Cooperative interactions between PBX, PREP, and HOX proteins modulate the activity of the alpha 2(V) collagen (COL5A2) promoter

Cell type-specific expression of the human (alpha 2(V) collagen (COL5A2) ge ne depends on a cis-acting element that consists of two contiguous protein binding sites (FPA and FPB) located between nucleotides -149 and -95, relat ive to the transcription start site. The present study focused on the chara cterization of the FPB-bound complex. DNA binding assays and cell transfect ion experiments revealed that the bipartite core sequence of FPB (5'-ATCAAT CA-3') binds the PBX1/2, PREP1, and HOXB1 proteins, and this in turn leads to promoter transactivation. In the presence of all three nuclear factors, cooperative interactions between recombinant PBX1 and PREP1 or PBX1 and HOX B1 result in binding of the heterodimers to FPB in vitro. Similarly, overex pression of different combinations of PBX1, PREP1, and HOXB1 transactivates FPB driven transcription. In contrast to the composition of the FPB comple x purified from COL5A2-positive cells, the FPB complex from COL5A2-negative cells contains PBX2 and PREP1 but lacks PBX1. However, PBX1 exogenously in troduced into COL5A2-negative cells cannot stimulate FPB-driven transcripti on unless co-expressed with PREP1. Within the intrinsic limitations of the experimental model, our results indicate that combinatorial interactions am ong PBX and PREP or HOX proteins are involved in regulating tissue-specific production of collagen V.