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First published online 13 April 2005
doi: 10.1242/dev.01824

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The t(8;21) translocation converts AML1 into a constitutive transcriptional repressor

¹ Center for Neurobiology and Behavior, Columbia University Medical School, New York, NY 10032, USA
² Department of Biochemistry and Molecular Biophysics, Columbia University Medical School, New York, NY 10032 USA

View larger version (34K): [in a new window]   Fig. 1. Schematic of RD- and ETO-related proteins. (A) The RD proteins (blue) contain the RD, which binds DNA and interacts with CBFß/Bro/Bgb, and a C-terminal VWRPY motif. ETO and Nervy (green) share four conserved domains [NHR 1-4; NHR 4 is the zinc-finger (ZF) domain]. enR (red) is from the Engrailed protein and includes its repressor domain. (B) Wild-type AML1 (blue) interacts with CBFß (purple) and can bind either transcriptional activators (yellow triangle) or repressors (red hexagon) to regulate gene expression. There are two models to describe how AML1-ETO (blue+green) could be interfering with endogenous AML1 target gene expression to cause leukemia. First, AML1-ETO, which recruits transcriptional repressors through its C terminus, could repress the expression of all AML1 targets (constitutive-repressor model). Alternatively, AML1-ETO might titrate away AML1 co-factors, such as CBFß, preventing AML1 from activating and repressing gene expression (dominant-negative model). In contrast to the predictions of the constitutive repressor model, negatively regulated targets would be de-repressed in the dominant-negative model. (C) A wild-type ommatidium contains eight photoreceptors (1-8; circles), four cone cells (c; ovals), eleven pigment cells (not shown) and three bristles (not shown). lz (blue), which expressed in photoreceptors 1, 6 and 7 and the cone cells, regulates the expression of svp (green), Drosophila Pax2 (red) and dpn (yellow) as indicated.

View larger version (68K): [in a new window]   Fig. 2. Adult eye phenotypes resulting from altering RD gene expression. Photographs of adult eyes, except for E, F and J, which were dissected from unhatched pupae. For clarity, the boundary of eye tissue is outlined in the GMR-Gal4 UAS-AML1-ETO and lz-Gal4 UAS-AML1-ETO flies. Two different driver lines (lz-Gal4, B-F; GMR-Gal4, H-K) were used to express ectopically different fly and mammalian RD proteins. The eye color differences are due to the presence or absence of white (or a mini-white transgene), which is necessary for eye pigmentation. The relevant genotypes of the flies are as follows: (A) wild type; (B) lz-Gal4; UAS-lz; (C) lz-Gal4; UAS-run; (D) lz-Gal4; UAS-AML1; (E) lz-Gal4; UAS-AML1-ETO; (F) lz-Gal4; UAS-lz-enR; (G) lznull; (H) GMR-Gal4; UAS-lz; (I) GMR-Gal4; UAS-run; (J) GMR-Gal4; UAS-AML1; (K) GMR-Gal4 UAS-AML1-ETO.

View larger version (54K): [in a new window]   Fig. 3. elav and svp-lacZ expression in larval eye discs expressing RD proteins. Discs were stained for Elav (red) and ß-gal (green). The ommatidia in the posterior of the GMR-Gal4 UAS-AML1 eye disc (arrow in G) are more disorganized and degenerated than in GMR-Gal4 UAS-AML1-ETO discs (E). In the higher magnification photographs (B,D,F,H), a single ommatidium is circled. The eye discs were taken from larvae of the following genotypes: (A,B) wild type, four cells express svp-lacZ per ommatidium; (C,D) lznull, more than four cells express svp-lacZ per ommatidium; (E,F) GMR-Gal4 UAS-AML1-ETO, the majority of ommatidia have only two svp-lacZ-expressing cells; (G,H) GMR-Gal4 UAS-AML1, many ommatidia have four cells that express svp-lacZ.

View larger version (108K): [in a new window]   Fig. 4. The AML1-ETO-induced phenotype is modified by changes in bro, bgb and lz levels. Photographs of adult eyes raised at 22°C (B,C,E-G) and 25°C (A,D,H). The eye tissue of the GMR-Gal4 UAS-AML1-ETO (D) and GMR-Gal4 UAS-AML1-ETO UAS-bro (F) flies is outlined. The eye color differences are due to the presence or absence of white (or a mini-white transgene), which is necessary for eye pigmentation. The genotypes of the flies are as follows: (A) wild type; (B) GMR-Gal4; UAS-bro; (C) bgbnull/+; (D) GMR-Gal4 UAS-AML1-ETO; (E) GMR-Gal4 UAS-AML1-ETO; (F) GMR-Gal4 UAS-AML1-ETO; UAS-bro; (G) GMR-Gal4 UAS-AML1-ETO; bgbnull/+; (H) GMR-Gal4 UAS-AML1-ETO; UAS-lz.

View larger version (97K): [in a new window]   Fig. 5. The ETO region of AML1-ETO is required for activity. Larval eye discs are stained for AML1 (red), ß-gal (green) and Elav (blue). (A,B) Wild-type eye (A) and larval eye disc (B), showing the normal expression pattern of SME-lacZ. anti-AML1 does not recognize any of the endogenous fly RD proteins. (C,D) lz-Gal4 UAS-NLS-AML1ETO eyes appear wild type (C) and expression of NLS-AML1ETO does not affect SME-lacZ expression or eye disc development (D). (D, inset) An ommatidium is enlarged to show that NLS-AML1ETO colocalizes with Elav in the nucleus.

View larger version (149K): [in a new window]   Fig. 6. Expression of AML1-ETO and Lz-enR inhibits SME-lacZ expression in larval and pupal eye discs. The eye discs are stained for Cut (green) and ß-gal (red). Larval eye discs are shown in A,B,E,F,I,J,M,N and pupal eye discs are shown in C,D,G,H,K,L,O,P. (A-D) In wild type, SME-lacZ is expressed in cone cells, which also express cut, during larval and pupal stages (the green and red channels are separated). (E-H) SME-lacZ is directly activated by Lz, and is therefore not expressed in lznull eye discs, which also do not have cone cells. (I-L) SME-lacZ levels are significantly reduced in lz-Gal4 UAS-AML1-ETO eye discs. This effect is more obvious at pupal stages (K,L). Cut expression remains. (M-P) Ectopic expression of Lz-enR also inhibits SME-lacZ expression (lz-Gal4 UAS-lz-enR). Cut expression remains.

View larger version (103K): [in a new window]   Fig. 7. Effects of AML1-ETO and AML1-ETOZF expression on dpn. Discs were stained for Elav (red) and Dpn (green). The identity of the dpn-expressing photoreceptors is indicated to the right of the panels and an arrow indicates an individual R7 cell in each photograph. The asterisk next to B indicates cone cells differentiating as photoreceptors in lznull eye discs. (A) dpn is normally expressed in differentiating R3, R4 and R7 photoreceptors (see also Fig. 1C). (B) In lznull eye discs, dpn is expressed in the transformed cone cells (arrowhead). (C) In GMR-Gal4 UAS-AML1-ETO eye discs, dpn is repressed in R7 (although an occasional R7 cell weakly expresses dpn). No ectopic Dpn expression is observed. (D) dpn is not expressed in R7 in GMR-Gal4 UAS-lz-enR eye discs. (E) In GMR-Gal4 UAS-AML1-ETOZF eye discs there are more dpn-expressing R7 cells than in GMR-Gal4 UAS-AML1-ETO eye discs, suggesting that the zinc-finger domain is required for complete dpn repression in this assay.