Endogenous P300 Research Articles

Exploration of bromodomain ligand-linker conjugation sites for efficient CBP/p300 heterobifunctional degrader activity

Synthesis of proteolysis targeting chimeras (PROTACs) involves conjugation of an E3 ligase binding ligand to a ligand targeting a protein of interest via a rigid or flexible chemical linker. The choice of linker conjugation site on these ligands can be informed by structural analysis of ligand-target binding modes, the feasibility of synthetic procedures to access specific sites, and computational modeling of predicted ternary complex formations. Small molecules that target bromodomains - epigenetic readers of lysine acetylation - typically offer several potential options for linker conjugation sites. Here we describe how varying the linker attachment site (exit vector) on a CBP/p300 bromodomain ligand along with linker length affects PROTAC degradation activity and ternary complex formation. Using kinetic live cell assays of endogenous CBP and p300 protein abundance and bead-based proximity assays for ternary complexes, we describe the structure-activity relationships of a diverse library of CBP/p300 degraders (dCBPs).

Screening of subclinical P300 event‑related potentials changes in childhood acute lymphoblastic leukemia survivors

Modern treatment of childhood acute lymphoblastic leukemia (ALL) has resulted in a high cure rate; however, it can cause central nervous system toxicity. In the present study, a group of 136 ALL survivors were screened for changes in P300. Therapy was conducted according to a modified New York (NY) protocol (30 patients) and two subsequent revisions of a modified Berlin-Frankfurt-Münster (BFM) protocol (32 and 74 patients). The control group consisted of 58 patients. The survivors had significantly prolonged mean latency of P300 (331.31±28.71 vs. 298.14±38.76 msec, P<0.001) and reaction time (439.51±119.86 vs. 380.11±79.94 msec, P=0.002) compared with in the control group. Abnormalities in the endogenous evoked potentials were observed in 36 patients (26.5%). The mean latency time was significantly longer in the treatment groups compared with in the control group (NY: 329.13±28.07 msec, P=0.001; pBFM: 332.97±23.97 msec, P<0.001; BFM95: 331.47±31.05 msec, P<0.001). The reaction time was equally prolonged in both groups. In comparisons between the studied groups and the control group the most significant prolongation was recorded in the NY group (461.8±140.3 vs. 380.1±78.04 msec, P=0.039). Significantly higher frequency of prolonged reaction time in non-irradiated patients that received BFM95 was also revealed (21.62 vs. 15.85%, P=0.007). In addition, radiotherapy significantly reduced the P300 wave amplitude (mean values: 10.395±5.727 vs. 12.739±6.508 mV, P=0.027). In conclusion, endogenous P300 event-related potentials may be a useful tool in screening of subclinical cognitive changes in ALL survivors.

Pregnancy-related hormones, progesterone and human chorionic gonadotrophin, upregulate expression of maternal plasma gelsolin.

Plasma gelsolin (pGSN), a protein primarily involved in clearance of circulating actin filaments, is an upcoming novel biomarker. Its level changes in multiple disease and injury conditions, attributable mainly to its consumption during actin clearance; the endogenous regulation of its expression, however, remains elusive as well as unexplored. Here, we are reporting the first isolation of the promoter region of pGSN gene and investigation of its transcriptional regulation during pregnancy (a natural process associated with a well-programmed injury course of parturition). Interestingly, two of the pregnancy-related hormones, human chorionic gonadotrophin (hCG) and progesterone, significantly upregulated pGSN promoter activity in muscle cells. This action of both hormones was found to mediate through their respective cellular receptors and involved a contribution of multiple signaling pathways including those of protein kinase A, protein kinase C, epidermal growth factor receptor and prostaglandin-endoperoxidase synthase 2 in the case of hCG-mediated upregulation. This novel upregulation was further supported by elevated levels of endogenous pGSN transcripts as well as secreted protein upon hormonal treatments of muscle cells compared with untreated controls. A participation of pGSN promoter cis-elements, capable of interacting with endogenous transcription factors, Ap1, Sp1, and p300, was also observed during this hormonal upregulation. Additionally, the augmented pGSN levels observed in pregnant mice compared with the control animals further supported an upregulation of this protein during pregnancy, implicating vital role(s) played by pGSN during this period in mammals.

21. Generators of oddball P300 to visual omitted stimuli: A simultaneous EEG-fMRI study

The present study aimed at investigating the cerebral sources of the visual P300 event-related potential (ERP) by the simultaneous recording of EEG and functional magnetic resonance imaging (fMRI). In addition to a standard two-stimuli visual “oddball” paradigm we applied a similar paradigm, in which the rare (target) stimuli were omitted in a sequence of regularly presented visual stimuli. Both conditions were preceded by a trial in which the subject gave no attention to the stimuli (passive condition). The EEG showed a clear N1/P3 complex during the standard “oddball” paradigm, whereas during the “omitted stimulus oddball” only a P300 component, with reduced amplitude and increased latency (about 700 msec, compared to 400 msec in the standard paradigm) was observed. fMRI showed, during the standard “oddball”, activations in lateral frontal, fronto- operculum, anterior insula regions, prevailing on the right. Minor activations were observed bilaterally in the parietal cortex near the intraparietal sulcus and in posterior temporal regions, also more evident on the right hemisphere. The “omitted stimuli” paradigm showed fMRI activations in the very same locations as those obtained in the standard “oddball”. These results highlight the “real” cortical sources of the endogenous P300, not contaminated by sensory activations.

P300-Dependent Chromatin Remodelling Regulates Multiple Liver Functions

Liver is a one of the largest tissues that performs a variety of complex functions which support body homeostasis including detoxification and providing essential molecules to the blood. Disorganization of liver functions is one of the main characteristics of several severe diseases including liver cancer and non-alcoholic fatty liver disease (NAFLD). Understanding the mechanisms which control functions of the healthy liver is highly important for development of approaches for treatments of liver diseases. Although the role of chromatin remodeling in liver biology has been documented by many reports, the precise mechanisms of this regulation are not well understood. We have recently determined these mechanisms using transgenic mice which express a dominant negative p300 molecule, dnp300 mice. This animal model is an excellent tool for the examination of the role of p300 since activities of the endogenous p300 are inhibited in these mice. An examination of global changes in expression of genes in livers of dnp300 mice revealed that p300 regulates multiple pathways. These pathways include chromatin remodeling, DNA damage, fatty liver, oncogenes, apoptosis, cell cycle and translation. One of the key specific pathways of p300-dependent regulation of liver functions is the co-operation of p300 with C/EBP family proteins. Our long term studies of C/ EBPα-p300 complexes in biological processes of the liver included investigations in wild type mice and in genetically modified animal models with elevated or reduced amounts of C/EBPα-p300 complexes. Examination of these animal models elucidated mechanisms by which C/EBPα-p300 complexes are involved in development of NAFLD and in regulation of liver proliferation/regeneration and liver injury. This review summarizes the knowledge obtained in these genetically-modified animal models with altered chromatin structure.

Abstract 5120: Using flow-proteometric platform to analyze individual signaling complexes in tumor tissue

Abstract Cellular signaling complexes, which are made up of mostly proteins and nucleic acids, play a major role in signal transduction by carrying and delivering messages that coordinate basic biological functions. These processes are mainly relayed through protein-protein and protein-nucleic acid interactions. Delineating the underlying mechanisms of cancer signal transduction, which is often deregulated, relies highly on the analysis of specific protein-protein and protein-nucleic acid interactions. Conventional methods for detecting protein-protein and protein-nucleic acid interactions mostly rely on cells collected from two-dimensional cell cultures, which are very different from the natural cellular environment in organs or tissues. Therefore, we developed a multiplex flow-proteometric platform that can analyze individual signaling complexes directly from tissue to enable us to accurately acquire the information on in vivo signal transduction. To demonstrate that single signaling complexes can be directly detected in lysates from fluorescent-labeled frozen tissue sections, we selected STAT3, p300, and genomic DNA as targets to detect and quantify individual complexes in xenograft tumor tissues. Endogenous STAT3 and p300 in frozen tumor tissue were immunolabeled with A488 and QD605, respectively, and with TOTO3 to label genomic DNA. All the detected events were presented in a 3D fluorescence plot, revealing seven different types of events. A minimal number of non-specific control IgG events were detected, and no interactions events were observed, indicating low background noise. Among all of the detected STAT3 events, on average 7.04% interacted with both p300 and genomic DNA in the same complex, 2.99% interacted with p300 only, and 15.23% interacted with DNA only. Thus, in the frozen xenograft tissue section, around 22% of STAT3 (15.23% STAT3−DNA + 7.04% STAT3−p300 −DNA) interacted with DNA and 7.04% of STAT3 interacted with p300 when bound on DNA to activate gene transcription. Looking at the data from the standpoint of p300, on average 5.88% of total p300 protein molecules interacted with STAT3 and genomic DNA, 2.5% with STAT3 only, and 3.06% with DNA only. All these data simultaneously obtained in each experiment not only identified STAT3, p300, and DNA in a single complex but also quantified the distribution of lone proteins and population complexes, which cannot be analyzed by conventional methods. Thus, we expect that this technique may reveal new aspects of molecular interaction in tissue. Citation Format: Chao-Kai Chou, Heng-Huan Lee, Pei-Hsiang Tsou, Chun-Te Chen, Jung-Mao Hsu, Hirohito Yamaguchi, Ying-Nai Wang, Jennifer L. Hsu, Jin-Fong Lee, Jun Kameoka, Mien-Chie Hung. Using flow-proteometric platform to analyze individual signaling complexes in tumor tissue. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5120. doi:10.1158/1538-7445.AM2015-5120

MiR-150 regulates high glucose-induced cardiomyocyte hypertrophy by targeting the transcriptional co-activator p300

p300, a transcriptional co-activator with histone acetyl transferase (HAT) activity, plays an essential role in the pathogenesis of cardiomyocyte hypertrophy in response to multiple pro-hypertrophic stimuli including hyperglycemia. However, the precise mechanisms by which p300 expression is regulated remain unclear. The purpose of this study was to investigate the role of miR-150, a potential p300-targeting microRNA (miRNA), in the post-transcriptional control of p300 expression and cardiomyocyte hypertrophy induced by high glucose. We observed that the expression of miR-150 was significantly reduced, whereas the expression of p300 was strongly elevated, concomitant with cardiomyocyte hypertrophy, in the hearts of diabetic rats compared with normal controls. Similar alterations were observed in neonatal rat cardiomyocytes that had been exposed to high levels of glucose. miR-150 mimics inhibited p300 3′-UTR luciferase reporter activity, as well as endogenous p300 expression. In addition, miR-150 mimics prevented glucose-induced cardiomyocyte hypertrophy. Co-transfection with a p300 expression vector and miR-150 mimics reversed the protective effect of miR-150 on cardiomyocyte hypertrophy. We further showed that the high glucose-mediated activation of PKCβ2 in turn mediated the down-regulation of miR-150 expression. These data demonstrated a novel upstream role for miR-150 in p300-mediated cardiomyocyte hypertrophy and revealed a previously uncharacterized miRNAs and HATs cross-talk mechanism for the hypertrophic phenotype induced by high glucose.

P300 acetyltransferase activity differentially regulates the localization and activity of the FOXO homologues in skeletal muscle

The Forkhead Box O (FOXO) transcription factors are both necessary and sufficient for skeletal muscle atrophy. Therefore understanding the mechanisms of FOXO regulation in skeletal muscle is of significant importance. The objective of the current study was to determine if FOXO signaling is regulated in skeletal muscle via the acetyltransferase (HAT) activities of p300/CBP. Gene transfer of rat soleus muscle with a dominant‐negative (d.n.) p300, which lacks HAT activity and inhibits endogenous p300 HAT activity, increased FOXO reporter activity and induced transcription from the promoter of the atrophy‐related gene, atrogin‐1. Conversely, gene transfer of either WT p300 or WT CBP repressed FOXO activation and atrogin‐1 in response to muscle disuse, and in C2C12 cells in response to acute starvation. Co‐transfection of FOXO1, FOXO3a or FOXO4 with the p300 constructs further identified p300 HAT activity to differentially regulate the activity of the FOXO factors. Decreased HAT activity potently increased FOXO3a transcriptional activity, while increased HAT activity repressed FOXO3a activity and prevented its nuclear localization in response to nutrient deprivation. In contrast, p300 increased FOXO1 nuclear localization. In summary, this study provides the first evidence to support the acetyltransferase activities of p300/CBP in regulating FOXO signaling in skeletal muscle.

P300 Acetyltransferase activity differentially regulates the localization and activity of the FOXO homologues in skeletal muscle

The Forkhead Box O (FOXO) transcription factors regulate diverse cellular processes, and in skeletal muscle are both necessary and sufficient for muscle atrophy. Although the regulation of FOXO by Akt is well evidenced in skeletal muscle, the current study demonstrates that FOXO is also regulated in muscle via the histone acetyltransferase (HAT) activities of p300/CREB-binding protein (CBP). Transfection of rat soleus muscle with a dominant-negative p300, which lacks HAT activity and inhibits endogenous p300 HAT activity, increased FOXO reporter activity and induced transcription from the promoter of a bona fide FOXO target gene, atrogin-1. Conversely, increased HAT activity via transfection of either wild-type (WT) p300 or WT CBP repressed FOXO activation in vivo in response to muscle disuse, and in C2C12 cells in response to dexamethasone and acute starvation. Importantly, manipulation of HAT activity differentially regulated the expression of various FOXO target genes. Cotransfection of FOXO1, FOXO3a, or FOXO4 with the p300 constructs further identified p300 HAT activity to also differentially regulate the activity of the FOXO homologues. Markedly, decreased HAT activity strongly increased FOXO3a transcriptional activity, while increased HAT activity repressed FOXO3a activity and prevented its nuclear localization in response to nutrient deprivation. In contrast, p300 increased FOXO1 nuclear localization. In summary, this study provides the first evidence to support the acetyltransferase activities of p300/CBP in regulating FOXO signaling in skeletal muscle and suggests that acetylation may be an important mechanism to differentially regulate the FOXO homologues and dictate which FOXO target genes are activated in response to varying atrophic stimuli.

HDAC Class I Inhibition Acetylates a Non-Histone Protein STAT3 by Modulating p300-STAT3-HDAC1 Interaction In Activated B- Cell Like (ABC) Diffuse Large B Cell Lymphoma

AbstractAbstract 115Patients with diffuse large B- cell lymphoma (DLBCL) tumors that have an activated B-cell like (ABC) gene expression profile have a poorer prognosis. Understanding the mechanism(s) used by ABC tumor cells to resist the effects of common chemotherapy agents may lead to alternative approaches for the treatment of these tumors. ABC cell lines have been shown to have high levels of phosphorylated STAT3 (pSTAT3); however, the mechanisms that regulate STAT3 signaling in ABC DLBCL remain unclear. Histone deacetylases (HDACs) are enzymes that can deacetylate both non-histone and histone substrates. In this study we tested the hypothesis that HDACs in the tumor cells target a non-histone protein STAT3 in ABC DLBCL.In studies of HDAC expression in DLBCL tumors, we found over-expression of the type 1 HDACs, specifically HDAC1and HDAC3, in the pSTAT3- positive ABC tumors as compared to germinal centre B like (GCB) tumors. We then performed a co-immunoprecipitation (Co-IP) assay to learn the functional interaction between STAT3 and HDAC1. We found that STAT3 formed complexes with HDAC1 or HDAC3. Further Co-IP studies demonstrated that p300, a histone acetyltransferase (HAT), STAT3, and HDAC1 are all in the same complex. To determine whether p300 acetylates STAT3 in ABC cells, we immuno-precipitated endogenous p300 and blotted with acetylated STAT3 and showed that p300 acetylates STAT3 at lysine 685. We next tested whether HDAC inhibition could affect p300 mediated STAT3 acetylation in ABC cells. Inhibition of HDAC activity through the HDAC inhibitor LBH589 (LBH, Novartis Pharmaceuticals) increased STAT3 acetylation in a dose- dependent manner. Similar results were obtained when we used antiacetyl- lysine antibody. Furthermore HDAC1 over-expression inhibits STAT3 acetylation at lysine 685. This data implies a tight regulation of STAT3 acetylation and deacetylases in vivo in ABC lymphoma. In addition to acetylation, STAT3 can be modified by phosphorylation, thus the effect of HDAC inhibition on pSTAT3 both at serine and tyrosine residues was studied. We observed a dose-dependent decrease in pSTAT3 with some inhibitory effect on total STAT3. LBH was found to mediate STAT3 dephosphorylation by inhibiting the tyrosine phosphorylation of JAK2 and TYK2, the STAT3 upstream activators, in a dose- dependent manner. Since ABC lymphoma has higher levels of HDAC1 or HDAC3 and pSTAT3/STAT3 than GCB, we hypothesized that ABC cells will be more sensitive to HDAC inhibition than GCB. In fact, when ABC and GCB DLBCL cells were treated with LBH we observed that LBH was more cytotoxic to ABC than GCB as evidenced by annexin/PI staining and PARP cleavage. LD90 was 25 nM for ABC cells, however GCB cells required 5 times more LBH to kill 90% cells. STAT3 activation regulates genes involved in cell survival, including Bcl-2, Mcl-1, Bcl-XL, and c-Myc. LBH treatment resulted in down-regulation of Mcl-1 and c-Myc in ABC cells but has no effect in GCB cells; however, Bcl-2 and Bcl-XL levels were not decreased in both the subtype. Having established that HDAC1 physically associated with STAT3 and that LBH treatment elevated STAT3 acetylation in ABC cells, we proceeded to deplete endogenous HDAC1 with siRNA in Ly3 cells and found that HDAC1 knockdown up-regulated STAT3 acetylation indicating that HDAC1 negatively regulates the acetylation in vivo. HDAC1 inhibition also prevented phopshorylation of STAT3 and induces aopotosis in ABC cells.In summary, we have demonstrated that a key consequence of HATs and HDACs expression and activity is modulation of the STAT3 pathway in ABC lymphoma. Inhibition of this pathway with the HDAC inhibitor LBH inhibits constitutive STAT3 signaling and induces Mcl-1 mediated apoptosis. These studies provide the rationale for targeting the poorly responsive ABC-type DLBCL by inhibiting HDAC activity with epigenetic inhibitors such as LBH. We are currently testing LBH589 in relapsed DLBCL in a phase I clinical trial. Disclosures:No relevant conflicts of interest to declare.

Dual roles for MEF2A and MEF2D during human macrophage terminal differentiation and c-Jun expression

Recent reports have evidenced a role for MEF2C (myocyte enhancer factor 2C) in myelopoiesis, although the precise functions of this transcription factor are still unclear. We show in the present study that MEF2A and MEF2D, two other MEF2 family members, are expressed in human primary monocytes and in higher amounts in monocyte-derived macrophages. High levels of MEF2A-MEF2D heterodimers are found in macrophage-differentiated HL60 cells. Chromatin immunoprecipitations demonstrate that MEF2A is present on the c-Jun promoter, both in undifferentiated and in macrophage-differentiated cells. Moreover, c-Jun expression is derepressed in undifferentiated cells in the presence of HDAC (histone deacetylase) inhibitor, indicating the importance of chromatin acetylation in this process. We show that MEF2A/D dimers strongly interact with HDAC1, and to a lesser extent with HDAC7 in macrophages, whereas low levels of MEF2A/D-HDAC1 complexes are found in undifferentiated cells or in monocytes. Since trichostatin A does not disrupt MEF2A/D-HDAC1 complexes, we analysed the potential interaction of MEF2A with p300 histone acetyltransferase, whose expression is up-regulated in macrophages. Interestingly, endogenous p300 only associates with MEF2A in differentiated macrophages, indicating that MEF2A/D could activate c-Jun expression in macrophages through a MEF2A/D-p300 activator complex. The targets of MEF2A/D-HDAC1-HDAC7 multimers remain to be identified. Nevertheless, these data highlight for the first time the possible dual roles of MEF2A and MEF2D in human macrophages, as activators or as repressors of gene transcription.

AP-2γ promotes proliferation in breast tumour cells by direct repression of the CDKN1A gene

Overexpression of the activator protein (AP)-2gamma transcription factor in breast tumours has been identified as an independent predictor of poor outcome and failure of hormone therapy. To understand further the function of AP-2gamma in breast carcinoma, we have used an RNA interference and gene expression profiling strategy with the MCF-7 cell line as a model. Gene expression changes between control and silenced cells implicate AP-2gamma in the control of cell cycle progression and developmental signalling. A function for AP-2gamma in cell cycle control was verified using flow cytometry: AP-2gamma silencing led to a partial G1/S arrest and induction of the cyclin-dependent kinase inhibitor, p21cip/CDKN1A. Reporter and chromatin immunoprecipitation assays demonstrated a direct, functional interaction by AP-2gamma at the CDKN1A proximal promoter. AP-2gamma silencing coincided with acquisition of an active chromatin conformation at the CDKN1A locus and increased gene expression. These data provide a mechanism whereby AP-2gamma overexpression can promote breast epithelial proliferation and, coupled with previously published data, suggest how loss of oestrogen regulation of AP-2gamma may contribute to the failure of hormone therapy in patients.

Zinc-Induced Formation of a Coactivator Complex Containing the Zinc-Sensing Transcription Factor MTF-1, p300/CBP, and Sp1

Herein, the mechanisms of transactivation of gene expression by mouse metal response element-binding transcription factor 1 (MTF-1) were investigated. Evidence obtained from coimmunoprecipitation assays revealed that exposure of the cells to zinc resulted in the rapid formation of a multiprotein complex containing MTF-1, the histone acetyltransferase p300/CBP, and the transcription factor Sp1. Down-regulation of endogenous p300 expression by small interfering RNA transfection significantly decreased zinc-dependent metallothionein I (MT-I) gene transcription without altering induction of zinc transporter 1 (ZnT1). MTF-1 independently facilitated the recruitment of Sp1 and p300 to the protein complex in response to zinc. Mutagenesis demonstrated that the acidic domain, one of three transactivation domains of MTF-1, is required for recruitment of p300 but not Sp1 as well as for zinc-dependent activation of MT-I gene transcription. Furthermore, mutation of leucine residues (L-->A) within a nuclear exclusion signal in the MTF-1 acidic domain impaired recruitment of p300 and zinc-dependent activation of the MT-I gene. Nuclear magnetic resonance characterization of an isolated protein fragment corresponding to the MTF-1 acidic region demonstrated that this region is largely unstructured in the presence and absence of excess stoichiometric amounts of zinc. This suggests that the mechanism by which MTF-1 recruits p300 to this complex involves extrinsic-zinc-dependent steps. These studies reveal a novel zinc-responsive mechanism requiring an acidic region of MTF-1 that functions as a nuclear exclusion signal as well as participating in formation of a coactivator complex essential for transactivation of MT-I gene expression.

The use of endogenous P300 event-related potentials of the brain for assessing cognitive functions in healthy subjects and in clinical practice

Assessment of higher mental functions, objective detection of cognitive impairments, and investigation of pathophysiological mechanisms underlying these impairments in various neuropsychological diseases are of great importance for neuropsychophysiology. The endogenous event-related potential (ERP) approach is one of the instrumental neurophysiological methods that are currently used for assessing these complicated processes because recorded potentials reflect the intrinsic brain activity and changes in these potentials are caused by endogenous factors of the brain activity. The P300 cognitive evoked potential, induced by selective attention to a stimulus, has been the most widely used endogenous ERP. This potential may be helpful for studying mechanisms of mental disturbances, as it reflects neuronal processes connected with nonspecific activating reticulothalamic systems, as well as with limbic and neocortical mechanisms of selective attention and short-term memory.

SRC-p300 Coactivator Complex Is Required for Thyroid Hormone-induced Amphibian Metamorphosis

Gene activation by the thyroid hormone (T3) receptor (TR) involves the recruitment of specific coactivator complexes to T3-responsive promoters. A large number of coactivators for TR have been isolated and characterized in vitro. However, their roles and functions in vivo during development have remained largely unknown. We have utilized metamorphosis in Xenopus laevis to study the role of these coactivators during post-embryonic development. Metamorphosis is totally dependent on the thyroid hormone, and TR mediates a vast majority, if not all, of the developmental effects of the hormone. We have previously shown that TR recruits the coactivator SRC3 (steroid receptor coactivator-3) and that coactivator recruitment is essential for metamorphosis. To determine whether SRCs are indeed required, we have analyzed the in vivo role of the histone acetyltransferase p300/CREB-binding protein (CBP), which was reported to be a component of the SRC.coactivator complexes. Chromatin immunoprecipitation revealed that p300 is recruited to T3-responsive promoters, implicating a role of p300 in TR function. Further, transgenic tadpoles overexpressing a dominant negative form of p300, F-dnp300, containing only the SRC-interacting domain, displayed arrested or delayed metamorphosis. Molecular analyses of the transgenic F-dnp300 animals showed that F-dnp300 was recruited by TR (displacing endogenous p300) and inhibited the expression of T3-responsive genes. Our results thus suggest that p300 and/or its related CBP is an essential component of the TR-signaling pathway in vivo and support the notion that p300/CBP and SRC proteins are part of the same coactivator complex in vivo during post-embryonic development.

Gain of function of mutant p53: The mutant p53/NF-Y protein complex reveals an aberrant transcriptional mechanism of cell cycle regulation

This article investigates the mechanistic aspects of mutant p53 "gain of function" in response to DNA damage. We show that mutant forms of p53 protein interact with NF-Y. The expression of cyclin A, cyclin B1, cdk1, and cdc25C, as well as the cdk1-associated kinase activities, is upregulated after DNA damage, provoking a mutant p53/NF-Y-dependent increase in DNA synthesis. Mutant p53 binds NF-Y target promoters and, upon DNA damage, recruits p300, leading to histone acetylation. The recruitment of mutant p53 to the CCAAT sites is severely impaired upon abrogation of NF-YA expression. Endogenous NF-Y, mutant p53, and p300 proteins form a triple complex upon DNA damage. We demonstrate that aberrant transcriptional regulation underlies the ability of mutant p53 proteins to act as oncogenic factors.

Role of the N-Terminal Activation Domain of the Coiled-Coil Coactivator in Mediating Transcriptional Activation by β-Catenin

The coiled-coil coactivator (CoCoA) is involved in transcriptional activation of target genes by nuclear receptors and the xenobiotic aryl hydrocarbon receptor, as well as target genes of the Wnt signaling pathway, which is mediated by the lymphocyte enhancer factor (LEF)/T cell factor transcription factors and the coactivator beta-catenin. The recruitment of CoCoA by nuclear receptors is accomplished by the interaction of the central coiled-coiled domain of CoCoA with p160 coactivators; the C-terminal activation domain (AD) of CoCoA is used for downstream signaling, whereas the function of the N-terminal region is undefined. Here we report that the N terminus of CoCoA contains another AD, which is necessary and sufficient for synergistic activation of LEF1-mediated transcription by CoCoA and beta-catenin. The N-terminal AD contains a p300 binding motif, which is important for synergistic cooperation of CoCoA and p300 as coactivators for LEF1 and beta-catenin. p300 contributes to the function of the CoCoA N-terminal AD primarily through its histone acetyltransferase activity. Moreover, in cultured cells, endogenous p300 is recruited to the promoter of an integrated reporter gene by the N terminus of CoCoA. Thus, the coactivator function of CoCoA for nuclear receptors and LEF1/beta-catenin involves differential utilization of two different CoCoA ADs.

Hormonal regulation of assembly of the pepck gene transcription complex

Phosphoenolpyruvate carboxykinase (PEPCK) gene transcription is activated in response to glucocorticoids, such as dexamethasone (Dex). Previous studies, using a variety of techniques, including reporter gene assays, showed that the induction of PEPCK gene transcription by Dex requires a complex array of DNA accessory elements and associated DNA-binding transcription factors. These, in turn, bind a set of transcription coactivators, including CREB binding protein (CBP) and p300. We have now used the chromatin immunoprecipitation assay (ChIP) to assess promoter occupancy of these proteins, in vivo, in the presence and absence of the ligand, Dex. Dex causes a remarkable increase in the binding of glucocorticoid receptor, polymerase II, CBP and p300 to the promoter. However, even though both CBP and p300 are expressed in the nucleus of H4IIE cells, they do not co-exist in the same transcription complex on the PEPCK gene promoter. Furthermore, the selective reduction of either p300 or CBP, using small interfering RNAs (siRNAs), partially abolishes the Dex response of the PEPCK gene. Reduction of both CBP and p300 totally abolishes the Dex response of the PEPCK gene. Although endogenous p300 and CBP are not capable of compensating for the loss of CBP and p300, respectively, the overexpression of either of these proteins compensates for the loss of the other, and both coactivators support the dominant suppression of the gene by insulin.

P300 Modulates ATF4 Stability and Transcriptional Activity Independently of Its Acetyltransferase Domain

ATF4 plays a crucial role in the cellular response to stress and multiple stress responses pathways converge to the translational up-regulation of ATF4. ATF4 is a substrate of the SCF(betaTrCP) ubiquitin ligase that binds to betaTrCP through phosphorylation on a DSGXXXS motif. We show here that ATF4 stability is also modulated by the histone acetyltransferase p300, which induces ATF4 stabilization by inhibiting its ubiquitination. Despite p300 acetylates ATF4, we found that p300-mediated ATF4 stabilization is independent of p300 catalytic activity, using either the inactive form of p300 or the acetylation mutant ATF4-K311R. ATF4 deleted of its p300 binding domain is no more stabilized by p300 nor recruited into nuclear speckles. In consequence of ATF4 stabilization, both p300 and the catalytically inactive enzyme increase ATF4 transcriptional activity.

A Nuclear Function of β-Arrestin1 in GPCR Signaling: Regulation of Histone Acetylation and Gene Transcription

Chromatin modification is considered to be a fundamental mechanism of regulating gene expression to generate coordinated responses to environmental changes, however, whether it could be directly regulated by signals mediated by G protein-coupled receptors (GPCRs), the largest surface receptor family, is not known. Here, we show that stimulation of delta-opioid receptor, a member of the GPCR family, induces nuclear translocation of beta-arrestin 1 (betaarr1), which was previously known as a cytosolic regulator and scaffold of GPCR signaling. In response to receptor activation, betaarr1 translocates to the nucleus and is selectively enriched at specific promoters such as that of p27 and c-fos, where it facilitates the recruitment of histone acetyltransferase p300, resulting in enhanced local histone H4 acetylation and transcription of these genes. Our results reveal a novel function of betaarr1 as a cytoplasm-nucleus messenger in GPCR signaling and elucidate an epigenetic mechanism for direct GPCR signaling from cell membrane to the nucleus through signal-dependent histone modification.