Several neuron-specific genes placed in the AT-rich genomic environment are regulated by DNA topoisomerase Ⅱbeta (topo Ⅱbeta).HnRNP U/SAF-A/SP120 is a multifunctional abundant nuclear protein that directly binds to DNA and RNA.We have identified SP 120 as an AT-rich Matrix attachment region (MAR)-binding protein in a nuclear matrix fraction.As SP 120 forms a complex with topo Ⅱbeta, SP120 may assist topo Ⅱbeta to target the neuron-specific genes through its DNA-binding.The N-terminal domain termed SAF-box or SAP domain is required for DNA binding and the C-terminal domain called RGG-box is essential for the interaction with RNA.We and others have reported that SP120 specifically and cooperatively binds AT-rich MAR although the precise mechanism is not known.To understand the mechanism, we constructed several mutant proteins of SP120 and analyzed their DNA-binding activities and specificities for AT-rich MAR.The results showed that a C-terminal domain enriched with Arg-Gly (RG-domain) was predominantly important for the MAR-specific binding activity.RG-domain directory bound to MAR and the binding was inhibited by netropsin, a minor groove binder with AT pair preference.Unexpectedly, however, netropsin stimulated the binding of SAF-box to MAR.These data suggest that SAF-box and RG-domain associate with duplex DNA on opposite surfaces probably by a cooperative mechanism to achieve the MAR-specific binding of SP 120.