Intratumoral heterogeneity of esophageal squamous cell carcinoma and its clinical significance.

Recent studies have shown that intratumoral heterogeneity is prevalent in esophageal squamous cell cancer (ESCC) based on DNA sequencing and chromosome analysis in multiple regions from the same tumor. This study aimed to investigate the expression of ZNF750, EP300, MTOR and KMT2D and their intratumoral heterogeneity (ITH) in patients with ESCC. A total of 106 cases, who underwent esophagectomy from 2008 to 2010, with two foci from each case, were tested by immunohistochemistry(IHC) as well as 12 cases were tested by RNAscope in this study.We found that 58/106 (54.72%), 66/106 (62.26%), 75/106 (70.75%%) of ESCC showed high expression of ZNF750, EP300, MTOR, respectively by IHC, and 8/12 (66.67%), 10/12 (83.33%), 4/12 (33.33%) and 6/12 (50%) showed high expression of ZNF750, EP300, MTOR and KMT2D, respectively by RNAscope. Multivariate analysis showed that MTOR expression was an independent infavorable prognostic factor of overall survival (OS) (HR = 1.921; P = 0.000). This study also found that 44/106(4151%), 37/106 (34.91%), 39/106(36.79%) of ESCC showed heterogeneous expression of ZNF750, EP300 and MTOR respectively by IHC, 8/12(66.67%), 8/12(66.67%), 4/12(33.33%), 4/12(33.33%) of ZNF750, EP300, MTOR and KMT2D respectively by RNAscope, IHC and RNAscope could successfully detect a high prevalence of ITH. In conclusion, findings of this study showed that ZNF750, EP300, MTOR and KMT2D heterogeneously expressed in ESCC. High expression of ZNF750 related to a better outcome, while EP300 and MTOR related to a poor prognosis.

Subcellular localization of ß-arrestin1 and its prognostic value in lung adenocarcinoma.

ß-Arrestins play important roles in cancer progression, and the subcellular localization of ß-arrestin1 has been receiving increasingly more attention. Intriguingly, several studies, including some of our previous work, showed that the effects of ß-arrestin1 on outcomes of cancer patients were controversial.Specimens were obtained from 133 patients with lung adenocarcinoma. Immunohistochemistry was used to detect the expression of ß-arrestin1 and p300 in the collected tissues. The Kaplan-Meier analysis and Cox proportional hazards regression were used to examine the relationship between ß-arrestin1 and patient survival.We found no significant association between ß-arrestin1 and clinicopathological variables. The Kaplan-Meier plot showed that patients with high expression of ß-arrestin1 (especially in the nucleus) had a poorer overall survival (OS) and shorter disease-free survival (DFS) (P = .026, P = .015). Additionally, high p300 expression also resulted in worse OS (P = .039). Following the univariate analysis, high expressions of nuclear ß-arrestin1 and p300 were classed as poor prognostic factors for both OS (P = .016) and DFS (P = .025).The expression of ß-arrestin1 in the nucleus is associated with increased malignant tendency of lung adenocarcinoma, and the predictive value of ß-arrestin1 may be optimized by combining information about the expression of p300 acetyltransferase.

Octreotide inhibits the proliferation of gastric cancer cells through P300-HAT activity and the interaction of ZAC and P300.

Somatostatin (SST) exhibits a wide range of physiological functions, including the regulation of tumor cell growth. Octreotide (OCT) is a synthetic analogue of SST that can be used to slow gastrointestinal bleeding, inhibit the release of growth hormone and impede gastrointestinal tumor growth. The aim of the present study was to investigate the molecular mechanism of OCT underlying the inhibition of gastric cancer cell proliferation. Proteins of interest were detected using western blotting, and the zinc finger protein (ZAC)-P300 complex was quantified using co-immunoprecipitation. P300-histone acetyltransferase (P300-HAT) activity was determined spectrophotometrically. The results showed that OCT decreased the phosphorylation of Akt which caused the level of ZAC to increase. In turn, the interaction between ZAC and P300 increased the activity of P300-HAT; ultimately, the phosphorylation of serine 10 in histone H3 (pS10-H3) was decreased and the acetylation of lysine 14 in histone H3 (acK14-H3) was increased. These results suggest that OCT attenuates SGC-7901 cell proliferation by enhancing P300-HAT activity through the interaction of ZAC and P300, causing a reduction in pS10-H3 and an increase in acK14-H3. These findings provide insight for future research on OCT and further demonstrate the potential of OCT to be used as a therapeutic agent for gastric cancer.

Expression of p300 and p300/CBP associated factor (PCAF) in actinic keratosis and squamous cell carcinoma of the skin.

p300 and p300/CBP-associated factor (PCAF) are histone modifiers and transcriptional co-factors involved in a number of cell processes. We investigated their expression patterns in 79 actinic keratoses (AK), 45 cases of Bowen's disease (BD), and 168 invasive squamous cell carcinomas of the skin (SCC). Using tissue microarray and immunohistochemistry, we evaluated p300 and PCAF expression in relation to the type of the lesion and SCC prognostic parameters (grade, diameter, thickness and level of invasion). High nuclear expression of p300 (>60% of positive cells) (p=0.001) and absent cytoplasmic expression (p=0.026) were more frequent in SCC compared to AK and BD, respectively. Cytoplasmic expression of p300 was associated with the SCC invasion of subcutaneous fat and deeper tissues (p=0.049). Diffuse distribution of cells with p300 nuclear expression was more commonly seen in BD and SCC compared to AK (p<0.001), in moderately- and poorly-differentiated SCC compared to well-differentiated SCC (p<0.001), in tumors thicker than 6mm (p<0.001), and in deeply invading tumors (p=0.001). More frequent loss of PCAF nuclear expression was observed in SCC than in AK and BD (p<0.001). Diffuse distribution of cells with PCAF cytoplasmic expression was more common in BD and SCC compared to AK (p<0.001), and in poorly-differentiated SCC compared to well- and moderately-differentiated SCC (p<0.001). Our results suggest that increase in nuclear expression of p300, as well as the presence of cytoplasmic but loss of nuclear expression of PCAF, could play an important role in the development and progression of cutaneous SCC.

Cooperation of Sox4 with ß-catenin/p300 complex in transcriptional regulation of the Slug gene during divergent sarcomatous differentiation in uterine carcinosarcoma.

BACKGROUND: Uterine carcinosarcoma (UCS) represents a true example of cancer associated with epithelial-mesenchymal transition (EMT), which exhibits cancer stem cell (CSC)-like traits. Both Sox and ß-catenin signal transductions play key roles in the regulation of EMT/CSC properties, but little is known about their involvement in UCS tumorigenesis. Herein, we focused on the functional roles of the Sox/ß-catenin pathway in UCSs. METHODS: EMT/CSC tests and transfection experiments were carried out using three endometrial carcinoma (Em Ca) cell lines. Immunohistochemical investigation was also applied for a total of 32 UCSs. RESULTS: Em Ca cells cultured in STK2, a serum-free medium for mesenchymal stem cells, underwent changes in morphology toward an EMT appearance through downregulation of E-cadherin, along with upregulation of Slug, known as a target gene of ß-catenin. The cells also showed CSC properties with an increase in the aldehyde dehydrogenase (ALDH) 1(high) activity population and spheroid formation, as well as upregulation of Sox4, Sox7, and Sox9. Of these Sox factors, overexpression of Sox4 dramatically led to transactivation of the Slug promoter, and the effects were further enhanced by cotransfection of Sox7 or Sox9. Sox4 was also able to promote ß-catenin-mediated transcription of the Slug gene through formation of transcriptional complexes with ß-catenin and p300, independent of TCF4 status. In clinical samples, both nuclear ß-catenin and Slug scores were significantly higher in the sarcomatous elements as compared to carcinomatous components in UCSs, and were positively correlated with Sox4, Sox7, and Sox9 scores. CONCLUSIONS: These findings suggested that Sox4, as well as Sox7 and Sox9, may contribute to regulation of EMT/CSC properties to promote development of sarcomatous components in UCSs through transcriptional regulation of the Slug gene by cooperating with the ß-catenin/p300 signal pathway.

The epigenetic regulators CBP and p300 facilitate leukemogenesis and represent therapeutic targets in acute myeloid leukemia.

Growing evidence links abnormal epigenetic control to the development of hematological malignancies. Accordingly, inhibition of epigenetic regulators is emerging as a promising therapeutic strategy. The acetylation status of lysine residues in histone tails is one of a number of epigenetic post-translational modifications that alter DNA-templated processes, such as transcription, to facilitate malignant transformation. Although histone deacetylases are already being clinically targeted, the role of histone lysine acetyltransferases (KAT) in malignancy is less well characterized. We chose to study this question in the context of acute myeloid leukemia (AML), where, using in vitro and in vivo genetic ablation and knockdown experiments in murine models, we demonstrate a role for the epigenetic regulators CBP and p300 in the induction and maintenance of AML. Furthermore, using selective small molecule inhibitors of their lysine acetyltransferase activity, we validate CBP/p300 as therapeutic targets in vitro across a wide range of human AML subtypes. We proceed to show that growth retardation occurs through the induction of transcriptional changes that induce apoptosis and cell-cycle arrest in leukemia cells and finally demonstrate the efficacy of the KAT inhibitors in decreasing clonogenic growth of primary AML patient samples. Taken together, these data suggest that CBP/p300 are promising therapeutic targets across multiple subtypes in AML.

MiR-106b~25 cluster regulates multidrug resistance in an ABC transporter-independent manner via downregulation of EP300.

MicroRNA (miR)-106b~25 cluster regulates bypass of doxorubicin and γ-radiation induced senescence by downregulation of the E-cadherin transcriptional activator EP300. We asked whether upregulation of miR-106~25 cluster generates cells with a truly multidrug resistant (MDR) phenotype and whether this is due to upregulation of the ATP-binding cassette (ABC) transporter P-glycoprotein. We used minimally transformed mammary epithelial breast cancer cells (MTMECs) in which the miR-106b~25 cluster was experimentally upregulated by lentiviral transfection or in which hairpins targeting either EP300 or E-cadherin mRNAs have been expressed with lentiviruses. We find that overexpression of miR-106b~25 cluster led to the generation of MDR MTMECs (resistant to etoposide, colchicine and paclitaxel). Paclitaxel resistance was also studied after experimental downregulation of EP300 or E-cadherin. However none of these cells overexpressed P-glycoprotein or where able to efflux a fluorescent derivative of paclitaxel, making this phenotype drug-transporter independent. Paclitaxel treatment in MTMECs led to an increase in early apoptotic cells (Annexin V-positive), activation of caspase-9 and increase in the proportion of cells at the G2/M phase of the cell cycle. However, MTMEC overexpressing miR-106b~25 cluster, or with EP300 or E-cadherin downregulated, showed less activation of apoptosis, caspase-9 and caspase-3/-7 activities. Thus, miR-106b~25 cluster controls transporter-independent MDR by apoptosis evasion via downregulation of EP300.

Poly(ADP-Ribosyl)ation Affects Histone Acetylation and Transcription.

Poly(ADP-ribosyl)ation (PARylation) is a posttranslational protein modification catalyzed by members of the poly(ADP-ribose) polymerase (PARP) enzyme family. PARylation regulates a wide variety of biological processes in most eukaryotic cells including energy metabolism and cell death, maintenance of genomic stability, chromatin structure and transcription. Inside the nucleus, cross-talk between PARylation and other epigenetic modifications, such as DNA and histone methylation, was already described. In the present work, using PJ34 or ABT888 to inhibit PARP activity or over-expressing poly(ADP-ribose) glycohydrolase (PARG), we show decrease of global histone H3 and H4 acetylation. This effect is accompanied by a reduction of the steady state mRNA level of p300, Pcaf, and Tnfα, but not of Dnmt1. Chromatin immunoprecipitation (ChIP) analyses, performed at the level of the Transcription Start Site (TSS) of these four genes, reveal that changes in histone acetylation are specific for each promoter. Finally, we demonstrate an increase of global deacetylase activity in nuclear extracts from cells treated with PJ34, whereas global acetyltransferase activity is not affected, suggesting a role for PARP in the inhibition of histone deacetylases. Taken together, these results show an important link between PARylation and histone acetylation regulated transcription.

miRNA-132-3p inhibits osteoblast differentiation by targeting Ep300 in simulated microgravity.

Recent studies have demonstrated that miRNAs can play important roles in osteoblast differentiation and bone formation. However, the function of miRNAs in bone loss induced by microgravity remains unclear. In this study, we investigated the differentially expressed miRNAs in both the femur tissues of hindlimb unloading rats and primary rat osteoblasts (prOB) exposed to simulated microgravity. Specifically, miR-132-3p was found up-regulated and negatively correlated with osteoblast differentiation. Overexpression of miR-132-3p significantly inhibited prOB differentiation, whereas inhibition of miR-132-3p function yielded an opposite effect. Furthermore, silencing of miR-132-3p expression effectively attenuated the negative effects of simulated microgravity on prOB differentiation. Further experiments confirmed that E1A binding protein p300 (Ep300), a type of histone acetyltransferase important for Runx2 activity and stability, was a direct target of miR-132-3p. Up-regulation of miR-132-3p by simulated microgravity could inhibit osteoblast differentiation in part by decreasing Ep300 protein expression, which, in turn, resulted in suppression of the activity and acetylation of Runx2, a key regulatory factor of osteoblast differentiation. Taken together, our findings are the first to demonstrate that miR-132-3p can inhibit osteoblast differentiation and participate in the regulation of bone loss induced by simulated microgravity, suggesting a potential target for counteracting decreases in bone formation.

Mechanisms of endothelial to mesenchymal transition in the retina in diabetes.

PURPOSE: Hyperglycemia-induced endothelial damage is a key pathogenetic factor in diabetic retinopathy. Endothelial damage may lead to phenotypic changes in the cells manifested by reduced expression of endothelial markers and increased expression of mesenchymal markers, termed endothelial to mesenchymal transition (EndMT). We investigated mechanisms of such changes in the retinal endothelial cells and in the retina of diabetic animals. METHODS: Human retinal microvascular endothelial cells were grown in medium containing 5 mM glucose or 25 mM glucose with or without TGFß1 peptide or TGFß1 inhibitor or miR-200b mimic transfection. Messenger RNA levels of endothelial markers, mesenchymal markers, and specific signaling molecules of TGFß pathway were quantified. Expression of miR-200b and histone acetylator p300 was quantified. Retinal tissues from mice with endothelial-specific overexpression of miR-200b, with or without streptozotocin-induced diabetes, were similarly examined. RESULTS: Glucose caused decreased expression of mRNA and protein levels of endothelial markers and increased expression of mesenchymal markers with reduced miR-200b. A glucose-like effect was seen using TGFß1 peptide. Such changes were mediated by miR-200b and p300. In the retinas of wild-type diabetic mice, EndMT was observed, which was prevented in miR-200b transgenic mice with diabetes. CONCLUSIONS: These data indicate glucose-induced EndMT in vitro and in vivo is possibly mediated through TGFß and regulated by miR-200b and p300.

A combination of p300 and Braf expression in the diagnosis and prognosis of melanoma.

BACKGROUND: To date only a handful of drugs are available for the treatment of melanoma. Among them vemurafenib, a BrafV600E specific inhibitor, showed promising results in terms of response rate and increase in median survival time. However, its effectiveness is limited by development of resistance and the search for additional drugs for melanoma treatment is ongoing. The present study was performed to analyze the correlation between Braf expression and the expression of p300, a known down stream target of the mitogen activated protein kinase (MAPK) pathway, which was recently shown by us to be a prognostic marker for melanoma progression and patient survival. METHODS: The expression of Braf and p300 expression were correlated and analyzed by Chi-square test. A total of 327 melanoma patient cases (193 primary melanoma and 134 metastatic melanoma) were used for the study. Classification & regression tree (CRT), Kaplan-Meier, and multivariate Cox regression analysis were used to elucidate the significance of the combination of Braf and p300 expression in the diagnosis and prognosis of melanoma. RESULTS: Our results demonstrate that Braf expression is inversely correlated with nuclear p300 and positively correlated with cytoplasmic p300 expression. Braf and cytoplasmic p300 were found to be associated with melanoma progression, tumor size and ulceration status. CRT analysis revealed that a combination of Braf and p300 expression (nuclear and cytoplasmic), could be used to distinguish between nevi and melanoma, and primary from metastatic melanoma lesions. The combination of Braf and nuclear p300 was significantly associated with patient survival and nuclear p300 was found to be an independent predictor of patient survival. CONCLUSION: Our results indicate a cross-talk between Braf and p300 in melanoma and demonstrate the importance Braf and p300 expression in the diagnosis and prognosis of melanoma.

An epigenetic mechanism underlying doxorubicin induced EMT in the human BGC-823 gastric cancer cell.

The epithelial to mesenchymal transition (EMT) is a key step during embryonic morphogenesis and plays an important role in drug resistance and metastasis in diverse solid tumors. We previously reported that 48 h treatment of anti-cancer drug doxorubicin could induce EMT in human gastric cancer BGC-823 cells. However, the long term effects of this transient drug treatment were unknown. In this study we found that after 48 h treatment with 0.1 µg/ml doxorubicin, most cells died during next week, while a minor population of cells survived and formed colonies. We propagated the surviving cells in drug free medium and found that these long term cultured drug survival cells (abbreviated as ltDSCs) retained a mesenchymal-like cell morphology, and expressed high levels of EMT-related molecules such as vimentin, twist and ß-catenin. The expression of chromatin reprogramming factors, Oct4 and c-myc, were also higher in ltDSCs than parental cells. We further demonstrated that the protein level of p300 was upregulated in ltDSCs, and inhibition of p300 by siRNA suppressed the expression of vimentin. Moreover, the ltDSCs had higher colony forming ability and were more drug resistant when compared to parental cells. Our results suggested that an epigenetic mechanism is involved in the EMT of ltDSCs.

A specific CBP/p300-dependent gene expression programme drives the metabolic remodelling in late stages of spermatogenesis.

Histone hyperacetylation is thought to drive the replacement of histones by transition proteins that occur in elongating spermatids (ElS) after a general shut down of transcription. The molecular machineries underlying this histone hyperacetylation remain still undefined. Here, we focused our attention on the role of Cbp and p300 in histone hyperacetylation and in the preceding late-gene transcriptional activity in ElS. A strategy was designed to partially deplete Cbp and p300 in ElS. These cells progressed normally through spermiogenesis and showed normal histone hyperacetylation and removal. However, a genome-wide transcriptomic analysis, performed in the round spermatids (RS) and ElS, revealed the existence of a gene regulatory circuit encompassing genes presenting high expression levels in pre-meiotic cells, undergoing a repressed state in spermatocytes and early post-meiotic cells, but becoming reactivated in ElS, just prior to the global shutdown of transcription. Interestingly, this group of genes was over-represented within the genes affected by Cbp/p300 knock down and were all involved in metabolic remodelling. This study revealed the occurrence of a tightly regulated Cbp/p300-dependent gene expression programme that drives a specific metabolic state both in progenitor spermatogenic cells and in late transcriptionally active spermatids and confirmed a special link between Cpb/p300 and cell metabolism programming previously shown in somatic cells.

Downregulation of p300 gene expression in airway mesenchyme of nitrofen-induced hypoplastic lungs.

PURPOSE: Congenital diaphragmatic hernia (CDH) is a relatively common developmental abnormality causing life-threatening respiratory distress at birth. The nitrofen model has been widely used to investigate the pathogenesis of hypoplastic lungs associated with CDH. Embryos lacking p300 and CBP genes are significantly smaller in lung formation. We hypothesized that pulmonary gene expression of p300 and CBP is downregulated during late gestation in the nitrofen-induced CDH model. METHODS: Time-pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9). Fetal lungs were harvested on D18 and D21 (n = 8 at each time point). Pulmonary gene expression of p300 and CBP was analyzed by quantitative real-time PCR. Immunohistochemistry was performed to investigate expression and localization of pulmonary p300 and CBP proteins. RESULTS: Relative mRNA expression levels of p300 were significantly decreased in nitrofen-induced hypoplastic lungs on D18 compared to controls (3.00 ± 0.20 vs. 3.76 ± 0.14; p = 0.0039), while CBP levels were not altered. p300 immunoreactivity was markedly diminished in surrounding mesenchymal compartments and nuclei of proximal and distal airway cells, while CBP expression was not altered. CONCLUSION: Downregulation of p300 gene expression during the early canalicular stage may disrupt epithelial-mesenchymal signaling interactions, contributing to the development of hypoplastic lungs in the nitrofen-induced CDH model.

High expression of p300 in HCC predicts shortened overall survival in association with enhanced epithelial mesenchymal transition of HCC cells.

P300 impacts the transcription of several genes involved in biological behavior of human malignancies including hepatocellular carcinomas (HCC). We found p300 is highly expressed in 47% of surgically resected HCC specimens by immunohistochemistry, which correlated with advanced TNM staging (P = 0.034), vascular invasion (P = 0.036), intrahepatic metastasis (P = 0.001) and shortened overall survival (P = 0.028). In vitro study, knocking down of p300 expression in hepatoma cells recovered E-cadherin expression, inhibited the translocation of beta (ß)-catenin into the nuclei, decreased cyclin D1 activity and suppressed the migration/invasion of HCC cells. Furthermore, suppression of p300 led to down-regulation of epithelial-mesenchymal transition (EMT)-related molecules such as Snail, Twist and HIF-1 alpha. These observations suggest that p300 contributes to the EMT-related progression of HCCs.

Expression of ERß and its co-regulators p300 and NCoR in human transitional cell bladder cancer.

OBJECTIVE: Several data support a possible role of estrogens in bladder carcinogenesis, mediated mainly through estrogen receptor-ß (ERß). We study the expression of ERß and its co-regulators p300 and nuclear co-repressor (NCoR) in patients with bladder cancer. PATIENTS AND METHODS: One hundred and eleven consecutive patients (74 males and 37 females), aged 23-90 years (mean 70 ± 10) diagnosed with transitional cell bladder cancer were included in this study. The control group consisted of 29 patients that underwent transurethral prostatectomy and consented to simultaneous bladder biopsies. Immunohistochemical studies took place on formalin-fixed, paraffin-embedded sections from the TUR (transurethral resection) specimens. We studied the expression of ERß, p300 and NCoR.χ(2) test was used to evaluate the relationship between the histological grade and ERß expression, grade and co-regulators expression and grade and gender. Spearman rank correlation coefficient (r) was used in order to estimate the direction and strength of correlations between histological grade and ERß-p300-NCoR expressions. The Cochran-Armitage test for trend was applied in order to examine possible trends across the ordered levels of histological grade. RESULTS: ERß was more frequently expressed in the nucleus of normal bladder epithelium compared to malignant bladder epithelium with statistical significant association (r = -0.25, p = 0.003). The p300 was expressed only in the nucleus of bladder cancer cells and a positive correlation between molecular expression and cancer progression was demonstrated (r = 0.55, p < 0.001). NCoR immunostaining was demonstrated in the nuclei of bladder cells. Nuclear staining was significantly higher in normal tissue than in cancer cells (r = -0.33, p < 0.001), with negative correlation. Furthermore, its expression in grade I tumors was significantly higher than in grade II (r = -0.46, p < 0.001) and grade III tumors (r = -0.51, p < 0.001). Thus, like ERß, NCoR expression in bladder epithelium decreased during cancer progression and loss of cell differentiation. There was no correlation between the levels of expression of the three proteins in normal bladder epithelium, but there was an inverse correlation between the nuclear expression of ERß and p300 in carcinomas (r = -3.88, p = 0.042). Statistical significant association was established when correlating ERß expression with NCoR expression (r = 0.273, p = 0.005), while co-regulators' nuclear expression did not correlate with each other (p > 0.05). CONCLUSIONS: In bladder carcinogenesis, we demonstrated inhibition in the expression of ERß and its co-repressor NCoR as well as increased expression of the co-activator p300.

Efficient in vivo manipulation of alternative pre-mRNA splicing events using antisense morpholinos in mice.

Mammalian pre-mRNA alternative splicing mechanisms are typically studied using artificial minigenes in cultured cells, conditions that may not accurately reflect the physiological context of either the pre-mRNA or the splicing machinery. Here, we describe a strategy to investigate splicing of normal endogenous full-length pre-mRNAs under physiological conditions in live mice. This approach employs antisense vivo-morpholinos (vMOs) to mask cis-regulatory sequences or to disrupt splicing factor expression, allowing functional evaluation of splicing regulation in vivo. We applied this strategy to gain mechanistic insight into alternative splicing events involving exons 2 and 16 (E2 and E16) that control the structure and function of cytoskeletal protein 4.1R. In several mouse tissues, inclusion of E16 was substantially inhibited by interfering with a splicing enhancer mechanism using a target protector morpholino that blocked Fox2-dependent splicing enhancers in intron 16 or a splice-blocking morpholino that disrupted Fox2 expression directly. For E2, alternative 3'-splice site choice is coordinated with upstream promoter use across a long 5'-intron such that E1A splices almost exclusively to the distal acceptor (E2dis). vMOs were used to test the in vivo relevance of a deep intron element previously proposed to determine use of E2dis via a two-step intrasplicing model. Two independent vMOs designed against this intronic regulatory element inhibited intrasplicing, robustly switching E1A splicing to the proximal acceptor (E2prox). This finding strongly supports the in vivo physiological relevance of intrasplicing. vMOs represent a powerful tool for alternative splicing studies in vivo and may facilitate exploration of alternative splicing networks in vivo.

Spatial memory consolidation is associated with induction of several lysine-acetyltransferase (histone acetyltransferase) expression levels and H2B/H4 acetylation-dependent transcriptional events in the rat hippocampus.

Numerous genetic studies have shown that the CREB-binding protein (CBP) is an essential component of long-term memory formation, through its histone acetyltransferase (HAT) function. E1A-binding protein p300 and p300/CBP-associated factor (PCAF) have also recently been involved in memory formation. By contrast, only a few studies have reported on acetylation modifications during memory formation, and it remains unclear as to how the system is regulated during this dynamic phase. We investigated acetylation-dependent events and the expression profiles of these HATs during a hippocampus-dependent task taxing spatial reference memory in the Morris water maze. We found a specific increase in H2B and H4 acetylation in the rat dorsal hippocampus, while spatial memory was being consolidated. This increase correlated with the degree of specific acetylated histones enrichment on some memory/plasticity-related gene promoters. Overall, a global increase in HAT activity was measured during this memory consolidation phase, together with a global increase of CBP, p300, and PCAF expression. Interestingly, these regulations were altered in a model of hippocampal denervation disrupting spatial memory consolidation, making it impossible for the hippocampus to recruit the CBP pathway (CBP regulation and acetylated-H2B-dependent transcription). CBP has long been thought to be present in limited concentrations in the cells. These results show, for the first time, that CBP, p300, and PCAF are dynamically modulated during the establishment of a spatial memory and are likely to contribute to the induction of a specific epigenetic tagging of the genome for hippocampus-dependent (spatial) memory consolidation. These findings suggest the use of HAT-activating molecules in new therapeutic strategies of pathological aging, Alzheimer's disease, and other neurodegenerative disorders.

p300 expression is related to high-risk human papillomavirus infections and severity of cervical intraepithelial neoplasia but not to viral or disease outcomes in a longitudinal setting.

To evaluate the role of the expression of the transcription factor p300 as an independent predictor of high-risk human papillomavirus (HR-HPV) infections and outcome of the cervical disease.Cervical biopsy samples taken at enrolment from 225 women of the Latin American Screening study cohort were analyzed for p300 using immunohistochemistry to assess its value as predictor of (a) cervical intraepithelial neoplasia (CIN) grade, and (b) HR-HPV at baseline, as well as (c) outcomes of HR-HPV infections, and (d) development of incident CIN as surrogate endpoints of progressive disease.There was a significant linear trend in increasing upregulation (=pattern shift) of p300 (P=0.0001) in parallel with increasing grade of CIN. When dichotomized (normal/moderately increase vs. strong-intense), upregulated p300 expression predicted CIN3+ with odds ratio=4.16 (95% confidence interval: 1.95-8.86) (P=0.0001) and CIN2+ with odds ratio=3.48 (95% confidence interval: 1.86-6.48) (P=0.0001). p300 was upregulated more often in HR-HPV+ lesions than in those remaining negative. Semiquantitative viral loads were also directly related to upregulation of p300 (P=0.036), but p300 was not a significant predictor of disease progression to either CIN1+ or CIN2+.p300 expression was upregulated in CIN lesions and related to detection and viral load of HR-HPV but not to their outcome or to incident CIN.

Myocardial regulation of p300 and p53 by doxorubicin involves ubiquitin pathways.

BACKGROUND: Doxorubicin (Dox) depletes p300 from cardiac myocytes and induces apoptosis of these cells. p300 protein possesses ubiquitin ligase activity for the p53 tumor suppressor gene product, catalyzes p53 polyubiqutination, and facilitates p53 degradation in an ubiquitin-dependent manner. The present study investigated the ubiquitin-dependent regulation of p53 by Dox and p300 in cardiac myocytes. METHODS AND RESULTS: Primary cardiac myocytes from neonatal rats were exposed to a proteasome inhibitor, MG132, in culture. MG132 increased both p300 and p53 protein levels in these cells, suggesting that ubiquitin-dependent degradation is involved in the homeostasis of these proteins. Notably, treatment of cardiac myocytes with Dox decreased the protein levels of p300 but markedly increased those of p53. By immunoprecipitation-Western blotting, it was shown that treatment with Dox decreased poly-ubiquitinated p53 but increased that of p300 in cardiac myocytes. Finally, the overexpression of p300 in cardiomyocytes suppressed the Dox-mediated increase in the p53 level in addition to inhibiting Dox-induced apoptosis. CONCLUSION: Dox reciprocally regulates p300 and p53 through ubiquitin-dependent pathways and that p300, by its ubiquitin ligase activity, is partially involved in the ubiquitin-dependent degradation of p53 in cardiac myocytes.