The Homeodomain Protein Barx2 Contains Activator and Repressor Domains and Interacts with Members of the CREB Family

Abstract

Barx1 and Barx2 are homeodomain proteins originally identified using regulatory elements of genes encoding certain cell adhesion molecules (CAMs). In the present study, we characterize regions of Barx2 that bind to regulatory elements of genes encoding three CAMs, L1, neuron-glia CAM (Ng-CAM), and neural CAM (N-CAM), and identify domains of Barx2 that regulate N-CAM transcription. The homeodomain of Barx2 was sufficient for binding to homeodomain binding sites (HBS) from all three CAM genes. The presence of a 17-amino acid Barx basic region resulted in a 2-fold decrease in binding to HBS sequences from the Ng-CAM and L1 genes, whereas it led to a 6.5-fold increase in binding to the HBS from the N-CAM promoter. Thus, the Barx basic region influences the strength and specificity of Barx2 binding to DNA. In co-transfection experiments, Barx2 repressed N-CAM promoter activity. A 24-residue N-terminal region of Barx2 was essential for repression. When this region was absent, Barx2 activated the N-CAM promoter. A 63-residue C-terminal domain was required for this activation. In GST pull-down experiments, Barx2 bound to proteins of the CREB family, CREB1 and ATF2. Overall, these findings provide a framework for understanding developmental and physiological contexts that influence repressor or activator functions of Barx2.

Identification of Novel Binding Elements and Gene Targets for the Homeodomain Protein BARX2

“…All probes were of comparable specific activity. EMSAs used 25,000 cpm of each probe and in vitro translated proteins, as described previously (3). Relative binding of the protein to the HBS probes was determined by measuring the intensity of the probe/protein complexes using a PhosphorImager (Molecular Dynamics).…”

“…Fragments-Like most homeodomain proteins, BARX2 binds to motifs that contain the core sequence, TAAT (3,6,19) however, further requirements for BARX2 binding have not been well defined. Earlier studies showed that BARX2 bound with high affinity to the HBS element from the murine Ncam1 promoter (3).…”

“…In most cases, the identities of the target genes that link homeodomain proteins to these processes have not been elucidated. Previous work characterized the role of homeodomain and paired domain transcription factors in the temporal and spatial regulation of cell adhesion molecules (CAMs) 1 (3)(4)(5). A Southwestern screen to identify proteins that bound to a homeobox recognition motif from the chicken Ng-CAM gene, led to the discovery of the murine homeodomain protein, Barx2 (6).…”

“…2,3 Our recent studies indicate that Barx2 influences cellular processes that control cell adhesion and remodeling of the actin cytoskeleton seen in myoblast fusion (10), and chondrogenesis. 3 Human BARX2 has also been described as a tumor suppressor gene; loss of BARX2 expression correlates with invasiveness in ovarian cancer, and ectopic expression of BARX2 inhibits the invasiveness of an ovarian cancer cell line (11).…”

“…A Southwestern screen to identify proteins that bound to a homeobox recognition motif from the chicken Ng-CAM gene, led to the discovery of the murine homeodomain protein, Barx2 (6). Related binding sites for BARX2 were subsequently identified in the mouse L1cam and Ncam1 genes, and these sites were shown in transgenic mice to be required for the native pattern of gene expression (3,(5)(6)(7).…”