Interaction of EVI1 with cAMP-responsive element-binding protein-binding protein (CBP) and p300/CBP-associated factor (P/CAF) results in reversible acetylation of EVI1 and in co-localization in nuclear speckles.
EVI1 is a very complex protein with two domains of zinc fingers and is inappropriately expressed in many types of human myeloid leukemias. Using reporter gene assays, several investigators showed that EVI1 is a transcription repressor, and recently it was shown that EVI1 interacts with the co-repressor carboxyl-terminal binding protein 1 (CtBP1). Earlier, we showed that the inappropriate expression of EVI1 in murine hematopoietic precursor cells leads to their abnormal differentiation and to increased proliferation. Using biochemical assays, we have identified two groups of transcription co-regulators that associate with EVI1 presumably to regulate gene expression. One group of co-regulators includes the CtBP1 and histone deacetylase. The second group includes the two co-activators cAMP-responsive element-binding protein-binding protein (CBP) and p300/CBP-associated factor (P/CAF), both of which have histone acetyltransferase (HAT) activity. All of these proteins require separate regions of EVI1 for efficient interaction, and they divergently affect the ability of EVI1 to regulate gene transcription in reporter gene assays. Confocal microscopy analysis shows that in the majority of the cells, EVI1 is nuclear and diffused, whereas in about 10% of the cells EVI1 localizes in nuclear speckles. However, in the presence of the added exogenous co-repressors histone deacetylase or CtBP1, all of the nuclei have a diffuse EVI1 staining, and the proteins do not appear to reside together in obvious nuclear structures. In contrast, when CBP or P/CAF are added, defined speckled bodies appear in the nucleus. Analysis of the staining pattern indicates that EVI1 and CBP or EVI1 and P/CAF are contained within these structures. These nuclear structures are not observed when CBP is substituted with a point mutant HAT-inactive CBP with which EVI1 also physically interacts. Finally, we show that the interaction of EVI1 with either CBP or P/CAF leads to acetylation of EVI1. These results suggest that the assembly of EVI1 in nuclear speckles requires the intact HAT activity of the co-activators.
Chakraborty
S
S
Department of Pathology and The Cancer Center, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
Senyuk
V
V
Sitailo
S
S
Chi
Y
Y
Nucifora
G
G
D000107:Acetylation; D000123:Acetyltransferases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000502:physiology*; D050881:E1A-Associated p300 Protein; D000939:Epitopes; D017930:Genes, Reporter; D051548:Histone Acetyltransferases; D006801:Humans; D007938:Leukemia / Q000378:metabolism; D000074008:MDS1 and EVI1 Complex Locus Protein; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011519:Proto-Oncogenes; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation; D016335:Zinc Fingers; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor
D000123:Acetyltransferases; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050881:E1A-Associated p300 Protein; D000939:Epitopes; D051548:Histone Acetyltransferases; D000074008:MDS1 and EVI1 Complex Locus Protein; D009687:Nuclear Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor; C063575:MECOM protein, human; C499305:Mecom protein, mouse; C494361:Ep300 protein, mouse