Identification and analysis of Hoxc8 downstream target genes during mouse development

Abstract

[한글]

[영문]Hox genes are transcription factors that control patterns along the anterior-posterior body axis and perform important functions in the regulation of gene expression during embryogenesis in vertebrates. Hox proteins are expressed in spatiotemporal way to form body pattern specific places (or regions). These Hox proteins are evolutionary conserved and harbor a helix-turn-helix DNA binding motif that binds to specific DNA sequences within their target genes. Therefore, Hox proteins regulate target gene expression during embryogenesis by functioning either as activators or repressors. However, the target genes downstream of Hoxc8 and their physiological significance remain to be clearly elucidated. In this study, 12 putative downstream target genes of Hoxc8 were identified via the ChIP (Chromatin ImmunoPrecipitation)-Cloning method. This technique is based on the specific binding of a protein directly to its target genes. We employed E11.5 mouse embryos in which Hoxc8 was expressed at rather high levels and isolated naive chromatin; following immunoprecipitation with specific Hoxc8 antibody, some plausible downstream target genes of Hoxc8 were identified. The zinc finger protein 804a (Zfp804a; NM_175513) gene, a strong candidate protein and a critical downstream target of Hoxc8, is known to harbor a large number of Hox binding sites (TAAT/ATTA/TTAT/ATAA). In this study, we confirmed the binding capability of the intronic region of Zfp804a with Hoxc8 in mouse E11.5 embryos in vivo via ChIP-PCR, and determined whether or not Hoxc8 overexpression induced Zfp804a expression in vitro. Additionally, Zfp804a and Hoxc8 were found to have been co-expressed not only in E11.5 mouse embryos; Hoxc8 was also overexpressed in an F9 embryonic teratocarcinoma cell line. Hoxc8 upregulated Zfp804a mRNA levels and increased minimal promoter activity in vitro, according to the results of a dual luciferase assay. Recently, Zfp804a (or ZNF804a in humans) was associated with susceptibility to schizophrenia, according to the results of a genome-wide association (GWA) study. Therefore, we hypothesize this embryogenic regulatory control is mediated by the binding of the Hoxc8 protein to the first intron sequence of Zfp804a, which may also affect the adult mouse brain during the expression of the Zfp804a gene. Collectively, the results of this study demonstrate that Zfp804a is one of the downstream target genes of Hoxc8 in the mouse embryo and the adult mouse brain.