ARID domain of H3K4 demethylase RBP2 binds to GC rich DNA

Abstract

In an effort to identify human trithorax response elements (TREs) and study transcription regulation by histone H3K4 demethylase RBP2, a trithorax group protein, we studied RBP2 by DNA binding, solution NMR, immunofluorescence, and reporter assays. In this study, PCR‐assisted DNA binding selection revealed that RBP2 DNA binding domain ARID selectively binds to GC rich DNA. Subsequent electrophoretic mobility‐shift assays verified RBP2 ARID binds to BRD2 promoter via a consensus G/C run motif. Solution NMR structure of RBP2 ARID showed it had 6 helices and 2 loops. Further NMR titration on ARID‐DNA complex identified that the binding interface is consisted of loop 1, helix 4, loop 2, and helix 5. Importantly, we found loop 2 contributes significantly to DNA binding affinity. One residue in loop 2 could be used to predict ARID DNA binding specificity. Moreover, in vivo immnofluorescence and reporter assays indicated that ARID is necessary for proper RBP2 H3K4 demethylase activity. In summary, the RBP2 ARID has regulatory roles and its consensus motif identified here is one of the first human TRE motifs. Funds from NIH and Academia Sinica