• Biomedical Science Letters
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• v.17 no.1
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• pp.1-5
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• 2011

Previously, we have identified 14 putative downstream target genes of Hoxc8 homeoprotein in F9 murine embryonic teratocarcinoma cells through proteomics analysis. Among those, we tested a possibility of a DNA-k type molecular chaperone, Grp78, as a direct downstream target of Hoxc8, by cloning a 2.4 kb upstream region of murine Grp78 into a reporter plasmid and by testing if Hoxc8 can regulate its expression. We observed that Hoxc8 proteins could transactivate the reporter gene, which was affected by small interference RNAs (siRNAs) against to Hoxc8, suggesting that Grp78 is a novel downstream target of Hoxc8 in vivo.

• Biomedical Science Letters
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• v.13 no.3
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• pp.239-244
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• 2007

Hoxc8 is one of the homeotic developmental control genes regulating the expression of many downstream target genes, through which animal body pattern is established during embryonic development. In previous proteomics analysis, proliferating cell nuclear antigen (PCNA) which is also known as cyclin, has been implied to be regulated by Hoxc8 in F9 murine embryonic teratocarcinoma cell. When the 5' upstream region of PCNA was analyzed, it turned out to contain 20 Hox core binding sites (ATTA) in about 1.17 kbp (kilo base pairs) region ($-520{\sim}-1690$). In order to test whether this region is responsible for Hoxc8 regulation, the upstream 2.3 kbp fragment of PCNA was amplified through PCR and then cloned into the pGL3 basic vector containing a luciferase gene as a reporter. When the luciferase activity was measured in the presence of effector plasmid (pcDNA : c8) expressing murine Hoxc8, the PCNA promoter driven reporter activity was reduced. To confirm whether this reduction is due to the Hoxc8 protein, the siRNA against Hoxc8 (5'-GUA UCA GAC CUU GGA ACU A-3' and 5'-UAG UUC CAA GGU CUG AUA C-3') was prepared. Interestingly enough, siRNA treatment up regulated the luciferase activity which was down regulated by Hoxc8, indicating that Hoxc8 indeed regulates the expression of PCNA, in particular, down regulation in NIN3T3 cells. These results altogether indicate that Hoxc8 might orchestrate the pattern formation by regulating PCNA which is one of the important proteins involved in several processes such as DNA replication and methylation, chromatin remodeling, cell cycle regulation, differentiation, as well as programmed cell death.

• Molecules and Cells
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• v.39 no.5
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• pp.395-402
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• 2016

Identifying Hoxc8 target genes is at the crux of understanding the Hoxc8-mediated regulatory networks underlying its roles during development. However, identification of these genes remains difficult due to intrinsic factors of Hoxc8, such as low DNA binding specificity, context-dependent regulation, and unknown cofactors. Therefore, as an alternative, the present study attempted to test whether the roles of Hoxc8 could be inferred by simply analyzing genes frequently coexpressed with Hoxc8, and whether these genes include putative target genes. Using archived gene expression datasets in which Hoxc8 was differentially expressed, we identified a total of 567 genes that were positively coexpressed with Hoxc8 in at least four out of eight datasets. Among these, 23 genes were coexpressed in six datasets. Gene sets associated with extracellular matrix and cell adhesion were most significantly enriched, followed by gene sets for skeletal system development, morphogenesis, cell motility, and transcriptional regulation. In particular, transcriptional regulators, including paralogs of Hoxc8, known Hox co-factors, and transcriptional remodeling factors were enriched. We randomly selected Adam19, Ptpn13, Prkd1, Tgfbi, and Aldh1a3, and validated their coexpression in mouse embryonic tissues and cell lines following $TGF-{\beta}2$ treatment or ectopic Hoxc8 expression. Except for Aldh1a3, all genes showed concordant expression with that of Hoxc8, suggesting that the coexpressed genes might include direct or indirect target genes. Collectively, we suggest that the coexpressed genes provide a resource for constructing Hoxc8-mediated regulatory networks.

• Biomedical Science Letters
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• v.14 no.1
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• pp.47-53
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• 2008

Hox genes are known to be transcription factors controlling vertebrate pattern formation along the anteroposterior body axis by regulating many target gene expressions during vertebrate embryogenesis. In order to isolate in vivo Hox responsive target genes, ChIP-cloning technique has been applied using Hoxc8 antibody. Here murine embryo of day 11.5 post coitum (E11.5) highly expressing Hoxc8 gene was used after removing head and tail portions where Hoxc8 is rarely expressing. After fixation with formaldehyde, the chromatin DNAs harboring bound proteins were isolated. After sonication, about 0.5- to 1 Kb chromatin DNAs were immunoprecipitated with anti Hoxc8 antibody. After removing the bound proteins with proteinase K, DNAs were isolated, cloned into the pBluescsript II SK vector, and then sequenced. Total 33 random clones sequenced were anlalyzed to be located at 12 different genomic regions. Among these, 8 turned out to be introns and 4 were intergenic regions localized in random chromosomes. The base composition of total cloned genomic sequences (6608 bp) were AT-rich, i.e., 40% GC. When the Hoxc8 core binding sites, such as TAAT, ATTA, TTAT, and ATAA were analyzed total number of 55, 45, 54, and 55 were found, respectively, which are than twice as many as expected number of 26. Although this in silico analysis does not mean that the ChIP-cloned sequence is real Hoxc8 regulatory element in vivo, these results strongly imply that the DNA fragments cloned through chromatin immunoprecipitation could be very much likely the putative Hoxc8 downstream target genes.

• Biomedical Science Letters
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• v.11 no.3
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• pp.311-318
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• 2005

Grp78, discovered as one of the putative target genes of Hoxc8, is a highly conserved stress protein and functions as a molecular chaperone in the endoplasmic reticulum (ER). In order to see the stage-specific expression pattern of Grp78 during development, mouse embryos from day 7.5 to 17.5 p.c. were isolated, and RT-PCR as well as in situ hybridization was performed. When RT-PCR was performed using Grp78 specific primers, periodic expression pattern was detected. And also a region-specific expression pattern was detected with a strong expression in the trunk part of day 11.5 p.c. embryo, like that of Hoxc8. When in situ hybridization was performed, Grp78 was revealed to be expressed in the endoderm, somite, neuroepithelium cells of neural tube in early embryos. In the case of late embryos, Grp78 expression was detected in the liver, segmental bronchus within cranial lobe of lung, ossification center within the cartilage primordium of rib and vertebra, submandibular gland, as well as metanephros. These expression patterns are very much similar to those of Hoxc8. Since Hoxc8 has been reported to regulate apoptosis during organogenesis, it might be possible that the apoptotic function could have been conveyed through the expression of Grp78, implying that the Grp78 is one of the Hoxc8 downstream target genes.

• Proceedings of the Zoological Society Korea Conference
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• 1999.04a
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• pp.63.2-63
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• 1999

No Abstract, See Full Text

• Molecules and Cells
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• v.40 no.4
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• pp.314-314
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• 2017