Human Transcriptional Coactivator Research Articles

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

Structure of H3K36-methylated nucleosome-PWWP complex reveals multivalent cross-gyre binding.

Recognition of histone-modified nucleosomes by specific reader domains underlies the regulation of chromatin-associated processes. Whereas structural studies revealed how reader domains bind modified histone peptides, it is unclear how reader domains interact with modified nucleosomes. Here we report the cryo-electron microscopy (cryo-EM) structure of the PWWP reader domain of human transcriptional coactivator LEDGF in complex with a H3K36-methylated nucleosome at 3.2 Å resolution. The structure reveals multivalent binding of the reader domain to the methylated histone tail and to both gyres of nucleosomal DNA, explaining the known cooperative interactions. The observed cross-gyre binding may contribute to nucleosome integrity during transcription. The structure also explains how human PWWP domain-containing proteins are recruited to H3K36-methylated regions of the genome for transcription, histone acetylation and methylation, and for DNA methylation and repair.

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.

Nonhistone human chromatin protein PC4 is critical for genomic integrity and negatively regulates autophagy.

Multifunctional human transcriptional positive co-activator 4 (PC4) is a bonafide nonhistone component of the chromatin and plays a pivotal role in the process of chromatin compaction and functional genome organization. Knockdown of PC4 expression causes a drastic decompaction which leads to open conformation of the chromatin, and thereby altered nuclear architecture, defects in chromosome segregation and changed epigenetic landscape. Interestingly, these defects do not induce cellular death but result in enhanced cellular proliferation, possibly through enhanced autophagic activity. Moreover, PC4 depletion confers significant resistance to gamma irradiation. Exposure to gamma irradiation further induced autophagy in these cells. Inhibition of autophagy by small molecule inhibitors as well as by silencing of a critical autophagy gene drastically reduces the ability of PC4 knockdown cells to survive. On the contrary, complementation with wild-type PC4 could reverse this phenomenon, confirming the process of autophagy as the key mechanism for radiation resistance in the absence of PC4. These data connect the unexplored role of chromatin architecture in regulating autophagy during stress conditions such as radiation.

Multifunctional human transcriptional coactivator protein PC4 is a substrate of Aurora kinases and activates the Aurora enzymes.

Positive coactivator 4 (PC4), a human transcriptional coactivator, is involved in diverse processes like chromatin organization and transcription regulation. It is hyperphosphorylated during mitosis, with unknown significance. For the first time, we demonstrate the function of PC4 outside the nucleus upon nuclear envelope breakdown. A fraction of PC4 associates with Aurora A and Aurora B and undergoes phosphorylation, following which PC4 activates both Aurora A and B to sustain optimal kinase activity to maintain the phosphorylation gradient for the proper functioning of the mitotic machinery. This mitotic role is evident in PC4 knockdown cells where the defects are rescued only by the catalytically active Aurora kinases, but not the kinase-dead mutants. Similarly, the PC4 phosphodeficient mutant failed to rescue such defects. Hence, our observations establish a novel mitotic function of PC4 that might be dependent on Aurora kinase-mediated phosphorylation.

Human positive coactivator 4 (PC4) is involved in the progression and prognosis of astrocytoma

Astrocytoma is the most common primary brain tumor and it is associated with poor prognosis. Accumulating evidences suggest that certain molecular abnormalities or genetic mutations are associated with its progression and prognosis. Human transcriptional coactivator 4 (PC4), originally identified as a transcriptional coactivator then as a DNA replication and repair factor has been shown to be involved in chromatin organization. Recently, it is reported to function both as tumor suppressor and promoter depending on the cellular settings. In the present study, we for the first time demonstrated that the expression of PC4 in astrocytoma was upregulated as assessed by western blot and immunohistochemical staining. Moreover, elevated PC4 expression was strongly correlated with the progression of astrocytoma. Furthermore, high PC4 expression was also associated with poor overall survival. Finally, in vitro study demonstrated that siRNA mediated PC4 downregulation significantly inhibited the proliferation and invasiveness of human glioma cells. These results suggested that PC4 might play a role in human astrocytoma progression and may be used as a novel indicator for the prognosis of astrocytoma patient.

Structural Insights into Acetylated-Histone H4 Recognition by the Bromodomain-PHD Finger Module of Human Transcriptional Coactivator CBP

Bromodomain functions as the acetyl-lysine bindingdomains to regulate gene transcription in chromatin. Bromodomains are rapidly emerging as new epigenetic drug targets for human diseases. However, owing to their transient nature and modest affinity, histone-binding selectivity of bromodomains has remained mostly elusive. Here, we report high-resolution crystal structures of the bromodomain-PHD tandem module of human transcriptional coactivator CBP bound to lysine-acetylated histone H4 peptides. The structures reveal that the PHD finger serves astructural role in the tandem module and that thebromodomain prefers lysine-acetylated motifs comprising a hydrophobic or aromatic residue at -2 and a lysine or arginine at -3 or -4 position from the acetylated lysine. Our study further provides structural insights into distinct modes of singly and diacetylated histone H4 recognition by the bromodomains of CBP and BRD4 that function differently as a transcriptional coactivator and chromatin organizer, respectively, explaining their distinct roles in control of gene expression in chromatin.

Peptide-Protein Interactions Suggest That Acetylation of Lysines 381 and 382 of p53 Is Important for Positive Coactivator 4-p53 Interaction

The human transcriptional positive coactivator 4 (PC4) activates several p53-dependent genes. It has been demonstrated that this is a consequence of direct interaction with p53. Previously, we have concluded that PC4 interacts mainly with the C-terminal negative regulatory domain of p53 through its DNA binding C-terminal half. NMR chemical shift perturbation studies with peptide fragments indicated that amino acids 380-386 of p53 are crucial for interaction with PC4. This was verified by fluorescence anisotropy and sedimentation velocity studies. A peptide consisting of p53-(380-386) sequence, when attached to a cell penetration tag and nuclear localization signal, localizes to the nucleus and inhibits luciferase gene expression from a transfected plasmid carrying a Luc gene under a p53-dependent promoter. Acetylation of lysine 382/381 enhanced the binding of this peptide to PC4 by about an order of magnitude. NMR and mutagenesis studies indicated that serine 73 of PC4 is an important residue for recognition of p53. Intermolecular nuclear Overhauser effect placed aspartate 76 in the vicinity of lysine 381, indicating that the region around residues 73-76 of PC4 is important for p53 recognition. We conclude that the 380-386 region of p53 interacts with the region around residues 73-76 of PC4, and acetylation of lysine 382/381 of p53 may play an important role in modulating p53-PC4 interaction and as a consequence PC4 mediated activation of p53 target genes.

The role of T-cell reactivity towards the autoantigen α-NAC in atopic dermatitis

A subgroup of patients with atopic dermatitis (AD) produces IgE autoantibodies to human proteins which may be present in inflamed skin and perpetuate cutaneous inflammation. In order to investigate mechanisms of 'autoallergy' for AD we studied T-cell responses to the autoallergen Hom s 2, the human transcriptional coactivator α-nascent polypeptide-associated complex (α-NAC). Specific proliferation of blood lymphocytes from 30 patients and 12 healthy control individuals was investigated by flow cytometry. The proliferation of skin- and blood-derived T cells was assessed in limiting-dilution assays. T-cell clones (TCC) were generated from peripheral blood and from biopsies of lesional skin of patients with AD and the phenotype and cytokine patterns were determined. α-NAC-specific T-cell responses were detected in patients and control individuals. α-NAC induced a significantly higher proliferation of CCR4+ (compared with CCR4-) and CLA+ (compared with CLA-) T cells from the circulation. Limiting-dilution assays revealed a high proliferation of blood and skin-infiltrating lymphocytes in the presence of α-NAC compared with control cultures. α-NAC-specific TCC generated from lesional skin of AD predominantly produced interferon-γ and some TCC also produced interleukin-17. The cytokine pattern of α-NAC TCC may contribute to keratinocyte apoptosis and eczema formation in AD. α-NAC-specific TCC can be generated from blood and lesional skin of patients with AD. These TCC produce not only Th2 but also Th1 cytokines which may explain the Th1 phenotype of inflammation in AD.

Rational Design of Cyclic Peptide Modulators of the Transcriptional Coactivator CBP: A New Class of p53 Inhibitors

The CREB binding protein (CBP) is a human transcriptional coactivator consisting of several conserved functional modules, which interacts with distinct transcription factors including nuclear receptors, CREB, and STAT proteins. Despite the importance of CBP in transcriptional regulation, many questions regarding the role of its particular domains in CBP functions remain unanswered. Therefore, developing small molecules capable of selectively modulating a single domain of CBP is of invaluable aid at unraveling its prominent activities. Here we report the design, synthesis, and biological evaluation of conformationally restricted peptides as novel modulators for the acetyl-lysine binding bromodomain (BRD) of CBP. Utilizing a target structure-guided and computer-aided rational design approach, we developed a series of cyclic peptides with affinity for CBP BRD significantly greater than those of its biological ligands, including lysine-acetylated histones and tumor suppressor p53. The best cyclopeptide of the series exhibited a K(d) of 8.0 μM, representing a 24-fold improvement in affinity over that of the linear lysine 382-acetylated p53 peptide. This lead peptide is highly selective for CBP BRD over BRDs from other transcriptional proteins. Cell-based functional assays carried out in colorectal carcinoma HCT116 cells further demonstrated the efficacy of this compound to modulate p53 stability and function in response to DNA damage. Our results strongly argue that these CBP modulators can effectively inhibit p53 transcriptional activity by blocking p53K382ac binding to CBP BRD and promoting p53 instability by changes of its post-translational modification states, a different mechanism than that of the p53 inhibitors reported to date.

Human Transcriptional Coactivator with PDZ-Binding Motif (TAZ) Is Downregulated During Decidualization1

Transcriptional coactivator with PDZ-binding motif (TAZ) is known to bind to a variety of transcription factors to control cell differentiation and organ development. However, its role in uterine physiology has not yet been described. To study its regulation during the unique process of differentiation of fibroblasts into decidual cells (decidualization), we utilized the human uterine fibroblast (HuF) in vitro cell model. Immunocytochemistry data demonstrated that the majority of the TAZ protein is localized in the nucleus. Treatment of HuF cells with the embryonic stimulus cytokine interleukin 1 beta in the presence of steroid hormones (estradiol-17 beta and medroxyprogesterone acetate) for 13 days did not cause any apparent TAZ mRNA changes but resulted in a significant TAZ protein decline (approximately 62%) in total cell lysates. Analysis of cytosolic and nuclear extracts revealed that the decline of total TAZ was caused primarily by a drop of TAZ protein levels in the nucleus. TAZ was localized on the peroxisome proliferator-activated receptor response element site (located at position -1200 bp relative to the transcription start site) of the genomic region of decidualization marker insulin-like growth factor-binding protein 1 (IGFBP1) in HuF cells as detected by chromatin immunoprecipitation. TAZ is also present in human endometrium tissue as confirmed by immunohistochemistry. During the secretory phase of the menstrual cycle, specific TAZ staining particularly diminishes in the stroma, suggesting its participation during the decidualization process, as well as implantation. During early baboon pregnancy, TAZ protein expression remains minimal in the endometrium close to the implantation site. In summary, the presented evidence shows for the first time to date TAZ protein in the human uterine tract, its downregulation during in vitro decidualization, and its localization on the IGFBP1 promoter region, all of which indicate its presence in the uterine differentiation program during pregnancy.

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.

Eya4 regulation of Na+/K+-ATPase is required for sensory system development in zebrafish

To investigate the mechanisms by which mutations in the human transcriptional co-activator EYA4 gene cause sensorineural hearing loss that can occur in association with dilated cardiomyopathy, we studied eya4 expression during zebrafish development and characterized eya4 deficiency. eya4 morphant fish embryos had reduced numbers of hair cells in the otic vesicle and lateral line neuromasts with impaired sensory responses. Analyses of candidate genes that are known to be expressed in a temporal and spatial pattern comparable to eya4 focused our analyses on atp1b2b, which encodes the beta2b subunit of the zebrafish Na+/K+-ATPase. We demonstrate atp1b2b levels are reduced in eya4 morphant fish and that morpholino oligonucleotides targeting the atp1b2b gene recapitulated the eya4 deficiency phenotypes, including heart failure, decreased sensory hair cell numbers in the otic vesicle and neuromasts, and abnormal sensory responses. Furthermore, atp1b2b overexpression rescued these phenotypes in eya4 morphant fish. We conclude that eya4 regulation of Na+/K+-ATPase is crucial for the development of mechanosensory cells and the maintenance of cardiac function in zebrafish.

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.

Structure-Guided Optimization of Small Molecules Inhibiting Human Immunodeficiency Virus 1 Tat Association with the Human Coactivator p300/CREB Binding Protein-Associated Factor

Human immunodeficiency virus 1 (HIV-1) trans-activator Tat recruits the human transcriptional coactivator PCAF (p300/CREB binding protein-associated factor) to facilitate transcription of the integrated HIV-1 provirus. We report here structure-based lead optimization of small-molecule inhibitors that block selectively Tat and PCAF association in cells. Our lead optimization was guided by grand-canonical ensemble simulation of the receptor/lead complex that leads to definition of chemical modifications with improved lead affinity through displacing weakly bound water molecules at the ligand-receptor interface.

Hot Spots in Ag Core−Au Shell Nanoparticles Potent for Surface-Enhanced Raman Scattering Studies of Biomolecules

The vicinity of metallic nanostructures that provides intense optical fields is termed as “hot spot”. Any molecule in close proximity to these hot spots will give rise to an increased surface-enhanced Raman spectroscopic (SERS) signal. We have synthesized Ag core−Au shell (core−shell) nanoparticles (NP) with nanopores, which act as hot spots in the SERS measurements. We have demonstrated a large enhancement in SERS studies of various molecules using core−shell NP with hot spots, which is better than using silver nanoparticles. The core−shell NP with hot spots can be used for ultratrace analysis of important biomolecules such as histone acetyltranferase p300, a human transcriptional coactivator protein. The core−shell NP does not change the biomolecule's physical and chemical property upon adsorption, which makes it biocompatible. The core−shell NP carrying hot spots with high SERS enhancement would be ideally suited for in vivo studies of biological systems.

Surface-Enhanced Raman Scattering Studies of Human Transcriptional Coactivator p300

We report for the first time the surface-enhanced Raman scattering (SERS) studies on p300, a large multidomain transcriptional coactivator protein. Vibration spectral analysis has been performed in an attempt to understand the structure of the p300 in the absence of its crystal structure. Strong Raman bands associated with amides I-III have been observed in the protein spectra. This has been confirmed by performing SERS on deuterated p300. We also observe Raman bands associated with the alpha-helix, tryptophan, phenylalanine, tyrosine, and histidine. These bands will provide an ideal tool to study the drug-protein interactions in therapeutics using SERS. We have successfully demonstrated the chloride ion effect on the SERS of p300. The Raman intensity increases in the SERS spectra upon addition of chloride ion along with appearance of new modes. We have developed a new method, namely, the "sandwich technique", which could be used to perform SERS experiments on proteins in dry conditions.

Mutation in the transcriptional coactivator EYA4 causes dilated cardiomyopathy and sensorineural hearing loss

We identified a human mutation that causes dilated cardiomyopathy and heart failure preceded by sensorineural hearing loss (SNHL). Unlike previously described mutations causing dilated cardiomyopathy that affect structural proteins, this mutation deletes 4,846 bp of the human transcriptional coactivator gene EYA4. To elucidate the roles of eya4 in heart function, we studied zebrafish embryos injected with antisense morpholino oligonucleotides. Attenuated eya4 transcript levels produced morphologic and hemodynamic features of heart failure. To determine why previously described mutated EYA4 alleles cause SNHL without heart disease, we examined biochemical interactions of mutant Eya4 peptides. Eya4 peptides associated with SNHL, but not the shortened 193-amino acid peptide associated with dilated cardiomyopathy and SNHL, bound wild-type Eya4 and associated with Six proteins. These data define unrecognized and crucial roles for Eya4-Six-mediated transcriptional regulation in normal heart function.

Selective Small Molecules Blocking HIV-1 Tat and Coactivator PCAF Association

Development of drug resistance from mutations in the targeted viral proteins leads to continuation of viral production by chronically infected cells, contributing to HIV-mediated immune dysfunction. Targeting a host cell protein essential for viral reproduction, rather than a viral protein, may minimize the viral drug resistance problem as observed with HIV protease inhibitors. We report here the development of a novel class of N1-aryl-propane-1,3-diamine compounds using a structure-based approach that selectively inhibit the activity of the bromodomain of the human transcriptional co-activator PCAF, of which association with the HIV trans-activator Tat is essential for transcription and replication of the integrated HIV provirus.