematology.
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  P. Modena, E. Lualdi, F. Facchinetti, L. Galli, M. R. Teixeira, S. Pilotti, and G. Sozzi SMARCB1/INI1 Tumor Suppressor Gene Is Frequently Inactivated in Epithelioid Sarcomas Cancer Res., May 15, 2005; 65(10): 4012 - 4019. [Abstract] [Full Text] [PDF]
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 C. B. Bittner, D. T. Zeisig, B. B. Zeisig, and R. K. Slany Direct Physical and Functional Interaction of the NuA4 Complex Components Yaf9p and Swc4p Eukaryot. Cell, August 1, 2004; 3(4): 976 - 983. [Abstract] [Full Text] [PDF]
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 A. Maitra, D. E. Hansel, P. Argani, R. Ashfaq, A. Rahman, A. Naji, S. Deng, J. Geradts, L. Hawthorne, M. G. House, et al. Global Expression Analysis of Well-Differentiated Pancreatic Endocrine Neoplasms Using Oligonucleotide Microarrays Clin. Cancer Res., December 1, 2003; 9(16): 5988 - 5995. [Abstract] [Full Text] [PDF]
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 Z. Nie, Z. Yan, E. H. Chen, S. Sechi, C. Ling, S. Zhou, Y. Xue, D. Yang, D. Murray, E. Kanakubo, et al. Novel SWI/SNF Chromatin-Remodeling Complexes Contain a Mixed-Lineage Leukemia Chromosomal Translocation Partner Mol. Cell. Biol., April 15, 2003; 23(8): 2942 - 2952. [Abstract] [Full Text] [PDF]
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L. Perrin, S. Bloyer, C. Ferraz, N. Agrawal, P. Sinha, and J. M. Dura The Leucine Zipper Motif of the Drosophila AF10 Homologue Can Inhibit PRE-Mediated Repression: Implications for Leukemogenic Activity of Human MLL-AF10 Fusions Mol. Cell. Biol., January 1, 2003; 23(1): 119 - 130. [Abstract] [Full Text]
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Top
Abstract
Introduction
-gal activity and for growth on fully
selective medium. As negative control, the pBD-Gal4 vector fused to
Lamin C was used.
as GST
fusion proteins after 3 hours of induction with 0.1 mM isopropyl
thiogalactoside. Cells were harvested, resuspended in 1×
phosphate-buffered saline (PBS; 80 mM Na2HPO4,
20 mM NaH2PO4, 100 mM NaCl, pH 7.5), and lysed
by mild sonication. The cleared lysate was immobilized on affinity
Glutathione-Sepharose beads. The cDNAs for the partner proteins were
cloned in the pCR 2.1 vector (Invitrogen, Carlsbad, CA), and the
proteins were synthesized and radiolabeled with 20 µCi (0.74 MBq) 35S-Met (TNT Quick Coupled Transcription/Translation
System Kit; Promega, Madison, WI) in a coupled
transcription-translation system. About 25 µg of each immobilized GST
chimeric fusion protein was incubated at 4°C for 2 hours with one
fifth of the labeled polypeptides in binding buffer (20 mM Tris-HCl pH
8.0, 0.2% Triton X-100, 2 mM EDTA pH 8.0, 300 mM NaCl, 1 µg/mL
aprotinin, 1 µg/mL leupeptin, and 1 mM phenyl-methyl sulfonyl
fluoride [PMSF]). After extensive washing in RIPA buffer (10 mM
Tris-HCl pH 7.5, 0.2% NP-40, 1 mM EDTA pH 8.0, 400 mM NaCl, 1 µg/mL
aprotinin, 1 µg/mL leupeptin, and 1 mM PMSF) bound proteins were
eluted by boiling in 2× sodium dodecyl sulfate (SDS) sample buffer
(125 mM Tris-HCl pH 6.8, 20% glycerol, 4% SDS, 0.05% bromophenol
blue, and 10% 
