p300/CREB-binding Protein-associated Factor Research Articles

Overexpression of Neurogenin 1 Negatively Regulates Osteoclast and Osteoblast Differentiation.

Neurogenin 1 (Ngn1) belongs to the basic helix–loop–helix (bHLH) transcription factor family and plays important roles in specifying neuronal differentiation. The present study aimed to determine whether forced Ngn1 expression contributes to bone homeostasis. Ngn1 inhibited the p300/CREB-binding protein-associated factor (PCAF)-induced acetylation of nuclear factor of activated T cells 1 (NFATc1) and runt-related transcription factor 2 (Runx2) through binding to PCAF, which led to the inhibition of osteoclast and osteoblast differentiation, respectively. In addition, Ngn1 overexpression inhibited the TNF-α- and IL-17A-mediated enhancement of osteoclast differentiation and IL-17A-induced osteoblast differentiation. These findings indicate that Ngn1 can serve as a novel therapeutic agent for treating ankylosing spondylitis with abnormally increased bone formation and resorption.

CircKIAA0368 Promotes Proliferation, Migration, and Invasion by Upregulating HOXA10 in Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) represents a head and neck cancer caused by cancerization of nasal epithelial cells. HOXA10 has been identified to promote proliferation and invasion of NPC cells, but its regulatory mechanism has not been well discussed. Published research work has also pointed out that circular RNAs (circRNAs) could regulate mRNAs to affect NPC tumorigenesis and development. To explore the roles of HOXA10 and its specific regulatory mechanism regarding circRNAs in NPC. Reverse transcription polymerase chain reaction and western blot were applied to test gene expression. Functional assays were used to evaluate changes in NPC cell phenotypes. Mechanism assays were done to verify RNA-RNA or RNA-protein interaction. HOXA10 was highly expressed in NPC tissues and cell lines. Moreover, HOXA10 knockdown could restrict NPC cell proliferation, invasion, migration, and epithelial-mesenchymal transition. CircKIAA0368 was upregulated in NPC cells and could elevate HOXA10 expression by sponging miR-6838-5p. Furthermore, circKIAA0368 was unveiled to competitively bind to p300/CREB-binding protein-associated factor (PCAF) to repress acetylation and degradation of HOXA10 protein. CircKIAA0368 upregulates HOXA10 expression via miR-6838-5p and PCAF, consequently promoting NPCcell and tumor growth.

Acetylation of the influenza A virus polymerase subunit PA in the N-terminal domain positively regulates its endonuclease activity.

The post-translational acetylation of lysine residues is found in many nonhistone proteins and is involved in a wide range of biological processes. Recently, we showed that the nucleoprotein of the influenza A virus is acetylated by histone acetyltransferases (HATs), a phenomenon that affects viral transcription. Here, we report that the PA subunit of influenza A virus RNA-dependent RNA polymerase is acetylated by the HATs, P300/CREB-binding protein-associated factor (PCAF), and general control nonderepressible 5 (GCN5), resulting in accelerated endonuclease activity. Specifically, the full-length PA subunit expressed in cultured 293T cells was found to be strongly acetylated. Moreover, the partial recombinant protein of the PA N-terminal region containing the endonuclease domain was also acetylated by PCAF and GCN5 invitro, which facilitated its endonuclease activity. Mass spectrometry analyses identified K19 as a candidate acetylation target in the PA N-terminal region. Notably, the substitution of the lysine residue at position 19 with glutamine, a mimic of the acetyl-lysine residue, enhanced its endonuclease activity invitro; this point mutation also accelerated influenza A virus RNA-dependent RNA polymerase activity in the cell. Our findings suggest that PA acetylation is important for the regulation of the endonuclease and RNA polymerase activities of the influenza A virus.

Histone Acetyltransferase p300/CREB-binding Protein-associated Factor (PCAF) Is Required for All-trans-retinoic Acid-induced Granulocytic Differentiation in Leukemia Cells

Differentiation therapy with all-trans-retinoic acid (ATRA) improves the treatment outcome of acute promyelocytic leukemia (APL); however, the molecular mechanism by which ATRA induces granulocytic differentiation remains unclear. We previously reported that the inhibition of the NAD-dependent histone deacetylase (HDAC) SIRT2 induces granulocytic differentiation in leukemia cells, suggesting the involvement of protein acetylation in ATRA-induced leukemia cell differentiation. Herein, we show that p300/CREB-binding protein-associated factor (PCAF), a histone acetyltransferase (HAT), is a prerequisite for ATRA-induced granulocytic differentiation in leukemia cells. We found that PCAF expression was markedly increased in leukemia cell lines (NB4 and HL-60) and primary APL cells during ATRA-induced granulocytic differentiation. Consistent with these results, the expression of PCAF was markedly up-regulated in the bone marrow cells of APL patients who received ATRA-containing chemotherapy. The knockdown of PCAF inhibited ATRA-induced granulocytic differentiation in leukemia cell lines and primary APL cells. Conversely, the overexpression of PCAF induced the expression of the granulocytic differentiation marker CD11b at the mRNA level. Acetylome analysis identified the acetylated proteins after ATRA treatment, and we found that histone H3, a known PCAF acetylation substrate, was preferentially acetylated by the ATRA treatment. Furthermore, we have demonstrated that PCAF is required for the acetylation of histone H3 on the promoter of ATRA target genes, such as CCL2 and FGR, and for the expression of these genes in ATRA-treated leukemia cells. These results strongly support our hypothesis that PCAF is induced and activated by ATRA, and the subsequent acetylation of PCAF substrates promotes granulocytic differentiation in leukemia cells. Targeting PCAF and its downstream acetylation targets could serve as a novel therapeutic strategy to overcome all subtypes of AML.

Fli1 deficiency and beyond: a unique pathway linking peripheral vasculopathy and dermal fibrosis in systemic sclerosis

Fli1 deficiency and beyond: a unique pathway linking peripheral vasculopathy and dermal fibrosis in systemic sclerosis

The Transcription Factor GLI1 Interacts with SMAD Proteins to Modulate Transforming Growth Factor β-Induced Gene Expression in a p300/CREB-binding Protein-associated Factor (PCAF)-dependent Manner

The biological role of the transcription factor GLI1 in the regulation of tumor growth is well established; however, the molecular events modulating this phenomenon remain elusive. Here, we demonstrate a novel mechanism underlying the role of GLI1 as an effector of TGFβ signaling in the regulation of gene expression in cancer cells. TGFβ stimulates GLI1 activity in cancer cells and requires its transcriptional activity to induce BCL2 expression. Analysis of the mechanism regulating this interplay identified a new transcriptional complex including GLI1 and the TGFβ-regulated transcription factor, SMAD4. We demonstrate that SMAD4 physically interacts with GLI1 for concerted regulation of gene expression and cellular survival. Activation of the TGFβ pathway induces GLI1-SMAD4 complex binding to the BCL2 promoter whereas disruption of the complex through SMAD4 RNAi depletion impairs GLI1-mediated transcription of BCL2 and cellular survival. Further characterization demonstrated that SMAD2 and the histone acetyltransferase, PCAF, participate in this regulatory mechanism. Both proteins bind to the BCL2 promoter and are required for TGFβ- and GLI1-stimulated gene expression. Moreover, SMAD2/4 RNAi experiments showed that these factors are required for the recruitment of GLI1 to the BCL2 promoter. Finally, we determined whether this novel GLI1 transcriptional pathway could regulate other TGFβ targets. We found that two additional TGFβ-stimulated genes, INTERLEUKIN-7 and CYCLIN D1, are dependent upon the intact GLI1-SMAD-PCAF complex for transcriptional activation. Collectively, these results define a novel epigenetic mechanism that uses the transcription factor GLI1 and its associated complex as a central effector to regulate gene expression in cancer cells.

PCAF Impairs Endometrial Receptivity and Embryo Implantation by Down-Regulating β3-Integrin Expression via HOXA10 Acetylation

Homeobox A10 (HOXA10), a key transcription factor, plays a critical role in endometrial receptivity by regulating the expression of downstream target genes, such as β3-integrin (ITGB3), but little is understood about the mechanisms of the posttranslational modification of HOXA10 during embryo implantation. The aim of this study was to assess the effect of HOXA10 acetylation by p300/CREB-binding protein-associated factor (PCAF) in the embryo implantation process. The association of HOXA10 with PCAF was detected by coimmunoprecipitation, Western blotting, and confocal immunofluorescent assays. A luciferase reporter assay, Western blotting, quantitative real-time PCR, and chromatin immunoprecipitation techniques were used to determine the effect of PCAF on HOXA10 protein stability and the HOXA10-mediated regulation of ITGB3 expression. HOXA10-PCAF association on embryo implantation was evaluated using a BeWo spheroid attachment assay. PCAF expression in the eutopic endometrium of women with endometriosis and fertile controls was measured by Western blotting technique. PCAF was identified as an HOXA10-interacting protein and inhibited HOXA10-mediated ITGB3 transcription via acetylating HOXA10 at K338 and K339. Overexpressing or knocking down PCAF in Ishikawa cells showed that PCAF not only down-regulated HOXA10-mediated ITGB3 protein expression but also diminished HOXA10-mediated embryo adhesiveness by acetylating HOXA10 (P < .05). Furthermore, we found aberrantly high PCAF expression in the eutopic endometrium of women with a diagnosis of endometriosis compared with the fertile controls (P < .05). These observations demonstrate that 1) HOXA10 associates with and is acetylated by PCAF at lysines K338 and K339 in Ishikawa cells and 2) HOXA10-PCAF association impairs embryo implantation by inhibiting ITGB3 protein expression in endometrial epithelial cells.

Phosphorylation of Fli1 at Threonine 312 by Protein Kinase C δ Promotes Its Interaction with p300/CREB-Binding Protein-Associated Factor and Subsequent Acetylation in Response to Transforming Growth Factor β

Previous studies have shown that transforming growth factor beta (TGF-beta)-induced collagen gene expression involves acetylation-dependent dissociation from the human alpha2(I) collagen (COL1A2) promoter of the transcriptional repressor Fli1. The goal of this study was to elucidate the regulatory steps preceding the acetylation of Fli1. We first showed that TGF-beta induces Fli1 phosphorylation on a threonine residue(s). The major phosphorylation site was localized to threonine 312 located in the DNA binding domain of Fli1. Using several independent approaches, we demonstrated that Fli1 is directly phosphorylated by protein kinase C delta (PKC delta). Additional experiments showed that in response to TGF-beta, PKC delta is recruited to the collagen promoter to phosphorylate Fli1 and that this step is a prerequisite for the subsequent interaction of Fli1 with p300/CREB-binding protein-associated factor (PCAF) and an acetylation event. The phosphorylation of endogenous Fli1 preceded its acetylation in response to TGF-beta stimulation, and the blockade of PKC delta abrogated both the phosphorylation and acetylation of Fli1 in dermal fibroblasts. Promoter studies showed that a phosphorylation-deficient mutant of Fli1 exhibited an increased inhibitory effect on the COL1A2 gene, which could not be reversed by the forced expression of PCAF or PKC delta. These data strongly suggest that the phosphorylation-acetylation cascade triggered by PKC delta represents the primary mechanism whereby TGF-beta regulates the transcriptional activity of Fli1 in the context of the collagen promoter.

Hepatic insulin resistance induced by prenatal alcohol exposure is associated with reduced PTEN and TRB3 acetylation in adult rat offspring

Prenatal alcohol exposure (EtOH) results in insulin resistance in rats of both sexes with increased expression of hepatic gluconeogenic genes and glucose production. To investigate whether hepatic insulin signaling is defective, we studied 3-mo-old female offspring of dams that were given EtOH during pregnancy compared with those from pair-fed and control dams. We performed an intraperitoneal pyruvate tolerance test, determined the phosphorylation status of hepatic phosphoinositide-dependent protein kinase-1 (PDK1), Akt, and PKCzeta before and after intravenous insulin bolus, and measured mRNA and in vivo acetylation of TRB3 (tribbles 3) and PTEN (phosphatase and tensin homolog deleted on chromosome ten) as well as the expression of the histone acetylase (HAT) PCAF (p300/CREB-binding protein-associated factor), histone deacetylase-1 (HDAC1), and HAT and HDAC activities. In EtOH compared with pair-fed and control offspring, basal and pyruvate-induced blood glucose was increased, insulin-induced PDK1, Akt, and PKCzeta phosphorylation was reduced, and expression of PTEN and TRB3 was increased while their acetylation status was decreased in association with increased HDAC and decreased HAT activities. Thus female adult rats prenatally exposed to EtOH have increased gluconeogenesis, reduced insulin signaling, and increased PTEN and TRB3 expression in the liver. In addition, PTEN and TRB3 are hypoacetylated, which can contribute to Akt-inhibiting activity. These results suggest that hepatic insulin resistance in rats prenatally exposed to EtOH is explained, at least in part, by increased PTEN and TRB3 activity due to both increased gene expression and reduced acetylation.

The Werner Syndrome Helicase Is a Cofactor for HIV-1 Long Terminal Repeat Transactivation and Retroviral Replication

The Werner syndrome helicase (WRN) participates in DNA replication, double strand break repair, telomere maintenance, and p53 activation. Mutations of wrn cause Werner syndrome (WS), an autosomal recessive premature aging disorder associated with cancer predisposition, atherosclerosis, and other aging related symptoms. Here, we report that WRN is a novel cofactor for HIV-1 replication. Immortalized human WRN(-/-) WS fibroblasts, lacking a functional wrn gene, are impaired for basal and Tat-activated HIV-1 transcription. Overexpression of wild-type WRN transactivates the HIV-1 long terminal repeat (LTR) in the absence of Tat, and WRN cooperates with Tat to promote high-level LTR transactivation. Ectopic WRN induces HIV-1 p24(Gag) production and retroviral replication in HIV-1-infected H9(HIV-1IIIB) lymphocytes. A dominant-negative helicase-minus mutant, WRN(K577M), inhibits LTR transactivation and HIV-1 replication. Inhibition of endogenous WRN, through co-expression of WRN(K577M), diminishes recruitment of p300/CREB-binding protein-associated factor (PCAF) and positive transcription elongation factor b (P-TEFb) to Tat/transactivation response-RNA complexes, and immortalized WRN(-/-) WS fibroblasts exhibit comparable defects in recruitment of PCAF and P-TEFb to the HIV-1 LTR. Our results demonstrate that WRN is a novel cellular cofactor for HIV-1 replication and suggest that the WRN helicase participates in the recruitment of PCAF/P-TEFb-containing transcription complexes. WRN may be a plausible target for antiretroviral therapy.

Kinetic and Mass Spectrometric Analysis of p300 Histone Acetyltransferase Domain Autoacetylation

Acetylation of proteins by p300 histone acetyltransferase plays a critical role in the regulation of gene expression. The prior discovery of an autoacetylated regulatory loop in the p300 histone acetyltransferase (HAT) domain prompted us to further explore the mechanisms of p300 autoacetylation. Here we have described a kinetic and mass spectrometric analysis of p300 HAT autoacetylation. The rate of p300 HAT autoacetylation was approximately fourth order with respect to p300 HAT domain concentration and thus appeared to be a highly cooperative process. By showing that a catalytically defective p300 HAT domain could be efficiently acetylated by active p300 HAT, we deduced that autoacetylation occurs primarily by an intermolecular mechanism. This was further confirmed using a semisynthetic biotinylated p300 HAT domain that could be physically separated from the catalytically defective p300 HAT by avidin affinity chromatography. Autoacetylation catalyzed by p300 HAT was approximately 1000-fold more efficient than PCAF (p300/CREB-binding protein-associated factor)-mediated acetylation of catalytically defective p300 HAT. Using a novel tandem mass spectrometric approach, it was found to be possible to observe up to 17 autoacetylation events within the intact p300 regulatory loop. Kinetic analysis of the site specificity of p300 autoacetylation reveals a class of rapid events followed by a slower set of modifications. Several of these rapid autoacetylation sites correlate with an acetyltransferase-activating function based on prior mutagenesis analysis.

HIV-1 Tat Interactions with p300 and PCAF Transcriptional Coactivators Inhibit Histone Acetylation and Neurotrophin Signaling through CREB

The human immunodeficiency virus type-1 (HIV-1) infects microglia, macrophages, and astrocytes in the central nervous system (CNS) and may cause severe neurological diseases, such as AIDS-related dementias or progressive encephalopathies, as a result of CNS inflammation and neurotrophin signaling defects associated with expression of viral antigens and HIV-1 replication in the brain. The HIV Tat protein can be endocytosed by surrounding uninfected cells; interacts with transcriptional coactivators/acetyltransferases, p300/CREB-binding protein, and p300/CREB-binding protein-associated factor (PCAF); and induces neuronal apoptosis. Since nerve growth factor (NGF) receptor and brain-derived neurotrophic factor receptor signaling through CREB requires p300 and PCAF histone acetyltransferases, we sought to determine whether HIV-1 Tat coactivator interactions interfere with neurotrophin receptor signaling in neuronal cells. Here, we demonstrate that Tat-coactivator interactions inhibit NGF- and brain-derived neurotrophic factor-responsive CRE trans-activation and neurotrophin protection against apoptosis in PC12 and IMR-32 neuroblastoma cells. Purified recombinant Tat or Tat-derived synthetic peptides, spanning p300- and PCAF-binding sequences, inhibit histone H3/H4 acetylation in vitro. A Tat mutant, TatK28A/K50A, defective for binding p300 and PCAF, neither repressed NGF-responsive CRE transactivation nor inhibited histone acetylation. HIV-1 Tat interacts in PCAF complexes in post-mortem CNS tissues from donor neuro-AIDS patients, as determined by fluorescence resonance energy transfer immunoconfocal microscopy. Importantly, these findings suggest that HIV-1 Tat-coactivator interactions may contribute to neurotrophin signaling impairments and neuronal apoptosis associated with HIV-1 infections of the CNS.

Nerve Growth Factor Receptor Signaling Induces Histone Acetyltransferase Domain-dependent Nuclear Translocation of p300/CREB-binding Protein-associated Factor and hGCN5 Acetyltransferases

The transcriptional coactivators, p300/CREB-binding protein-associated factor (PCAF) and hGCN5, are recruited to chromatin-remodeling complexes on enhancers of various gene promoters in response to growth factor stimulation. However, the molecular mechanisms by which surface receptor signals modulate the assembly of nuclear transcription complexes are not fully understood. Here we report that nerve growth factor receptor signaling induces nuclear translocation of PCAF and hGCN5 dependent upon the phosphorylation of Ser and Thr residues within their histone acetyltransferase domains, which requires activation of PI3K, Rsk2(pp90), and MSK-1. Neurotrophin stimulation induces p53(K320) acetylation by PCAF and transcriptionally activates p53-responsive enhancer elements within the p21(WAF/CIP1) promoter associated with G(1)/S arrest during neuronal differentiation. Most importantly, these findings represent the first evidence for signal-dependent nuclear translocation of PCAF and hGCN5 acetyltransferases and allude to a novel mechanism for ligand/receptor modulation of nuclear chromatin-remodeling complexes in neurons.

MDM2 Mediates p300/CREB-binding Protein-associated Factor Ubiquitination and Degradation

We recently reported that MDM2, a negative feedback regulator of the tumor suppressor p53, inhibits p300/CREB-binding protein-associated factor (PCAF)-mediated p53 acetylation. Our further study showed that MDM2 also regulates the stability of PCAF. MDM2 ubiquitinated PCAF in vitro and in cells. PCAF ubiquitination occurred at the N terminus and in the nucleus, as the nuclear localization signal sequence-deletion mutant of MDM2, which localized in the cytoplasm and degraded p53, was unable to degrade nuclear PCAF. Restriction of PCAF in the nucleus by leptomycin B did not affect MDM2-mediated PCAF degradation. Consistently, overexpression of MDM2 in p53 null cells caused the reduction of the protein level of PCAF, but not the mRNA level. Conversely, PCAF levels were higher in MDM2-deficient mouse p53(-/-)/mdm2(-/-) embryonic fibroblast (MEF) cells than that in MDM2-containing MEF cells. Furthermore, MDM2 reduced the half-life of PCAF by 50%. These results demonstrate that MDM2 regulates the stability of PCAF by ubiquitinating and degrading this protein.

MDM2 Inhibits PCAF (p300/CREB-binding Protein-associated Factor)-mediated p53 Acetylation

Our previous study shows that MDM2, a negative feedback regulator of the tumor suppressor p53, inhibits p300-mediated p53 acetylation. Because PCAF (p300/CREB-binding protein-associated factor) also acetylates and activates p53 after DNA damage, in this study we have examined the effect of MDM2 on PCAF-mediated p53 acetylation. We have found that MDM2 inhibited p53 acetylation by PCAF in vitro. In addition, when overexpressed, MDM2 inhibited PCAF-mediated p53 acetylation in cells. MDM2 interacted with PCAF both in vitro and in cells, as assessed using GST fusion protein interaction and immunoprecipitation assays, respectively. Consistent with the above results, MDM2 significantly repressed the activation of p53 transcriptional activity by PCAF without apparently affecting the level of p53. In addition, MDM2 co-resided with p53 at the p53-responsive mdm2 and p21(waf1/cip1) promoters, inhibiting expression of the endogenous p21(waf1/cip1). These results demonstrate that MDM2 can inhibit PCAF-mediated p53 acetylation and activation.

Regulation of the homeodomain CCAAT displacement/cut protein function by histone acetyltransferases p300/CREB-binding protein (CBP)-associated factor and CBP.

The CCAAT displacement protein/cut homologue (CDP/cut) is a divergent homeodomain protein that is highly conserved through evolution and has properties of a potent transcriptional repressor. CDP/cut contains three conserved cut-repeat domains and a conserved homeobox, each involved in directing binding specificity to unique nucleotide sequence elements. Furthermore, CDP/cut may play a role as a structural component of chromatin through its direct interaction with nucleosomal DNA and association with nuclear matrix attachment regions. CDP/cut is cell-cycle regulated through interactions with Rb, p107, specific kinases and phosphatases directing the transcriptional activity of CDP/cut on such genes encoding p21(WAF1,CIP1), c-myc, thymidine kinase, and histones. Our previous studies indicate that CDP/cut is associated with histone deacetylase activity and is associated with a corepressor complex through interactions with histone deacetylases. Here, we report the interaction of CDP/cut with CBP and p300/CREB-binding protein-associated factor (PCAF) along with the modification of CDP/cut by the histone acetyltransferase PCAF. Acetylation of CDP/cut by PCAF is directed at conserved lysine residues near the homeodomain region and regulates CDP/cut function. These observations are consistent with the ability of CDP/cut to regulate genes as a transcriptional repressor, suggesting acetylation as a mechanism that regulates CDP/cut function.

The Mechanism of Action of Thyroid Hormones

Thyroid hormone is essential for normal development, differentiation, and metabolic balance. Thyroid hormone action is mediated by multiple thyroid hormone receptor isoforms derived from two distinct genes. The thyroid hormone receptors belong to a nuclear receptor superfamily that also includes receptors for other small lipophilic hormones. Thyroid hormone receptors function by binding to specific thyroid hormone-responsive sequences in promoters of target genes and by regulating transcription. Thyroid hormone receptors often form heterodimers with retinoid X receptors. Heterodimerization is regulated through distinct mechanisms that together determine the specificity and flexibility of the sequence recognition. Amino-terminal regions appear to modulate thyroid hormone receptor function in an isoform-dependent manner. Unliganded thyroid hormone receptor represses transcription through recruitment of a corepressor complex, which also includes Sin3A and histone deacetylase. Ligand binding alters the conformation of the thyroid hormone receptor in such a way as to release the corepressor complex and recruit a coactivator complex that includes multiple histone acetyltransferases, including a steroid receptor family coactivator, p300/CREB-binding protein-associated factor (PCAF), and CREB binding protein (CBP). The existence of histone-modifying activities in the transcriptional regulatory complexes indicates an important role of chromatin structure. Stoichiometric, structural, and sequence-specific rules for coregulator interaction are beginning to be understood, as are aspects of the tissue specificity of hormone action. Moreover, knockout studies suggest that the products of two thyroid hormone receptor genes mediate distinct functions in vivo. The increased understanding of the structure and function of thyroid hormone receptors and their interacting proteins has markedly clarified the molecular mechanisms of thyroid hormone action.