Identification of an upstream enhancer in the mouse laminin α1 gene defining its high level of expression in parietal endoderm cells

Laminin-1 is the major component of the embryonic basement membrane and consists of alpha1, beta1, and gammal chains. The expression of laminin-1 is induced in mouse F9 embryonal carcinoma cells upon differentiation into parietal endoderm through transcriptional up-regulation of the genes encoding these subunits. Here, we identified a 435-bp enhancer in the 5'-flanking region of the mouse laminin alpha1 (LAMA1) gene that activated its transcription in a differentiation-dependent manner. This enhancer was also active in PYS-2 parietal yolk sac-derived cells but not in NIH/3T3 fibroblasts, indicating that it was a parietal endoderm-specific enhancer. This enhancer was also active in Engelbreth-Holm-Swarm (EHS) tumor-derived cells characterized by excessive production of laminin-1 and other basement membrane components, suggesting that EHS tumors have a transcriptional control mechanism similar to that of parietal endoderm cells. Electrophoretic mobility shift analyses revealed four protein binding sites (PBS1-PBS4) in the 435-bp region. However, these DNA-binding proteins were detected not only in parietal endoderm cells (i.e. differentiated F9 cells, PYS-2 cells, and EHS tumor-derived cells) but also in undifferentiated F9 cells and NIH/3T3 cells. Mutational analyses revealed that three of these binding sites (PBS2, PBS3, and PBS4) function synergistically to confer the parietal endoderm-specific enhancer activity. The proteins binding to PBS2 and PBS4 were identified as the Sp1/Sp3 family of transcription factors and YY1, respectively.