HOXB6 protein is bound to CREB-binding protein and represses globin expression in a DNA binding-dependent, PBX interaction-independent process.
Although HOXB6 and other HOX genes have previously been associated with hematopoiesis and leukemias, the precise mechanism of action of their protein products remains unclear. Here we use a biological model in which HOXB6 represses alpha- and gamma-globin mRNA levels to perform a structure/function analysis for this homeodomain protein. HOXB6 protein represses globin transcript levels in stably transfected K562 cells in a DNA-binding dependent fashion. However, the capacity to form cooperative DNA-binding complexes with the PBX co-factor protein is not required for HOXB6 biological activity. Neither the conserved extreme N-terminal region, a polyglutamic acid region at the protein C terminus, nor the Ser(214) CKII phosphorylation site was required for DNA binding or activity in this model. We have previously reported that HOX proteins can inhibit CREB-binding protein (CBP)-histone acetyltransferase-mediated potentiation of reporter gene transcription. We now show that endogenous CBP is co-precipitated with exogenous HOXB6 from nuclear and cytoplasmic compartments of transfected K562 cells. Furthermore, endogenous CBP co-precipitates with endogenous HOXB6 in day 14.5 murine fetal liver cells during active globin gene expression in this tissue. The CBP interaction motif was localized to the homeodomain but does not require the highly conserved helix 3. Our data suggest that the homeodomain contains most or all of the important structures required for HOXB6 activity in blood cells.
Department of Medicine, University of California Veterans Affairs Medical Center, San Francisco, California 94121, USA.
Weifang
W
Shen
Daniel
D
Chrobak
Keerthi
K
Krishnan
H Jeffrey
HJ
Lawrence
Corey
C
Largman
D000123:Acetyltransferases / Q000378:metabolism; D000818:Animals; D050882:CREB-Binding Protein; D004247:DNA / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D005914:Globins / Q000235:genetics*; D051548:Histone Acetyltransferases; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D008099:Liver / Q000166:cytology; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D011233:Precipitin Tests; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D012694:Serine / Q000235:genetics; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection
C093252:HOXB6 protein, human; D018398:Homeodomain Proteins; C490537:Hoxb6 protein, mouse; D009687:Nuclear Proteins; C106682:PKNOX1 protein, human; C491412:Pknox1 protein, mouse; D012333:RNA, Messenger; D015534:Trans-Activators; D012694:Serine; D005914:Globins; D004247:DNA; D000123:Acetyltransferases; D050882:CREB-Binding Protein; C494363:CREBBP protein, human; C494364:Crebbp protein, mouse; D051548:Histone Acetyltransferases