Homeobox genes play key roles in specifying body part identity during vertebrate embryonic development. Retinoids are signaling molecules involved in the regulation of expression of homeobox genes. We have previously identified an retinoic acid (RA)-inducible enhancer (RAIDR5) located approximately 6.5 kb 3' of the coding region of the murine Hoxb1 gene. This 3' enhancer contains three sequences that are highly conserved in similar RA-inducible enhancers identified in the murine and human Hoxa1 genes and in the chicken Hoxb1 gene. One element, a DR5 RA response element, contributes to the RA inducibility of a Hoxb1 reporter gene construct in F9 cells. In this report, further analysis of the other two elements of the Hoxb1 3' enhancer is reported. The two other sequences, conserved element (CE) 1 and CE2, act as negative elements in cultured F9 cells; when either is mutated, an increase in the beta-galactosidase activity of a Hoxb1 reporter gene construct results. A single Hoxb1 CE2 DNA element:protein binding complex was detected in F9 stem cells, and experiments suggest that this is the same binding protein that recognizes the CE2 element of Hoxa1. In a variant F9 cell line in which both allelic copies of the RA receptor gamma (RARgamma) gene are disrupted, the CE2 binding complex is absent, and this absence correlates with the inability of the CE2 element to function as a repressor of Hoxb1 reporter gene expression in these cells. A single Hoxb1 CE1 binding complex is also detected by gel shift assays in nuclear extracts prepared from both stem and RA-treated F9 cells. This complex contains an Mr approximately 200,000 protein as shown by UV cross-linking. Although the sequences of the CE1 elements of Hoxb1 and Hoxa1 are highly conserved, they differ by two nucleotides. Gel shift analysis shows that either of these nucleotide changes prevents binding of F9 cell protein extracts. When gel shift assays were performed using nuclear extracts prepared from mouse embryos at a time when Hoxb1 mRNA is expressed, i.e., day 9.0, CE1 and CE2 binding complexes identical in mobility to those detected in F9 cells were observed. This suggests roles for both the CE1 and CE2 elements in regulating Hoxb1 gene expression during development.
