The GHR is essential for the actions of GH on growth and metabolism. We had previously identified and partially characterized a 42-bp regulatory element in the 5' -flanking region of the L1 transcript of the murine GHR gene that interacted with both double (DSBP) and single- strand (SSBP) DNA binding proteins. Indirect evidence suggested that the SSBP functions as a repressor of GHR gene transcription. Our recent efforts were directed towards establishing the identity of these regulatory proteins. To determine the identity of the SSBP we used a Southwestern technique to screen a cDNA expression library. A single cDNA clone that specifically interacted with the coding strand of the enhancer element was thus identified. DNA sequencing of the entire 1.5 KB insert established that the clone coded for a protein termed MSY-1, a DNA/RNA-binding protein that is evolutionarily conserved from prokaryotes to eukaryotes. Translation of the 1.5kb cDNA insert resulted in a protein with an apparent molecular weight of 31-35 kDa on PAGE. An array of experiments including supershift electromobility shift assay (EMSA), Southwestern blot analysis and transient transfection analysis confirmed the identity of the SSBP binding to the regulatory element of the GHR gene to be MSY-1. Consistent with our previous findings, transient co-transfection experiments indicated that MSY-1 functions as a repressor of GHR gene transcription. Inhibition of binding of the DSBP to the minor groove of the DNA double-helix by distamycin altered the DNA binding of MSY-1, indicating that these two proteins conjointly regulate expression of the GHR gene. MSY-1 represents the first transcription factor identified to regulate expression of the GHR gene. Because the L1-GHR transcript is expressed in the liver only during pregnancy, we postulate that MSY-1 may be important in the up-regulation of hepatic GHR gene expression during pregnancy and are currently investigating this hypothesis.
