UBIQUITOUS AND NEURONAL DNA-BINDING PROTEINS INTERACT WITH A NEGATIVEREGULATORY ELEMENT OF THE HUMAN HYPOXANTHINE PHOSPHORIBOSYLTRANSFERASE GENE

The hypoxanthine phosphoribosyltransferase (HPRT) gene is constitutive ly expressed at low levels in all tissues but at higher levels in the brain; the significance and mechanism of this differential expression are unknown. We previously identified a 182-bp element (hHPRT-NE) with in the 5'-flanking region of the human HPRT (hHPRT) gene, which is inv olved not only in conferring neuronal specificity but also in repressi ng gene expression in nonneuronal tissues. Here we report that this el ement interacts with different nuclear proteins, some of which are pre sent specifically in neuronal cells (complex I) and others of which ar e present in cells showing constitutive expression of the gene (comple x II). In addition, we found that complex I factors are expressed in h uman NT2/D1 cells following induction of neuronal differentiation by r etinoic acid. This finding correlates with an increase of HPRT gene tr anscription following neuronal differentiation. We also mapped the bin ding sites for both complexes to a 60-bp region (Ff; positions -510 to -451) which, when analyzed in transfection assays, functioned as a re pressor element analogous to the full-length hHPRT-NE sequence. Methyl ation interference footprintings revealed a minimal unique DNA motif, 5'-GGAAGCC-3', as the binding site for nuclear proteins from both neur onal and nonneuronal sources, However, site-directed mutagenesis of th e footprinted region indicated that different nucleotides are essentia l for the associations of these two complexes. Moreover, UV cross-link ing experiments showed that both complexes are formed by the associati on of several different proteins, Taken together, these data suggest t hat differential interaction of DNA-binding factors with this regulato ry element plays a crucial role in the brain-preferential expression o f the gene, and they should lead to the isolation of transcriptional r egulators important in neuronal expression of the HPRT gene.