lozenge

EVOLUTIONARY HOMOLOGS

See Drosophila Runt for more information about Lozenge mammalian homologs.

To investigate the normal biologic function of AML1 (also known as Core-binding factor) alpha subunit 2 (CBFA2) mice carrying a disrupted AML1 allele were generated using gene targeting in embryonic stem (ES) cells. Mice lacking aml1 died during midembryonic development, secondary to the complete absence of fetal liver-derived hematopoiesis. Homozygous aml1-deficient cells failed to contribute to hematopoiesis in chimeric animals. These findings indicate that aml1-regulated target genes are essential for definitive hematopoiesis of all lineages. Mice lacking AML1 died between embryonic days 11.5 and 12.5 due to hemorrhaging in the central nervous system, at the nerve/CNS interfaces of cranial and spinal nerves, and in somitic/intersomitic regions along the presumptive spinal cord. Hemorrhaging was preceded by symmetric, bilateral necrosis in these regions. Definitive erythropoiesis and myelopoiesis did not occur in AML1-deficient embryos, and in heterozygotes there was a significant reduction in the number of progenitors for erythroid and myeloid cells (Okuda, 1996 and Wang, 1996).

LEF-1 (see Drosophila Pangolin) is a transcription factor that participates in the regulation of the T-cell receptor alpha (TCRalpha) enhancer by facilitating the assembly of multiple proteins into a higher order nucleoprotein complex. The function of LEF-1 is dependent, in part, on the HMG domain that induces a sharp bend in the DNA helix, and on an activation domain that stimulates transcription only in a specific context of other enhancer-binding proteins. ALY, a novel LEF-1-interacting protein was cloned in order to gain insight into the function of context-dependent activation domains. ALY is a ubiquitously expressed, nuclear protein that specifically associates with the activation domains of LEF-1 and AML-1, which is another protein component of the TCRalpha enhancer complex. In addition, ALY can increase DNA binding by both LEF-1 and AML proteins. Overexpression of ALY stimulates the activity of the TCRalpha enhancer complex reconstituted in transfected nonlymphoid HeLa cells, whereas down-regulation of ALY by anti-sense oligonucleotides virtually eliminates TCRalpha enhancer activity in T cells. Similar to LEF-1, ALY can stimulate transcription in the context of the TCRalpha enhancer but apparently not when tethered to DNA through an heterologous DNA-binding domain. It is proposed that ALY mediates context-dependent transcriptional activation by facilitating the functional collaboration of multiple proteins in the TCRalpha enhancer complex (Bruhn, 1997).

Mammalian Runt domain genes encode the alpha subunit of the heterometric DNA-binding factor PEBP2/CBF. The unrelated PEBP2/CBF beta protein interacts with the Runt domain to increase its affinity for DNA. The conserved ability of the Drosophila Runt protein to respond to the stimulating effect of mammalian PEBP2/CBF beta indicates that flies are likely to have a homologous beta protein. Using the yeast two-hybrid system to isolate cDNAs for Runt-interacting proteins, two Drosophila genes have been identifed: Brother and Big-brother. These genes have substantial sequence homology with PEBP2/CBF beta. Yeast two-hybrid experiments as well as in vitro DNA-binding studies confirm the functional homology of Brother, Big-brother, and PEBP2/CBF beta proteins, and demonstrates that the conserved regions of the Runt and Brother proteins are required for their heterodimeric interaction. The DNA-bending properties of Runt domain proteins, both in the presence and absence of their partners, has been examined. Runt domain proteins bend DNA: this bending is influenced by Brother protein family members, supporting the idea that heterodimerization is associated with a conformational change in the Runt domain. Analysis of expression patterns in Drosophila embryos reveals that Brother and Big-brother are likely to interact with Runt in vivo and further suggests that the activity of these proteins is not restricted to their interaction with Runt (Grolling, 1996).

The t(8;21) translocation converts AML1, a mammalian Lozenge homolog, into a constitutive transcriptional repressor

The human translocation (t8;21) is associated with ~12% of the cases of acute myelogenous leukemia. Two genes, AML1 and ETO, are fused together at the translocation breakpoint, resulting in the expression of a chimeric protein called AML1-ETO. AML1-ETO is thought to interfere with normal AML1 function, although the mechanism by which it does so is unclear. Here, Drosophila genetics was used to investigate two models of AML1-ETO function. In the first model, AML1-ETO is a constitutive transcriptional repressor of AML1 target genes, regardless of whether they are normally activated or repressed by AML1. In the second model, AML1-ETO dominantly interferes with AML1 activity by, for example, competing for a common co-factor. To discriminate between these models, the effects of expressing AML1-ETO were characterized and compared with loss-of-function phenotypes of lozenge (lz), an AML1 homolog expressed during Drosophila eye development. Results of genetic interaction experiments with AML1 co-factors are presented that are not consistent with AML1-ETO behaving as a dominant-negative factor. Instead, the data suggest that AML1-ETO acts as a constitutive transcriptional repressor (Wildonger, 2005b).

AML1-ETO has been widely studied using both cell culture and animal models. Several AML1-ETO knock-in murine models now exist, including an inducible knock-in that bypasses the embryonic lethality observed in other AML1-ETO knock-in mice. Surprisingly, these knock-in murine models indicate that AML1-ETO alone is not sufficient to cause leukemia. The AML1-ETO-expressing mice develop leukemia only after additional mutations are induced. The need to induce secondary mutations to trigger the disease state could complicate the use of mice as a model organism to study how AML1-ETO acts at a mechanistic level. Complementary animal models to investigate AML1-ETO function in vivo may provide additional insights into the activity of this oncogene. For example, AML1-ETO has recently been expressed in zebrafish embryos, resulting in abnormal hematopoiesis. This study describes the phenotypes resulting from the expression of AML1-ETO in flies, establishing another animal model in which to study AML1-ETO function. Drosophila is a particularly attractive model organism, given the relative ease of performing large-scale genetic screens in flies. In support of this idea, it is shown that the AML1-ETO eye phenotype is sensitive to changes in the levels of brother (bro) and big brother (bgb) Thus, it should be possible to perform a modifier screen to identify additional genes that interact with AML1-ETO. In fact,an initial screen was performed and several genomic regions were identified that show a genetic interaction with AML1-ETO (Wildonger, 2005b).

The Drosophila eye was used 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. In addition, AML1-ETO has been shown to compete with AML1 for CBFß. In flies, the CBFß homologs bro and bgb are required for runt domain (RD function). However, in contrast to the prediction of a dominant-negative model, it was found that supplying higher levels of Bro (or Bgb) increases 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, 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 show an effect only when AML1-ETO or Lz are present in limiting amounts (lz^ts 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, it was found that expression of an AML1-ETO truncation that still contains the Bro- and Bgb-interaction domain has 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 (Wildonger, 2005b).

By contrast, these 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. 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. In these experiments, it is proposed 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, it is suggested 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. In sum, the 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 (Wildonger, 2005b). 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). 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. Since the current experiments specifically examined the regulation of previously characterized lz target genes, for which it is not known if there is an E protein input, at present it is not possible to distinguish between these two possibilities. However, it is emphasized that these mechanisms are not mutually exclusive and that both may be operating in vivo (Wildonger, 2005b).

The results also tested if the zinc fingers are necessary for AML1-ETO to inhibit gene expression. When compared with AML1-ETO, it was found that AML1-ETODeltaZF 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, since multiple transcriptional repressor complexes can interact with different regions of ETO. 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. Furthermore, an attempt to define a single region of ETO that disrupts its function in vivo was unsuccessful. 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 (Wildonger, 2005b).

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, it is suggested that a deeper understanding of how AML1-ETO contributes to AML will require the identification of genes that are normally activated by AML1 (Wildonger, 2005b).

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