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.

These authors contributed equally to this work.Keywords: core promoter elements/motifs, DPE, embryonic development, histone gene cluster, RNAP II transcription, ribosomal protein genes, spermiogenesis, TBP-related factors, TRF2, TCT Transcriptional regulation is pivotal for development and differentiation of organisms. Transcription of eukaryotic protein-coding genes by RNA polymerase II (RNAP II) initiates at the core promoter. Core promoters, which encompass the transcription start site, may contain functional core promoter elements, such as the TATA box, initiator, TCT and downstream core promoter element. TRF2 (TATA-box-binding protein-related factor 2) does not bind TATA box-containing promoters. Rather, it is recruited to core promoters via sequences other than the TATA box. We review the recent findings implicating TRF2 as a basal transcription factor in the regulation of diverse biological processes and specialized transcriptional programs.

The core promoter, which is generally defined as the region to which RNA Polymerase II is recruited to initiate transcription, plays a pivotal role in the regulation of gene expression. The core promoter consists of different combinations of several short DNA sequences, termed core promoter elements or motifs, which confer specific functional properties to each promoter. Earlier studies that examined the ability to modulate gene expression levels via the core promoter, led to the design of strong synthetic core promoters, which combine different core elements into a single core promoter. Here, we designed a new core promoter, termed super core promoter 3 (SCP3), which combines four core promoter elements (the TATA box, Inr, MTE and DPE) into a single promoter that drives prolonged and potent gene expression. We analyzed the effect of core promoter architecture on the temporal dynamics of reporter gene expression by engineering EGFP expression vectors that are driven by distinct core promoters. We used live cell imaging and flow cytometric analyses in different human cell lines to demonstrate that SCPs, particularly the novel SCP3, drive unusually strong long-term EGFP expression. Importantly, this is the first demonstration of long-term expression in transiently transfected mammalian cells, indicating that engineered core promoters can provide a novel non-viral strategy for biotechnological as well as gene-therapy-related applications that require potent expression for extended time periods.

Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression.

26Background: Diverse biological processes and transcriptional programs are 27 regulated by RNA polymerase II (Pol II), which is recruited by the general transcription 28 machinery to the core promoter to initiate transcription. TRF2 (TATA-box-binding 29 protein-related factor 2) is an evolutionarily conserved general transcription factor that 30 is essential for embryonic development of Drosophila melanogaster, C. elegans, 31 zebrafish and Xenopus. Nevertheless, the cellular processes that are regulated by 32 TRF2 are largely underexplored. 33Results: Here, using Drosophila Schneider cells as a model, we discovered that TRF2 34 regulates apoptosis and cell cycle progression. We show that TRF2 knockdown 35 results in increased expression of distinct pro-apoptotic genes and induces apoptosis. 36Using flow cytometry, high-throughput microscopy and advanced imaging-flow 37 cytometry, we demonstrate that TRF2 regulates cell cycle progression and exerts 38 distinct effects on G1 and specific mitotic phases. RNA-seq analysis revealed that 39 TRF2 controls the expression of Cyclin E and the mitotic cyclins, Cyclin A, Cyclin B 40 and Cyclin B3, but not Cyclin D or Cyclin C. To identify proteins that could account for 41 the observed regulation of these cyclin genes, we searched for TRF2-interacting 42 proteins. Interestingly, mass spectrometry analysis of TRF2-containing complexes 43 identified GFZF, a nuclear glutathione S-transferase implicated in cell cycle regulation, 44and Motif 1 binding protein (M1BP). TRF2 has previously been shown to interact with 45 M1BP and M1BP has been shown to interact with GFZF. Furthermore, available ChIP-46 exo data revealed that TRF2, GFZF and M1BP co-occupy the promoters of TRF2-47 with TRF2, it is TRF2, rather than GFZF or M1BP, that is the main factor regulating 50 the expression of Cyclin E and the mitotic cyclins. 51 Conclusions: Our findings uncover a critical and unanticipated role of a general 52 transcription factor as a key regulator of cell cycle and apoptosis. 53 54 Keywords 55 Basal transcription machinery, RNA polymerase II, gene expression, TATA box-56 binding protein (TBP), TBP-related factor 2 (TRF2), cyclin genes. 57 58 BACKGROUND 59 Multiple biological processes and transcriptional programs are regulated by RNA 60 polymerase II (Pol II). The initiation of transcription of protein-coding genes and 61 distinct non-coding RNAs occurs following the recruitment of Pol II to the core 62 promoter region by the general/basal transcription machinery (1-4). The core 63promoter, which directs accurate initiation of transcription and encompasses the 64 transcription start site (TSS), may contain short DNA sequence elements/motifs, 65 which confer specific properties to the core promoter (1, 4-10). The first step in the 66 recruitment of Pol II to initiate transcription is the binding of TFIID, which is 67 composed of TATA-box-binding protein (TBP) and TBP-associated factors. 68Remarkably, although TBP is considered a universal general transcription factor, 69 robust Pol II transc...