Abstract
The POU-domain transcription factor Brn3b/ POU4f2 is an essential regulator of gene expression in mouse retinal ganglion cells. Although Brn3b's importance in the differentiation of these cells has been firmly established, the regions on Brn3b where transcriptional activation and/or repression domains reside are only vaguely defined, and conflicting publications report both activation and repression activities for Brn3b. To clarify its function, we monitored the transcriptional activity of Brn3b and Gal4 DNA-binding domain (DBD)-Brn3b fusion proteins in cotransfection experiments using either Brn3-consensus or Gal4 DNA-binding sites to drive reporter gene expression. At Gal4 DNA-binding sites, transrepression activity mapping to the POU domain within Brn3b's C-terminal region masked any transactivation activity. More detailed experiments revealed that expressing abnormally high levels of POU homeodomain- or other homeodomain-containing sequences caused fortuitous transrepression in the cotransfection assay. To avoid transrepression, Brn3b sequences lacking Brn3b's POU domain were fused to the Gal4 DBD to allow identification of regions that were responsible for transcriptional activation. Considerable transactivation activity was located between amino acid residues 100 and 239, although other regions also had activity. The transactivation domain synergized strongly with another transcription factor, LexA-VP16. At Brn3 DNA-binding sites, full-length Brn3b increased transcription more than 25-fold, and similar activation was observed with the closely related factor Brn3a/POU4f1. No transactivation activity was associated with the C-terminal POU domain-containing portion of Brn3b. The results demonstrate that Brn3b regulates gene expression through the action of a strong transcriptional activation domain within its N-terminal sequence.
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