Recently, the Nrf1 transcription factor (TCF11) was shown to be important for induction of proteasome gene transcription in mammalian cells ( 41, 45). However, it has been shown that proteasome genes are not coregulated by the same transcription factor, Hsf1, as heat shock proteins in response to cellular stress in mammalian cells ( 49). Heat stress results in strong upregulation of proteasomal activity in human fibroblasts ( 2). We propose that this single cnc gene in Drosophila resembles the ancestral gene family of mammalian nuclear factor erythroid-derived 2-related transcription factors, which are essential in regulating oxidative stress and proteolysis. Expression of a V5-His-tagged form of Cnc-C revealed that the transcription factor is itself a proteasome substrate that is stabilized when the proteasome is inhibited. Quantitative reverse transcription-PCR confirmed the role of Cnc-C in activation of transcription of genes encoding proteasome components. Transcript-specific silencing revealed that the longest of the seven transcripts for the cnc gene, cnc-C, was needed for proteasome and p97 ATPase production. Immunoblot assays against proteasome components revealed a general decline in both 19S regulatory complex and 20S proteasome subunits after RNAi depletion of this transcription factor. In fact, 20S proteasome activity was reduced in cells depleted of cnc. This screen identified Cnc (cap 'n’ collar basic region leucine zipper) as a candidate transcriptional regulator of proteasome component expression. We used an RNAi library against 993 Drosophila transcription factor genes to identify genes whose suppression in Schneider 2 cells stabilized a ubiquitin-green fluorescent protein reporter protein. Here we describe an RNA interference (RNAi) screen of Drosophila melanogaster that was used to identify transcription factors that may play a role in maintaining levels of the 26S proteasome.
While the 26S proteasome is a key proteolytic complex, little is known about how proteasome levels are maintained in higher eukaryotic cells.
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