Regulation of NF-kappaB by cyclin-dependent kinases associated with the p300 coactivator.

The nuclear factor kappaB (NF-kappaB) transcription factor is responsive to specific cytokines and stress and is often activated in association with cell damage and growth arrest in eukaryotes. NF-kappaB is a heterodimeric protein, typically composed of 50- and 65-kilodalton subunits of the Rel family, of which RelA(p65) stimulates transcription of diverse genes. Specific cyclin-dependent kinases (CDKs) were found to regulate transcriptional activation by NF-kappaB through interactions with the coactivator p300. The transcriptional activation domain of RelA(p65) interacted with an amino-terminal region of p300 distinct from a carboxyl-terminal region of p300 required for binding to the cyclin E-Cdk2 complex. The CDK inhibitor p21 or a dominant negative Cdk2, which inhibited p300-associated cyclin E-Cdk2 activity, stimulated kappaB-dependent gene expression, which was also enhanced by expression of p300 in the presence of p21. The interaction of NF-kappaB and CDKs through the p300 and CBP coactivators provides a mechanism for the coordination of transcriptional activation with cell cycle progression.

Specificity of cyclin E-Cdk2, TFIIB, and E1A interactions with a common domain of the p300 coactivator.

The p300 and CREB binding protein (CBP) transcriptional coactivators interact with a variety of transcription factors and regulate their activity. Among the interactions that have been described, the COOH-terminal region of p300 binds to cyclin E-cyclin-dependent kinase 2 (cyclin E-Cdk2) and TFIIB, as well as to the E1A gene products of adenovirus. Inhibition of Cdk activity by Cdk inhibitors, such as p21 or p27, potentiates NF-kappaB activity and provides a mechanism to coordinate cell cycle progression with the transcription of genes expressed during growth arrest. In this report, we analyze the specific domains of p300 required for the binding of p300 to cyclin E-Cdk2, TFIIB, and E1A and the ability of these proteins to interact with p300, alone or in combination. 12S E1A, an inhibitor of p300-dependent transcription, reduces the binding of TFIIB, but not that of cyclin E-Cdk2, to p300. In contrast, 13S E1A, a pleiotropic transcriptional activator, does not inhibit TFIIB binding to p300, although it enhances the interaction of cyclin E-Cdk2 with p300. Modification of cyclin E-Cdk2 is most likely required for association with p300 since the interaction is observed only with cyclin E-Cdk2 purified from mammalian cells. Domain swap studies show that the cyclin homology domain of TFIIB is involved in interactions with p300, although the homologous region from cyclin E does not mediate this interaction. These findings suggest that p300 or CBP function is regulated by interactions of various proteins with a common coactivator domain.

Early phosphorylation of the retinoblastoma gene product regulates protein binding to the c-fos retinoblastoma control element during T cell activation.

Function of the retinoblastoma tumor suppressor protein [pRb] is regulated by phosphorylation during the G1 and S phases of the cell cycle. pRb regulates transcription of several genes, including c-fos. However, since c-fos is regulated during exit from G0, it has remained unclear how pRb participates in c-fos regulation. We have identified a protein complex, the retinoblastoma control factor A [RCF-A] which binds to the c-fos retinoblastoma control element [RCE] and is regulated by pRb within 10 min after T cell activation. We demonstrate that pRb control of RCF-A is dependent upon the state of phosphorylation of pRb. pRb becomes hyperphosphorylated on specific peptides at 10 min after mitogenic stimulation and pRb is dephosphorylated by 30 min. This time course coincides with RCF-A DNA binding. RCF-A binds RCE DNA longer when cells are treated with okadaic acid, and okadaic acid prevents pRb dephosphorylation. Dephosphorylated pRb inhibits RCF-A binding in vitro but phosphorylated pRb does not. Thus, in addition to the described G1/S regulation of pRb, transient inactivation by phosphorylation of pRb in T cells may also be important as resting cells leave G0.

Increased chemokine gene expression during aging in the murine brain.

Normal aging results in changes in the brain that contribute to the decline of various functions, including learning and memory. Mechanisms causing this decline have not been clearly established. Activation of microglia is associated with the normal aging process in rodents and primates. Microglial activation is regulated by chemokine gene expression, and activated microglia produce substances that can be detrimental to surrounding cells. In this study we determined whether changes in chemokine expression occur during normal aging in the mouse brain. RNA samples taken from the cortex, midbrain, hippocampus, and cerebellum of 4-, 10-, 21- and 30-month-old C57BL6/DBA2 mice were analyzed for changes in gene expression. RNase protection assays were used to examine a panel of chemokines. Increased expression of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES occurred in all four regions of the brains in the oldest mice. These increases were first detectable at 21 months of age. Increases in MIP-1alpha, MIP-1beta, and RANTES protein levels were also detected in the brains of old mice, as measured by ELISA. Increased microglial activation in the brains of 30-month-old mice, as detected by immunohistochemistry using F4/80 antibodies, correlated with increases in chemokine expression. The observed increases in chemokine gene expression that occur in conjunction with increased microglial activation suggest that chemokines may contribute to the decreased brain function that occurs during normal aging.

Modulation of cytokine-induced HIV gene expression by competitive binding of transcription factors to the coactivator p300.

The host response to viral infection involves the secretion of multiple cytokines which alter immune function and viral replication. These proteins activate several signal transduction pathways in infected cells which must be integrated to regulate cellular and viral gene expression. In this report, we demonstrate that specific transcription factors induced by distinct cytokines regulate HIV transcription by competitive binding to the p300 coactivator. Interferon-alpha (IFN-alpha) was found to inhibit NF-kappaB-dependent HIV gene expression stimulated by tumor necrosis factor-alpha (TNF-alpha). This inhibition was mediated by binding of the IFN-alpha signal transducer and activator of transcription 2, Stat2, to a specific domain of p300 which also binds to the RelA (p65) subunit of NF-kappaB. p300 was found to be limiting with respect to RelA (p65) and Stat2, and this effect was reversed by overexpression of p300. Inhibition by Stat2 was specific for NF-kappaB and was not mediated by Stat1, which is also induced by IFN-alpha. Gene activation induced by the Stat2 transcription domain was also inhibited by expression of RelA. These results demonstrate that HIV transcription can be regulated in the nucleus by competitive binding of specific cytokine-induced transcription factors to a discrete domain of a transcriptional coactivator.

Distinct domains of adenovirus E1A interact with specific cellular factors to differentially modulate human immunodeficiency virus transcription.

Transcription of human immunodeficiency virus (HIV) type 1 and other viruses is regulated by the transcription factor NF-kappaB, which interacts with the multifunctional cellular protein p300. p300, originally identified by its ability to bind adenovirus early region 1A (E1A), has also been shown to regulate HIV transcription through its interaction with NF-kappaB. The 13S form of E1A activates HIV gene expression, while the 12S form represses its transcription. In this report, we have investigated whether these divergent effects of E1A are dependent upon common or distinct cellular cofactors, including p300, pRb, and the TATA box-binding protein (TBP). Unlike activation in the absence of E1A, cooperative stimulation of HIV gene expression by 13S E1A and RelA was independent of the ability of E1A to bind p300 but was critically dependent on the E1A CR3 region which associates with TBP. In contrast, inhibition of basal HIV gene expression by the 12S form of E1A was dependent on p300 but not pRb or TBP. Interestingly, mutation of the CR2 region of 12S E1A responsible for pRb binding abolished the repression of HIV transcription stimulated by tumor necrosis factor alpha, suggesting that repression of cytokine-activated transcription involves cofactors different from those used in unstimulated cells. Repression and activation of HIV transcription by different forms of E1A are mediated by distinct sets of cellular cofactors. These findings suggest that E1A has evolved to interact by alternative mechanisms with a transcriptional coactivator and its associated cofactors to differentially modulate cellular and viral gene expression.

HIV transcriptional activation by the accessory protein, VPR, is mediated by the p300 co-activator.

The accessory protein, Vpr, is a virion-associated protein that is required for HIV-1 replication in macrophages and regulates viral gene expression in T cells. Vpr causes arrest of cell cycle progression at G2/M, presumably through its effect on cyclin B1.Cdc2 activity. Here, we show that the ability of Vpr to activate HIV transcription correlates with its ability to induce G2/M growth arrest, and this effect is mediated by the p300 transcriptional co-activator, which promotes cooperative interactions between the Rel A subunit of NF-kappaB and cyclin B1.Cdc2. Vpr cooperates with p300, which regulates NF-kappaB and the basal transcriptional machinery, to increase HIV gene expression. Similar effects are seen in the absence of Vpr with a kinase-deficient Cdc2, and overexpression of p300 increases levels of HIV Vpr+ replication. Taken together, these data suggest that p300, through its interactions with NF-kappaB, basal transcriptional components, and Cdks, is modulated by Vpr and regulates HIV replication. The regulation of p300 by Vpr provides a mechanism to enhance viral replication in proliferating cells after growth arrest by increasing viral transcription.