The chimeric oncoproteins E2A-PBX1 and E2A-HLF are concentrated within spherical nuclear domains

Abstract

Oncogenic mutation of nuclear transcription factors often is associated with altered patterns of subcellular localization that may be of functional importance. The leukemogenic transcription factor gene E2A-PBX1 is created through fusion of the genes E2A and PBX1 as a result of t(1;19) in acute lymphoblastic leukemia. We evaluated subcellular localization patterns of E2A-PBX1 protein in transfected cells using immuno¯uorescence. Full-length E2A-PBX1 was exclusively nuclear and was concentrated in spherical domains denoted chimeric-E2A oncoprotein domains (CODs). In contrast, nuclear uorescence for wild-type E2A or PBX1 proteins was diuse. Enhanced concentrations of RNA polymerase II within many CODs and the requirement for an E2A-encoded activation domain suggested transcriptional relevance. However, in situ co-detection of nascent transcripts labeled with bromouridine failed to con®rm altered transcriptional activity in relation to CODs. CODs also failed to co-localize with other proteins known to occupy functional nuclear compartments, including the transcription factor PML, the spliceosome-associated protein SC-35 and the adenovirus replication factor DBP, or with foci of DNA replication. Co-transfection of Hoxb7, a homeodomain protein capable of enhancing DNA binding by PBX1, impaired COD formation, suggesting that CODs contain E2A-PBX1 protein not associated with DNA. We conclude that, as a`gain of function' phenomenon requiring protein elements from both E2A and PBX1, COD formation may be relevant to the biology of E2A-PBX1 in leukemogenesis.

Functional Cloning and Characterization of a Novel Nonhomeodomain Protein That Inhibits the Binding of PBX1-HOX Complexes to DNA

“…Our subcellular localization studies show HPIP to be localized mainly in the cytoplasm, although a small amount was found in the nucleus. This finding, together with the knowledge that PBX is expressed in both nuclei and cytosol (48), implies that HPIP could in principle interact with PBX in either compartment. If the initial interaction occurs in the cytoplasm, the heterodimer would either stay in the cytosol or enter the nucleus as a complex.…”

“…PBX, in contrast with E2A-PBX, is unable to activate transcription by itself (8,10,46); therefore, and based on our deletion data pointing at HPIP as a potential E2A-PBX binding protein, we studied the effect of HPIP on the ability of E2A-PBX to induce a luciferase reporter gene driven by seven copies of a E2A-PBX binding site (TGATTGAT) (47) upstream of the minimal FOS promoter. Co-expression of the E2A-PBX reporter plasmid, E2A-PBX, and HPIP decreased the E2A-PBX-mediated tran- 6 and 10 6 cells, respectively) stably expressing FLAG-HPIP were resolved on a 7.5% SDS-PAGE.…”

Role for Homodimerization in Growth Deregulation by E2a Fusion Proteins

“…Two days after infection the coverslips were washed briefly in PBS, fixed for 10 min in 3.7% formaldehyde in PBS, and stored for various periods of time at 4°C immersed in 70% ethanol. Recombinant proteins were detected by indirect immunofluorescence using the YAE primary antibody at a 1:100 dilution and a fluorescein isothiocyanate-labeled secondary antibody (Jackson) at a 1:200 dilution according to a previously published protocol (32). Expression of the proteins E2A-GCN4 250-281 1-273 and VP16-NLS-Pbx1a was ascertained using monoclonal antibody clone G193-86 (Pharmingen, San Diego, Calif.) and a monoclonal antibody directed against the C terminus of Pbx1a (kindly provided by Michael Cleary, Stanford University), respectively.…”

Evi9 Encodes a Novel Zinc Finger Protein That Physically Interacts with BCL6, a Known Human B-Cell Proto-Oncogene Product

“…The speckled hybridization pattern of Evi9a and Evi9b suggested that they are located within nuclear bodies or other subnuclear compartments (8,11,20,33). Many proteins are known to localize to these nuclear compartments.…”