The p300 transcriptional coactivator has been implicated in the development of a large number of malignancies; however, the precise mechanism of p300-associated tumorigenesis remains unclear. Here, we demonstrate the functional impact of p300 in human melanomas using both genetic and chemical approach. Depletion of p300 in human melanoma cells was associated with cellular growth arrest and senescence. Microarray analysis identified the Microphthalmia-associated transcription factor (MITF), a critical lineage-specific transcription factor in melanocytes and melanomas, as a major downstream target of p300 in human melanoma. Ectopic expression of MITF in p300-depleted melanoma cells allowed rescue of the p300-silencing phenotype, suggesting a critical regulatory axis involving p300 and MITF. Chromatin immunoprecipitation studies revealed direct regulation of MITF transcription through p300 acetylation of proximal regulatory domains. Critically, we identified that Forkhead Box M1 (FOXM1), a potent pro-proliferation transcription factor, is a target of the p300-MITF signaling axis. Further evaluation of p300 regulation of melanoma cell growth was performed using a highly selective p300/CBP HAT inhibitor, 228-1. Inhibition of p300/CBP histone acetyltransferase (HAT) activity was found to significantly inhibit proliferation of multiple melanoma lines in an MITF-dependent fashion. Together, these data support the role of p300 as a promising therapeutic target in human melanoma and suggest particular therapeutic efficacy of small molecule inhibitors of p300 HAT activity in tumors expressing high levels of MITF. / 2018-12-14T00:00:00Z

The purpose of this study was to determine the effects of task difficulty on the amplitude and latency of the P300 by altering the intensity of the oddball stimulus. A P300 was obtained on 22 adult subjects ranging in age from 21 to 34 years of age (mean = 24 years) with normal hearing. The "frequent stimulus" was a 1000 Hz or 4000 Hz tone burst, gated with a rise and fall time of 10 msec and 20 msec plateau, presented at 75 dBn HL The "oddball stimulus" was a tone burst of the same frequency (1000 Hz or 4000 Hz)presented at 77, 79, or 81 dBn HL. A four-channel recording was made with linked reference electrodes and the following montages:Cz-A1+A2, Pz-A1+A2, and Fz-A1+A2. The fourth channel was used to monitor "eye blink" activity. The investigation tested the null hypothesis that changing the intensity of the oddball stimuli would not result in a significant change in either the amplitude or latency of the P300. Analyses of Variance (ANOVA) indicate that P300 latency and amplitude did not differ significantly by run, stimulus frequency, intensity of the oddball, or montage. Thus the null hypothesis was supported.

Background – Previous reports of abnormal auditory N2 and P3 event-related potentials (ERPs) suggest impaired discrimination, evaluation or context updating for infrequent target stimuli in post-traumatic stress disorder (PTSD). This study examines each of these processes by investigating high-resolution ERP topography during target detection for visual word stimuli. Method – ERPs were recorded at 124 electrodes from 10 PTSD patients and 10 matched controls. Target detection tasks comprised blocks of equally probable red and blue words, with low probability target events. Detection of fixed target words in one color provided the basis for measurement of selective attention for color, stimulus evaluation and target detection processing. Alternative task instructions, with the same stimuli, required detection of any consecutive word repeats in an attended color, which demands working memory updating for nontarget words. Comparison of attended non-target words from each task indicates the extra activity for updating working memory representations of target attributes. Thus, specific condition comparisons provide measures of stimulus discrimination and evaluation, working memory updating and target detection. Results – PTSD patients had slower and less accurate motor responses in both tasks, with greater inaccuracy during the variable target task. There was abnormal ERP activity in PTSD at 200-300 ms in the left posterior temporal region during stimulus discrimination and target recognition. During evaluation of attended non-target words, PTSD patients demonstrate deficits in frontal and parietal regions at 400-500 ms. During working memory updating, at 400-600 ms, there was a delay in frontal activation, followed by smaller activity in parietal areas in PTSD. During target word recognition, PTSD patients demonstrate deficits in frontal activity, with greater occipital and parietal activity. Conclusions – These findings indicate impaired evaluation and integration of new information in working memory. In particular, the results suggest failure in frontal executive systems, with greater dependence on visual processing for effective target detection. The current findings are consistent with neuropsychology studies that identify deficits of attention and memory for verbal information in PTSD. This study provides insight into the temporal components of attention and working memory in PTSD. It is proposed that working memory deficits arise from disruption to synchronized activity in distributed networks engaged in working memory processes.

Le p300/CBP acétyltransférase est un co-activateur transcriptionnel très important qui est impliqué dans la régulation d'un grand nombre de processus biologiques, comme la transcription d'ADN, le développement et l'immunité innée. Jusqu'à présent, le rôle de p300/CBP dans la régulation de l'expression des gènes a été largement étudiée, mais les mécanismes qui régulent son activité enzymatique sont encore peu connus. Des études ont montré que le dysfonctionnement de p300/CBP est associé à plusieurs formes de cancer et de maladies neurodégénératives. Dés lors, chaque progrès concernant les mécanismes de régulation de p300/CBP est devenu primordial pour le développement de nouvelles thérapies. Le 'noyau' de p300/CBP contient deux domaines pour la reconnaissance des modifications post-traductionnelles (MPTs), un bromodomaine et un PHD finger (le module BP), adjacent à un domaine HAT (ou domaine histone acétyltransférase). Plusieurs enzymes, modifiant la chromatine, contiennent des domaines de reconnaissance des MPTs. Fréquemment des groupements particuliers de ces domaines sont très conservés et liés, au sein de la même protéine ou du même complexe protéique, suggérant qu'ils réalisent des fonctions coordonnées. Ces domaines adjacents peuvent agir en concertation dans la reconnaissance simultanée de différents MPTs ou peuvent exercer des fonctions différentes de celles qui sont effectuées par ces deux domaines particuliers, tels que les fonctions de régulation enzymatique. Plusieurs études suggèrent que les cycles acétylation/désacétylation dans la boucle d'auto-inhibition, à l'intérieur du domaine HAT, jouent un rôle important dans la régulation de l'activité enzymatique de p300/CBP. La proximité du module BP et du domaine HAT suggère que la spécificité de liaison, appartenant au module BP, peut être intrinsèquement liée à la régulation de l'activité du domaine HAT. L'objectif de ma thèse est de déterminer le rôle du module BP dans la régulation de l'activité du domaine HAT. Je propose que le module BP soit impliqué dans la régulation de p300/CBP de deux façons. La première consiste à établir un lien avec le domaine HAT qui stabilise la conformation auto-inhibée de l'enzyme. La deuxième exige que le module BP joue un rôle dans le choix des substrats de p300/CBP. J'ai été en mesure de montrer que BP peut se lier au domaine HAT et à la chromatine modifiée et qu'il peut reconnaître les modifications effectuées par p300/CBP lui-même. Les données obtenues indiquent que le module BP peut être impliqué dans la régulation de l'activité de p300/CBP et dans son ciblage à la chromatine.

Forkhead box Class O (FOXO), one subfamily of the Forkhead box (Fox) family, which is featured by the Forkhead (FH) DNA-binding domain, includes four human transcription factors: FOXO1, FOXO3a, FOXO4, and FOXO6. The tumor suppressor FOXO3a is involved in multiple physiological and pathological processes, such as breast cancer and acute myeloid leukemia, and is related to human longevity. It plays essential role in metabolism, cell cycle arrest, DNA repair, and apoptosis. Besides the FH domain, FOXO3a contains three other regions (CR1-3), conserved within FOXO subfamily. It specifically binds a consensus Forkhead response element (FRE) DNA sequence through the FH domain, and recruits transcriptional coactivator CBP/p300 to activate gene transcription. FOXO3a functions through interacting with other proteins as well. FOXO3a binds p53 through the FH domain and the CR3 region, which are also engaged in an intramolecular interaction, and the solution structure of the former one was determined. This intramolecular interaction regulates coactivator recruitment and is disrupted by FRE DNA. A novel transactivation domain (TAD) CR2C was identified in addition to the known TAD CR3, both of which promiscuously associate with the KIX domain of CBP/p300 in equilibrium between two conformational states, the structures of which were determined by NMR spectroscopy. These two TADs of FOXO3a form additional multivalent binding to the TAZ1 and TAZ2 domains of CBP/p300, further increasing the promiscuity and complexity of the interaction. The coactivator recruitment is modulated by AMPK phosphorylation, which enhances the multivalent interaction between FOXO3a and CBP/p300, and thus the transactivation. These results indicate the significance of intrinsically disordered regions (IDRs) of FOXO3a in transcriptional activation and protein interaction, provide insight of the role of FOXO3a in gene transcription and apoptosis under various conditions, and potentially contribute to the cancer therapy.

Severe motor impairments can severely restrict interaction with one's surroundings. Brain computer interfaces combined with text-based communication systems, such as the P300 Speller, have allowed individuals with motor disabilities to spell messages with their EEG signals. Although providing full composition flexibility, they enable communication rates of only a few characters per minute. Utterance-based communication systems have been developed for individuals with disability and have greatly increased communication speeds, but have yet to be applied to BCIs. This paper proposes an utterance-based communication system using the P300-BCI in which words are organized in a network structure that facilitates rapid retrieval. In trials with able-bodied participants, the proposed system achieved greater message speeds, but rated lower in effectiveness than the P300 Speller. Nonetheless, subject preferences and reports of self-perceived effectiveness suggested an inclination towards the proposed word system and thus further investigation of word-based networks is warranted in brain-controlled AAC systems.

Forkhead box Class O (FOXO), one subfamily of the Forkhead box (Fox) family, which is featured by the Forkhead (FH) DNA-binding domain, includes four human transcription factors: FOXO1, FOXO3a, FOXO4, and FOXO6. The tumor suppressor FOXO3a is involved in multiple physiological and pathological processes, such as breast cancer and acute myeloid leukemia, and is related to human longevity. It plays essential role in metabolism, cell cycle arrest, DNA repair, and apoptosis. Besides the FH domain, FOXO3a contains three other regions (CR1-3), conserved within FOXO subfamily. It specifically binds a consensus Forkhead response element (FRE) DNA sequence through the FH domain, and recruits transcriptional coactivator CBP/p300 to activate gene transcription. FOXO3a functions through interacting with other proteins as well. FOXO3a binds p53 through the FH domain and the CR3 region, which are also engaged in an intramolecular interaction, and the solution structure of the former one was determined. This intramolecular interaction regulates coactivator recruitment and is disrupted by FRE DNA. A novel transactivation domain (TAD) CR2C was identified in addition to the known TAD CR3, both of which promiscuously associate with the KIX domain of CBP/p300 in equilibrium between two conformational states, the structures of which were determined by NMR spectroscopy. These two TADs of FOXO3a form additional multivalent binding to the TAZ1 and TAZ2 domains of CBP/p300, further increasing the promiscuity and complexity of the interaction. The coactivator recruitment is modulated by AMPK phosphorylation, which enhances the multivalent interaction between FOXO3a and CBP/p300, and thus the transactivation. These results indicate the significance of intrinsically disordered regions (IDRs) of FOXO3a in transcriptional activation and protein interaction, provide insight of the role of FOXO3a in gene transcription and apoptosis under various conditions, and potentially contribute to the cancer therapy.

<p>Regulation of cell growth is controlled by a variety of factors, including a number of oncogenes and tumor suppressors. PTEN is an inositol phosphatase that regulates cell growth by hydrolyzing the phospholipid products of PI3K. PTEN is mutated in a number of cancers, leading to its characterization as an important tumor suppressor. Recent data indicate that PTEN may also perform important functions that are independent of its phosphatase activity, most notably within the nucleus. Studies in this thesis addressed a novel role for PTEN in the regulation of the cellular response to hypoxia.</p> <p>PTEN overexpression significantly increased hypoxic gene expression independent of its catalytic activity, while shRNA-mediated silencing of PTEN significantly inhibited hypoxia-mediated HRE-luciferase activity. Nuclear-localized PTEN was more effective in promoting HRE activity than nuclear-excluded PTEN. These results suggested a scaffolding function of PTEN in the hypoxic nucleus. To identify specific gene targets regulated by PTEN in hypoxia, a custom oligo-array consisting of 46 hypoxia-responsive genes was utilized following both gain- and loss-of- PTEN function. Based on real-time quantitative results, PTEN positively regulated genes involved in metabolism (PFKFB3, PFKFB4, ALDOA, PGK-1), oxygen supply (VEGFA, EPO), cell growth (Tgf-a, TERT, cyclin D1, BNIP3), motility (E-cadherin) and transcription (DEC2). A single missense mutation at isoleucine 224 (I224M) of PTEN, however, abrogated the ability of PTEN to regulate the hypoxia response without affecting its lipid phosphatase activity. PTEN has previously been shown to bind to the co-activator p300 and to affect p53 acetylation and stabilization. As p300 is also a co-activator for the HIF proteins, we hypothesized that PTEN's association with p300 would promote the HIF/p300 complex to positively regulate hypoxic gene transcription. Overexpression of PTEN-WT extended the half-life of p300 and histone acetyltransferase activity of p300 in hypoxia, while overexpression of PTEN-I224M or PTEN silencing decreased both. In vivo, these effects resulted in a significant increase in hypoxic area in PTEN-null tumors compared to tumors expressing endogenous levels of PTEN, suggesting an inability to mount a hypoxia response necessary for revascularization of the tissue. PTEN's effect on p300 extended to other functions of p300 outside of the hypoxia response, most notably p300's role in p53 stability and p53-mediated gene transcription. Overexpression of PTEN resulted in an increase in p53 reporter activity following DNA damage (mitomycin C treatment). PTEN silencing or overexpression of PTEN-I224M resulted in abrogation of these effects. Taken together, these findings demonstrate that PTEN is required for the hypoxia response and they suggest that PTEN acts as a scaffold for p300 and the HIF machinery in the hypoxic nucleus independent of its canonical lipid phosphatase activity. These results may have important implications for the treatment of tumors in which PTEN is lost or mutated. The potential use of PTEN-I224M as a therapeutic is also discussed</p> / Dissertation

The current study investigated the cognitive organization of alcohol expectancies using event-related potentials (ERPs). Building on previous behavioral and ERP paradigms, the goal of the current study was to quantify the relationship among alcohol expectancies using ERP indices of salience, congruence, and cognitive distance. The ERP components being evaluated fit perfectly into the alcohol expectancy theory and research; however, implementing specific paradigms to reliably measure individual differences in alcohol expectancies using ERPs has proven to be more elusive than originally thought. This study utilized established cognitive modeling techniques coupled with ERP responses to linguistic stimuli. In essence, this study provides an implicit measure of how particular types of words, in the context of alcohol, are categorized and integrated into individuals' expectancy frameworks. The study looked at two specific ERP components, the P300 and the N400, that have been shown to be sensitive to expectancy violations. In a sentence processing task the P300 was predicted to be related to individuals' alcohol expectancies and in a word pair task the N400 was predicted to index these expectancies. Results indicated that the P300 and N400 were both related to alcohol expectancies in the sentence task and the N400 was related to alcohol expectancies in the word pair task. While the results supported parts of the hypotheses, they were not unequivocal endorsements of the hypothesized relationships, perhaps highlighting the countervailing forces of salience and expectancy congruence. Furthermore, there were unexpected differences between males and females in the sample that interacted with the effect of expectancy on ERPs. In sum, prior research has highlighted individuals' expectations about alcohol as a mediator of biopsychosocial risk for alcohol use disorders (Goldman, 2002), and the results of this study provide a model for how ERP measures of expectancy could capture an aspect of individuals' risk based on reactions to expectancy related stimuli

The aim of this study was to investigate how young adults who stutterer allocate attentional resources during two linguistic stages in picture naming, specifically lemma and lexeme retrieval. This study reports on behavioral and brain electrophysiological data collected during a simple auditory oddball task and a Dual Picture-Word Interference/Tone Monitoring Task.