Transcription factor FOXA2-centered transcriptional regulation network in non-small cell lung cancer.

Lung cancer is the leading cause of cancer-mediated death. Although various therapeutic approaches are used for lung cancer treatment, these mainly target the tumor suppressor p53 transcription factor, which is involved in apoptosis and cell cycle arrest. However, p53-targeted therapies have limited application in lung cancer, since p53 is found to be mutated in more than half of lung cancers. In this study, we propose tumor suppressor FOXA2 as an alternative target protein for therapies against lung cancer and reveal a possible FOXA2-centered transcriptional regulation network by identifying new target genes and binding partners of FOXA2 by using various screening techniques. The genes encoding Glu/Asp-rich carboxy-terminal domain 2 (CITED2), nuclear receptor subfamily 0, group B, member 2 (NR0B2), cell adhesion molecule 1 (CADM1) and BCL2-associated X protein (BAX) were identified as putative target genes of FOXA2. Additionally, the proteins including highly similar to heat shock protein HSP 90-beta (HSP90A), heat shock 70 kDa protein 1A variant (HSPA1A), histone deacetylase 1 (HDAC1) and HDAC3 were identified as novel interacting partners of FOXA2. Moreover, we showed that FOXA2-dependent promoter activation of BAX and p21 genes is significantly reduced via physical interactions between the identified binding partners and FOXA2. These results provide opportunities to understand the FOXA2-centered transcriptional regulation network and novel therapeutic targets to modulate this network in p53-deficient lung cancer.

The chromodomain-containing histone acetyltransferase TIP60 acts as a code reader, recognizing the epigenetic codes for initiating transcription.

TIP60 can act as a transcriptional activator or a repressor depending on the cellular context. However, little is known about the role of the chromodomain in the functional regulation of TIP60. In this study, we found that TIP60 interacted with H3K4me3 in response to TNF-α signaling. TIP60 bound to H3K4me3 at the promoters of the NF-κB target genes IL6 and IL8. Unlike the wild-type protein, a TIP60 chromodomain mutant did not localize to chromatin regions. Because TIP60 binds to histones with specific modifications and transcriptional regulators, we used a histone peptide assay to identify histone codes recognized by TIP60. TIP60 preferentially interacted with methylated or acetylated histone H3 and H4 peptides. Phosphorylation near a lysine residue significantly reduced the affinity of TIP60 for the modified histone peptides. Our findings suggest that TIP60 acts as a functional link between the histone code and transcriptional regulators.

The expression of p21 is upregulated by forkhead box A1/2 in p53-null H1299 cells.

The expression of the cell cycle inhibitor p21 is increased in response to various stimuli and stress signals through p53-dependent and independent pathways. We demonstrate in this study that forkhead box A1/2 (FOXA1/2) is a crucial transcription factor in the activation of p21 transcription via direct binding to the p21 promoter in p53-null H1299 lung carcinoma cells. In addition, histone deacetylase inhibitor trichostatin A (TSA)-mediated upregulation of p21 expression was repressed by knockdown of FOXA1/2 in H1299 cells. Consequently, these results suggest that FOXA1/2 is required for p53-independent p21 expression.

Drought stress-induced compositional changes in tolerant transgenic rice and its wild type.

Comparing well-watered versus deficit conditions, we evaluated the chemical composition of grains harvested from wild-type (WT) and drought-tolerant, transgenic rice (Oryza sativa L.). The latter had been developed by inserting AtCYP78A7, which encodes a cytochrome P450 protein. Two transgenic Lines, '10B-5' and '18A-4', and the 'Hwayoung' WT were grown under a rainout shelter. After the harvested grains were polished, their levels of key components, including proximates, amino acids, fatty acids, minerals and vitamins were analysed to determine the effect of watering system and genotype. Drought treatment significantly influenced the levels of some nutritional components in both transgenic and WT grains. In particular, the amounts of lignoceric acid and copper in the WT decreased by 12.6% and 39.5%, respectively, by drought stress, whereas those of copper and potassium in the transgenics rose by 88.1-113.3% and 10.4-11.9%, respectively, under water-deficit conditions.

Sox4-mediated caldesmon expression facilitates differentiation of skeletal myoblasts.

Caldesmon (CaD), which was originally identified as an actin-regulatory protein, is involved in the regulation of diverse actin-related signaling processes, including cell migration and proliferation, in various cells. The cellular function of CaD has been studied primarily in the smooth muscle system; nothing is known about its function in skeletal muscle differentiation. In this study, we found that the expression of CaD gradually increased as differentiation of C2C12 myoblasts progressed. Silencing of CaD inhibited cell spreading and migration, resulting in a decrease in myoblast differentiation. Promoter analysis of the caldesmon gene (Cald1) and gel mobility shift assays identified Sox4 as a major trans-acting factor for the regulation of Cald1 expression during myoblast differentiation. Silencing of Sox4 decreased not only CaD protein synthesis but also myoblast fusion in C2C12 cells and myofibril formation in mouse embryonic muscle. Overexpression of CaD in Sox4-silenced C2C12 cells rescued the differentiation process. These results clearly demonstrate that CaD, regulated by Sox4 transcriptional activity, contributes to skeletal muscle differentiation.

Transcription factor Sox4 is required for PUMA-mediated apoptosis induced by histone deacetylase inhibitor, TSA.

PUMA is a crucial regulator of apoptotic cell death mediated by p53-dependent and p53-independent mechanisms. In many cancer cells, PUMA expression is induced in response to DNA-damaging reagent in a p53-dependent manner. However, few studies have investigated transcription factors that lead to the induction of PUMA expression via p53-independent apoptotic signaling. In this study, we found that the transcription factor Sox4 increased PUMA expression in response to trichostatin A (TSA), a histone deacetylase inhibitor in the p53-null human lung cancer cell line H1299. Ectopic expression of Sox4 led to the induction of PUMA expression at the mRNA and protein levels, and TSA-mediated up-regulation of PUMA transcription was repressed by the knockdown of Sox4. Using luciferase assays and chromatin immunoprecipitation, we also determined that Sox4 recruits p300 on the PUMA promoter region and increases PUMA gene expression in response to TSA treatment. Taken together, these results suggest that Sox4 is required for p53-independent apoptotic cell death mediated by PUMA induction via TSA treatment.

Transcriptional activity of paired homeobox Pax6 is enhanced by histone acetyltransferase Tip60 during mouse retina development.

Pax6 is a member of the Pax family of transcription factors that contains a DNA binding paired-box and homeobox domain. In animals, including humans, Pax6 plays a key role in development, regulating organogenesis of the eye and brain. The current data show that histone acetyltransferase Tip60 physically interacts with Pax6 in developing post-natal day 4 (P4) mouse retinas. We also found that Tip60 binds with paired-domain of Pax6 using its chromo- and zinc-finger-containing regions, and that these protein interactions were needed for the effective full-transcriptional activation of Pax6. Furthermore, among the combinations of Pax6-target gene interactions using its two DNA binding domain, paired- and homeobox domain, Tip60 significantly enhanced the transcriptional activity of Pax6 on the paired-domain binding sequence (P6CON) containing reporter construct (pCON) than other homeo domain and chimera binding containing pP3 and pCON/P3 constructs. Taken together, these results suggest that Tip60 binds with Pax6 and that this physical interaction leads to the full-transcriptional activation of Pax6 during retina development.

Transdermal drug delivery using disk microneedle rollers in a hairless rat model.

BACKGROUND: Transdermal drug delivery systems (TDDSs) represent more reliable and consistent methods of drug dosing than oral administration. However, TDDSs can administer only low molecular weight (MW) drugs and require a power source. Disk microneedle rollers facilitate the passage of low and high MW substances through the direct perforation of the stratum corneum and dermis, without stimulating dermal nerves. OBJECTIVES: We investigated in vitro whether disk microneedle rollers, developed for the Diskneedle Therapy System (DTS™) in South Korea, can deliver drugs effectively through the skin of hairless rats. METHODS: The disk microneedle rollers used in the DTS™ are metal and consist of several plates bearing microneedles of graded lengths (0.15 mm, 0.25 mm, 0.50 mm). To test in vitro permeation, the skin of a hairless rat was mounted in a Franz diffusion cell system and rolled with a disk roller without microneedles and with rollers fitted with microneedles of each size. Rhodamine B base (80 µl) was applied to the skin for 24 hours, 48 hours, and 72 hours, and dye permeation was detected at 543 nm. Dye binding to the skin was also confirmed using fluorescence microscopy at six hours after the application of rhodamine B. RESULTS: Use of the disk microneedle roller increased the skin penetrance of rhodamine B base in hairless rats in accordance with microneedle length, as assessed using a fluorescence penetration test. CONCLUSIONS: Disk microneedle rollers, as designed for the DTS™, can be used for transdermal drug delivery. Microneedles can be selected according to the length appropriate for each application.

Transcriptional activity of neural retina leucine zipper (Nrl) is regulated by c-Jun N-terminal kinase and Tip60 during retina development.

Neural retina leucine zipper (Nrl), a key basic motif leucine zipper (bZIP) transcription factor, modulates rod photoreceptor differentiation by activating rod-specific target genes. In searching for factors that might couple with Nrl to modulate its transcriptional activity through posttranslational modification, we observed the novel interactions of Nrl with c-Jun N-terminal kinase 1 (JNK1) and HIV Tat-interacting protein 60 (Tip60). JNK1 directly interacted with and phosphorylated Nrl at serine 50, which enhanced Nrl transcriptional activity on the rhodopsin and Ppp2r5c promoters. Use of an inactive JNK1 mutant or treatment with a JNK inhibitor (SP600125) significantly reduced JNK1-mediated phosphorylation and transcriptional activity of Nrl in cultured retinal explants. We also found that Nrl activated rhodopsin and Ppp2r5c transcription by recruiting Tip60 to promote histone H3/H4 acetylation. The binding affinity of phospho-Nrl for Tip60 was significantly greater than that of the unphosphorylated Nrl. Thus, the histone acetyltransferase-containing Tip60 behaved as a coactivator in the Nrl-dependent transcriptional regulation of the rhodopsin and Ppp2r5c genes in the developing mouse retina. A transcriptional network of interactive proteins, including Nrl, JNK1, and Tip60, may be required to precisely control spatiotemporal photoreceptor-specific gene expression during retinal development.

New molecular bridge between RelA/p65 and NF-κB target genes via histone acetyltransferase TIP60 cofactor.

The nuclear factor-κB (NF-κB) family is involved in the expressions of numerous genes, in development, apoptosis, inflammatory responses, and oncogenesis. In this study we identified four NF-κB target genes that are modulated by TIP60. We also found that TIP60 interacts with the NF-κB RelA/p65 subunit and increases its transcriptional activity through protein-protein interaction. Although TIP60 binds with RelA/p65 using its histone acetyltransferase domain, TIP60 does not directly acetylate RelA/p65. However, TIP60 maintained acetylated Lys-310 RelA/p65 levels in the TNF-α-dependent NF-κB signaling pathway. In chromatin immunoprecipitation assay, TIP60 was primarily recruited to the IL-6, IL-8, C-IAP1, and XIAP promoters in TNF-α stimulation followed by acetylation of histones H3 and H4. Chromatin remodeling by TIP60 involved the sequential recruitment of acetyl-Lys-310 RelA/p65 to its target gene promoters. Furthermore, we showed that up-regulated TIP60 expression was correlated with acetyl-Lys-310 RelA/p65 expressions in hepatocarcinoma tissues. Taken together these results suggest that TIP60 is involved in the NF-κB pathway through protein interaction with RelA/p65 and that it modulates the transcriptional activity of RelA/p65 in NF-κB-dependent gene expression.

Tip60 regulates myoblast differentiation by enhancing the transcriptional activity of MyoD via their physical interactions.

The progression of muscle differentiation is tightly controlled by multiple groups of transcription factors and transcriptional coregulators. MyoD is a transcription factor of the myogenic basic helix-loop-helix family required for the process of muscle cell differentiation. We now show that Tip60 is required for myoblast differentiation via enhancement of the transcriptional activity of MyoD. Knockdown of Tip60 in C2C12 cells leads to a lack of ability to switch from proliferating myoblasts to differentiated myotubes. Ectopic expression of Tip60 increased MyoD-mediated luciferase activity on the myogenic regulatory gene, myogenin. We also found that Tip60 physically interacts with MyoD using its chromo- and Zn-finger-containing region, and that these protein interactions were required for the effective transcriptional activation of MyoD. Furthermore, a chromatin immunoprecipitation assay revealed that Tip60 recruits MyoD on the myogenin promoter, and Tip60 also increases the levels of acetylated histones H3 and H4 during myogenic differentiation. Taken together, these findings suggest that Tip60 is an important co-activator for MyoD-mediated myogenesis in mouse myoblast C2C12 cells.

Gelsolin negatively regulates the activity of tumor suppressor p53 through their physical interaction in hepatocarcinoma HepG2 cells.

As a transcription factor, p53 modulates several cellular responses including cell-cycle control, apoptosis, and differentiation. In this study, we have shown that an actin regulatory protein, gelsolin (GSN), can physically interact with p53. The nuclear localization of p53 is inhibited by GSN overexpression in hepatocarcinoma HepG2 cells. Additionally, we demonstrate that GSN negatively regulates p53-dependent transcriptional activity of a reporter construct, driven by the p21-promoter. Furthermore, p53-mediated apoptosis was repressed in GSN-transfected HepG2 cells. Taken together, these results suggest that GSN binds to p53 and this interaction leads to the inhibition of p53-induced apoptosis by anchoring of p53 in the cytoplasm in HepG2 cells.

Neural retina leucine-zipper regulates the expression of Ppp2r5c, the regulatory subunit of protein phosphatase 2A, in photoreceptor development.

Protein phosphatase 2A plays an important role in balancing phosphorylation signals that are critical for cell proliferation and differentiation. Here, we report that Ppp2r5c (regulatory subunit of protein phosphatase 2A) expression was regulated by the transcription factor neural retina leucine-zipper (Nrl) through enhancement of its transcriptional activity on the Ppp2r5c promoter. Using electrophoretic mobility shift assays and chromatin immunoprecipitation, we also found that Nrl bound directly to the Nrl-response element on the Ppp2r5c promoter. The affinity of binding of Nrl to the Ppp2r5c promoter was tightly regulated during mouse photoreceptor development. Overall, these results suggest that Ppp2r5c expression is regulated by Nrl during retinogenesis through direct binding to the promoter region of Ppp2r5c.

Control of transferrin expression by ß-amyloid through the CP2 transcription factor.

Accumulation of ß-amyloid protein (Aß) is one of the most important pathological features of Alzheimer's disease. Although Aß induces neurodegeneration in the cortex and hippocampus through several molecular mechanisms, few studies have evaluated the modulation of transcription factors during Aß-induced neurotoxicity. Therefore, in this study, we investigated the transcriptional activity of transcription factor CP2 in neuronal damage mediated by Aß (Aß(1-42) and Aß(25-35) ). An unbiased motif search of the transferrin promoter region showed that CP2 binds to the transferrin promoter, an iron-regulating protein, and regulates transferrin transcription. Ectopic expression of CP2 led to increased transferrin expression at both the mRNA and protein levels, whereas knockdown of CP2 down-regulated transferrin mRNA and protein expression. Moreover, CP2 trans-activated transcription of a transferrin reporter gene. An electrophoretic mobility shift assay and a chromatin immunoprecipitation assay showed that CP2 binds to the transferrin promoter region. Furthermore, the binding affinity of CP2 to the transferrin promoter was regulated by Aß, as Aß (Aß(1-42) and Aß(25-35) ) markedly increased the binding affinity of CP2 for the transferrin promoter. Taken together, these results suggest that CP2 contributes to the pathogenesis of Alzheimer's disease by inducing transferrin expression via up-regulating its transcription.

Microtubule-associated protein 1B light chain (MAP1B-LC1) negatively regulates the activity of tumor suppressor p53 in neuroblastoma cells.

The tumor suppressor and transcription factor p53 is a key modulator of cellular stress responses and can trigger apoptosis in many cell types, including neurons. In this study, we have shown that the Microtubule-Associated Protein 1B (MAP1B) light chain can interact with the tumor suppressor p53. We also demonstrate that both p53 and the MAP1B light chain (MAP1B-LC1) alter their localization from the cytoplasm to the nucleus when neuroblastoma cells, SH-SY5Y, are treated with doxorubicin. Additionally, we demonstrate that the MAP1B-LC1 negatively regulates p53-dependent transcriptional activity of a reporter construct driven by the p21 promoter. Consequently, MAP1B-LC1 binds to p53 and this interaction leads to the inhibition of doxorubicin-induced apoptosis in SH-SY5Y cells.

TIP60 represses transcriptional activity of p73beta via an MDM2-bridged ternary complex.

TIP60, a histone acetyl transferase, acts as a p53 coactivator by interfering with MDM2-mediated degradation of p53. However, little is known about its functional regulation of p73, which has structural features similar to p53. In this study we found that TIP60 represses apoptosis, which is induced by exogenous and endogenous p73beta. TIP60 also negatively regulated the expression of p73beta downstream target genes such as p21 and Bax. Moreover, the specific repression of p73beta-mediated transactivation by TIP60 was independent of p53 expression and not due to histone deacetylase recruiting transcriptional machinery. Transcriptional activities of both p73 splicing variants, p73alpha and p73beta, were also repressed by TIP60. Furthermore, TIP60 markedly enhanced p73beta binding affinity to MDM2 and physically associated with MDM2 through its zinc finger domain, which is specifically localized in the nucleus. Therefore, we demonstrate that TIP60 forms a ternary complex with p73beta, which is directly bridged by MDM2. It is important to note that our findings contribute to a functional linkage between TIP60 and p73beta through MDM2 in the transcriptional regulation of cellular apoptosis.

Functional cross-talk between p73beta and NF-kappaB mediated by p300.

p73beta is associated with induction of apoptosis or cellular growth arrest, while NF-kappaB is closely related with promotion of resistance to programmed cell death. These biologically opposing activities between p73beta and NF-kappaB propose a regulatory mechanism of critical turning on/off in cellular apoptotic or survival responses. In this study, we demonstrate that NF-kappaB-mediated transactivation is specifically downregulated by p73beta; conversely, p73beta-transactivation is negatively regulated by functional expression of p65, NF-kappaB RelA subunit. The p73beta transactivation domain (TA) and p65 NH2-terminus are crucial for their negative regulation of p65- and p73beta-mediated transactivation, respectively. Furthermore, p65- or p73beta-interaction with p300 is reciprocally inhibited by their competitive binding to p300 in a restrict amount-dependent manner. Likewise, both p73beta-activated apoptosis and p65-dependent increase of cell viability are reciprocally repressed by p65 and p73beta, respectively. These results have important implications for p300-mediated regulatory mechanism between p73beta- and p65-transactivation, by which both p73beta and NF-kappaB could mutually affect on their biological activities. Therefore, we propose that p300 is a transactivational regulator of competitively balanced cross-talk between p73beta and p65.