CHARACTERIZATION OF 2 DISTINCT FAMILIES OF TRANSCRIPTION FACTORS THATBIND TO THE CCAAT BOX REGION OF THE HUMAN COL1A2 GENE

Both the mouse and human alpha 2(I) procollagen promoters contain an i nverted CCAAT box at -80, but only the human promoter contains an addi tional regulatory element the collagen modulating element (CME)I immed iately downstream of the CCAAT box [Collins et at. (1997): Biochem J 3 22:199-206]. In this study, the transcription factors that bind to the G/CBE and CME within the human promoter were characterized in SVWI-38 and CT-1 nuclear extracts. Two distinct proteins bind to the CME, and both were identified as heat-labile factors that were sensitive to hi gh ionic strengths and required Zn2+ for DNA-binding activity. These p roteins had Stokes radii of 4.12 and 3.15 nm, sedimentation coefficien ts of 3.9 and 3.2 S and native molecular weights of 66 and 41 kDa, res pectively. On the basis of biochemical and DNA-binding properties, the CME binding proteins are probably novel factors involved in the regul ation of the human alpha 2(I) procollagen gene. By contrast, the C/CBE binding proteins were more resistant to heat, ionic strength, and div alent metal ion chelators, demonstrating that the C/CBE and CME bindin g proteins had distinct DNA-binding properties. The above properties s uggest that this factor is a member of the previously characterized fa mily of CCAAT box-binding factors, CBF, NF-Y, CP-1 and alpha-CP1. Take n together, these physicochemical properties of the COL1A2 CCAAT box a nd CME-binding proteins demonstrated that they were distinct unrelated transcription factors. These results also suggest that there is a dis tinct difference in the DNA-binding activity between the equivalent re gion of the mouse and human alpha 2(I) procollagen promoters. J. Cell. Biochem. 70:455-467, 1998. (C) 1998 Wiley-Liss, Inc.