Transcriptional regulation of thyroid transcription factor-1 (TTF-1) (vol 93, pg A 14, 1999)

TTF-1 is a regulator of lung-specific genes such as surfactant proteins A, B and C and Clara cell secretory protein. It is also vital for the normal d evelopment of the lungs, thyroid and parts of the forebrain. TTF-1 gene kno ckout mice die at birth with primitive lung buds containing no differentiat ed epithelium. The aim of this work is to investigate the transcriptional regulation of th e TTF-1 gene in particular high order regulatory sequences distal to the pr oximal promoter. DNase I hypersensitive (HS) sites represent a marker for o pen and potentially active chromatin and are likely to be of particular imp ortance in gene regulation, being associated with many DNA sequences that r egulate gene expression. We have performed DNase I hypersensitivity assays to locate HS sites in a 2 4kb DNA region containing the 3kb TTF-1 gene. Five DNase 1 HS sites have be en identified. There is one intragenic site and four sites upstream of the TTF-1 transcription start site. Transgenic mice have been generated using a 70kb subclone of a 120kb CYPAC clone containing the human TTF-1 gene. Tran sgenic status was confirmed by PCR on tail DNA and Southern bolts, using a TTF-1 probe. RNA expression of the TTF-1 transgene was detected by RT-PCR w ith a radiolabelled PCR primer. Human TTF-1 was expressed in the lungs and brains of adult transgenic mice. The functional significance of the HS sites will be assessed in cell cultur e and transgenic animal systems. If the identified control elements could b e condensed into a suitable gene delivery vector, such as those based on ad enoviruses, the system could potentially provide the mechanism for regulate d, lung-specific gene expression for gene therapy purposes.