Oligomerization of ETO Is Obligatory for Corepressor Interaction

Abstract

Nearly 40% of cases of acute myelogenous leukemia (AML) of the M2 subtype are due to a chromosomal translocation that combines a sequence-specific DNA binding protein, AML1, with a potent transcriptional repressor, ETO. ETO interacts with nuclear receptor corepressors SMRT and N-CoR, which recruit histone deacetylase to the AML1-ETO oncoprotein. SMRT-N-CoR interaction requires each of two zinc fingers contained in C-terminal Nervy homology region 4 (NHR4) of ETO. However, here we show that polypeptides containing NHR4 are insufficient for interaction with SMRT. NHR2 is also required for SMRT interaction and repression by ETO, as well as for inhibition of hematopoietic differentiation by AML1-ETO. NHR2 mediates oligomerization of ETO as well as AML1-ETO. Fusion of NHR4 polypeptide to a heterologous dimerization domain allows strong interaction with SMRT in vitro. These data support a model in which NHR2 and NHR4 have complementary functions in repression by ETO. NHR2 functions as an oligomerization domain bringing together NHR4 polypeptides that together form the surface required for high-affinity interaction with corepressors. As nuclear receptors also interact with corepressors as dimers, oligomerization may be a common mechanism regulating corepressor interactions.Nearly 40% of cases of acute myeloid leukemia (AML) M2 are associated with the t(8;21)(q22;q22) chromosome translocation (39). This translocation creates a fusion between the AML1 gene on chromosome 21 and the ETO gene (also known as MTG8/CDR) on chromosome 8. The resulting chimeric protein AML1-ETO contains the DNA-binding domain (DBD) of AML1 and nearly all of ETO (5,24,38,42). The underlying mechanism of AML1-ETO leukemogenic activity is not fully understood.AML1 is a hematopoietic cell-specific transcription factor and is essential for definitive hematopoietic development (43,44,51). At least two potentially related mechanisms have been proposed for the AML1 function. The first is that AML1 synergistically interacts with other adjacent transcription factors, including C/EBP (55) and myb (2, 53). The second is that AML1 is able to recruit the p300/CBP coactivator complex to activate its target gene expression (23). In t(8;21), the activation domain of AML1 is replaced by the ETO protein. Transient-transfection assays indicate that AML1-ETO interferes with AML1 transactivation from certain potential AML1 target genes (9, 36).ETO is expressed at high levels in brain (38) and has also been detected in hematopoietic cells (6). Recently, we and others discovered that ETO as well as AML1-ETO physically associates with nuclear receptor corepressors called N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoid and thyroid receptors) (11,33,49). This discovery provides a mechanism for the repression activity of AML1-ETO. N-CoR and SMRT recruit class I and class II histone deacetylases (HDACs) (19)(20)(21), and this enzyme activity leads to a repressive chromatin state. Consistent with this mechanism, ETO and AML1-ETO are both associ...

Decreased intranuclear mobility of acute myeloid leukemia 1-containing fusion proteins is accompanied by reduced mobility and compartmentalization of core binding factor β

“…However, we observed that the AML1-ETO(DNHR2)/DNA complex moved much faster than that of the AML1-ETO(DNHR34) (Figure 1e,upper panel). As the molecular mass of AML1-ETO(DNHR2) is greater than that of AML1-ETO(DNHR34) (Figure 1e, bottom panel), these results suggest that while AML1-ETO and AML-ETO(DNHR34) bind the AML1 consensus sequence as a dimer, AML1-ETO(DNHR2) most likely binds to this element as a monomer, in agreement with the NHR2 domain of ETO being responsible for the formation of AML1-ETO homodimer (Minucci et al, 2000;Zhang et al, 2001;Davis et al, 2003).…”

“…It was reported that removal of the NHR2 domain diminished the ability of AML1-ETO to block hematopoietic differentiation of U937 cells (Zhang et al, 2001;Hug et al, 2002). Sin3 is directly dependent on this domain for interactions with ETO, whereas corepressors for nuclear hormone receptor, SMRT and NCoR, are indirectly dependent on this domain to facilitate interactions with the NHR4 domain of ETO (Zhang et al, 2001). In the studies reported here, we demonstrated that the NHR2 domain of ETO was responsible for the reduction of intranuclear mobility of AML1-ETO, which is reminiscent of that for NuMARARa, in which the coiled-coil domain of NuMA was found to be responsible for the mobility reduction of NuMA-RARa .…”

“…The NHR2 domain, which mediates homodimerization and heterodimerization of ETO family proteins, may itself be sufficient for the oncogenic activity of AML1-ETO (Minucci et al, 2000) and is important for the association of ETO with several proteins (Davis et al, 2003;Peterson and Zhang, 2004). It was reported that removal of the NHR2 domain diminished the ability of AML1-ETO to block hematopoietic differentiation of U937 cells (Zhang et al, 2001;Hug et al, 2002). Sin3 is directly dependent on this domain for interactions with ETO, whereas corepressors for nuclear hormone receptor, SMRT and NCoR, are indirectly dependent on this domain to facilitate interactions with the NHR4 domain of ETO (Zhang et al, 2001).…”

“…In contrast, the mobility of GFP-AML1-ETO(DNHR34) was very close to AML1-ETO (P ¼ 0.34). Therefore, similar to its contribution to other oncogenic activities of AML1-ETO (Minucci et al, 2000;Zhang et al, 2001), the NHR2 dimerization also plays a key role in the mobility reduction of AML1-ETO.…”

“…Nervy homology region 2 domain is responsible for the formation of acute myeloid leukemia 1-eight twenty-one homodimer detected by gel-shift assays It is well known that ETO protein is composed of four evolutionarily conserved NHR domains (Minucci et al, 2000;Zhang et al, 2001;Davis et al, 2003). The NHR2 domain is the dimerization domain, which plays an important role in homodimerization and heterodimerization between the ETO family members as well as ETO and AML1-ETO.…”