Transcriptional Coactivator PC4 Research Articles

Disrupting the interaction between a p53 gain-of-function mutant and the transcriptional co-activator PC4 reverses drug resistance in cancer cells.

PC4 is a chromatin-associated protein and transcriptional coactivator whose role in gene regulation by wild-type p53 is now well known. Little is known about the roles of PC4 in tumor cells bearing mutant p53 genes. We show that PC4 associates with one of the tumor-associated gain-of-function p53 mutants, R273H. This association drives its recruitment to two promoters, UBE2C and MDR1, known to be responsible for imparting aggressive growth and resistance to many drugs. Here, we introduced a peptide that disrupts the PC4-R273Hp53 interaction to tumor cells bearing the R273HTP53 gene, which led to a lowering of MDR1 expression and abrogation of drug resistance in a mutant-specific manner. The results suggest that the PC4-R273Hp53 interaction may be a promising target for reducing proliferation and drug resistance in tumors.

Correction for Batta and Kundu, "Activation of p53 Function by Human Transcriptional Coactivator PC4: Role of Protein-Protein Interaction, DNA Bending, and Posttranslational Modifications".

This article refers to:Activation of p53 Function by Human Transcriptional Coactivator PC4: Role of Protein-Protein Interaction, DNA Bending, and Posttranslational Modifications

Chromatin protein PC4 is downregulated in breast cancer to promote disease progression: Implications of miR-29a.

The human transcriptional coactivator PC4 has numerous roles to play in the cell. Other than its transcriptional coactivation function, it facilitates chromatin organization, DNA damage repair, viral DNA replication, etc. Although it was found to be an essential protein in vivo, the importance of this multifunctional protein in the regulation of different cellular pathways has not been investigated in details, particularly in oncogenesis. In this study, PC4 downregulation was observed in a significant proportion of mammary tissues obtained from Breast cancer patient samples as well as in a subset of highly invasive and metastatic Breast cancer patient-derived cell lines. We have identified a miRNA, miR-29a which potentially reduce the expression of PC4 both in RNA and protein level. This miR-29a was found to be indeed overexpressed in a substantial number of Breast cancer patient samples and cell lines as well, suggesting one of the key mechanisms of PC4 downregulation. Stable Knockdown of PC4 in MCF7 cells induced its migratory as well as invasive properties. Furthermore, in an orthotopic breast cancer mice model system; we have shown that reduced expression of PC4 enhances the tumorigenic potential substantially. Absence of PC4 led to the upregulation of several genes involved in Epithelial to Mesenchymal Transition (EMT), indicating the possible mechanism of uniform tumour progression in the orthotropic mice. Collectively these data establish the role of PC4 in tumour suppression.

Transcriptional Coactivator and Chromatin Protein PC4 Is Involved in Hippocampal Neurogenesis and Spatial Memory Extinction

Although the elaborate combination of histone and non-histone protein complexes defines chromatin organization and hence regulates numerous nuclear processes, the role of chromatin organizing proteins remains unexplored at the organismal level. The highly abundant, multifunctional, chromatin-associated protein and transcriptional coactivator positive coactivator 4 (PC4/Sub1) is absolutely critical for life, because its absence leads to embryonic lethality. Here, we report results obtained with conditional PC4 knock-out (PC4(f/f) Nestin-Cre) mice where PC4 is knocked out specifically in the brain. Compared with the control (PC4(+/+) Nestin-Cre) mice, PC4(f/f) Nestin-Cre mice are smaller with decreased nocturnal activity but are fertile and show no motor dysfunction. Neurons in different areas of the brains of these mice show sensitivity to hypoxia/anoxia, and decreased adult neurogenesis was observed in the dentate gyrus. Interestingly, PC4(f/f) Nestin-Cre mice exhibit a severe deficit in spatial memory extinction, whereas acquisition and long term retention were unaffected. Gene expression analysis of the dorsal hippocampus of PC4(f/f) Nestin-Cre mice revealed dysregulated expression of several neural function-associated genes, and PC4 was consistently found to localize on the promoters of these genes, indicating that PC4 regulates their expression. These observations indicate that non-histone chromatin-associated proteins like PC4 play a significant role in neuronal plasticity.

Selective Recognition of H3.1K36 Dimethylation/H4K16 Acetylation Facilitates the Regulation of All-trans-retinoic Acid (ATRA)-responsive Genes by Putative Chromatin Reader ZMYND8

ZMYND8 (zinc finger MYND (Myeloid, Nervy and DEAF-1)-type containing 8), a newly identified component of the transcriptional coregulator network, was found to interact with the Nucleosome Remodeling and Deacetylase (NuRD) complex. Previous reports have shown that ZMYND8 is instrumental in recruiting the NuRD complex to damaged chromatin for repressing transcription and promoting double strand break repair by homologous recombination. However, the mode of transcription regulation by ZMYND8 has remained elusive. Here, we report that through its specific key residues present in its conserved chromatin-binding modules, ZMYND8 interacts with the selective epigenetic marks H3.1K36Me2/H4K16Ac. Furthermore, ZMYND8 shows a clear preference for canonical histone H3.1 over variant H3.3. Interestingly, ZMYND8 was found to be recruited to several developmental genes, including the all-trans-retinoic acid (ATRA)-responsive ones, through its modified histone-binding ability. Being itself inducible by ATRA, this zinc finger transcription factor is involved in modulating other ATRA-inducible genes. We found that ZMYND8 interacts with transcription initiation-competent RNA polymerase II phosphorylated at Ser-5 in a DNA template-dependent manner and can alter the global gene transcription. Overall, our study identifies that ZMYND8 has CHD4-independent functions in regulating gene expression through its modified histone-binding ability.

Human Transcriptional Coactivator PC4 Stimulates DNA End Joining and Activates DSB Repair Activity

Human transcriptional coactivator PC4 is a highly abundant nuclear protein that is involved in diverse cellular processes ranging from transcription to chromatin organization. Earlier, we have shown that PC4, a positive activator of p53, overexpresses upon genotoxic insult in a p53-dependent manner. In the present study, we show that PC4 stimulates ligase-mediated DNA end joining irrespective of the source of DNA ligase. Pull-down assays reveal that PC4 helps in the association of DNA ends through its C-terminal domain. In vitro nonhomologous end-joining assays with cell-free extracts show that PC4 enhances the joining of noncomplementary DNA ends. Interestingly, we found that PC4 activates double-strand break (DSB) repair activity through stimulation of DSB rejoining in vivo. Together, these findings demonstrate PC4 as an activator of nonhomologous end joining and DSB repair activity.

Activation of p53 Function by Human Transcriptional Coactivator PC4: Role of Protein-Protein Interaction, DNA Bending, and Posttranslational Modifications

Tumor suppressor p53 controls cell cycle checkpoints and apoptosis via the transactivation of several genes that are involved in these processes. The functions of p53 are regulated by a wide variety of proteins, which interact with it either directly or indirectly. The multifunctional human transcriptional coactivator PC4 interacts with p53 in vivo and in vitro and regulates its function. Here we report the molecular mechanisms of the PC4-mediated activation of p53 function. PC4 interacts with the DNA binding and C-terminal domains of p53 through its DNA binding domain, which is essential for the stimulation of p53 DNA binding. Remarkably, ligation-mediated circularization assays reveal that PC4 induces significant bending in the DNA double helix. Deletion mutants defective in DNA bending are found to be impaired in activating p53-mediated DNA binding and apoptosis. Furthermore, acetylation of PC4 enhances, while phosphorylation abolishes, its ability to bend DNA, activate p53 DNA binding, and, thereby, regulate p53 functions. In conclusion, PC4 activates p53 recruitment to p53-responsive promoters (Bax and p21) in vivo through its interaction with p53 and by providing bent substrate for p53 recruitment. These results elucidate the general molecular mechanisms of activation of p53 function, mediated by its coactivators.

P53 regulates its own activator: transcriptional co-activator PC4, a new p53-responsive gene

The tumour suppressor protein p53 regulates the expression of several genes that mediate cell cycle arrest, apoptosis, DNA repair and other cellular responses. Recently, we have shown that human transcriptional co-activator PC4 is a unique activator of p53 function. In the present study, we report that PC4 is a p53-inducible gene. Bioinformatics analysis reveals multiple p53-binding sites in the PC4 promoter. We have found that indeed p53 binds to all the identified sites in vitro and in vivo with varying affinities. p53 acts as an activator of PC4 transcription. Both PC4 mRNA and protein levels increase in response to stimuli that result in p53 induction. Furthermore, PC4 enhances p53 recruitment to the PC4 promoter. Our results thus establish the first report of a positively regulated feedback loop to control p53 function.

Transcriptional Coactivator PC4, a Chromatin-Associated Protein, Induces Chromatin Condensation

Human transcriptional coactivator PC4 is a highly abundant multifunctional protein which plays diverse important roles in cellular processes, including transcription, replication, and repair. It is also a unique activator of p53 function. Here we report that PC4 is a bona fide component of chromatin with distinct chromatin organization ability. PC4 is predominantly associated with the chromatin throughout the stages of cell cycle and is broadly distributed on the mitotic chromosome arms in a punctate manner except for the centromere. It selectively interacts with core histones H3 and H2B; this interaction is essential for PC4-mediated chromatin condensation, as demonstrated by micrococcal nuclease (MNase) accessibility assays, circular dichroism spectroscopy, and atomic force microscopy (AFM). The AFM images show that PC4 compacts the 100-kb reconstituted chromatin distinctly compared to the results seen with the linker histone H1. Silencing of PC4 expression in HeLa cells results in chromatin decompaction, as evidenced by the increase in MNase accessibility. Knocking down of PC4 up-regulates several genes, leading to the G2/M checkpoint arrest of cell cycle, which suggests its physiological role as a chromatin-compacting protein. These results establish PC4 as a new member of chromatin-associated protein family, which plays an important role in chromatin organization.

Redox-mediated upregulation of hepatocyte iNOS transcription requires coactivator PC4

Redox-mediated upregulation of transcription of hepatocyte inducible nitric oxide synthase (iNOS) requires hepatocyte nuclear factor IV-alpha (HNF-4alpha). In this setting, PC4 is often isolated with HNF-4alpha in DNA-protein pull-down studies. Transcriptional coactivator PC4 facilitates activator-dependent transcription via interactions with basal transcriptional machinery that are independent of PC4-DNA binding. We hypothesized that PC4 is a necessary component of HNF-4alpha-regulated redox-sensitive hepatocyte iNOS transcription. Murine CCL9.1 hepatocytes were stimulated with interleukin-1beta (IL-1beta; 1000 U/mL) in the presence and absence of peroxide (H(2)O(2); 50 nmol/L). Antisense and sense oligonucleotides to HNF-4alpha and PC4 were added selectively. Coimmunoprecipitation (Co-IP) studies determined the association between HNF-4alpha and PC4. Transient transfection was performed with the use of a luciferase reporter construct containing the murine iNOS promoter (1.8 kb). Chromatin immunoprecipitation assays determined in vivo binding of PC4 and HNF-4alpha to the iNOS promoter region. Ablation of either HNF-4alpha or PC4 blunted the peroxide-mediated increase in the activation of the iNOS promoter. In IL-1beta+H(2)O(2) only, co-IP studies demonstrated the presence of an HNF-4alpha-PC4 protein complex, and chromatin immunoprecipitation assays demonstrated that this complex binds to the genomic iNOS promoter. Redox-mediated upregulation of hepatocyte iNOS transcription requires an HNF-4alpha-PC4 transcriptional complex.

Involvement of General Transcriptional Coactivator PC4 in the Transcription of Medaka Fish Intestine-Specific Membrane Guanylyl Cyclase Gene (OlGC6)

A recent study showed that the AGACCTTTGC nucleotides sequence (between -90 and -81) contained in the cis-regulatory element in an intestine-specific membrane guanylyl cyclase gene, OlGC6, of the medaka fish, Oryzias latipes, are important for the transcription of the gene in mammalian cultured cell line and in medaka fish. Using sequence-specific DNA affinity chromatography, we purified a cis-regulatory element-binding protein from a medaka fish intestinal nuclear extract and used mass spectrometry to identify it as a medaka fish homologue of general transcriptional coactivator PC4, which we designated as OlPC4. The expression of the OlPC4 gene was detected in embryos, as well as in a large variety of tissues of adult medaka fish. Using a 17-kDa recombinant OlPC4, we carried out an ultraviolet (UV) cross-linking experiment and an electrophoretic mobility shift assay (EMSA), and demonstrated that the recombinant OlPC4 can be substituted for native OlPC4 in medaka fish intestinal nuclear extracts. In CACO-2 cells, cotransfection of the OlGC6-luciferase fusion genes with an OlPC4 expression vector resulted in 1.5-fold stimulation of the OlGC6 promoter.

General transcriptional coactivator PC4 activates p53 function.

The function of p53 is modulated by several transcriptional coactivators that regulate its tumor suppressor activity. Here we report that human transcriptional coactivator PC4 enhances the DNA binding of p53 to its cognate site in vitro and directly interacts with p53 in vivo. In vitro interaction studies demonstrated that the C-terminal 30 amino acids (364 to 393) of p53 strongly interact with PC4. Surprisingly, PC4 also stimulates the sequence-specific DNA binding of p53 with the C-terminal 30 amino acids deleted (p53Delta30), suggesting that PC4 mediates enhancement of p53 DNA binding by a unique mechanism. We also demonstrated that PC4 can stimulate p53- and p53Delta30-mediated transactivation from a p53-responsive promoter. Furthermore, PC4 enhances p53- and p53Delta30-dependent apoptosis by inducing bax (a p53-targeted proapoptotic gene) gene expression. These results establish the first physiological role of PC4 as a transcriptional coactivator.

Evolutionarily Conserved Interaction between CstF-64 and PC4 Links Transcription, Polyadenylation, and Termination

Tight connections exist between transcription and subsequent processing of mRNA precursors, and interactions between the transcription and polyadenylation machineries seem especially extensive. Using a yeast two-hybrid screen to identify factors that interact with the polyadenylation factor CstF-64, we uncovered an interaction with the transcriptional coactivator PC4. Both human proteins have yeast homologs, Rna15p and Sub1p, respectively, and we show that these two proteins also interact. Given evidence that certain polyadenylation factors, including Rna15p, are necessary for termination in yeast, we show that deletion or overexpression of SUB1 suppresses or enhances, respectively, both growth and termination defects detected in an rna15 mutant strain. Our findings provide an additional, unexpected connection between transcription and polyadenylation and suggest that PC4/Sub1p, via its interaction with CstF-64/Rna15p, possesses an evolutionarily conserved antitermination activity.

Functional Interaction between the HIV Transactivator Tat and the Transcriptional Coactivator PC4 in T Cells

The human immunodeficiency virus (HIV) transactivator Tat is a potent activator of transcription from the HIV long terminal repeat and is essential for efficient viral gene expression and replication. Tat has been shown to interact with components of the basal transcription machinery and transcriptional activators. Here we identify the cellular coactivator PC4 as a Tat-interacting protein using the yeast two-hybrid system and confirmed this interaction both in vitro and in vivo by coimmunoprecipitation. We found that this interaction has a functional outcome in that PC4 overexpression enhanced activation of the HIV long terminal repeat in transient transfection studies in a Tat-dependent manner. The domains of PC4 and Tat required for the interaction were mapped. In vitro binding studies showed that the basic transactivation-responsive binding domain of Tat is required for the interaction with PC4. The minimum region of PC4 required for Tat binding was amino acids 22-91, whereas mutation of the lysine-rich domain between amino acids 22 and 43 prevented interaction with Tat. Tat-PC4 interactions may be controlled by phosphorylation, because phosphorylation of PC4 by casein kinase II inhibited interactions with Tat both in vivo and in vitro. We propose that PC4 may be involved in linking Tat to the basal transcription machinery.

The adeno-associated virus type 2 regulatory proteins rep78 and rep68 interact with the transcriptional coactivator PC4.

The adeno-associated virus type 2 (AAV-2) Rep78/Rep68 regulatory proteins are pleiotropic effectors of viral and cellular DNA replication, of cellular transformation by viral and cellular oncogenes, and of homologous and heterologous gene expression. To search for cellular proteins involved in mediating these functions, we used Rep68 as bait in the yeast two-hybrid system and identified the transcriptional coactivator PC4 as a Rep interaction partner. PC4 has been shown to mediate transcriptional activation by a variety of sequence-specific transcription factors in vitro. Rep amino acids 172 to 530 were sufficient and amino acids 172 to 224 were absolutely necessary for the interaction with PC4. The PC4 domains required for interaction were mapped to the C-terminal single-stranded DNA-binding domain of PC4. In glutathione S-transferase (GST) pull-down assays, in vitro-transcribed and -translated Rep78 or Rep68 proteins were bound specifically by GST-PC4 fusion proteins. Similarly, PC4 expressed in Escherichia coli was bound by GST-Rep fusion proteins, confirming the direct interaction between Rep and PC4 in vitro. Rep was found to have a higher affinity for the nonphosphorylated, transcriptionally active form of PC4 than for the phosphorylated, transcriptionally inactive form. The latter is predominant in nuclear extracts of HeLa or 293 cells. In the yeast system, but not in vitro, Rep-PC4 interaction was disrupted by a point mutation in the putative nucleotide-binding site of Rep68, suggesting that a stable interaction between Rep and PC4 in vivo is ATP dependent. This mutation has also been shown to impair Rep function in AAV-2 DNA replication and in inhibition of gene expression and inducible DNA amplification. Cytomegalovirus promoter-driven overexpression of PC4 led to transient accumulation of nonphosphorylated PC4 with concomitant downregulation of all three AAV-2 promoters in the absence of helper virus. In the presence of adenovirus, this effect was relieved. These results imply an involvement of the transcriptional coactivator PC4 in the regulation of AAV-2 gene expression in the absence of helper virus.

Properties of PC4 and an RNA Polymerase II Complex in Directing Activated and Basal Transcription in Vitro

A human RNA polymerase II (pol II) complex was isolated from a HeLa-derived cell line that conditionally expresses an epitope-tagged RPB9 subunit of human pol II. The isolated FLAG-tagged pol II complex (f:pol II) contains a subset of general transcription factors but is devoid of TFIID and TFIIA. In conjunction with TATA-binding protein (TBP) or TFIID, f:pol II is able to mediate both basal and activated transcription by Gal4-VP16 when a transcriptional coactivator PC4 is also provided. Interestingly, PC4, in the absence of a transcriptional activator, actually functions as a repressor to inhibit basal transcription. Remarkably, TBP is able to mediate activator function in this transcription system. The presence of TBP-associated factors, however, helps overcome PC4 repression and further enhance the level of activation mediated by TBP. Alleviation of PC4 repression can also be achieved by preincubation of the transcriptional components with the DNA template. Sarkosyl disruption of preinitiation complex formation further illustrates that PC4 can only inhibit transcription prior to the assembly of a functional preinitiation complex. These results suggest that PC4 represses basal transcription by preventing the assembly of a functional preinitiation complex, but it has no effect on the later steps of the transcriptional process.

High-affinity DNA binding by the C-terminal domain of the transcriptional coactivator PC4 requires simultaneous interaction with two opposing unpaired strands and results in helix destabilization

The general transcriptional cofactor PC4 enhances transcription from various promoters and functions with a wide range of transcriptional activators. Earlier studies have suggested that this enhancement originates mostly from stabilization of the TATA-box/TFIID/TFIIA complex by simultaneous interaction of PC4 with transactivation domains of upstream-binding factors and the basal factor TFIIA. However, the C-terminal half of the protein also has been shown to exhibit substantial ssDNA binding properties, to which as yet no clear function has been assigned. We have investigated the interaction of this domain with various DNA structures and report that high-affinity binding, characterized by an equilibrium dissociation constant in the nanomolar range, requires either a heteroduplex containing a minimum of about eight mismatches, or alternatively a single-stranded DNA molecule consisting of 16 to 20 nucleotides. Furthermore, both juxtaposed single strands of a heteroduplex are protected by the C-terminal domain of PC4 in DNase I footprinting experiments, whereas the double-stranded regions do not appear to be contacted. We conclude from these observations that the role of PC4 ssDNA binding is likely to involve simultaneous interaction with opposing strands in internally melted duplexes, or the induction of a pronounced distortion in the local structure of ssDNA that results in a similar juxtaposed arrangement of single strands. In addition, we have observed that both the PC4 C-terminal domain and the intact PC4 destabilize dsDNA and we discuss the possible involvement of PC4 in promoter opening and other strand displacement events.