Recent reports indicate that deacetylation of nucleosomal histones represents a major mechanism of transcriptional repression (34
). Nucleosomes deprived of acetyl groups may have altered interactions with DNA and other proteins that may hinder access of the RNA polymerase II transcriptional machinery to target gene sequences (50
). This fundamental mechanism has come under increased scrutiny with the discovery that several leukemogenic chimeric transcription factors are aberrant transcriptional repressors (17
). Prominent among these fusion products are the AML-1/ETO fusion generated in t(8;21) M2 leukemia and the RARα fusion proteins of M3, APL. Among these partners of RARα, PLZF stands out as a transcriptional repressor, usually expressed in myeloid progenitor cells. In t(11;17)-associated APL, PLZF-RARα attracts an HDAC-corepressor complex to RARα target promoters, resulting in a leukemia refractory to ATRA (18
). ETO, a protein whose exact role in cellular physiology remains uncertain, in a similar manner inappropriately forces a repressor to the promoters of complex AML-1 targets in t(8;21)-associated AML (43
We showed, for the first time, that ETO can associate with a sequence-specific DNA binding protein, namely PLZF. This indicates that, like N-CoR, SMRT, and Sin3A, ETO can be considered a transcriptional corepressor. This finding is particularly significant, since this interaction may be a marker for a repressor complex active in early myeloid cells, which disappears as cells differentiate. Expression of PLZF was shown to mediate growth suppression, inhibition of myeloid differentiation, and cell cycle arrest (56
). Such effects may be mediated by the ability of PLZF to repress target genes, such as those coding for cyclin A2 and the IL-3Rα chain, through the actions of complexes which contain ETO, other corepressors, and HDACs. Thus, further characterization of the transcriptional complex containing ETO and PLZF may contribute to understanding the role of transcriptional repression in myeloid progenitor cells. These statements are supported by several lines of evidence. (i) Both ETO and PLZF are expressed in CD34+
progenitor cells, but decrease as myeloid cells differentiate (12
; A. M. Melnick, G. Carlile, and J. D. Licht, unpublished data). (ii) Endogenous coexpressed ETO and PLZF interact in vivo, as shown by coimmunoprecipitation in a hematopoietic cell line. (iii) In contrast to PLZF, ETO is unable to bind specifically to DNA (11
). (iv) PLZF and ETO bind to a similar group of corepressor and HDAC proteins (8
). (v) ETO strongly and consistently potentiates transcriptional repression mediated by PLZF. (vi) The transcriptional repression mediated by PLZF plus ETO is abrogated by treatment with HDAC inhibitors.
It should be emphasized that, in this report, we indicated that the endogenous ETO and PLZF proteins can interact. In contrast, although N-CoR, SMRT, and Sin3A were shown to interact with PLZF in vitro (8
) and overexpressed PLZF-RARα and PLZF were found to interact with these corepressor molecules in immunoprecipitation experiments (8
), no experiments have yet been published indicating endogenous PLZF interacts with any of these corepressors. Although coimmunoprecipitations of overexpressed protein and yeast two-hybrid assays indicated a strong and consistent interaction between ETO and PLZF, similar studies of the native proteins in HEL cells indicate that only about 10% of endogenous PLZF coprecipitates with ETO (Fig. C). This finding may have several explanations. First, not all repressive complexes containing PLZF may require or contain ETO. Second, ETO may target other proteins to mediate repression. Furthermore, ETO may serve in other roles besides transcription. ETO binds to poly(G) RNA and can be localized in the nucleolus, consistent with a role for ETO in RNA metabolism (11
). PLZF may have other functions as well. PLZF can colocalize with the PML protein of t(15;17)-associated APL (32
) in the nuclear body structure. Therefore, PLZF may be involved in PML-mediated processes such as apoptosis and control of mRNA transport and translation (3
). Finally, the interaction between ETO and PLZF may depend on posttranscriptional modifications, which may change depending on the cell cycle, stage of differentiation, and cell growth conditions. Indeed, both ETO and PLZF proteins interact with regulatory kinases and are subject to phosphorylation (2
ETO consistently potentiated transcriptional repression mediated by native PLZF as well as the PLZF repression domains fused to a heterologous DBD. At least part of this transcriptional function is dependent on the ability of PLZF to associate with the same corepressors and HDACs as ETO (8
). The potentiation of transcriptional repression by ETO can be further enhanced by coexpression of the N-CoR or SMRT corepressors and HDACs, indicating a combinatorial effect in which all of these proteins functionally interact (data not shown). Finally, the transcriptional repression mediated by ETO and PLZF is blocked by TSA and sodium butyrate, indicating that the enhancement of PLZF repression mediated by ETO is dependent on HDAC activity. This suggests that ETO enhances the ability of PLZF to recruit a transcriptional repression complex which functions, at least in part, through histone deacetylation (Fig. A).
FIG. 7 Model of interaction between ETO and PLZF. (A) PLZF binds to specific sequences in its target promoters through its zinc finger (ZF) domains. PLZF then recruits ETO through sequences in the second repression domain of PLZF (residues 200 to 300), binding (more ...)
The POZ/BTB domain of PLZF mediates homodimerization, transcriptional repression, and heterologous protein-protein interactions, including those with corepressors (8
). ETO does not associate with the PLZF-POZ/BTB domain in yeast and cannot form a stable complex with this domain in mammalian cells. Furthermore, ETO can still bind to PLZF with the POZ/BTB domain deleted. Despite this fact, ETO still could enhance repression mediated by the POZ/BTB domain of PLZF. This paradox can be solved by hypothesizing that ETO is able to mediate this effect indirectly, through interaction with the N-CoR and SMRT or Sin3A corepressors, all of which can interact with the POZ/BTB domain (Fig. B). ETO fails to enhance transcription of PLZF mutants with either the POZ/BTB domain or the second repression domain deleted, and PLZF with the POZ/BTB domain deleted exhibited diminished binding of ETO. It therefore seems likely that the POZ/BTB domain is important for stabilizing a multiprotein complex containing ETO. The second repression domain of PLZF was absolutely required for interaction with ETO. This was intriguing, given that the mechanism of action of this domain of PLZF had not previously been characterized. A PLZF deletion mutant lacking the RD2 was severely deficient for transcriptional repression and was not significantly potentiated by ETO (Fig. A), while ETO was able to augment repression mediated by the second repression domain of PLZF fused to GAL4 (Fig. C). Together, this information indicates that there are multiple sites for corepressor interaction in the PLZF protein. The binding of N-CoR, SMRT, and Sin3A to the N-terminal POZ/BTB domain of PLZF and ETO to the more C-terminal repression domain may lead to the formation of a multiprotein repression complex, stabilized by multiple cross-protein contacts (Fig. A).
Surprisingly, none of the four conserved domains of ETO were required to associate with PLZF, while a previously poorly characterized, central region of ETO was sufficient to complex with PLZF. Interestingly, the Sin3A corepressor binds to ETO through similar sequences (B. Lutterbach and S. W. Hiebert, unpublished data). The functional relationship between Sin3A, ETO, and PLZF is underscored by the fact that ETO and Sin3A (44
), ETO and PLZF (Fig. C), and PLZF and Sin3A (A. M. Melnick, G. Carlile, and J. D. Licht, unpublished data) can be coimmunoprecipitated in HEL cells. Although our yeast two-hybrid (Fig. B) and protein affinity chromatography studies (Fig. C) could indicate a direct interaction between PLZF and ETO, it remains a strong possibility that Sin3A could act as a bridging factor between these proteins, since Sin3A is present in yeast and Sin3A can be detected in reticulocyte lysates (B. Lutterbach and S. W. Hiebert, unpublished data). It must be emphasized that the N-terminal half of ETO alone, despite its ability to bind to both PLZF and Sin3A, was unable to mediate corepression, suggesting that the participation of Sin3A is not sufficient for repression by ETO. The ability of ETO to augment repression by PLZF required C-terminal sequences of ETO. This suggests a model in which the central portion of ETO forms complexes with PLZF, while the C-terminal region binds to the N-CoR and SMRT corepressors, which in turn can bind to the POZ/BTB domain of PLZF. The resulting collection of proteins recruit HDACs and mediate transcriptional repression (Fig. A).
Given the fact that ETO and PLZF physically and functionally interact, how is this relationship disrupted in leukemia? If for example, the AML-1-ETO oncoprotein interacts with wild-type PLZF in myeloid progenitor cells, would this affect PLZF-mediated repression? Alternatively, does ETO participate in the repression of RARα target genes? In fact, our preliminary results indicate that AML-1–ETO is a dominant-negative inhibitor of PLZF repression. Hence in t(8;21)-associated AML, not only might AML-1 target genes be aberrantly repressed, but PLZF targets associated with control of cell growth might be derepressed as well. In the case of APL, our initial data indicate that ETO can potentiate repression of reporter genes containing RAR binding sites in the absence of retinoic acid. Whether PLZF-RARα might affect some other essential function of ETO or sequester ETO, making it unavailable for use by other transcriptional repressors or other cellular processes, is uncertain. ETO and PLZF may represent components of a functional repression complex present in myeloid cells. Disruption of this complex could represent a common pathway of malignant transformation. A fuller understanding of other components of this complex will lead to the identification of mechanisms of transcriptional repression and hence offer new opportunities for therapeutic intervention in leukemia.