The identification of <em>Hoxc8</em> target genes

Abstract

Hox genes regulate anterior/posterior developmental patterning in an extensive domain, extending from the midbrain/hindbrain junction to the tail. The individual genes are expressed in an overlapping array, each regulating differentiation and morphogenesis in their individual expression domains along the anterior/posterior axis. The Hoxc8 gene has been studied in considerable detail by us and others (1–4). Expression analysis shows that the gene is expressed initially at 8 days postconception (dpc) in the tail bud and then extends to an anterior position at the level of the forelimbs. A posterior limit of expression is defined later at the junction between the thoracic and lumbar regions. The gene is expressed in both the neural tube and the somites in the prospective thorax (5–7). Null mutants of Hoxc8 show neuromuscular defects in the forelimb and skeletal defects in the ribs and vertebrae of the thorax (8). We have shown recently that a retardation of Hoxc8 expression results in the phenocopy of Hoxc8-null mutations, demonstrating the criticality of expression timing for the Hox transcription factors (9).

The tissue-specific overexpression of Hoxc8 has been shown to inhibit chondrocyte maturation and stimulate chondrocyte proliferation (10). Bone morphogenetic protein (BMP) is a potent osteotropic protein that induces osteoblast differentiation and bone formation. Hox-binding elements (ATTA) are common in promoters of osteoblast differentiation marker genes, especially those that rapidly respond to BMP stimulation, such as osteoprotegerin, BMP-4, and osteonectin (11–14). Gel retardation studies have shown that Hoxc8 binds to ATTA-rich sites in the osteopontin (OPN) promoter domain (15), but evidence for functional interaction in vivo is lacking. BMP stimulation activates gene transcription by depressing Hoxc8 protein through the interaction of Smad1 and Hoxc8 proteins. These results suggest that direct interaction between Smad1 and Hoxc8 proteins represents a major mechanism of osteoblast differentiation in BMP-induced skeletal development (15).

Hox gene deregulation is implicated in human cancers including leukemia and colorectal, breast, renal, and lung cancers (16–21). Expression of Hoxc8 correlates with higher Gleason scores in prostate cancers (22). Hoxc8 is selectively activated in cervical cancer cells (23). Hematopoietic progenitor cells show abnormalities in Hoxc8-null mutant mice (24). However, it is not clear how Hox gene deregulation specifically effects neoplastic inception and progression. Few studies have established direct functional roles for Hox genes in carcinogenesis.

In the present study, we overexpressed mouse Hoxc8 gene in the C57BL/6J mouse embryo fibroblasts (MEF) cells and then applied microarray assay to identify possible Hoxc8 target genes. Expression of candidate genes was also examined by semiquantitative RT-PCR, and these data correlated well with the array data. Chromatin immunoprecipitation (ChIP) assay confirmed that OPN is a direct transcriptional target of Hoxc8 in vivo. Most of the 34 identified candidate target genes are involved in proliferation, adhesion, migration, metabolism, and related cellular processes, and can be viewed as global regulators of growth and differentiation (25). In general, our results suggest that Hox genes may play important roles in cancer progression by serving as modulators in neoplastic pathways.

Acknowledgments

Notes

Abbreviations: dpc, days postconception; BMP, bone morphogenetic protein; OPN, osteopontin; MEF, mouse embryo fibroblast; ChIP, chromatin immunoprecipitation; Fzd2, frizzled homolog 2.