TRANSCRIPTIONAL REGULATION OF A MOUSE CLARA-CELL-SPECIFIC PROTEIN (MCC10) GENE BY THE NKX TRANSCRIPTION FACTOR FAMILY MEMBERS THYROID TRANSCRIPTION FACTOR-1 AND CARDIAC MUSCLE-SPECIFIC HOMEOBOX PROTEIN (CSX)

This report defines the elements between bp -800 and -166 that regulat e the quantitative level of mouse CC10 (mCC10) transcription in the lu ngs, The elements in this promoter domain are the response elements fo r the NKx 2.1 homeobox protein, thyroid transcription factor 1 (TTF1), DNase I footprint analysis identified five binding sites for TTF1 bet ween bp -800 and -166, These sites are located at bp -344 to -335, -28 2 to -273, -268 to -263, -258 to -249, and -199 to -190, In addition t o these enhancer elements, two TTF1 binding sites were identified in t he proximal promoter region (bp -166 to +1), at bp -74 to -69 and -49 to -39, An identical footprint of the mCC10 promoter region was also o bserved,vith another member of the NKx family, NKx 2.5, the cardiac mu scle-specific homeobox protein (CSX), Deletion and linker-scanner muta tional analyses of the TTF1 binding sites in the mCC10 distal promoter region with transient cotransfection into CV1 cells with either TTF1 or CSX identified the site located between bp -282 and -273 as the maj or regulator of CC10 expression, with minor regulation by sites at bp -344 to -335 and -258 to -249, The importance of the NKx binding site at bp -282 to -273 was verified in vivo, Transgenic mice generated wit h the human growth hormone gene fused to 800 bp of the mCC10 promoter containing a mutation in the TTF1 binding site at bp -282 to -273 show ed a reduction in transgene expression equal to that of the mice gener ated with only 166 bp of 5'-flanking DNA, This report emphasizes the i mportance of TTF1 or related factors as major regulators of pulmonary gene expression and demonstrates the potential of NKx proteins to bind and activate heterologous target genes.