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

Abstract

Acute myeloid leukemia 1 (AML1) gene on chromosome 21 is involved in several chromosomal translocations, including t(8;21) and t(16;21), that produce chimeric fusion proteins AML1-eight twenty-one (ETO) and AML-myeloid transforming gene chromosome 16 (MTG16), which contribute to leukemogenesis. The molecular basis for the leukemogenic effects of these fusion proteins is incompletely understood. Using gel-shift assay, we showed that AML1-ETO and AML1-MTG16 bound to a series of AML1 consensus DNA-binding sites with different affinities. Using fluorescence recovery after photobleaching (FRAP), we demonstrated that a fusion of AML1 with ETO or MTG16 exhibits reduced intranuclear mobility compared with wild-type AML1 or either fusion partner. The dimerization domain (nervy homology region 2) of ETO is responsible for the reduced mobility of AML1-ETO. Dual FRAP studies revealed that CBFb colocalized with AML1-ETO within the nucleus, resulting in reduced mobility of CBFb. Therefore, AML1 fusion proteins may interfere with normal AML1 function due to aberrant nuclear dynamics, which leads to spatial and temporal sequestration of CBFb and perhaps other coregulators critical for myeloid differentiation.

The reduced and altered activities of PAX5 are linked to the protein–protein interaction motif (coiled-coil domain) of the PAX5–PML fusion protein in t(9;15)-associated acute lymphocytic leukemia

“…In our previous studies (Dong et al, 2004;Qiu et al, 2006), the decreasing intranuclear mobility of leukemia-related fusion proteins resulting from chromosomal translocation in acute myeloid leukemia (AML), namely PML-RARa or AML1-ETO, has been demonstrated compared with wild-type RARa or AML1, respectively. The CC domain of PML, like in PML-RARa oncogenic chimeric protein (Huang et al, 2008), was also shown to be responsible for the reduction of intranuclear mobility of PAX5-PML, implying its importance for this effect.…”

“…A rectangular region in the nucleus was bleached by applying maximal laser power for approximately 1 s. In all FRAP experiments, only cells expressing low levels of protein were examined to avoid artifacts of overexpression as previously described by us (Dong et al, 2004;Qiu et al, 2006). Figure 5 shows images taken right before bleaching (prebleach), immediately after bleaching (bleach) and at several time points after bleaching.…”

“…The role of the CC domain in the reduction of intranuclear mobility of PAX5-PML We recently reported that, in addition to being essential for other oncogenic functions, the CC dimerization domain of NuMA-RARa, PML-RARa and NHR2 dimerization domain of AML1-ETO was responsible for the reduced intranuclear mobility of NuMA-RARa, PML-RARa and AML1-ETO, respectively (Dong et al, 2004;Qiu et al, 2006;Huang et al, 2008). In this study, we next examined whether or not the dimerization domain (CC-domain) in PAX5-PML contributes to its mobility reduction within the nucleus.…”

“…Cell culture 293T cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and HeLa cells were maintained in Opti-MEM I media (Invitrogen, Grand Island, NY, USA) with 4% fetal bovine serum (Qiu et al, 2006(Qiu et al, , 2007. A03_1 cell line, which contains integrated lac operator (lacO) heterochromatic array arranged in a globular structure useful for studying protein-protein interaction in the context of chromatin, was cultured in F-12 Ham's medium (without hypoxanthine and thymidine) (Invitrogen) with 0.3 mM methotrexate and 10% dialyzed fetal bovine serum (HyClone, Logan, UT, USA) (Qiu et al, 2007).…”

“…Cells with transient expression of GFP-LacR-tagged plus ChFPtagged constructs (in A03_1 cell) and YFP-PAX5-PML, YFP-PAX5-PML(DCC), YFP-PAX5, YFP-5 0 -PAX5 or YFP-3 0 -PML (in HeLa and 293T cell) were fixed and deconvolution microscopy was performed as described previously (Qiu et al, 2006(Qiu et al, , 2007(Qiu et al, , 2010a.…”