Reduction of ZFX levels decreases histone H4 acetylation and increases Pol2 pausing at target promoters [GRO-seq]
The ZFX transcriptional activator binds to CpG island promoters, with a major peak at ~200-250bp downstream from transcription start sites. Because ZFX binds within the transcribed region, we investigated whether it regulates transcriptional elongation. We used GRO-seq to show that loss or reduction of ZFX increased the pause ratio at ZFX-regulated promoters. To further investigate the mechanisms by which ZFX regulates transcription, we determined regions of the protein needed for transactivation and for recruitment to the chromatin. Interestingly, although ZFX has 13 grouped zinc fingers, deletion of the first 11 fingers produces a protein that can still bind to chromatin and activate transcription. We next used TurboID-MS to detect ZFX-interacting proteins, identifying ZNF593, proteins that interact with the N-terminal transactivation domain (e.g. histone modifying proteins), and proteins that interact with ZFX when it is bound to the chromatin (e.g. TAFs and other histone modifying proteins). Our studies support a model in which ZFX enhances elongation at target promoters by recruiting H4 acetylation complexes and reducing pausing. Overall design: To profile the effects of ZFX on Pol2 pausing in human cell lines. siRNA knockdowns with siZFX and a control siRNA oligo were performed in LN-229 cells with 2 biological replicates. 293T cells and 293T cells with ZFX and ZNF711 knockouts (DKO) were also performed with 2 biological replicates.