Figure 6
Sensory-specific expression of a VP16-Brn3a constitutive transcriptional activator reveals possible direct regulatory targets. (A) Activation of transcription by VP16-Brn3a in transfection assays. Increased expression requires both the VP16-Brn3a protein and the presence of a consensus Brn3a binding site (3× Brn3a cons) in the reporter gene construct. (B) Structure of the VP16-Brn3a transgene. A dicistronic message encoding VP16-Brn3a and enhanced green fluorescent protein (eGFP) is driven by an 11-kb sensory enhancer from the Brn3a locus. (C) Expression of eGFP in the E13.5 dorsal root ganglia (DRG) of VP16-Brn3a transgenic founders by direct GFP fluorescence (whole mount) and immunofluorescence (sections). Appropriate axonal projections to the dorsal spinal cord (SC) are evident. The TG of these embryos were harvested for array analysis. (D) RT-PCR assays of Brn3a-VP16 expression in TG dissected from transgenic embryos, normalized to phosphoglycerate kinase (Pgk) expression. Embryos 11, 49, and 76 (asterisks) were selected for microarray analysis. (E) Microarray analysis of changes in gene expression in VP16-Brn3a transgenic embryos at E13.5. Changes in gene expression in the VP16-Brn3a transgenic TG were plotted (y-axis) against changes in the E13.5 Brn3a knockout (x axis). Only data for transcripts showing significantly increased or decreased expression in the Brn3a-/- TG relative to controls are shown, thus data points are missing from around the origin. For the entire group, the correlation was weak (slope = 0.126, R2 = 0.147), suggesting that many Brn3a targets are indirectly regulated. The microarray analyses of Brn3a-/- and control ganglia used for comparison purposes will be presented in detail elsewhere (Lanier et al., in preparation). (F) Changes in transcription factor expression in E13.5 Brn3a knockout ganglia and VP16-Brn3a ganglia, and the number of change calls (out of nine possible comparisons) for VP16-Brn3a versus control ganglia. 'A' indicates absent call. Neurod1 and Neurod4, previously shown to be directly repressed by Brn3a, were activated by VP16-Brn3a. Runx3 and Hes5 are candidates for direct positive regulation by Brn3a (expression increased by VP16-Brn3a, decreased in knockout), Neurog1, Insm1, Eya2, and Tcaf2b are further candidates for direct negative regulation (expression increased by VP16, increased in knockout).