Sang-Min Jang

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.

p19ras Represses proliferation of non-small cell lung cancer possibly through interaction with Neuron-Specific Enolase (NSE)

p19(ras) is an alternative splicing product of the proto-oncogene c-H-ras pre-mRNA. In this study, we identified a novel p19(ras)-binding protein, Neuron-Specific Enolase (NSE), using the yeast two-hybrid method. NSE is one of the enolase families that convert 2-phospho-d-glycerate (PGA) to phosphoenolpyruvate (PEP) in the glycolysis pathway. In both endogenous and over-expressed systems, we confirmed interactions between p19(ras) and NSE via co-immunoprecipitation assay. We also identified the interaction region of p19(ras), which is required for binding with NSE. When full-length p19(ras) and C-terminal region are bound to NSE, it inhibits the enzymatic activity of NSE. Furthermore, p19(ras) interacted with Enolase alpha (Enoalpha) and repressed its enzymatic activity in vitro. p19(ras) repressed lung cancer cell proliferation mostly increased by NSE in H1299 cells. Taken together, these results suggest that p19(ras) is a novel regulator to suppress cell proliferation in lung cancer through the interaction with NSE.

Transcriptional Activity of Neural Retina Leucine Zipper (Nrl) Is Regulated by c-Jun N-Terminal Kinase and Tip60 during Retina Development

ABSTRACT 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.

p19ras amplifies p73β-induced apoptosis through mitochondrial pathway

p73 and p53 have been known to play an important role in cellular damage responses such as apoptosis. Although p73 is a structural and functional homolog of p53 tumor suppressor gene, much less is known about the mechanism of p73-induced apoptotic cell death. In this study, we demonstrate that p19(ras) interaction with p73beta amplifies p73beta-induced apoptotic signaling responses including Bax mitochondrial translocation, cytochrome c release, increased production of reactive oxygen species (ROS) and loss of mitochondrial transmembrane potential (DeltaPsi(m)). Furthermore, endogenous expression of p19(ras) and p73beta is significantly increased by Taxol treatment, and Taxol-enhanced endogenous p73beta transcriptional activities are further amplified by p19(ras), which markedly increased cellular apoptosis in p53-null SAOS2 cancer cell line. These results have important implications for understanding the molecular events of p19(ras) to p73 functions in cancer cells.

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. As a code translator, TIP60 interacted to methylated or acetylated histone H3, H4, inducing open chromatin state for accessibility of transcription complexes.

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.

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.

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 β‐amyloid through the CP2 transcription factor

Accumulation of β‐amyloid protein (Aβ) is one of the most important pathological features of Alzheimer’s disease. Although Aβ induces neurodegeneration in the cortex and hippocampus through several molecular mechanisms, few studies have evaluated the modulation of transcription factors during Aβ‐induced neurotoxicity. Therefore, in this study, we investigated the transcriptional activity of transcription factor CP2 in neuronal damage mediated by Aβ (Aβ1–42 and Aβ25–35). 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 Aβ, as Aβ (Aβ1–42 and Aβ25–35) 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.