Based upon previous studies defining a specific role for the HNF-3 factors in CCSP gene expression and the evidence that additional factors are required for regulation of CCSP gene expression, we hypothesized that other winged helix/forkhead factors may be involved in regulating gene expression and cellular differentiation in the bronchiolar epithelium. Consistent with this hypothesis, a winged helix/forkhead sequence termed hepatocyte nuclear factor/forkhead homologue 4 (HFH-4) is expressed in the lung but has not been characterized in terms of cell-specific or developmental expression (12). In this study, we have isolated the full-length cDNA encoding HFH-4 and defined the pattern of tissue-and cell-specific expression during development in the rat and mouse.
MATERIALS AND METHODSIsolation and Characterization of Rat HFH-4. Oligonucleotides corresponding to the 5' (5'-AGCCACCCTACTCCTA-TGCCAC-3') and 3' (5'-GCAGCCTCCGCTTCTTGAAG-3') sequences of the murine HFH-4 forkhead domain (12) were used to PCR-amplify a 284-bp fragment from a rat lung cDNA library (Clontech). Sequence analysis confirmed that the isolated fragment encoded the rat homologue of the murine HFH-4 forkhead domain. After initial RNA blot analysis indicated that HFH-4 transcript was present in lung and testis, rat lung and testis cDNA libraries (Clontech) were screened at high stringency with a random-primed [32P]cDNA probe derived from the rat HFH-4 forkhead domain to obtain full-length clones encoding rat HFH-4. The nucleotide sequence of each clone was determined and the full-length sequence was determined from a series of overlapping clones.RNA Isolation and RNA Blot Analysis. Organs were isolated from rats or mice, rinsed in saline, and quick frozen in liquid nitrogen. Total RNA was isolated by dissolution of tissue in guanidinium isothiocyanate followed by cesium chloride density gradient centrifugation (13). RNA samples were denatured, electrophoresed, and transferred to nylon (Hybond-N, Amersham) as described (10). Abbreviations: CCSP, Clara cell secretory protein; HFH, hepatocyte nuclear factor/forkhead homologue; E, embryonic day; cRNA, complementary RNA.
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