The mechanisms that regulate gene expression during the transition from stem cell to transient amplifying cell to differentiated cell are not well understood. hINV expression is increased during this transition. A major goal of this study was to assess the role of AP1 factors in this regulation. Studies have shown that the hINV promoter distal regulatory region (DRR) encodes several potential transcriptional regulatory elements.
28 These include an AP1 factor–binding site, AP1-5. We began by performing experiments designed to assess the in vivo role of the AP1-5 site. For these studies, hINV promoter transgenic mice, including hINV H6B, hINV H6B(AP1-5mm) and hINV P3.4B, were constructed. hINV H6B encodes the full-length hINV promoter linked in its native context to the hINV protein coding sequences.
35 H6B(AP1-5mm) is identical with H6B, except that the AP1-5 site is mutated
(Fig. 1A) . P3.4B encodes the hINV gene minimal promoter. The authentic and mutated AP1-5 sequences are indicated in
Figure 1B . Each construct retains the authentic relationship between the upstream regulatory region (nucleotides −2473/−1), the hINV transcription start site (
Fig. 1A , arrow), and the hINV gene coding sequence (hINV box). Six independent transgenic lines were established for each construct.
Figure 1Cshows an immunoblot analysis of hINV protein content in punch biopsy tissue derived from the central corneal epithelium of mice representing two representative transgenic lines for each construct. Transgenic mice harboring H6B, the full-length transgene, express high levels of hINV in the central corneal epithelium. In contrast, no hINV was detected in mice harboring hINV H6B(AP1-5mm), in which the AP1-5 site was mutated. As a control, we also monitored corneal epithelial hINV content in mice harboring hINV P3.4B, a transgene that encodes only the hINV minimal promoter (nucleotides −41/−1). No expression was detected. To assess the status of expression as a function of corneal epithelial differentiation, we treated sections derived from central cornea epithelium with anti-hINV. As shown in
Figure 1D , hINV was expressed in the central cornea upper epithelial layers and also in selected basal cells. In contrast, and consistent with the immunoblot data
(Fig. 1C) , no hINV was detected in comparable sections prepared from hINV H6B(AP1-5mm) or hINV P3.4B mice.