Transcriptional coactivators such as p300 act as crucial elements in the eukaryotic gene regulation network. These proteins bind to various transcription factors which recruit them to specific gene regions whose chromatin structure subsequently is remodeled. Previously, we have shown that C/EBPβ recruits p300 by interacting with the E1A‐binding site of the coactivator. We now show that C/EBPβ not only binds to p300 but also triggers massive phosphorylation of p300. This novel activity of C/EBPβ is dependent on the E1A‐binding region of p300 as well as on several subdomains of C/EBPβ, all of which are involved in the p300–C/EBPβ interaction. We have identified several sites of C/EBPβ‐inducible phosphorylation within the C‐terminal domain of p300. Mutation of these sites substantially impairs the activity of p300 as a coactivator of C/EBPβ. Interestingly, phosphorylation of p300 is also triggered by other C/EBP family members as well as by various other transcription factors that interact with the E1A‐binding domain of p300, suggesting that this novel phosphorylation mechanism may be of general relevance.

The tumor suppressor protein Pdcd4 is thought to suppress translation of mRNAs containing structured 5'-UTRs by interacting with translation initiation factor eIF4A and inhibiting its helicase activity. However, natural target mRNAs regulated by Pdcd4 so far are mostly unknown. Here, we identified p53 mRNA as a translational target of Pdcd4. We found that Pdcd4 is associated with p53 mRNA and suppresses its translation. The inhibitory effect of Pdcd4 on the translation of p53 mRNA depends on the ability of Pdcd4 to interact with eIF4A and is mediated by the 5'-UTR of p53 mRNA, which is able to form a stable stem-loop structure. We show that treatment of cells with DNA-damaging agents decreases the expression of Pdcd4. This suggests that translational suppression by Pdcd4 plays a role in maintaining a low level of p53 in unstressed cells and that this suppression is abrogated due to low levels of Pdcd4 after DNA damage. Overall, our work demonstrates for the first time that Pdcd4 is directly involved in translational suppression of a natural mRNA with a 5'-structured UTR and provides novel insight into the translational control of p53 expression.

The transcription factor c-Myb is highly expressed in hematopoietic progenitor cells and controls the transcription of genes important for lineage determination, cell proliferation, and differentiation. Deregulation of c-Myb has been implicated in the development of leukemia and certain other types of human cancer. c-Myb activity is highly dependent on the interaction of the c-Myb with the KIX domain of the coactivator p300, making the disruption of this interaction a reasonable strategy for the development of Myb inhibitors. Here, we have used bacterial Autodisplay to develop an in vitro binding assay that mimics the interaction of Myb and the KIX domain of p300. We have used this binding assay to investigate the potential of Naphthol AS-E phosphate, a compound known to bind to the KIX domain, to disrupt the interaction between Myb and p300. Our data show that Naphthol AS-E phosphate interferes with the Myb–KIX interaction in vitro and inhibits Myb activity in vivo. By using several human leukemia cell lines, we demonstrate that Naphthol AS-E phosphate suppresses the expression of Myb target genes and induces myeloid differentiation and apoptosis. Our work identifies Naphthol AS-E phosphate as the first low molecular weight compound that inhibits Myb activity by disrupting its interaction with p300, and suggests that inhibition of the Myb–KIX interaction might be a useful strategy for the treatment of leukemia and other tumors caused by deregulated c-Myb. Mol Cancer Ther; 14(6); 1276–85. ©2015 AACR. See related commentary by Liu et al., p. 1273

The transcription factor c-Myb is essential for the proliferation of hematopoietic cells and has been implicated in the development of leukemia and other human cancers. Pharmacologic inhibition of Myb is therefore emerging as a potential therapeutic strategy for these diseases. By using a Myb reporter cell line, we have identified plumbagin and several naphthoquinones as potent low-molecular weight Myb inhibitors. We demonstrate that these compounds inhibit c-Myb by binding to the c-Myb transactivation domain and disrupting the cooperation of c-Myb with the coactivator p300, a major driver of Myb activity. Naphthoquinone-induced inhibition of c-Myb suppresses Myb target gene expression and induces the differentiation of the myeloid leukemia cell line HL60. We demonstrate that murine and human primary acute myeloid leukemia cells are more sensitive to naphthoquinone-induced inhibition of clonogenic proliferation than normal hematopoietic progenitor cells. Overall, our work demonstrates for the first time the potential of naphthoquinones as small-molecule Myb inhibitors that may have therapeutic potential for the treatment of leukemia and other tumors driven by deregulated Myb. Mol Cancer Ther; 15(12); 2905–15. ©2016 AACR.