Transient retinoic acid signaling confers anterior-posterior polarity to the inner ear

Vertebrate hearing and balance are based in complex asymmetries of inner ear structure. Here, we identify retinoic acid (RA) as an extrinsic signal that acts directly on the ear rudiment to affect its compartmentalization along the anterior-posterior axis. A rostrocaudal wave of RA activity, generated by tissues surrounding the nascent ear, induces distinct responses from anterior and posterior halves of the inner ear rudiment. Prolonged response to RA by posterior otic tissue correlates with Tbx1 transcription and formation of mostly nonsensory inner ear structures. By contrast, anterior otic tissue displays only a brief response to RA and forms neuronal elements and most sensory structures of the inner earope

Post-transcriptional Regulation of Gcn5, a Putative Regulator of Hox in Mouse Embryonic Fibroblast Cells

Hox proteins containing DNA-binding homedomain act as transcription factors important for anteroposterior body patterning during vertebrate embryogenesis. However, the precise mechanisms by which signal pathways are transduced to regulate the Hox gene expression are not clear. In the course of an attempt to isolate an upstream regulatory factor(s) controlling Hox genes, protein kinase B alpha (Akt1) has been identified as a putative regulator of Hox genes through in silico analysis (GEO profile). In the Gene Expression Omnibus (GEO) dataset GDS1784 at the NCBI (National Center for Biotechnology Information) site, Hox genes were differentially expressed depending on the presence or absence of Akt1. Since it was not well known how Akt1 regulates the specific Hox genes, whose transcription was reported to be regulated by epigenetic modifications such as histone acetylation, methylation etc., the expression of Gcn5, a histone acetyltransferase (HAT), was analyzed in wild type (WT) as well as in Akt1-/- mouse embryonic fibroblast (MEF) cells. RT-PCR analysis revealed that the amount of Gcn5 mRNA was similar in both WT and Akt1-/- MEFs. However, the protein level of Gcn5 was significantly increased in Akt1-/- MEF cells. The half life of Gcn5 was 1 hour in wild type whereas 8 hours in Akt1-/- MEF. These data all together, indicate that Gcn5 is post-transcriptionally down-regulated and the protein stability is negatively regulated by Akt1 in MEF cells.ope

Direct Interaction Between Akt1 and Gcn5 and its Plausible Function on Hox Gene Expression in Mouse Embryonic Fibroblast Cells

Hox genes encode transcription factors important for anterior‐posterior body patterning at early stages of embryonic development. However, the precise mechanisms by which signal pathways are stimulated to regulate Hox gene expression are not clear. In the previous study, protein kinase B alpha (Akt1) has been identified as a putative upstream regulator of Hox genes, and Akt1 has shown to regulate Gcn5, a prototypical histone acetyltransferase (HAT), in a negative way in mouse embryonic fibroblast (MEF) cells. Since the activity of HAT such as the CBP/p300, and PCAF (a Gcn5 homolog), was down‐regulated by Akt through a phosphorylation at the Akt consensus substrate motif (RXRXXS/T), the amino acid sequence of Gcn5 protein was analyzed. Mouse Gcn5 contains an Akt consensus substrate motif as RQRSQS sequence while human Gcn5 does not have it. In order to see whether Akt1 directly binds to Gcn5, immunoprecipitation with anti‐Akt1 antibody was carried out in wild‐type (WT) mouse embryonic fibroblast (MEF) cells, and then western blot analysis was performed with anti‐Akt1 and anti‐Gcn5 antibodies. Gcn5 protein was detected in the Akt1 immunoprecipitated samples of MEFs. This result demonstrates that Akt1 directly binds to Gcn5, which might have contributed the down regulation of the 5’ Hoxc gene expressions in wild type MEF cells.ope

생쥐 배아 섬유아세포에서 Akt1에 의한 혹스 유전자 발현의 조절

Dept. of Medical Science/박사In mammals, precise spatiotemporal expressions of Hox genes control body pattern and provide positional information along the body axis during embryogenesis. However, the mechanism by which the Hox genes are regulated is poorly understood. To search for novel regulators of the Hox genes, archived gene expression profiles were analyzed from the Gene Expression Omnibus (GEO) database. In a particular dataset, clustered Hox gene expressions were largely altered in Akt1-/- mouse embryonic fibroblasts (MEFs) compared to the wild type, suggesting the hypothesis that Akt1 is required for the proper expression of Hox genes during mouse embryonic development. Therefore, the expressions of all 39 Hox genes were examined with quantitative RT-PCR and it was found that the transcripts of the 5′ Hoxc genes, Hoxc10, 11, 12 and 13 including a noncoding RNA, were upregulated in Akt1 null MEFs. Particularly, the upregulation of Hoxc11 was further confirmed in the Akt1 null embryonic limbs with quantitative RT-PCR and in situ hybridization. To elucidate the molecular mechanism, first, whether forkhead box O (FoxO), an Akt downstream transcription factor, was involved in the expression of the 5’ Hoxc gene in Akt1-/- MEFs was investigated. Knockdown of Foxo1 and Foxo3 failed to repress Hox gene expression in Akt null MEFs. However, epigenetic modifications correlated with Hox gene transcription; DNA hypomethylation at promoter regions and hyperacetylation of histone H3K9, underlined the upregulation of Hoxc11 and 12 in Akt1 null MEFs. From the inhibitor-treated experiment, histone deacetylases (HDAC) might be required for the repression of 5’ Hoxc transcription in Akt1+/+ MEFs. Moreover, histone H3K9 acetyltransferase, GCN5 might have an effect on Hoxc11 expression in Akt1 null MEFs. These results suggest that Akt1 is necessary for the epigenetic regulation of a group of Hox genes during embryogenesis.restrictio

Akt1 as a putative regulator of Hox genes

In mammals, precise spatiotemporal expressions of Hox genes control the main body axis during embryogenesis. However, the mechanism by which Hox genes are regulated is poorly understood. To discover the putative regulator of Hox genes, in silico analyses were performed using GEO profiles, and Akt1 emerged as a candidate regulator of Hox genes in E13.5 MEFs. The results of the RT-PCR showed that 5′ Hoxc genes, including ncRNA were upregulated in Akt1 null MEF. Combined bisulfite restriction analysis (COBRA) and bisulfite sequencing showed that the CpG island of a 5′ Hoxc gene was hypomethylated in Akt1 null cells. These results indicate that Hox expression could be controlled by the function of Akt1 through epigenetic modification such as DNA methylation.ope

A novel protein Jpk induces bacterial cell death through reactive oxygen species.

Jpk, a trans-acting regulatory factor associating with the position-specific regulatory element of Hoxa-7, has been reported to induce cell death in both prokaryotic and eukaryotic cells upon overexpression. The N- and C-terminal deleted variants of Jpk were constructed and then the toxicity of each construct was analyzed by checking the viability of the cells and the concomitant morphological changes through electron microscopy following the expression. The N-terminus of Jpk harboring transmembrane domain seemed to be more toxic to bacterial cell than C-terminus and the morphology of bacterial cells expressing N-terminal Jpk was similar to that induced by full length Jpk. The toxicity caused by Jpk protein in bacterial cell was through the production of ROS, which was decreased by an antioxidant (DTT) in a concentration dependent manner. The finding described in this study provides valuable clues on the relationship between Jpk toxicity and ROS generation.ope

ER Stress-Induced Jpk Expression and the Concomitant Cell Death

A Jopock (Jpk), a trans-acting factor associating with the position-specific regulatory element of murine Hoxa-7, has shown to have a toxicity to both prokaryotic and eukaryotic cells when overexpressed. Since Jpk protein harbors a transmembrane domain and a putative endoplasmic reticulum (ER)-retention signal at the N-terminus, a subcellular localization of the protein was analyzed after fusing it into the green fluorescent protein (GFP): Both N-term (Jpk-EGFP) and C-term tagged-Jpk (EGFP-Jpk) showed to be localized in the ER when analyzed under the fluorescence microscopy after staining the cells with ER- and MitoTracker. Since ER stress triggers the ER-stress mediated apoptosis to eliminate the damaged cells, we analyzed the expression pattern of Jpk under ER-stress condition. When MCF7 cells were treated with the ER-stress inducer such as DTT and EGTA, the expression of Jpk was upregulated at the transcriptional level like that of Grp78, a molecular chaperone well known to be overexpressed under ER-stress condition. In the presence of high concentration of ER-stress inducer (10 mM), about 70 (DTT) to 95% (EGTA) of cells died stronly expressing (10~12 fold) Jpk. Whereas at the low concentration (0.001~1.0 mM) of the inducer, the expression of Jpk was increased about 2.5 (EGTA) to 5 fold (DTT), which is rather similar to those of ER chaperone protein Grp78. These results altogether indicate that the ER-stress upregulated the expression of Jpk and the excess stress induces the ER-stress induced apoptosis and the concomitant expression of Jpk.ope

A Novel Gene, Jpk, Induces Apoptosis in F9 Murine Teratocarcinoma Cell through ROS Generation

A novel gene Jpk (Jopock) has been originally isolated through yeast 1 hybridization technique as a trans-acting factor interacting with the position-specific regulatory element of a murine Hoxa-7. Northern analysis revealed that the Jpk was expressed at day 7.0 post coitum (p.c.) during early gastrulation. Previously it has been shown that a trace amount of JPK protein led bacterial cells to death. In eukaryotic F9 cells, Jpk also led the cell to death-generating DNA ladder: fewer than 50% of the cells survived after 72-h transfection. Flow cytometric analysis with cells stained with each Annexin V/7-amino-actinomycin D (7-AAD), MitoTracker, and hydroethidine (HE) revealed that Jpk induced apoptotic cell death in a time-dependent manner, reduced mitochondrial membrane potential, and increased ROS (reactive oxygen species) production, respectively. Additionally, Jpk seemed to regulate the Bcl family at the transcriptional level when RT-PCR was performed. Although the precise mechanism is not clear, these results altogether suggest that Jpk is a potent inducer of apoptosis through generation of ROS as well as concomitant reduction of mitochondrial membrane potential.ope

The third helix of the Hoxc8 homeodomain peptide enhances the efficiency of gene transfer in combination with lipofectamine.

Protein transduction domains (PTDs) have been shown to cross the biological cell membranes efficiently through a receptor and energy independent mechanism. Because of its ease in membrane transducing ability, PTDs could be used as a gene delivery vector. Since we already have shown that purified Hoxc8 homeoprotein has the ability to cross the cellular membrane, we analyzed the possibility of the third helix of the Hoxc8 homeodomain as a useful gene delivery vector. For that purpose, a 16-aa long synthetic oligopeptide Hoxc8 Protein Transduction Domain (HPTD) was chemically synthesized and then tested to see whether the HPTD could form a complex with DNA or not. Gel retardation analysis revealed that the HPTD interacts with plasmid DNA efficiently but failed to transfer the DNA into the cells. However, HPTD can enhance the efficiency of gene transfer in combination with Lipofectamine which doubled the gene transfer rate into COS-7 cells compared with the DNA/Lipofectamine control. An MTT assay indicated that the amount of HPTD used in the complex for the transfection did not show any cytotoxicty in COS-7 cells. The TEM studies showed compact particle formation in the presence of HPTD. These results indicate that the HPTD could be a good candidate adjuvant molecule to enhance the gene transfer efficiency of Lipofectamine in eukaryotic cellsope

ER Stress Induces the Expression of Jpk, which Inhibits Cell Cycle Progression in F9 Teratocarcinoma Cell

Jopock (Jpk), a transacting factor associated with the position-specific regulatory element of murine Hoxa-7, has shown to induce cell death in both prokaryotic and eukaryotic cells when introduced and overexpressed. Since Jpk protein harbors a transmembrane domain (TM) and a putative endoplasmic reticulum (ER) -retention signal at the N terminus, a subcellular localization of the protein was analyzed after fusing it into the green fluorescent protein (GFP). Both N–term- (Jpk-EGFP) and C–term-fused Jpk (EGFP-Jpk) showed to be localized in the ER when analyzed under the fluorescence microscope after staining the cells with ER- and Mito-Tracker. Through deletion analysis TM turned out to be important for ER localization of Jpk. When flow cytometric analysis was performed, both cells expressing Jpk-EGFP and EGFP-Jpk led cell cycle arrest and subsequent apoptotic cell death. In order to see whether Jpk is expressed during ER stress-mediated apoptosis, F9 cells were treated with DTT, an ER stress inducer. In the presence of 4 mM of DTT, about 50% of cells died strongly expressing Jpk (sevenfold) as well as Grp78, a molecular chaperone, and CHOP-10, a well-known apoptotic protein. When cells were transfected with both pEGFP-Jpk and pJpk-EGFP, cell cycle progression was interrupted compared to those of control cells. In summary, excess ER stress upregulated the expression of Jpk, which seemed to inhibit the cell cycle progression. These results altogether suggest that Jpk could be a useful cell death-triggering molecule applicable for cancer therapy as well as a useful target molecule for the treatment of certain neurodegenerative diseases caused by ER stress.ope