Nianli Sang

p300 is required for MyoD-dependent cell cycle arrest and muscle-specific gene transcription.

The nuclear phosphoprotein p300 is a new member of a family of ‘co‐activators’ (which also includes the CREB binding protein CBP), that directly modulate transcription by interacting with components of the basal transcriptional machinery. Both p300 and CBP are targeted by the adenovirus E1A protein, and binding to p300 is required for E1A to inhibit terminal differentiation in both keratinocytes and myoblasts. Here we demonstrate that, in differentiating skeletal muscle cells, p300 physically interacts with the myogenic basic helix–loop–helix (bHLH) regulatory protein MyoD at its DNA binding sites. During muscle differentiation, MyoD plays a dual role: besides activating muscle‐specific transcription, it induces permanent cell cycle arrest by up‐regulating the cyclin‐dependent kinase inhibitor p21. We show that p300 is involved in both these activities. Indeed, E1A mutants lacking the ability to bind p300 are greatly impaired in the repression of E‐box‐driven transcription, and p300 overexpression rescues the wild‐type E1A‐mediated repression. Moreover, p300 potentiates MyoD‐ and myogenin‐dependent activation of transcription from E‐box‐containing reporter genes. We also provide evidence, obtained by microinjection of anti‐p300 antibodies, that p300 is required for MyoD‐dependent cell cycle arrest in either myogenic cells induced to differentiate or in MyoD‐converted C3H10T1/2 fibroblasts, but is dispensable for maintenance of the post‐mitotic state of myotubes.

Histone Deacetylase Inhibitors Induce VHL and Ubiquitin-Independent Proteasomal Degradation of Hypoxia-Inducible Factor 1α

ABSTRACT Adaptation to hypoxic microenvironment is critical for tumor survival and metastatic spread. Hypoxia-inducible factor 1α (HIF-1α) plays a key role in this adaptation by stimulating the production of proangiogenic factors and inducing enzymes necessary for anaerobic metabolism. Histone deacetylase inhibitors (HDACIs) produce a marked inhibition of HIF-1α expression and are currently in clinical trials partly based on their potent antiangiogenic effects. Although it has been postulated that HDACIs affect HIF-1α expression by enhancing its interactions with VHL (von Hippel Lindau), thus promoting its ubiquitination and degradation, the actual mechanisms by which HDACIs decrease HIF-1α levels are not clear. Here, we present data indicating that HDACIs induce the proteasomal degradation of HIF-1α by a mechanism that is independent of VHL and p53 and does not require the ubiquitin system. This degradation pathway involves the enhanced interaction of HIF-1α with HSP70 and is secondary to a disruption of the HSP70/HSP90 axis function that appears mediated by the activity of HDAC-6.

Carboxyl-Terminal Transactivation Activity of Hypoxia-Inducible Factor 1α Is Governed by a von Hippel-Lindau Protein-Independent, Hydroxylation-Regulated Association with p300/CBP

ABSTRACT Hypoxia-inducible factor 1 complex (HIF-1) plays a pivotal role in oxygen homeostasis and adaptation to hypoxia. Its function is controlled by both the protein stability and the transactivation activity of its alpha subunit, HIF-1α. Hydroxylation of at least two prolyl residues in the oxygen-dependent degradation domain of HIF-1α regulates its interaction with the von Hippel-Lindau protein (VHL) that targets HIF-1α for ubiquitination and proteasomal degradation. Several prolyl hydroxylases have been found to specifically hydroxylate HIF-1α. In this report, we investigated possible roles of VHL and hydroxylases in the regulation of the transactivation activity of the C-terminal activating domain (CAD) of HIF-1α. We demonstrate that regulation of the transactivation activity of HIF-1α CAD also involves hydroxylase activity but does not require functional VHL. In addition, stimulation of the CAD activity by a hydoxylase inhibitor, hypoxia, and desferrioxamine was severely blocked by the adenoviral oncoprotein E1A but not by an E1A mutant defective in targeting p300/CBP. We further demonstrate that a hydroxylase inhibitor, hypoxia, and desferrioxamine promote the functional and physical interaction between HIF-1α CAD and p300/CBP in vivo. Taken together, our data provide evidence that hypoxia-regulated stabilization and transcriptional stimulation of HIF-1α function are regulated through partially overlapping but distinguishable pathways.

Histone Deacetylase Inhibitors Repress the Transactivation Potential of Hypoxia-inducible Factors Independently of Direct Acetylation of HIF-α

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors regulating the oxygen supply, glucose metabolism, and angiogenesis. HIF function requires the recruitment of p300/CREB-binding protein, two coactivators with histone acetyltransferase activity, by the C-terminal transactivation domain of HIF-α (HIF-αCAD). Histone deacetylase inhibitors (HDAIs) induce differentiation or apoptosis and repress tumor growth and angiogenesis, hence being explored intensively as anti-cancer agents. Using combined pharmacological, biochemical, and genetic approaches, here we show that HDAIs repress the transactivation potential of HIF-αCAD. This repression is independent of the function of tumor suppressors von Hippel-Lindau or p53 or the degradation of HIF-α. We also demonstrate the sufficiency of low concentrations of HDAIs in repression of HIF target genes in tumor cells. We further show that HDAIs induce hyperacetylation of p300 and repress the HIF-1α·p300 complex in vivo. In vitro acetylation analysis reveals that the p300CH1 region, but not HIF-αCAD, is susceptible to acetylation. Taken together, our data demonstrate that a deacetylase activity is indispensable for the transactivation potential of HIF-αCAD and support a model that acetylation regulates HIF function by targeting HIF-α·p300 complex, not by direct acetylating HIF-α. The demonstration that HDAIs repress both HIF-1α and HIF-2α transactivation potential independently of von Hippel-Lindau tumor suppressor and p53 function indicates that HDAIs may have biological effects in a broad range of tissues in addition to tumors.

Histone Deacetylase Inhibitors Synergize p300 Autoacetylation that Regulates Its Transactivation Activity and Complex Formation

p300/cyclic AMP-responsive element binding protein-binding protein (CBP) are general coactivators for multiple transcription factors involved in various cellular processes. Several highly conserved domains of p300/CBP serve as interacting sites for transcription factors and regulatory proteins. Particularly, the intrinsic histone acetyltransferase (HAT) activity and transactivation domains (TAD) play essential roles for their coactivating function. Autoacetylation of p300/CBP is commonly observed in cell-free HAT assays and has been implicated in the regulation of their HAT activity. Here, we show that six lysine-rich regions in several highly conserved functional domains of p300 are targeted by p300HAT for acetylation in cell-free systems. We show that p300 is susceptible to acetylation in cultured tumor cells and that its acetylation status is affected by histone deacetylase inhibitor trichostatin A. We further show that either treatment with deacetylase inhibitors or coexpression of Gal4-p300HAT, which alone has no transactivation activity, stimulates the activity of the COOH-terminal TAD of p300 (p300C-TAD). We have defined the minimal p300C-TAD and show that it is sufficient to respond to deacetylase inhibitors and is a substrate for p300HAT. Finally, we show that acetylated p300 possesses enhanced ability to interact with p53. Taken together, our data suggest that acetylation regulates p300C-TAD and that acetylation of p300/CBP may contribute to the dynamic regulation of their complex formation with various interacting partners.

Roles of p300, pocket proteins, and hTBP in E1A‐mediated transcriptional regulation and inhibition of p53 transactivation activity

The conserved region 1 and the extreme N‐terminus of adenoviral oncoprotein E1A are essential for transforming activity. They also play roles in the interaction of E1A with p300/CBP and pRb and are involved in both transactivation and repression of host gene expression. It was reported recently that p53‐mediated transactivation is specifically repressed by E1A and that p53‐induced apoptosis can be protected by pRb. In this report, we investigated the roles of pRb and p300 in the N‐terminus of E1A‐mediated transcriptional regulation. We demonstrate here that p300 and pRb have no effect on DBD.1‐70 transactivation and that overexpression of p300 or pRb failed to relieve the repression by E1A. Repression of p53 transactivation requires both the extreme amino terminus and CR1 but not CR2. This repressive activity of E1A specifically correlates with E1As ability to bind p300 and TBP. On the other hand, E1A inhibited the transactivation activity of a fusion construct containing the DNA binding domain of yeast Gal4 and the transactivation domain of p53. When p53 was cotransfected with E1A, similar inhibition was found in Saos‐2 cells that lack endogenous pRb and p53 activity. Introduction of pRb into Saos‐2 cells did not affect p53 transcription activity. E1A‐mediated repression can be relieved by overexpression of either p300, hTBP, or TFIIB but cannot be released by overexpression of pocket proteins. Our data suggest that p300/CBP and TBP but not the pocket proteins, pRb, p107, and pRb2/p130 are functional targets of E1A in transcriptional regulation and that p53 transactivation requires the function of the p300/TBP/TFIIB complex, thus delineating a new pathway by which E1A may exert its transforming activity. J. Cell. Biochem. 66:277–285, 1997.

Adenoviral E1A: everlasting tool, versatile applications, continuous contributions and new hypotheses.

Adenoviral E1A is an indispensable protein for virus-host interaction. To provide a suitable environment for viral replication, E1A physically interacts with multiple cellular proteins to reprogram gene expression and other processes of the host cells. Proteins targeted by E1A include the pRb family of pocket proteins, p300/CBP, cyclin/Cdk, the carboxyl terminal binding protein (CtBP), transcriptional regulator YY1, and the recently identified RACK1 and SWI/SNF complex. Reprogramming activity of E1A and the host cell response to this reprogramming lead to transformation, growth arrest or apoptosis. Based on the ability of E1A to override the fundamental controls of host cells, E1A has been being utilized to make continuous contributions not only to a better understanding of the molecular mechanisms underlying the regulation of transcription, cell division, apoptosis and tumorigenesis but also to new therapeutics such as gene therapy.

Extreme N terminus of E1A oncoprotein specifically associates with a new set of cellular proteins.

By interacting with key regulatory proteins such as the pRb family, cyclins, cyclin‐dependent kinases and p300/CBP of host cells, adenoviral E1A interferes with various cellular processes to provide a suitable environment for the replication of viruses. E1A may promote DNA synthesis and cell cycle progression, immortalize rodent cells in culture and transform cultured cells in cooperation with E1B, Ras, or other oncoproteins. Both extreme N terminus and conserved region 1 of E1A are required for the immortalization and the transformation of rodent cells, transcriptional repression and specific induction of the expression of cellular genes such as the proliferating cell nuclear antigen (PCNA) and heat shock protein 70 (HSP70). Although the molecular mechanisms of these functions of E1A are not fully understood, it is believed that protein‐protein interactions may play essential roles. In this communication, we report that a new set of cellular proteins with apparent molecular weight of 200, 90, 45, 30, and 28 specifically associate with the extreme N terminus of E1A. Further analysis demonstrate that these associations do not depend on E1As association with p300 or pRB. Neither the 30 kDa nor the 28 kDa polypeptide is identical to Cdc2 or Cdk2. The region of E1A required for the protein interaction is also required for the recently identified N‐terminal transactivation activity of E1A. Our observations suggest that in addition to p300/CBP, the new set of cellular proteins may be involved in the functional complexity of the N terminus of E1A, thus predicting a p300/CBP independent pathway. J. Cell. Physiol. 170:182–191, 1997.

RACK1 is a functional target of the E1A oncoprotein

The adenoviral E1A proteins have been implicated in promotion of proliferation and transformation, inhibition of differentiation, induction of apoptosis, regulation of transcription, and suppression of tumor growth. The ability of E1A to override the fundamental controls of host cells is based on its ability to physically interact with several cellular proteins. We recently characterized RACK1 as a new E1A‐interacting protein. In this report, we show that the extreme N‐terminal region of E1A, spanning from aminoacids 1–36, and the conserved WD regions of RACK1 are responsible for this interaction. We also demonstrate that E1A and RACK1 colocalize at the perinuclear membrane in the cells. Furthermore, we provide evidence that E1A is able to antagonize the inhibitory effects of RACK1 on Src activity. These results suggest that RACK1 signaling pathway may be a functional target of E1A, contributing to E1A oncogenic effect in the host cells. J. Cell. Physiol. 199: 134–139, 2004© 2003 Wiley‐Liss, Inc.

A gene highly expressed in tumor cells encodes novel structure proteins

We isolated several related but distinct cDNA clones encoding novel structure proteins (NSP) when screening a cDNA library. Analysis revealed that these cDNAs and several similar ESTs in the public databases are derived from a single gene of 17 exons that span a minimum of 227-kb region. This gene is located at chromosome 17p11.2, a region frequently amplified in human gliomas and osteosarcomas, and involved in Birt–Hogg–Dube syndrome, a tumor-prone syndrome. The major coding sequences shared by all isolated transcripts are predicted to encode SMC (structural maintenance of chromosome)/SbcC ATPase motifs and coiled-coil domains commonly seen in motor or structure proteins. Two 5′-end and two 3′-end variants (type 5α/β and 3α/β, respectively) were identified, making a total of four possible transcripts. Both 5α and 5β variants were detected in human testis mRNA, but only type 5α was detectable in RNA samples extracted from HeLa cells. The unique carboxyl-terminus of 3β contains a Ca2+-dependent actin-binding domain. Immunohistochemistry studies revealed that NSPs were mostly localized to nuclei. Northern blot analysis demonstrated two major bands and the expression levels are tremendously high in testis while barely detectable in other normal tissues examined. Interestingly, NSP5α3α is highly expressed in some tumor cell lines. These results suggest that NSPs represent a new family of structure proteins with a possible role in nuclear dynamics during cell division, and that NSP5α3α may serve as a tumor marker.