E2A expression, nuclear localization, and in vivo formation of DNA- and non-DNA-binding species during B-cell development.

Jacobs, Yakop; Vierra, Craig; Nelson, Christine

doi:10.1128/mcb.13.12.7321

Cited by 65 publications

(58 citation statements)

References 67 publications

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“…For E14.5 dpc mouse embryos, cell suspensions were prepared in phosphate-buffered saline with a tissue grinder prior to hypotonic lysis. EMSA of nuclear proteins was performed as described elsewhere (15).…”

Section: Methodsmentioning

confidence: 99%

Meis Proteins are Major In Vivo DNA Binding Partners for Wild-Type but Not Chimeric Pbx Proteins

Chang

Jacobs

Nakamura

et al. 1997

Molecular and Cellular Biology

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The Pbx1 and Meis1 proto-oncogenes code for divergent homeodomain proteins that are targets for oncogenic mutations in human and murine leukemias, respectively, and implicated by genetic analyses to functionally collaborate with Hox proteins during embryonic development and/or oncogenesis. Although Pbx proteins have been shown to dimerize with Hox proteins and modulate their DNA binding properties in vitro, the biochemical compositions of endogenous Pbx-containing complexes have not been determined. In the present study, we demonstrate that Pbx and Meis proteins form abundant complexes that comprise a major Pbx-containing DNA binding activity in nuclear extracts of cultured cells and mouse embryos. Pbx1 and Meis1 dimerize in solution and cooperatively bind bipartite DNA sequences consisting of directly adjacent Pbx and Meis half sites. Pbx1-Meis1 heterodimers display distinctive DNA binding specificities and cross-bind to a subset of Pbx-Hox sites, including those previously implicated as response elements for the execution of Pbx-dependent Hox programs in vivo. Chimeric oncoprotein E2a-Pbx1 is unable to bind DNA with Meis1, due to the deletion of amino-terminal Pbx1 sequences following fusion with E2a. We conclude that Meis proteins are preferred in vivo DNA binding partners for wild-type Pbx1, a relationship that is circumvented by its oncogenic counterpart E2a-Pbx1.Hox proteins make critical contributions to cell fate and segmental patterning during embryonic development (30). As targets of oncogenic mutations in human and murine leukemias, they are also implicated in cancer pathogenesis (3,4,21,34,35), which likely reflects perturbations of their roles in normal hematopoietic cell differentiation (23). In these capacities, they are presumed to function as transcription factors whose DNA binding activities are mediated through a conserved motif known as the homeodomain, which is structurally related to the bacterial helix-turn-helix motif (48). However, at a molecular level, the contributions of Hox proteins to developmental processes and disease pathogenesis are inadequately explained, given their disappointingly poor in vitro DNA binding affinities and specificities as monomeric proteins. This has led to the proposal that additional factors are required to modulate the DNA binding and transcriptional properties of Hox proteins (13), which would be consistent with models for achievement of specificity by other classes of transcriptional proteins.Genetic and biochemical studies support the argument for a role for members of the Pbx, exd, and ceh-20 subfamily (5) of divergent homeodomain proteins as potential Hox cofactors. In Drosophila melanogaster, exd is required for the execution of genetic programs that are also dependent on Hox proteins for appropriate segment-specific expression (40,45,46). A similar role for mammalian Pbx proteins is suggested by genetic analyses demonstrating that sequence elements with features of Pbx-Hox consensus sites are required for appropriate expression of murine Hoxb-1 in th...

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Section: Methodsmentioning

confidence: 99%

Meis Proteins are Major In Vivo DNA Binding Partners for Wild-Type but Not Chimeric Pbx Proteins

Chang

Jacobs

Nakamura

et al. 1997

Molecular and Cellular Biology

Self Cite

241

263

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“…Cell lysates were examined by Western blot to determine the kinetics of E2a-Pbx1b induction. E2a-Pbx1b was detected using an anti-E2a monoclonal antibody (Jacobs et al, 1993) which also recognizes the endogenous, wild type E2a proteins whose migrations are indicated to the right the MT1 control cells whose survival was maintained at greater than 90% throughout the 72 h treatment period.…”

Section: E2a-pbx1b Induces Apoptosis Of Reh Cellsmentioning

confidence: 99%

“…The lysates were sonicated and the OD 280 was determined. Ten micrograms of total cellular protein were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) followed by Western blot analysis using an anti-E2a monoclonal antibody as previously described (Jacobs et al, 1993). The homeodomain lacking construct (1bDHD) was detected using a Pbx1b-speci®c monoclonal antibody (YJ and MLC, unpublished).…”

Section: Expression and Immunoblotting Of Proteinmentioning

confidence: 99%

Chimeric oncoprotein E2a-Pbx1 induces apoptosis of hematopoietic cells by a p53-independent mechanism that is suppressed by Bcl-2

et al. 1997

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The chimeric oncoprotein E2a-Pbx1 results from fusion of the E2A and PBX1 genes following t(1;19) chromosomal translocations in B cell precursor acute leukemias. Experimentally B cell progenitors do not tolerate constitutive expression of E2a-Pbx1 which contrasts with transformation of several other cell types following its stable expression both in vitro and in vivo. To further investigate the e ects of E2a-Pbx1 on the B cell progenitors, we conditionally expressed E2a-Pbx1 under control of a metal response element in hematopoietic precursor cell lines in vitro. Inducible expression of E2a-Pbx1 resulted in cell death with the morphologic and molecular features of apoptosis. A structure-function analysis demonstrated that induction of apoptosis was not a dominant-negative e ect of the E2a moiety but, rather, required the DNA-binding homeodomain of Pbx1. E2a-Pbx1-induced apoptosis proceeded through a BCL2-responsive checkpoint eventuating in PARP inactivation but did require p53. Constitutive expression of E2a-Pbx1 did not induce apoptosis or continued cycling of Rat-1 ®broblasts in low serum conditions. These studies demonstrate that E2a-Pbx1 initiates programmed cell death of hematopoietic precursers by a mechanism that requires its chimeric transcriptional properties, but, unlike other nuclear oncoproteins, is independent of p53.

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“…Isolation of nuclear extracts was performed as previously described (Jacobs et al, 1993). [ 32 P]-labeled double-stranded oligonucleotides, POSHwt (5 0 -CTTGAAATGAGTAAGACTGAGT-3 0 ), POSH-mt (5 0 -CTTGAAATGCGTAAGACTGAGT-3 0 ), were mixed with in vitro-translated EWS/ATF-1 or nuclear extract and analyzed as previously described (Fujino et al, 2001).…”

Section: Emsamentioning

confidence: 99%

Identification of target genes for EWS/ATF-1 chimeric transcription factor

et al. 2003

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E2A expression, nuclear localization, and in vivo formation of DNA- and non-DNA-binding species during B-cell development.

Cited by 65 publications

References 67 publications

Meis Proteins are Major In Vivo DNA Binding Partners for Wild-Type but Not Chimeric Pbx Proteins

Meis Proteins are Major In Vivo DNA Binding Partners for Wild-Type but Not Chimeric Pbx Proteins

Chimeric oncoprotein E2a-Pbx1 induces apoptosis of hematopoietic cells by a p53-independent mechanism that is suppressed by Bcl-2

Identification of target genes for EWS/ATF-1 chimeric transcription factor

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