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
* Author for correspondence (e-mail: rsm10{at}columbia.edu)
Accepted 10 March 2005
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SUMMARY |
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Key words: AML1 (RUNX1), AML1-ETO, MTG8, Lozenge, CBFß, Drosophila eye, RUNX1T1
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Introduction |
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We used the Drosophila eye as an in vivo system to distinguish
between these two models of AML1-ETO function. We chose the fly eye for these
studies for two reasons. First, the eye is an experimentally accessible tissue
that is well suited for analyzing gene function. Second, the role of the
AML1 homolog lz has been extensively studied in fly eye
development (Daga et al.,
1996; Batterham et al.,
1996
; Crew et al.,
1997
; Xu et al.,
2000
; Flores et al.,
2000
; Siddall et al.,
2003
; Canon and Banerjee,
2003
). The Drosophila eye has also been used to analyze
other human disease genes, including those that contribute to
neurodegeneration such as spinocerebellar ataxia and huntingtin
(Jackson et al., 1998
;
Kazantsev et al., 2002
)
(reviewed by Bonini and Fortini,
2002
). In Drosophila, there are two characterized RD
family members, lz and runt (run), and two
uncharacterized genes that are predicted to encode RD transcription factors
(Fig. 1A)
(Rennert et al., 2003
). There
are also two well-conserved CBFß homologs in flies, called
brother (bro) and big brother (bgb), which
are able to stimulate the ability of both fly and mammalian RD proteins to
bind DNA (Golling et al.,
1996
; Li and Gergen,
1999
; Kaminker et al.,
2001
). In vitro, RD factors from different species are able to
recognize similar binding sites, suggesting that they may also recognize
similar sites in vivo (Pepling and Gergen,
1995
; Golling et al.,
1996
; Xu et al.,
2000
; Flores et al.,
2000
). Consistent with this molecular conservation, RD factors in
different species also have analogous roles during development. For example,
like AML1 in humans, lz is expressed in the fly
hematopoietic lineage, where it is necessary for the specification of a subset
of hematopoietic cell types (Lebestky et
al., 2000
; Waltzer et al.,
2003
).
We found that the phenotypes resulting from AML1-ETO expression in the fly eye differ from those produced by expressing AML1 and from the lznull phenotype, indicating that the effects of AML1-ETO on Drosophila eye development are distinct from these other genetic alterations. Furthermore, we show that expression of AML1-ETO represses the expression of Drosophila Pax2 (sv FlyBase) which is a directly activated target gene of Lz. These data suggest that AML1-ETO is able to repress the expression of RD targets in vivo. AML1-ETO is also able to block the expression of deadpan (dpn), a gene that is normally repressed by Lz. This result is particularly informative for distinguishing between the dominant-negative and constitutive repressor models, because we predict that a negatively regulated target would be de-repressed if AML1-ETO acts by dominantly interfering with Lz function, but would remain repressed if AML1-ETO behaves as a constitutive repressor. Finally, genetic interaction experiments with the RD co-factors Bro and Bgb are inconsistent with a dominant-negative model of AML1-ETO function. Together, these results support a constitutive repressor model of AML1-ETO function.
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Materials and methods |
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Immunofluorescence
Eye discs were dissected from 3rd instar larvae or pupae raised at
25°C. The discs were fixed in 4% formaldehyde in 1 xPBS, incubated
overnight in primary antibody diluted in 1 xPBT (0.1% Triton X-100 in 1
xPBS), washed and then incubated with fluorescently-conjugated secondary
antibodies from Jackson ImmunoResearch. The following primary antibodies were
used: mouse anti-ß-gal (1/1000; Sigma), rabbit anti-ßgal (1/1500;
Cappell), rat anti-Elav 7E8A10 (1/50; Developmental Studies Hybridoma Bank),
mouse anti-Cut (1/10; Developmental Studies Hybridoma Bank), rabbit anti-AML1
AP1651 (1/50; generously shared by P. Erickson) and rabbit anti-Dpn (1/200;
kind gift of H. Vaessin).
UAS-NLS-AML1ETO, UAS-AML1-ETO
ZF and UAS-lz-enR plasmids
UAS-NLS-AML1ETO was created by PCR amplifying
nucleotides 1-531 of AML1-ETO from an AML1-ETO cDNA template
kindly supplied by B. Mathey-Prevot. Nucleotide 531 corresponds to the last
nucleotide of the RD, which is also the last AML1 nucleotide in
AML1-ETO. The resulting AML1
ETO PCR product
was cloned into a modified pUASt vector that fuses an NLS in
frame to 5' of AML1
ETO.
UAS-AML1-ETO
ZF was generated using two Xcm
restriction digest sites that flank the zinc fingers. This removes amino acids
591-698 of AML1-ETO. The digested AML1-ETO cDNA was incubated with T4
DNA polymerase to generate in-frame blunt ends that were then ligated
together. AML1-ETO
ZF was sequenced and cloned in
pUASt. UAS-lz-enR was created by substituting the engrailed
repressor domain for nucleotides 1624-2259 of lz (lz cDNA
gift of the Banerjee laboratory) and then cloning lz-enR into
pUASt.
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Results |
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A wild-type adult Drosophila eye has 750 ommatidia organized
in a regular array. Each ommatidium has eight photoreceptors (R1 to R8), four
cone cells, eleven pigment cells and three interommatidial bristle cells
(reviewed by Wolff and Ready,
1993
). The eye forms from the eye imaginal disc, which is a
monolayer epithelium. In the eye imaginal disc the morphogenetic furrow (MF)
sweeps from the posterior to the anterior, orchestrating ommatidial
development as it progresses. Posterior to the MF the first cell to
differentiate is the R8 photoreceptor, which recruits additional cells to the
forming ommatidium from a pool of undifferentiated cells. The ommatidia form
in a step-wise manner: first, the photoreceptors join the ommatidium, followed
by the cone cells and, lastly, the pigment cells and interommatidial bristles.
lz is expressed in the undifferentiated precursors, photoreceptors
R1, R6, R7, cone cells and pigment cells and is necessary for the
differentiation of these cell types (Fig.
1C) (Daga et al.,
1996
; Batterham et al.,
1996
; Crew et al.,
1997
; Flores et al.,
1998
).
As a first step towards testing the dominant-negative model of AML1-ETO function, we analyzed the adult phenotypes resulting from the expression of AML1-ETO and other RD proteins (such as AML1 and Run) in the eye. We used two different Gal4 driver lines: lz-Gal4 and Glass Multimer Reporter (GMR)-Gal4. lz-Gal4 drives expression in cells that normally express lz, namely, the undifferentiated cells, R1, R6, R7 and cone and pigment cells, but is also expressed outside the eye. GMR-Gal4 is more eye specific and expressed in all cells that are within and posterior to the morphogenetic furrow.
lz-Gal4 UAS-lz eyes appear wild type, demonstrating that elevating Lz levels in lz-expressing cells has no effect on eye development (Fig. 2B). By contrast, the expression of either run or AML1 results in eyes that appear `glazed', meaning that individual ommatidium are virtually impossible to discern (Fig. 2C,D). This suggests that run and AML1 interfere with normal eye development and are not functionally equivalent to lz. However, lz-Gal4 UAS-run eyes are slightly more pigmented than the lz-Gal4 UAS-AML1 eyes, suggesting that run and AML1 may be functionally distinct from each other. Flies that express AML1-ETO via lz-Gal4 die during pupal stages, probably owing to expression outside the eye (for example, lz-Gal4 also drives expression in the hematopoietic system). However, in the eyes of lz-Gal4 UAS-AML1-ETO animals that survive to late pupal stages individual ommatidia cannot be observed and part of the eye is frequently covered by scar tissue (Fig. 2E). This phenotype is more severe than both the lz-Gal4 UAS-AML1 and lznull phenotypes. Owing to the pupal lethality of lz-Gal4; UAS-AML1-ETO flies, we also used GMR-Gal4 to express these proteins in the fly eye. GMR-Gal4 UAS-AML1-ETO eyes are greatly reduced in size and no ommatidia are visible (Fig. 2K). This phenotype is distinct from that produced by the expression of the other RD genes using this driver. The difference in phenotype resulting from AML1 versus AML1-ETO is not due to variations in expression levels, as antibody stains show that these two factors are expressed at similar levels (data not shown). GMR-Gal4 UAS-lz eyes are mildly rough and GMR-Gal4 UAS-run eyes have the same `glazed' appearance as lz-Gal4 UAS-run eyes (Fig. 2H,I). GMR-Gal4 UAS-AML1 eyes are reduced in size, although more eye tissue is present than in GMR-Gal4 UAS-AML1-ETO eyes (Fig. 2J). Thus, the ectopic expression of AML1-ETO in the eye via GMR-Gal4 results in a phenotype that is distinct from the phenotypes produced by other RD factors.
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The ETO region of AML1-ETO is required for its activity
Another test of the dominant-negative model is to determine if an AML1-ETO
truncation that still has the RD, and therefore retains the potential to
interact with Bro and Bgb, produces a similar phenotype as the full-length
protein. We truncated AML1-ETO C terminal to the RD, removing ETO completely
(NLS-AML1ETO) (Fig. 1A).
Similarly truncated AML1 proteins have been previously shown to maintain their
ability to bind CBFß, which interacts with residues present in the RD
(Tahirov et al., 2001
;
Warren et al., 2000
;
Kim et al., 1999
;
Kanno et al., 1998
). Because
AML1-ETO is a constitutively nuclear protein, we also added an exogenous NLS
to ensure that the truncated protein enters the nucleus (inset in
Fig. 5D). We expressed
NLS-AML1
ETO using lz-Gal4 so that its
expression would be restricted to lz-expressing cells in the eye.
lz-Gal4 UAS-NLS-AML1
ETO animals hatch and their eyes
appear wild type (Fig. 5C).
This is in contrast to lz-Gal4 UAS-AML1-ETO animals, which die during
pupation, and GMR-Gal4 UAS-AML1-ETO flies, which have very reduced
eyes (Fig. 2K). The expression
of two different markers of cell differentiation, elav and the cone
cell marker SME-lacZ (a direct target of Lz, see below), is also wild
type in lz-Gal4 UAS-NLS-AML1
ETO eye discs
(Fig. 5D). These results are
consistent with the idea that the RD domain in AML1-ETO is not titrating away
factor(s) that Lz requires to function and thus provides further evidence
against the dominant-negative model. These results also demonstrate that the
ETO region of the AML1-ETO chimera is necessary for the AML1-ETO-induced
phenotypes.
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|
AML1-ETO represses deadpan, a gene normally repressed by Lz
The results described above indicate that AML1-ETO is capable of inhibiting
the expression of Drosophila Pax2, a gene that is directly activated
by Lz. Our next question was whether expression of AML1-ETO also blocks the
expression of targets that Lz negatively regulates. The dominant-negative and
constitutive repressor models predict different outcomes for this experiment
(Fig. 1B). If AML1-ETO is
acting as a dominant-negative factor, a gene that Lz negatively regulates
should be de-repressed. In contrast, if AML1-ETO is acting as a constitutive
repressor this gene will remain repressed. dpn is directly and
negatively regulated by Lz in cone cells
(Canon and Banerjee, 2003).
dpn is normally expressed in the R3/R4 photoreceptors just posterior
to the furrow and is then transiently expressed in differentiating R7
photoreceptors (Fig. 7A; see
Fig. 1C for a summary of its
expression pattern) (Canon and Banerjee,
2003
). In lznull eye discs dpn is
expressed normally in R3, R4 and R7 but is also expressed in the transformed
cone cells (Fig. 7B)
(Canon and Banerjee, 2003
). In
GMR-Gal4 UAS-AML1-ETO eye discs, dpn expression in R7 is
virtually abolished and there is no ectopic expression in other cells
(Fig. 7C). dpn
expression in R7 is also repressed in GMR-Gal4 UAS-lz-enR eye discs,
similar to GMR-Gal4 UAS-AML1-ETO eye discs
(Fig. 7D). The expression of
dpn in R3/R4 is not affected, despite the fact that GMR-Gal4
is active in these cells. One explanation for this observation is that
bgb is not expressed in R3/R4 (although it is not known if Bro is
expressed in these cells) (Kaminker et
al., 2001
). The repression of dpn by AML1-ETO is
inconsistent with a model in which AML1-ETO inhibits Lz activity. Instead,
these results provide further support for the idea that AML1-ETO is a
constitutive transcriptional repressor.
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Discussion |
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AML1-ETO as a constitutive transcriptional repressor
We used the Drosophila eye to investigate two different models of
AML1-ETO function. Although previous studies showed that AML1-ETO
interferes with endogenous AML1 activity, it was unclear how AML1-ETO might
act in vivo. AML1-ETO contains the AML1 RD, which interacts with DNA and
co-factors such as CBFß. Thus, one plausible model is that AML1-ETO
titrates CBFß away from AML1, inhibiting AML1 from acting effectively.
CBFß is crucial for AML1 activity, as demonstrated by the fact that
Cbfb-null mice phenocopy AML1 mutants
(Wang et al., 1996b;
Sasaki et al., 1996
;
Niki et al., 1997
). In
addition, AML1-ETO has been shown to compete with AML1 for CBFß
(Meyers et al., 1995
;
Tanaka et al., 1998
). In
flies, the CBFß homologs bro and bgb are required for
RD function (Li and Gergen,
1999
; Kaminker et al.,
2001
). However, in contrast to the prediction of a
dominant-negative model, we found that supplying higher levels of Bro (or Bgb)
increased the severity of the AML1-ETO phenotype instead of suppressing it.
This result suggests that AML1-ETO uses these co-factors to generate the
observed phenotypes and that supplying additional Bro or Bgb results in a
higher concentration of functional AML1-ETO/co-factor complexes. In an
analogous manner, reducing the dose of Bgb enhances a lz hypomorphic
phenotype (Kaminker et al.,
2001
), consistent with the idea that Lz uses this co-factor and
that reducing its concentration results in lower amounts of functional
Lz/co-factor complexes. In both cases, changes in Bgb or Bro levels only show
an effect when AML1-ETO or Lz are present in limiting amounts
(lzts and GMR-Gal4; UAS-AML1-ETO at 22°C);
changing the levels of these co-factors does not produce a visible phenotype
in an otherwise wild-type background. Similarly, we found that expression of
an AML1-ETO truncation that still contains the Bro- and Bgb-interaction domain
had no effect on eye development in an otherwise wild-type background. Thus,
it appears that these co-factors are not normally present in limiting amounts,
but become limiting when their partners (e.g. AML1-ETO or Lz) are present at
low levels. Taken together, these results suggest that AML1-ETO does not
compete with endogenous RD factors for these co-factors and provide evidence
against a dominant-negative model of AML1-ETO function.
By contrast, our results, in particular showing that AML1-ETO represses
genes that are directly activated (Drosophila Pax2) or directly
repressed (dpn) by Lz, support the idea that AML1-ETO behaves as a
constitutive repressor. These results are also consistent with previous
findings showing that AML1-ETO represses gene expression
(Meyers et al., 1995;
Lutterbach et al., 1998a
).
Although AML1 functions as a transcriptional activator and repressor, neither
the AML1 transactivation domain nor repressor domain are present in AML1-ETO.
Instead, the RD is fused to nearly the entire ETO protein, which is capable of
recruiting several co-repressors through multiple domains
(Peterson and Zhang, 2004
;
Hug and Lazar, 2004
). In our
experiments, we propose that AML1-ETO binds to Lz-binding sites via its RD and
represses the expression of Lz target genes, regardless of whether these genes
are normally activated or repressed by Lz. By extension, we suggest that
AML1-ETO acts similarly to repress AML1 target genes when expressed in humans.
Although there are a few reports suggesting that AML1-ETO activates
transcription, it is unclear if this regulation is direct. Furthermore, for at
least one of these activated targets (bcl2), there is conflicting
evidence whether AML1-ETO causes an in increase in gene expression
(Klampfer et al., 1996
;
Banker et al., 1998
;
Shikami et al., 1999
;
Burel et al., 2001
). In sum,
our results support the idea that AML1-ETO is a constitutive transcriptional
repressor of AML1 targets and fit with a large body of evidence showing that
AML1-ETO represses transcription in a RD binding site-dependent manner
(Peterson and Zhang,
2004
).
Recently, AML1-ETO was also shown to affect transcription by interacting
with the basic helix-loop-helix factor called HeLa E-box binding factor (HEB)
(Zhang et al., 2004). In these
experiments, AML1-ETO and ETO were shown to block the transactivation activity
of HEB in cell culture assays by interfering with the ability of HEB to
recruit CBP/p300. For AML1 target genes that are activated by E proteins, the
mechanism defined by these experiments may be one way in which AML1-ETO causes
transcriptional repression. In addition, inhibition of E protein activity may
represent another mechanism by which AML1-ETO carries out its leukemogenic
functions. As our experiments specifically examined the regulation of
previously characterized lz target genes, for which it is not known
if there is an E protein input, we cannot at present distinguish between these
two possibilities. However, we emphasize that these mechanisms are not
mutually exclusive and that both may be operating in vivo.
Our results also tested if the zinc fingers are necessary for AML1-ETO to
inhibit gene expression. When compared with AML1-ETO, we found that
AML1-ETOZF is slightly less potent at repressing dpn
expression but is able to repress SME-lacZ equally well. These
results suggest that the zinc fingers may be more important for repressing
some target genes than others and that this domain might be functionally
redundant with other parts of the protein. This is not surprising, as multiple
transcriptional repressor complexes can interact with different regions of ETO
(Davis et al., 2003
). Although
the zinc fingers mediate an interaction with N-CoR/SMRT, the amino acids
surrounding NHR2, for example, are capable of recruiting HDAC-1, HDAC-3 and
Sin3a (Lutterbach et al.,
1998a
; Lutterbach et al.,
1998b
; Gelmetti et al.,
1998
; Wang et al.,
1998
; Amann et al.,
2001
; Hildebrand et al.,
2001
). Furthermore, an attempt to define a single region of ETO
that disrupts its function in vivo was unsuccessful
(Cao et al., 2002
). Thus,
although the zinc-finger domain is highly conserved, blocking its function may
only interfere with the repression of a small subset of AML1-ETO target
genes.
In conclusion, these data provide strong support for a model in which AML1-ETO is a constitutive transcriptional repressor rather than a factor that dominantly interferes with the activity of endogenous RD protein function. One implication from these findings is that AML might be caused by the repression of genes that AML1 normally activates, rather than a reduction of normal AML1 activity. Accordingly, we suggest that a deeper understanding of how AML1-ETO contributes to AML will require the identification of genes that are normally activated by AML1.
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ACKNOWLEDGMENTS |
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