Basis of gene-specific transcription regulation by the Inegrator Complex (RNA-Seq)
Integrator (INT) is a multi-subunit modular RNA processing complex, which exhibits both RNA endonuclease and protein phosphatase activity. It is responsible for transcription termination at the 3' ends of a diverse array of RNA polymerase II (RNAP2) transcribed non-coding RNAs, as well as transcription regulation at a large number of protein coding genes. There, it terminates RNAP2 at promoter proximal pausing sites, cleaves the nascent transcript and competes with elongation factors.This INT-mediated tapering of gene expression attenuates stimulus responsive genes and is essential for cell differentiation. Although INT affects transcription at varying classes of RNAs in diverse biological contexts, how it achieves specificity in a gene- and context-dependent manner has remained elusive. Using a combination of proteomics, interaction studies and structural characterization, we identified a diverse set of transcription factors (TFs) that associate directly with defined surfaces on INT. Stress conditions lead to changes in the types of TFs bound by INT, and quantitative binding studies suggest that TF affinities can be modulated by altering the phosphorylation states of specific residues in their INT-binding motifs. Integrated multi-omics data show that INT and its TF interactors regulate significantly overlapping sets of genes and indicate that these TFs recruit INT to specific genomic loci. Consistently, we find that starvation induced formation of primary cilia, which is a cellular stress response reliant on INT-mediated transcription regulation, depends on intact TF-INT binding. Taken together, our data suggest that TFs lend INT specificity to elicit targeted gene regulation as a transcriptional response in defined biological contexts. Overall design: To assess the functional relationship between INT and its TF factor interactors, we depleted one subunit of INT (INTS13) as well as each TF individually with siPOOLs in HEK293T cells and subjected these samples to total RNA-sequencing. In order to obtain efficient knockdown of each protein, we subjected the cells to double transfections with the first one occurring 24 hours after seeding and the second one occuring 24 hours later. The cells were then left to proliferate for 48 hours, after which RNA was extracted, Dnase treated. In order to determine the role of INTS13 and the TFs in glucose starvation response, we repeated the depletions of INTS13, HDGF and ZMYND8 under starved conditions, where growth medium was changes to glucose-free medium 24 hours after the second transfection. After DNase treatment, ribosomal RNAs were depleted and sequencing libraries were prepared using the Illumina TruSeq Stranded Total RNA kit. Libraries were sequenced on an Illumina NovaSeq 6000 using 100 bp single-end reads at a depth of 40M reads per library.