GCN5 inhibits XBP-1S-mediated transcription by antagonizing PCAF action.

Cellular unfolded protein response (UPR) is induced when endoplasmic reticulum (ER) is under stress. XBP-1S, the active isoform of X-box binding protein 1 (XBP-1), is a key regulator of UPR. Previously, we showed that a histone acetyltransferase (HAT), p300/CBP-associated factor (PCAF), binds to XBP-1S and functions as an activator of XBP-1S. Here, we identify general control nonderepressible 5 (GCN5), a HAT with 73% identity to PCAF, as a novel XBP-1S regulator. Both PCAF and GCN5 bind to the same domain of XBP-1S. Surprisingly, GCN5 potently blocks the XBP-1S-mediated transcription, including cellular UPR genes and latent membrane protein 1 of Epstein-Barr virus. Unlike PCAF, GCN5 acetylates XBP-1S and enhances nuclear retention and protein stability of XBP-1S. However, such GCN5-mediated acetylation of XBP-1S shows no effects on XBP-1S activity. In addition, the HAT activity of GCN5 is not required for repression of XBP-1S target genes. We further demonstrate that GCN5 inhibits XBP-1S-mediated transcription by disrupting the PCAF-XBP-1S interaction and preventing the recruitment of XBP-1S to its target genes. Taken together, our results represent the first work demonstrating that GCN5 and PCAF exhibit different functions and antagonistically regulate the XBP-1S-mediated transcription.

Inhibition of human cytomegalovirus replication by overexpression of CREB1.

Expression of the human cytomegalovirus (HCMV) major immediate-early (MIE) genes is regulated by a strong enhancer-containing promoter with multiple binding sites for various transcription factors, including cyclic AMP response element binding protein 1 (CREB1). Here we show that overexpression of CREB1 potently blocked MIE transcription and HCMV replication. Surprisingly, CREB1 still exhibited strong inhibition of the MIE promoter when all five CREB binding sites within the enhancer were mutated, suggesting that CREB1 regulated the MIE gene expression indirectly. Promoter deletion analysis and site-directed mutagenesis identified the region between -130 and -50 upstream of the transcription start site of the MIE gene as the "CREB1 responsive region". Mutations of SP1/3 and NF-κB binding sites within this region interrupted the inhibitory effect induced by CREB1 overexpression. Our findings suggest that overexpression of CREB1 can cause repression of HCMV replication and may contribute to the development of new anti-HCMV strategies.

HEXIM1, a New Player in the p53 Pathway.

Hexamethylene bisacetamide-inducible protein 1 (HEXIM1) is best known as the inhibitor of positive transcription elongation factor b (P-TEFb), which controls transcription elongation of RNA polymerase II and Tat transactivation of human immunodeficiency virus. Besides P-TEFb, several proteins have been identified as HEXIM1 binding proteins. It is noteworthy that more than half of the HEXIM1 binding partners are involved in cancers. P53 and two key regulators of the p53 pathway, nucleophosmin (NPM) and human double minute-2 protein (HDM2), are among the factors identified. This review will focus on the functional importance of the interactions between HEXIM1 and p53/NPM/HDM2. NPM and the cytoplasmic mutant of NPM, NPMc+, were found to regulate P-TEFb activity and RNA polymerase II transcription through the interaction with HEXIM1. Importantly, more than one-third of acute myeloid leukemia (AML) patients carry NPMc+, suggesting the involvement of HEXIM1 in tumorigenesis of AML. HDM2 was found to ubiquitinate HEXIM1. The HDM2-mediated ubiquitination of HEXIM1 did not lead to protein degradation of HEXIM1 but enhanced its inhibitory activity on P-TEFb. Recently, HEXIM1 was identified as a novel positive regulator of p53. HEXIM1 prevented p53 ubiquitination by competing with HDM2 in binding to p53. Taken together, the new evidence suggests a role of HEXIM1 in regulating the p53 pathway and tumorigenesis.

Identification of HEXIM1 as a positive regulator of p53.

Hexamethylene bisacetamide-inducible protein 1 (HEXIM1) is best known as the inhibitor of positive transcription elongation factor b (P-TEFb), which regulates the transcription elongation of RNA polymerase II and controls 60-70% of mRNA synthesis. Our previous studies show that HEXIM1 interacts with two key p53 regulators, nucleophosmin and human double minute-2 protein (HDM2), implying a possible connection between HEXIM1 and the p53 signaling pathway. Here we report the interaction between p53 and HEXIM1 in breast cancer, acute myeloid leukemia, and colorectal carcinoma cells. The C-terminal regions of p53 and HEXIM1 are required for the protein-protein interaction. Overexpression of HEXIM1 prevents the ubiquitination of p53 by HDM2 and enhances the protein stability of p53, resulting in up-regulation of p53 target genes, such as Puma and p21. Induction of p53 can be achieved by several means, such as UV radiation and treatment with anti-cancer agents (including doxorubicin, etoposide, roscovitine, flavopiridol, and nutlin-3). Under all the conditions examined, elevated protein levels of p53 are found to associate with the increased p53-HEXIM1 interaction. In addition, knockdown of HEXIM1 significantly inhibits the induction of p53 and releases the cell cycle arrest caused by p53. Finally, the transcription of the p53 target genes is regulated by HEXIM1 in a p53-dependent fashion. Our results not only identify HEXIM1 as a positive regulator of p53, but also propose a novel molecular mechanism of p53 activation caused by the anti-cancer drugs and compounds.