e20 Nucleic Acids Research, 2007, Vol. 35, No. 3 PAGE 6 OF 9 Table 1. Positional clustering based predictions of transcriptional regulatory sequences upstream of GABRs In total, we predict 15 orthologous pairs of regulatory sequences, representing 13 unique sequences. Comparing with known mammalian binding motifs, eight of the predictions show similarity to previously characterized TFBS, as indicated. Where no known binding motif exists, the corresponding in vitro EMSA and functional assay, if applicable, is indicated. Similar predictions are grouped together and aligned by hand. (31,35). We also included orthologous rodent promoters in the input sets (45). Orthologous promoters were included to pro- vide more instances of binding sites in the input set than would be expected by random, allowing for easier detection of the sites. Inclusion of orthologous promoters has the additional effect of selectively amplifying evolutionarily conserved binding sites. Such binding sites are more likely to have major functional roles in the regulation of the GABR receptor. Thus, sensitivity to such sequences is improved at the expense of sensitivity to species-speciﬁc binding sites. With this effect in mind, we require all GABR-binding site predictions to exist in orthologous promoters. Since the mechanisms of co-regulation for the seven GABRs are unknown, hypothetical co-regulation models were evaluated by querying all 27 possi- ble subsets of the seven GABRps. Clustering results on nucle- otide positions and selecting the most frequently occurring positions, we predicted 13 functional TF-binding sites. Predictions were compared with instances of known bind- ing motifs from TRANSFAC (40), and 8 of the 13 predictions (61.5%) resembled known binding sites for 10 TFs (Table 1). Of the 10 TFs, 7 have been identiﬁed in the CNS of rodents: SP-1 (46); AP-2, TST-1 (POU3F1), OCT-1 (POU2F1), OLF-1 (47); CP-2 (48); and RREB-1 (49). Furthermore, previous analyses of GABR promoter regions agree with our predic- tions that assign putative regulatory roles to SP-1, OCT-1, OLF-1 in the regulation of GABRs (29). We chose to validate novel motif predictions with EMSAs and functional studies in primary cultured neurons. EMSA (50) was performed with an excess of cold competi- tors to deﬁne speciﬁcity of protein binding in nuclear extracts derived from primary neocortical neurons and ﬁbroblasts (FIBs) kept in culture. As shown in Figures 4–6, out of six predicted binding sites found upstream of the (a, b, g and d) subunit genes, four (GABRA4, GABRB1, GABRB3 and GABRD) displayed speciﬁc binding. In addition to speciﬁc binding of neuronal extracts to novel GABRA4 motifs, we have evidence for speciﬁc binding using FIB extracts (Figure 5A and B), of especial interest given that the expres- sion of GABRs is restricted to the nervous system and repres- sors such as the RE1-silencing transcription factor (REST) (51,52) expressed in non-neuronal cells have been implicated in the neural speciﬁcity of gene expression. Clearly, protein binding to DNA does not always necessi- tate regulatory function. To begin to address the functional