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. 2017 Sep 6;37(36):8816–8829. doi: 10.1523/JNEUROSCI.2125-16.2017

Figure 1.

Figure 1.

Candidate DLX target sequences within Gad1 (GAD67) and Gad2 (GAD65) promoter regions were identified through the presence of canonical homeodomain DNA binding motifs within the Gad promoters and confirmed by chromatin immunoprecipitation (ChIP). A, The specific regions within the regulatory elements of the mouse GAD65/Gad2 (GenBank AB032757; Gad2 NM_008078.2) and GAD67/Gad1 (GenBank Z49978; Gad1 NM_008077.5) promoters, designated GAD65i/ii and GAD67i/ii, contain putative homeodomain DNA-binding sites (TAAT/ATTA), shown in filled boxes. DLX proteins occupy colored boxes and do not occupy black boxes, respectively, based on subsequent experiments. Nucleotide positions are indicated with respect to transcriptional start sites. B, DLX1 and DLX2 occupy the Gad1 and Gad2 promoters in E13.5 GE tissues using ChIP. ChIP assays of GAD65/Gad2 and GAD67/Gad1 promoter regions showed that DLX1 and DLX2 occupy both regions (region i and region ii) of each Gad gene promoter using E13 GE tissues and specific DLX1 and DLX2 antibodies. Negative controls used no addition of antibodies and/or E13.5 hindbrain tissues. The positive controls used E13.5 genomic DNA. PCR bands were subcloned and sequenced for confirmation.