Drosophila TRF2 is a preferential core promoter regulator

Abstract

Transcription of protein-coding genes is highly dependent on the RNA polymerase II core promoter. Core promoters, generally defined as the regions that direct transcription initiation, consist of functional core promoter motifs (such as the TATA-box, initiator [Inr], and downstream core promoter element [DPE]) that confer specific properties to the core promoter. The known basal transcription factors that support TATA-dependent transcription are insufficient for in vitro transcription of DPE-dependent promoters. In search of a transcription factor that supports DPE-dependent transcription, we used a biochemical complementation approach and identified the Drosophila TBP (TATA-boxbinding protein)-related factor 2 (TRF2) as an enriched factor in the fractions that support DPE-dependent transcription. We demonstrate that the short TRF2 isoform preferentially activates DPE-dependent promoters. DNA microarray analysis reveals the enrichment of DPE promoters among short TRF2 up-regulated genes. Using primer extension analysis and reporter assays, we show the importance of the DPE in transcriptional regulation of TRF2 target genes. It was previously shown that, unlike TBP, TRF2 fails to bind DNA containing TATA-boxes. Using microfluidic affinity analysis, we discovered that short TRF2-bound DNA oligos are enriched for Inr and DPE motifs. Taken together, our findings highlight the role of short TRF2 as a preferential core promoter regulator.

TRF2: TRansForming the view of general transcription factors

“…73 Interestingly, MEME analysis performed on the top TRF2-bound genes revealed enrichment of the DPE motif, while no enriched motif was identified in the least TRF2-bound genes. 74 Drosophila short TRF2 was recently identified by a biochemical complementation analysis as an enriched factor in fractions that support DPEdependent transcription. 74 Microarray analysis of Drosophila S2RC cells that overexpress inducible short TRF2 identified multiple DPE-dependent targets.…”

“…74 Drosophila short TRF2 was recently identified by a biochemical complementation analysis as an enriched factor in fractions that support DPEdependent transcription. 74 Microarray analysis of Drosophila S2RC cells that overexpress inducible short TRF2 identified multiple DPE-dependent targets. In vitro transcription followed by primer extension analysis of 4 TRF2 target genes (pvf2, sodh-2, bgm and inx3) indicated that their transcription was highly dependent on an intact DPE motif.…”

“…In vitro transcription followed by primer extension analysis of 4 TRF2 target genes (pvf2, sodh-2, bgm and inx3) indicated that their transcription was highly dependent on an intact DPE motif. 74 Furthermore, using microfluidics affinity analysis, protein extracts from S2RC cells that over-express inducible TRF2, were shown to bind these DPEcontaining promoters in a sequence-specific manner. Taken together, these findings suggest the existence of specialized transcriptional systems that do not involve TBP and regulate diverse biological pathways.…”

“…73 Microfluidic affinity analysis has demonstrated DNA binding of TRF2-containing complexes to DPE-containing promoters. 74 It is likely that there are TRF2-associated factors (like TBP-associated factors), which assist TRF2 in binding to its target promoters. Such TRF2-associated factors remain to be discovered.…”

Structure-Function Analysis of the Drosophila melanogaster Caudal Transcription Factor Provides Insights into Core Promoter-preferential Activation

“…We analyzed co-activation of gt reporter genes by the Bicoid transcription factor using firefly luciferase reporter genes driven by either a DPE-dependent (containing mTATA) or a TATA-dependent (containing mDPE) version of the gt transcriptional control region from Ϫ2031 to ϩ40 relative to the RNA start site (83). S2Rϩ cells were transfected with either a DPE-dependent or a TATA-dependent gt reporter construct as well as an expression vector for Bicoid and/or dCBP.…”