The histone acetylation mediated by Gcn5 regulates the Hoxc11 gene expression in MEFs.

Hox genes are responsible for encoding transcription factors that are essential for anterior-posterior body patterning at early stages of embryogenesis. However, detailed mechanisms of Hox genes are yet to be defined. Protein kinase B alpha (Akt1) was previously identified as a possible upstream regulator of Hox genes. Furthermore, the Hoxc11 gene has been upregulated in Akt1 null (Akt1-/-) mouse embryonic fibroblasts (MEFs), while repressed in wild-type MEFs. In this study, we propose to investigate the role of Gcn5, a histone acetyltransferase, in the regulation of Hoxc11 expression in MEFs. We showed that the H3 lysine 9 acetylation (H3K9ac) status has the same correlation with Hoxc11 expression and reported that Gcn5 is associated with the upregulation of Hoxc11 expression through H3K9ac in Akt1-/- MEFs. Since Hoxc11 was upregulated through histone acetylation in Akt1-/- MEFs, a functional role of Gcn5 on Hoxc11 expression was analyzed in Akt1-/- MEFs treated with Gcn5 specific inhibitor or transfected with Gcn5-small interfering RNA (Gcn5-siRNA). When the expression of Hoxc11 was analyzed using RT-PCR and real-time PCR, the Hoxc11 mRNA level was found to be similar in both Akt1-/- MEFs and control-siRNA transfected Akt1-/- MEFs. However, the Hoxc11 expression level was decreased in Gcn5-inhibited or Gcn5-knockdown Akt1-/- MEFs. Additionally, to analyze Gcn5-mediated histone acetylation status, chromatin immunoprecipitation assay was carried out in Gcn5-siRNA-transfected Akt1-/- MEFs. The H3K9ac at the Hoxc11 locus was decreased in Gcn5-knockdown Akt1-/- MEFs compared to controls. Based on these findings, we conclude that Gcn5 regulates Hoxc11 gene expression through mediating site-specific H3K9 acetylation in Akt1-/- MEFs.

CTCF-mediated Chromatin Loop for the Posterior Hoxc Gene Expression in MEF Cells.

Modulation of chromatin structure has been proposed as a molecular mechanism underlying the spatiotemporal collinear expression of Hox genes during development. CCCTC-binding factor (CTCF)-mediated chromatin organization is now recognized as a crucial epigenetic mechanism for transcriptional regulation. Thus, we examined whether CTCF-mediated chromosomal conformation is involved in Hoxc gene expression by comparing wild-type mouse embryonic fibroblast (MEF) cells expressing anterior Hoxc genes with Akt1 null MEFs expressing anterior as well as posterior Hoxc genes. We found that CTCF binding between Hoxc11 and -c12 is important for CTCF-mediated chromosomal loop formation and concomitant posterior Hoxc gene expression. Hypomethylation at this site increased CTCF binding and recapitulated the chromosomal conformation and posterior Hoxc gene expression patterns observed in Akt1 null MEFs. From this work we found that CTCF at the C12|11 does not function as a barrier/boundary, instead let the posterior Hoxc genes switch their interaction from inactive centromeric to active telomeric genomic niche, and concomitant posterior Hoxc gene expression. Although it is not clear whether CTCF affects Hoxc gene expression solely through its looping activity, CTCF-mediated chromatin structural modulation could be an another tier of Hox gene regulation during development. © 2016 IUBMB Life, 68(6):436-444, 2016.

Akt1 mediates the posterior Hoxc gene expression through epigenetic modifications in mouse embryonic fibroblasts.

The evolutionarily conserved Hox genes are organized in clusters and expressed colinearly to specify body patterning during embryonic development. Previously, Akt1 has been identified as a putative Hox gene regulator through in silico analysis. Substantial upregulation of consecutive 5' Hoxc genes has been observed when Akt1 is absent in mouse embryonic fibroblast (MEF) cells. In this study, we provide evidence that Akt1 regulates the 5' Hoxc gene expression by epigenetic modifications. Enrichment of histone H3K9 acetylation and a low level of the H3K27me3 mark were detected at the posterior 5' Hoxc loci when Akt1 is absent. A histone deacetylase (HDAC) inhibitor de-repressed 5' Hoxc gene expression when Akt1 is present, and a DNA demethylating reagent synergistically upregulated HDAC-induced 5' Hoxc gene expression. A knockdown study revealed that Hdac6 is mediated in the Hoxc12 repression through direct binding to the transcription start site (TSS) in the presence of Akt1. Co-immunoprecipitation analysis revealed that endogenous Akt1 directly interacted with Hdac6. Furthermore, exogenous Akt1 was enriched at the promoter region of the posterior Hoxc genes such as Hoxc11 and Hoxc12, not the Akt1-independent Hoxc5 and Hoxd10 loci. The regulation of the H3K27me3 mark by Ezh2 and Kdm6b at the 5' Hoxc gene promoter turned out to be Akt1 dependent. Taken together, these results suggest that Akt1 mediates the posterior 5' Hoxc gene expression through epigenetic modification such as histone methylation and acetylation, and partly through a direct binding to the promoter region of the 5' Hoxc genes and/or Hdac6 in mouse embryonic fibroblast cells.

Pax3 function is required specifically for inner ear structures with melanogenic fates.

Pax3 mutations result in malformed inner ears in Splotch mutant mice and hearing loss in humans with Waardenburg's syndrome type I. In the inner ear, Pax3 is thought to be involved mainly in the development of neural crest. However, recent studies have shown that Pax3-expressing cells contribute extensively to multiple inner ear structures, some of which were considered to be derived from the otic epithelium. To examine the specific functions of Pax3 during inner ear development, fate mapping of Pax3 lineage was performed in the presence or absence of functional Pax3 proteins using Pax3(Cre) knock-in mice bred to Rosa26 reporter (R26R) line. ß-gal-positive cells were widely distributed in Pax3(Cre/+); R26R inner ears at embryonic day (E) 15.5, including the endolymphatic duct, common crus, cristae, maculae, cochleovestibular ganglion, and stria vascularis. In the absence of Pax3 in Pax3(Cre/Cre); R26R inner ears, ß-gal-positive cells disappeared from regions with melanocytes such as the stria vascularis of the cochlea and dark cells in the vestibule. Consistently, the expression of Dct, a melanoblast marker, was also absent in the mutant inner ears. However, when examined at E11.5, ß-gal positive cells were present in Pax3(Cre/Cre) mutant otocysts, whereas Dct expression was absent, suggesting that Pax3 lineage with a melanogenic fate migrated to the inner ear, yet failed to differentiate and survive without Pax3 function. Gross inner ear morphology was generally normal in Pax3(Cre/Cre) mutants, unless neural tube defects extended to the cranial region. Taken together, these results suggest that despite the extensive contribution of Pax3-expressing cells to multiple inner ear tissues, Pax3 function is required specifically for inner ear components with melanogenic fates.

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 ear.

Clinical and molecular characterizations of novel POU3F4 mutations reveal that DFN3 is due to null function of POU3F4 protein.

X-linked deafness type 3 (DFN3), the most prevalent X-linked form of hereditary deafness, is caused by mutations in the POU3F4 locus, which encodes a member of the POU family of transcription factors. Despite numerous reports on clinical evaluations and genetic analyses describing novel POU3F4 mutations, little is known about how such mutations affect normal functions of the POU3F4 protein and cause inner ear malformations and deafness. Here we describe three novel mutations of the POU3F4 gene and their clinical characterizations in three Korean families carrying deafness segregating at the DFN3 locus. The three mutations cause a substitution (p.Arg329Pro) or a deletion (p.Ser310del) of highly conserved amino acid residues in the POU homeodomain or a truncation that eliminates both DNA-binding domains (p.Ala116fs). In an attempt to better understand the molecular mechanisms underlying their inner ear defects, we examined the behavior of the normal and mutant forms of the POU3F4 protein in C3H/10T1/2 mesodermal cells. Protein modeling as well as in vitro assays demonstrated that these mutations are detrimental to the tertiary structure of the POU3F4 protein and severely affect its ability to bind DNA. All three mutated POU3F4 proteins failed to transactivate expression of a reporter gene. In addition, all three failed to inhibit the transcriptional activity of wild-type proteins when both wild-type and mutant proteins were coexpressed. Since most of the mutations reported for DFN3 thus far are associated with regions that encode the DNA binding domains of POU3F4, our results strongly suggest that the deafness in DFN3 patients is largely due to the null function of POU3F4.

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 cells.

The third helix of the murine Hoxc8 homeodomain facilitates protein transduction in mammalian cells.

Previously, we have demonstrated that purified Hoxc8 homeoprotein has the ability to penetrate the cellular membrane and can be transduced efficiently into COS-7 cells. Moreover, the Hoxc8 protein is able to form a complex with DNA molecules in vitro and helps the DNA be delivered intracellularly, serving as a gene delivery vehicle. Here, we further analyzed the membrane transduction activity of Hoxc8 protein and provide the evidence that the 16 amino acid (a.a.191-206, 2.23 kDa) third helix of murine Hoxc8 protein is an efficient protein transduction domain (PTD). When the 16 amino acid peptide was fused at the carboxyl terminal of enhanced green fluorescence protein (EGFP), the fusion proteins were transduced efficiently into the primary pig fetal fibroblast cells. The transduction efficiency increased in a concentration-dependent manner up to 1 microM, and appeared to plateau above a concentration of 1 microM. When tandem multimers of PTD, EGFP-PTD(2), EGFP-PTD(3), EGFP-PTD(4), and EGFP-PTD(5), were analyzed at 500 nM of concentration, the penetrating efficiency increased in a dose-dependent manner. As the number of PTDs increased, the EGFP signal also increased, although the signal maintained plateau after EGFP-PTD(3). These results indicate that the 16 amino acid third helix is the key element responsible for the membrane transduction activity of Hoxc8 proteins, and further suggest that the small peptide could serve as a therapeutic delivery vehicle for large cargo proteins.

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.

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.

Identification of DC21 as a novel target gene counter-regulated by IL-12 and IL-4.

The Th1 vs. Th2 balance is critical for the maintenance of immune homeostasis. Therefore, the genes that are selectively-regulated by the Th1 and Th2 cytokines are likely to play an important role in the Th1 and Th2 immune responses. In order to search for and identify the novel target genes that are differentially regulated by the Th1/Th2 cytokines, the human PBMC mRNAs differentially expressed upon the stimulation with IL-4 or IL-12, were screened by employing the differential display polymerase chain reaction. Among a number of clones selected, DC21 was identified as a novel target gene that is regulated by IL-4 and IL-12. The DC21 gene expression was up-regulated either by IL-4 or IL-12, yet counterregulated by co-treatment with IL-4 and IL-12. DC21 is a dendritic cell protein with an unknown function. The sequence analysis and conserved-domain search revealed that it has two AU-rich motifs in the 3'UTR, which is a target site for the regulation of mRNA stability by cytokines, and that it belongs to the N-acetyltransferase family. The induction of DC21 by IL-12 peaked around 8-12 h, and lasted until 24 h. LY294002 and SB203580 significantly suppressed the IL-12-induced DC21 gene expression, which implies that PI3K and p38/JNK are involved in the IL-12 signal transduction pathway that leads to the DC21 expression. Furthermore, tissue blot data indicated that DC21 is highly expressed in tissues with specialized-resident macrophages, such as the lung, liver, kidney, and placenta. Together, these data suggest a possible role for DC21 in the differentiation and maturation of dendritic cells regulated by IL-4 and IL-12.

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.

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.

Membraneous localization of Jpk is not essential to exert cytotoxicity in F9 teratocarcinoma cells.

A novel gene, Jopock (Jpk), which was isolated as a trans-acting factor associating with the PSRE of murine Hoxa-7, has been shown to be toxic to both prokaryotic and eukaryotic cells when overexpressed. Here we demonstrate that the overexpression of enhanced green fluorescent protein (EGFP)-tagged Jpk in F9 cells results in the induction of apoptosis, as indicated by phosphatidylserine exposure, DNA fragmentation, and the alteration of mitochondria transmembrane potential. Fluorescence microscopy showed that EGFP-fused Jpk was mainly localized in the endoplasmic reticulum (ER) and a small amount was found in the mitochondria. Deletion mutants with a transmembrane (TM) domain showed a distribution similar to that of EGFP-Jpk, whereas constructs with a deletion of the TM domain localized in the whole cells. Deletion mapping experiments showed that Jpk with an N-terminal part deleted stimulated apoptosis to almost the same extent as that of the wild-type Jpk, indicating that the localization of Jpk in the ER and the TM domain does not appear to be essential for inducing cytotoxicity. Overall, these results suggest that Jpk, particularly the C-terminal part of Jpk and/or 3'UTR, triggers apoptosis through a perturbation of mitochondrial membrane permeabilization.