Comparative cofactor screens reveal the influence of transactivation domains and core promoters on the mechanisms of transcription [SLAM-seq]

Eukaryotic transcription factors (TFs) activate gene expression by recruiting cofactors to promoters. However, the relationships between TFs, promoters and their associated cofactors remain poorly understood. Here, we combine GAL4-transactivation assays with comparative CRISPR-Cas9 screens to identify the cofactors required by nine different TFs in human cells. Using this dataset, we associate key TFs with their cofactors, classify cofactors as ubiquitous or specific, discover novel transcriptional co-dependencies and demonstrate that certain TFs use the tail 2 and kinase submodules of Mediator to potentiate transcriptional elongation. By employing a reductionistic and comparative approach, we demonstrate that TFs do not display discrete mechanisms of activation. Instead, each TF is dependent on a unique combination of cofactors, which influences distinct steps in the transcriptional process. We also extend our screens to nine different core promoters to explore how core promoter elements influence cofactor dependence. Our data suggest that different classes of promoter are constrained by either initiation or pause release, which influences their dynamic range of gene expression and compatibility with specific cofactors. Overall, our comparative cofactor screens uncover the interplay between TFs, cofactors and core promoters and reveal general principles by which they influence transcription. A total of 10 million K562 cells were treated with dTAGV-1 (Tocris) for a total of 4 hours. In the final hour of treatment, cells were labelled with 200uM of 4-thioruidine (4sU; Cayman Chemical) to capture nascent mRNA transcripts. RNA extraction was performed using TRIzol (Ambion) following the manufacturer's instructions, with the addition of 1mM DTT to the isopropanol precipitation and ethanol wash steps. Total RNA was eluted in nuclease-free H2O containing 0.1mM DTT. 10ug of total RNA was treated with fresh 10mM iodoacetamide (Pierce) in reaction buffer containing 50mM Tris, pH 8.0 and 50% DMSO at 50oC for 15 minutes, light-protected and shaking at 1000rpm. The alkylation reaction was quenched with 20mM of fresh DTT, followed by adding 1ug of unlabelled Drosophila S2 RNA. Total RNA was cleaned up using the RNeasy MinElute Cleanup Kit (Qiagen) and DNAse treated. Libraries were prepared using 500ng of material using the QuantSeq 3’ mRNA-Seq Library Prep Kit FWD (Lexogen; V1 kit with single 6nt i7 indexes) according to the standard manufacturer’s protocol. Sequencing of cDNA libraries was performed on the Illumina NextSeq2000 with 100bp single-end configuration. SLAM-seq was performed in biological triplicate.