ANALYSIS OF DNA-BINDING PROTEINS ASSOCIATED WITH HEMIN-INDUCED TRANSCRIPTIONAL INHIBITION - THE HEMIN RESPONSE ELEMENT-BINDING PROTEIN IS AHETEROGENEOUS COMPLEX THAT INCLUDES THE KU PROTEIN

Hemin inhibits transcription of the tartrate resistant acid phosphatas e (TRAP) gene. Using deletion mutagenesis of the mouse TRAP 5'-flankin g region, we previously identified a 27-bp DNA segment containing a ce ntral GAGGC tandem repeat sequence (the hemin response element [HRE]), which bound nuclear proteins (hemin response element binding proteins [HREBPs]) from hemin-treated cells and appeared to be responsible for mediating transcriptional inhibition in response to hemin. We now hav e used affinity binding to HRE-derivatized beads to identify four HREB P components with apparent molecular masses of 133-, 90-, 80-, and 37- kD, respectively, The 80- and 90-kD components correspond to the p70 a nd p80/86 subunits of Ku antigen (KuAg) as documented by partial amino acid microsequencing of tryptic digests and immunologic reactivity. B ased on reactivity of the HREBP gel shift band with antibodies to the redox factor protein (ref1) in shift Western experiments, it is shown that the 37-kD component represents ref1. The 133-kD component appeare d to be a unique protein. KuAg participation in HREBP complexes was sp ecific as it was present in HREBPs bound to HRE microcircles. Results of depletion/reconstitution experiments suggested that KuAg does not b ind alone or directly to HRE DNA, but does so only in conjunction with the 133- and/or 37-kD proteins. We conclude that HREBP is a heterogen eous complex composed of KuAg, ref1, and a unique 133-kD protein. We s peculate that the role of heme may be to promote interactions among th ese components, thereby facilitating HRE binding and downregulation of hemin responsive genes. (C) 1998 by The American Society of Hematolog y.