Histone and RNA-binding protein interaction creates crosstalk network for regulation of alternative splicing.

Alternative splicing is an essential process in eukaryotes, as it increases the complexity of gene expression by generating multiple proteins from a single pre-mRNA. However, information on the regulatory mechanisms for alternative splicing is lacking, because splicing occurs over a short period via the transient interactions of proteins within functional complexes of the spliceosome. Here, we investigated in detail the molecular mechanisms connecting alternative splicing with epigenetic mechanisms. We identified interactions between histone proteins and splicing factors such as Rbfox2, Rbfox3, and splicing factor proline and glutamine rich protein (SFPQ) by in vivo crosslinking and immunoprecipitation. Furthermore, we confirmed that splicing factors were bound to specific modified residues of histone proteins. Additionally, changes in histone methylation due to histone methyltransferase inhibitor treatment notably affected alternative splicing in selected genes. Therefore, we suggested that there may be crosstalk mechanisms connecting histone modifications and RNA-binding proteins that increase the local concentration of RNA-binding proteins in alternative exon loci of nucleosomes by binding specific modified histone proteins, leading to alternative splicing. This crosstalk mechanism may play a major role in epigenetic processes such as histone modification and the regulation of alternative splicing.

Identification and characterization of the RNA-binding protein Rbfox3 in zebrafish embryo.

Rbfox3, an RNA-binding fox protein, binds to the antibody to pan-neuronal marker, neuronal nuclei (NeuN). Rbfox3 is expressed in neural tissues across a wide range of species including mammals, birds, and amphibians. However, the molecular identity of Rbfox3 in the zebrafish is largely unknown. In this study, we cloned two zebrafish Rbfox3 genes, Rbfox3a and Rbfox3b. We also cloned the Rbfox3-d31 isoform, which excludes a 93-nucleotide alternative exon within the RNA-recognition motif in both, Rbfox3a and Rbfox3b. Multiple protein sequence alignment revealed that the amino acid sequence for residues 1-20 of the zebrafish Rbfox3, which is the epitope region of NeuN antibody, was different from that of other species. Therefore, NeuN antibody lost its function as a neuronal marker antibody in zebrafish. Reverse transcriptase-polymerase chain reaction showed that both Rbfox3-d31 transcripts were abundant in the early blastula stage, after which they dramatically reduced, suggesting that these isoforms exist mainly as maternal transcripts. In contrast, full-length Rbfox3 transcripts were detected from the 24 h post-fertilization embryo, expression was also maintained at a constant level. Furthermore, full-length Rbfox3-expressing cells were located within the central nervous system during later stages of the zebrafish embryo. Our study provides insight into the molecular structure of zebrafish Rbfox3 as a step towards genetic association studies investigating the developmental role of Rbfox3.

Msx1 homeodomain transcription factor and TATA-binding protein interact to repress the expression of the glycoprotein hormone α subunit gene.

Studying the regulatory mechanism of the glycoprotein hormone α subunit (αGSU) gene in thyrotropes is essential for understanding the synthesis of functional thyroid-stimulating hormone (TSH). Here, we investigated the influence of a homeodomain transcription factor Msx1 (Msh homeobox 1) on αGSU expression in thyrotropes. The transient expression of Msx1 inhibited the activity of an αGSU reporter gene, as well as its endogenous mRNA level in thyrotrope-derived αTSH cells. Luciferase reporter assays with serial deletion constructs and a close examination of the sequences revealed that the putative Msx1 binding site (PMS) in the αGSU promoter is not responsible for Msx1-mediated transcriptional repression. We also identified the TATA-box binding protein (TBP) as an interacting protein in thyrotropes. Interaction of TBP with Msx1 attenuates the inhibitory effect of Msx1 on αGSU gene expression in a DNA binding-independent manner. Furthermore, transient transfection studies with mutant Msx1 revealed that the interaction of TBP and Msx1 is critical for Msx1-mediated transcriptional repression of the αGSU. These results suggest that Msx1 functions as a transcriptional repressor of αGSU and that its interaction with TBP is an integral part of the mechanism by which Msx1 regulates the inhibition of αGSU gene expression.

Eupafolin suppresses prostate cancer by targeting phosphatidylinositol 3-kinase-mediated Akt signaling.

Phosphatase and tensin homolog (PTEN) loss or mutation consistently activates the phosphatidylinositol 3-kinase (PI3-K)/Akt signaling pathway, which contributes to the progression and invasiveness of prostate cancer. Furthermore, the PTEN/PI3-K/Akt and Ras/MAPK pathways cooperate to promote the epithelial-mesenchymal transition (EMT) and metastasis initiated from prostate stem/progenitor cells. For these reasons, the PTEN/PI3-K/Akt pathway is considered as an attractive target for both chemoprevention and chemotherapy. Herein we report that eupafolin, a natural compound found in common sage, inhibited proliferation of prostate cancer cells. Protein content analysis indicated that phosphorylation of Akt and its downstream kinases was inhibited by eupafolin treatment. Pull-down assay and in vitro kinase assay results indicated that eupafolin could bind with PI3-K and attenuate its kinase activity. Eupafolin also exhibited tumor suppressive effects in vivo in an athymic nude mouse model. Overall, these results suggested that eupafolin exerts antitumor effects by targeting PI3-K.

A chrysin derivative suppresses skin cancer growth by inhibiting cyclin-dependent kinases.

Chrysin (5,7-dihydroxyflavone), a natural flavonoid widely distributed in plants, reportedly has chemopreventive properties against various cancers. However, the anticancer activity of chrysin observed in in vivo studies has been disappointing. Here, we report that a chrysin derivative, referred to as compound 69407, more strongly inhibited EGF-induced neoplastic transformation of JB6 P(+) cells compared with chrysin. It attenuated cell cycle progression of EGF-stimulated cells at the G1 phase and inhibited the G1/S transition. It caused loss of retinoblastoma phosphorylation at both Ser-795 and Ser-807/811, the preferred sites phosphorylated by Cdk4/6 and Cdk2, respectively. It also suppressed anchorage-dependent and -independent growth of A431 human epidermoid carcinoma cells. Compound 69407 reduced tumor growth in the A431 mouse xenograft model and retinoblastoma phosphorylation at Ser-795 and Ser-807/811. Immunoprecipitation kinase assay results showed that compound 69407 attenuated endogenous Cdk4 and Cdk2 kinase activities in EGF-stimulated JB6 P(+) cells. Pulldown and in vitro kinase assay results indicated that compound 69407 directly binds with Cdk2 and Cdk4 in an ATP-independent manner and inhibited their kinase activities. A binding model between compound 69407 and a crystal structure of Cdk2 predicted that compound 69407 was located inside the Cdk2 allosteric binding site. The binding was further verified by a point mutation binding assay. Overall results indicated that compound 69407 is an ATP-noncompetitive cyclin-dependent kinase inhibitor with anti-tumor effects, which acts by binding inside the Cdk2 allosteric pocket. This study provides new insights for creating a general pharmacophore model to design and develop novel ATP-noncompetitive agents with chemopreventive or chemotherapeutic potency.

Histone modification-mediated Lhx2 gene expression.

Lhx2, a member of LIM homeobox transcription factors, plays a key role in central nervous system (CNS) and embryonic tissue development. However, molecular mechanism of Lhx2 gene regulation remains largely unknown. Here, we identified and characterized a regulatory region of Lhx2 gene which mediates responses to two different signals such as inhibition of HDAC3 and stimulation by E2F1. In particular, the promoter region of -229 to -126 was responsible not only for basal expression but also for a inhibitor of histone deacetylase, trichostatin A (TSA)-mediated activation of Lhx2 gene. Intriguingly, transcription factor E2F1 also activates Lhx2 gene via direct binding to the same -229 to -126 region. Based on these observations, we could have demonstrated that E2F1 is necessary for TSA-mediated activation of Lhx2 gene and acetylation of histone 3 is involved in this event. This study provides evidence that the histone modification and E2F1 binding are integral parts of the mechanism for Lhx2 gene expression.

Insulin represses transcription of the thyroid stimulating hormone beta-subunit gene through increased recruitment of nuclear factor I.

Although the regulation of thyroid stimulating hormone ß-subunit gene (TSHß) has been intensively studied, the functions of transcription factors involved are not fully understood. The authors found that the -615/-516 promoter region of the TSHß interacts specifically with nuclear proteins derived from pituitary tissue or from cultured thyrotroph cells. The actual binding site at the nucleotide level, as revealed by DNase I protection assay, includes the consensus sequence for nuclear factor I (NFI). RT-PCR analysis indicated that NFI-B expression is restricted to thyrotroph cells in the anterior pituitary. EMSA and ChIP analysis showed that NFI-B binds most efficiently to the -588/-560 region of TSHß promoter. The forced expressions of NFI-B markedly reduced TSHß promoter activity and its mRNA expression. Furthermore, it was also shown that the -588/-560 region is involved in the insulin-mediated repression of the TSHß. It was of particular interest to observe that NFI-B was recruited to the -588/-560 region of the TSHß promoter in an insulin-dependent manner. Taken together, this study provides new insights of the delicate regulations of energy metabolism and hormonal homeostasis.

Pseudomonas aeruginosa eliminates natural killer cells via phagocytosis-induced apoptosis.

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes the relapse of illness in immunocompromised patients, leading to prolonged hospitalization, increased medical expense, and death. In this report, we show that PA invades natural killer (NK) cells and induces phagocytosis-induced cell death (PICD) of lymphocytes. In vivo tumor metastasis was augmented by PA infection, with a significant reduction in NK cell number. Adoptive transfer of NK cells mitigated PA-induced metastasis. Internalization of PA into NK cells was observed by transmission electron microscopy. In addition, PA invaded NK cells via phosphoinositide 3-kinase (PI3K) activation, and the phagocytic event led to caspase 9-dependent apoptosis of NK cells. PA-mediated NK cell apoptosis was dependent on activation of mitogen-activated protein (MAP) kinase and the generation of reactive oxygen species (ROS). These data suggest that the phagocytosis of PA by NK cells is a critical event that affects the relapse of diseases in immunocompromised patients, such as those with cancer, and provides important insights into the interactions between PA and NK cells.

In Vivo Crosslinking of Histone and RNA-Binding Proteins.

Protein-protein interactions are essential in various cellular processes including regulation of gene expression, formation of protein complexes, and cellular signaling transduction. In particular, several proteins in the nucleus interact to regulate transcription and RNA splicing. These protein-protein interactions are short and weak and occur through transient processes, making it difficult to identify these interactions. In addition, detection of interacting partners in vitro using cell lysates cannot provide complete information due to the loss of spatial organization and changes in protein modification. Here we describe an in vivo crosslinking technique using disuccinimidyl suberate (DSS), which is useful to capture and stabilize proteins to analyze the interacting proteins.

Staphylococcus aureus Specific FRET Probe-Based Antibacterial Susceptibility Testing (SF-AST) by Detection of Micrococcal Nuclease Activity.

In this study, we describe a simple and rapid antibacterial susceptibility testing (AST) method for Staphylococcus aureus called S. aureus specific fluorescence resonance energy transfer (FRET) probe-based AST (SF-AST), which is based on an S. aureus specific FRET probe (SF probe) that detects micrococcal nuclease (MNase) activity secreted from S. aureus. The SF-AST was tested with an S. aureus quality control (QC) strain against six relevant antibiotics, and the minimum inhibitory concentration (MIC) values obtained with the broth microdilution (BMD) method were compared, as a gold standard AST. Results were obtained with high accuracy in 4-6 h. The MIC for the methicillin resistance using 20 clinical S. aureus isolates of SF-AST showed 100% sensitivity, specificity, positive predictive value, and negative predictive value, as compared to BMD. Thus, the SF-AST method is a simple, rapid, and useful antibiotic resistance test for S. aureus, and it provides a basis for clinical treatment in a short time.

PAS1-modified optical SIS sensor for highly sensitive and specific detection of toluene.

We report on a novel solution immersed silicon (SIS) sensor modified with bio-receptor to detect toluene. To perform this approach, bio-receptor PAS1 which specifically interacts with toluene was chosen as a capture agent for SIS ellipsometric sensing. We constructed wild PAS1 and mutant PAS1 (F46A and F79Y) which are toluene binding-defective. Especially, we utilized an easily accessible capturing approach based on silica binding peptide (SBP) for direct immobilization of PAS1 on the SiO2 surfaces. After the immobilization of SBP-tagged PAS1 to the sensing layers, PAS1-based SIS sensor was evaluated for its ability to recognize toluene. As a result, a significant up-shift in Psi (Ψ) was clearly observed with a low limit of detection (LOD) of 0.1 µM, when treated with toluene on wild PAS1-surface, but not on mutant PAS1-sensing layers, indicating the selective interactions between PAS1 and toluene molecule. The PAS1-SIS sensor showed no changes in Psi (Ψ), if any, negligible, when exposed to benzene, phenol, xylene and 4-nitrophenol as negative controls, thereby demonstrating the specificity of interaction between PAS1 and toluene. Taken together, our results strongly indicate that PAS1-modified ellipsometry sensor can provide a high fidelity system for the accurate and selective detection of toluene.

Enhanced anticancer effects of a methylation inhibitor by inhibiting a novel DNMT1 target, CEP 131, in cervical cancer.

Methylation is a primary epigenetic mechanism regulating gene expression. 5-aza-2'-deoxycytidine is an FDA-approved drug prescribed for treatment of cancer by inhibiting DNA-Methyl-Transferase 1 (DNMT1). Results of this study suggest that prolonged treatment with 5-aza-2'-deoxycytidine could induce centrosome abnormalities in cancer cells and that CEP131, a centrosome protein, is regulated by DNMT1. Interestingly, cancer cell growth was attenuated in vitro and in vivo by inhibiting the expression of Cep131. Finally, Cep131-deficient cells were more sensitive to treatment with DNMT1 inhibitors. These findings suggest that Cep131 is a potential novel anti-cancer target. Agents that can inhibit this protein may be useful alone or in combination with DNMT1 inhibitors to treat cancer. [BMB Reports 2019; 52(5): 342-347].

Activation of the thyroid-stimulating hormone beta-subunit gene by LIM homeodomain transcription factor Lhx2.

Although there is evidence that the LIM homeodomain transcription factor, Lhx2, can stimulate transcription of the glycoprotein hormone alpha-subunit gene, the role of Lhx2 in regulating TSH beta-subunit has not been established. In the present studies, the ability of Lhx2 to regulate transcription of the TSH beta-subunit gene was examined. In the thyrotrope-derived TalphaT1 cell line, Lhx2 expression was found to be induced by treatment with either TRH or cAMP, consistent with the possibility that Lhx2 may play a role in mediating the ability of this signaling pathway to stimulate TSH gene expression. Transient, forced overexpression of Lhx2 stimulated activity of a TSH beta-subunit reporter gene. Deletion studies provided evidence that the -177 to -79 region of the TSH beta-subunit promoter was necessary for stimulation of reporter gene activity by Lhx2. A gel mobility shift assay provided the evidence that Lhx2 can bind to this region of DNA. DNase I footprinting studies demonstrated that two distinct regions of the TSHbeta promoter, -118 to -108 and -86 to -68, are protected by Lhx2 from nuclease digestion. These regions contain repeats of the sequence, 5'-(G/T)CAAT(T/A)-3'. Mutation of this sequence, especially in the -86 to -68 region, substantially decreased Lhx2 responsiveness of the TSH beta-subunit reporter gene. In addition, a DNA fragment containing the -177 to -79 region of the TSHbeta promoter was found to confer Lhx2 responsiveness to a minimal promoter. These results provide multiple lines of evidence consistent with a role for Lhx2 in modulating expression of the TSH beta-subunit gene.

A split enhanced green fluorescent protein-based reporter in yeast two-hybrid system.

We have developed a novel reporter system involving a yeast two-hybrid assay, which utilizes the reconstitution of the split EGFP reporter in order to characterize the relevant protein-protein interactions. To our knowledge, this study represents the first application of the split EGFP system as a read-out in a yeast two-hybrid assay. In comparison with the existing two-hybrid system, the bait and prey vectors were improved with regard to the reporter and the replication control element. As a result, the reconstituted EGFP has been observed to evidence a restored fluorescence upon protein-protein interactions in yeast, thereby allowing for the characterization of its interactor. The use of a split EGFP reporter has some salient advantages. Firstly, no substrates are required for the production of fluorescence. Secondly, low copy number plasmids may help to solve the protein toxicity problem, via the reduction of expression. Thirdly, this technique may prove useful in overcoming the autoactivation problem, due to the fact that the read-out of the yeast two-hybrid system is transcription-independent. Collectively, our results showed that the split EGFP reporter system might potentially be applied in yeast two-hybrid assays for the high-throughput screening of protein-protein interactions, with a simple and direct fluorescent read-out.

An in vitro technique to identify the RNA binding-site sequences for RNA-binding proteins.

RNA-protein interactions play a major role in gene regulation. Although many techniques to analyze RNA-protein interactions have been developed, noteworthy challenges such as determining the RNA sequences that bind RNA-binding proteins (RBPs) remain unsolved. Here, we describe a novel technique using a 4-thio-uridine-incorporated RNA pool to identify the RBP-binding consensus sequences for RBPs produced by in vitro transcription and translation. To confirm the fidelity of this approach, we determined the consensus RBP-binding sequence for RBFOX2, UGC(A/U)(A/U)NU, which is very similar to the known RBFOX2-binding sequence, UGCAUG. Using our method, consensus RBP-binding sequences were determined for three RBPs, namely FUS (fused in sarcoma), SFPQ (splicing factor proline and glutamine rich), and SAM68 (Src-Associated substrate in Mitosis 68 kDa). The consensus RBP-binding sequences for these RBPs were confirmed by RNA-protein complex immunoprecipitation-PCR analysis.

Transforming Growth Factor-ß-Induced RBFOX3 Inhibition Promotes Epithelial-Mesenchymal Transition of Lung Cancer Cells.

The RNA-binding protein Rbfox3 is a well-known splicing regulator that is used as a marker for post-mitotic neurons in various vertebrate species. Although recent studies indicate a variable expression of Rbfox3 in non-neuronal tissues, including lung tissue, its cellular function in lung cancer remains largely unknown. Here, we report that the number of RBFOX3-positive cells in tumorous lung tissue is lower than that in normal lung tissue. As the transforming growth factor-ß (TGF-ß) signaling pathway is important in cancer progression, we investigated its role in RBFOX3 expression in A549 lung adenocarcinoma cells. TGF-ß1 treatment inhibited RBFOX3 expression at the transcriptional level. Further, RBFOX3 depletion led to a change in the expression levels of a subset of proteins related to epithelial-mesenchymal transition (EMT), such as E-cadherin and Claudin-1, during TGF-ß1-induced EMT. In immunofluorescence microscopic analysis, mesenchymal morphology was more prominent in RBFOX3-depleted cells than in control cells. These findings show that TGF-ß-induced RBFOX3 inhibition plays an important role in EMT and propose a novel role for RBFOX3 in cancer progression.

Osteonectin-expressing cells in human stomach cancer and their possible clinical significance.

The clinical significance of osteonectin in human stomach cancer was examined immunohistochemically and molecular biologically in 31 differentiated and eight undifferentiated stomach adenocarcinomas and 19 non-cancer stomach tissues. Osteonectin-mAb-stained cells were observed in stroma of 90% differentiated and 63% undifferentiated adenocarcinomas, and of 26% non-cancer stomach tissues. Competitive reverse transcriptase polymerase chain reaction results generally coincided with immunohistochemical data. The present results suggest that osteonectin is highly expressed in reactive stroma associated with invasive differentiated adenocarcinomas and that it may serve as a useful clinical diagnostic marker for stomach cancer.

Gonadotropin-releasing hormone-induced stimulation of the rat secretogranin II promoter involves activation of CREB.

To investigate the events involved in regulation of the secretogranin II (SgII) gene, luciferase reporter constructs were transfected into gonadotrope-derived, alphaT3-1 cells. DNA between -91 and -60 relative to the transcription start site was found to be required for GnRH induced SgII reporter gene activation. This region contains a consensus cAMP response element (CRE) and disruption of this CRE reduced GnRH responsiveness of the SgII promoter. CREB was shown to bind to the SgII CRE and transfection studies with a dominant-negative CREB mutant provided evidence that CREB is required for GnRH responsiveness of the SgII promoter. An expression vector for an inhibitor of the cAMP-dependent protein kinase was found to reduce the ability of cAMP or GnRH to activate the SgII-luciferase reporter gene. These studies offer evidence that GnRH-induced activation of the SgII promoter in the alphaT3-1 cell line requires cAMP-dependent protein kinase activity and a functional CRE within the 5'-flanking region of the gene.

A direct repeat of N-type Ca2+ channel alpha1B gene functions as a negative regulatory element in HeLa cells.

The expression of the N-type voltage-gated calcium channel alpha1B gene is restricted to neurons by a 5'-upstream region (-3992 to -1788) that contains negative regulatory element(s) that are active in non-neuronal cells. A 39 bp DNA element, which is repeated nine times in a head-to-tail fashion, was found within the same region. To examine whether this direct repeat (DR) may function as a negatively acting cis-regulatory element, several fusion plasmids, DR-110alpha1BLUC (1X), DR-SV40LUC (IX, 2X), in which one or two copies of the DR fragment were subcloned upstream of the homologous and heterologous promoters, were transiently transfected into HeLa and NS20Y cells. The promoter activity of DR-110alpha1BLUC (1X) decreased to approximately 17% of the 110alph(a1B)LUC construct in HeLa cells. The expression of the DR-SV40LUC (1X) and DR-SV40LUC (2X) plasmids was also reduced to 50 to 23% of the levels that were observed in the pGL2-Promoter in the same cells. However, no repression of the DR constructs was observed in NS20Y cells. An electrophoretic mobility shift assay showed that two DR-specific complexes were detected in HeLa cells, but not in NS20Y cells. In addition, Southwestern blotting revealed the presence of approximately 33 and 43 kDa proteins in HeLa cells. Overall, these results suggest that a 39 bp DNA element might act as repressor in non-neuron cells through the specific interactions of the DNA-proteins.

Inhibition of TNF-α-Mediated NF-κB Transcriptional Activity by Dammarane-Type Ginsenosides from Steamed Flower Buds of Panax ginseng in HepG2 and SK-Hep1 Cells.

Panax ginseng is a medicinal herb that is used worldwide. Its medicinal effects are primarily attributable to ginsenosides located in the root, leaf, seed, and flower. The flower buds of Panax ginseng (FBPG) are rich in various bioactive ginsenosides, which exert immunomodulatory and anti-inflammatory activities. The aim of the present study was to assess the effect of 18 ginsenosides isolated from steamed FBPG on the transcriptional activity of NF-κB and the expression of tumor necrosis factor-α (TNF-α)-stimulated target genes in liver-derived cell lines. Noticeably, the ginsenosides Rk3 and Rs4 exerted the strongest activity, inhibiting NF-κB in a dose-dependent manner. SF and Rg6 also showed moderately inhibitory effects. Furthermore, these four compounds inhibited the TNF-α-induced expression of IL8, CXCL1, iNOS, and ICAM1 genes. Consequently, ginsenosides purified from steamed FBPG have therapeutic potential in TNF-α-mediated diseases such as chronic hepatic inflammation.