REST is a key regulator in brain-specific homeobox gene expression during neuronal differentiation

Brain-specific homeobox (Bsx) is specifically expressed at the early embryonic stages during brain development. Several studies show that Bsx plays important roles in brain development; however, the mechanisms of its transcriptional regulation remain to be established. In this study, we show that binding of repressor element silencing transcription factor (REST) to the neuron restrictive silencer element (NRSE) represses Bsx transcription in non-neuronal P19 cells. The Bsx promoter contains several putative binding sites for transcription factors, including NRSE for REST and the GC box for the transcriptional activator, Sp1. Upon neuronal differentiation of P19 cells with retinoic acid, Bsx gene expression increased, whereas that of the REST gene decreased. Electrophoretic mobility shift analyses demonstrated that recombinant REST proteins bound the NRSE region of the Bsx promoter. In neuronal NS20Y cells, transcriptional activity of the Bsx promoter was decreased upon expression of REST. Moreover, dominant-negative REST derepressed Bsx transcription in P19 cells. Sp1-mediated transcriptional activity of the Bsx promoter was attenuated by treatment with mithramycin A, a GC box-binding drug, but was enhanced upon mutation of NRSE. Co-immunoprecipitation and chromatin immunoprecipitation assays showed that the Bsx promoter appeared to be modulated by direct interactions between REST and Sp1. The CpG sites of NRSE and GC box were completely unmethylated, signifying no interference of DNA methylation. Our results suggest that binding of REST to NRSE suppresses the Sp1-mediated activation of Bsx in non-neuronal cells.