Abstract
Ecotropic viral integration site 1 (EVI1) is an important transcription factor for leukemogenesis. EVI1 is a member of a group of transcription factors with C-terminal binding protein (CtBP)-binding motifs that act as transcriptional co-repressors; however, we recently found that EVI1 directly activates GATA2 transcription, which is an important gene for the maintenance of hematopoietic stem cells. We show here that EVI1-activated GATA2 transcripts derive from exon 1S of GATA2, which is specifically activated in neural and hematopoietic cells. EVI1 was acetylated by the histone acetyltransferase p300/CBP association factor (P/CAF) in myeloid leukemia cells and hematopoietic progenitor cells. Acetylation at Lys(564), which is adjacent to the CtBP-binding consensus sequence of EVI1, was found to be important for transcriptional activation of GATA2. Mutation of Lys(564) to alanine (K564A) markedly reduced the ability of EVI1 to bind DNA and activate transcription of GATA2. Furthermore, we confirmed that Lys(564) in EVI1 was specifically acetylated in leukemia and primary hematopoietic cells by using an antibody directed against an acetylated Lys(564) EVI1 peptide. Moreover, co-transfection of P/CAF with EVI1 overcame the suppressive effect of the CtBP co-repressor and resulted in GATA2 transcriptional activation; nonetheless, CtBP2 was still included in the protein complex with EVI1 and P/CAF on the EVI1-binding site in the GATA2 promoter region. Thus, acetylation of EVI1 at Lys(564) by P/CAF enhances the DNA binding capacity of EVI1 and thereby contributes to the activation of GATA2.
Highlights
In Ecotropic viral integration site 1 (EVI1), two DNA binding domains with seven and three zinc finger repeats bind DNA through specific conserved GATA-like or ETS-like sequence motifs, and they have the potential to interact with both co-repressors and co-activators as a dual transcriptional factor (6 – 8)
EVI1 acetylation is due to its interaction with p300/cAMP-responsive element-binding proteinbinding protein (CBP)-associated factor (P/CAF), and the acetylation of Lys564 adjacent to the C-terminal binding protein (CtBP)-binding consensus sequence is an important modification for the transcriptional activation of GATA2
In the transcriptional activation complex on the EVI1 DNA-binding site, the co-repressor CtBP2 was still included with P/CAF and EVI1, but histone deacetylase complexes (HDACs) was not included, suggesting that this complex of EVI1, P/CAF, and CtBP2 may exist to aid in the transcriptional activation of GATA2
Summary
CtBP, C-terminal binding protein; P/CAF, p300/ CBP association factor; HDAC, histone deacetylase complex; shRNA, small hairpin RNA; CBP, cAMP-response element-binding protein-binding protein; TBS, Tris-buffered saline; RT, reverse transcriptase; ChIP, chromatin immunoprecipitation. We found that the histone acetyltransferase p300/CBPassociated factor (P/CAF) could bind to EVI1 and acetylated EVI1 in leukemia cells. Lys564 in EVI1 is one of the important residues for the activation of GATA2 transcription by P/CAF acetylation. Using chromatin immunoprecipitation PCR and gel mobility shift assays, we found that the binding of EVI1 to the GATA2 promoter region was clearly enhanced by EVI1 acetylation. The acetylation of EVI1 is an important modification that regulates the transcriptional activity and the DNA binding activity of EVI1
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