Abstract
Our study focuses on a family of ubiquitously expressed human C2H2 zinc finger proteins comprised of ZFX, ZFY and ZNF711. Although their protein structure suggests that ZFX, ZFY and ZNF711 are transcriptional regulators, the mechanisms by which they influence transcription have not yet been elucidated. We used CRISPR-mediated deletion to create bi-allelic knockouts of ZFX and/or ZNF711 in female HEK293T cells (which naturally lack ZFY). We found that loss of either ZFX or ZNF711 reduced cell growth and that the double knockout cells have major defects in proliferation. RNA-seq analysis revealed that thousands of genes showed altered expression in the double knockout clones, suggesting that these TFs are critical regulators of the transcriptome. To gain insight into how these TFs regulate transcription, we created mutant ZFX proteins and analyzed them for DNA binding and transactivation capability. We found that zinc fingers 11–13 are necessary and sufficient for DNA binding and, in combination with the N terminal region, constitute a functional transactivator. Our functional analyses of the ZFX family provides important new insights into transcriptional regulation in human cells by members of the large, but under-studied family of C2H2 zinc finger proteins.
Highlights
RNA Polymerase 2 (Pol2)-mediated gene regulation is achieved, in part, by transcription factors (TFs) binding to a core promoter, defined as a region ±50 bp from the transcription start site (TSS) of a gene [1,2,3,4]
We chose to use these cells because they express similar levels of ZFX and ZNF711 (Figure 2A) but lack ZFY
We identified multiple colonies that showed no expression of ZFX or ZNF711 (Figure 2C)
Summary
RNA Polymerase 2 (Pol2)-mediated gene regulation is achieved, in part, by transcription factors (TFs) binding to a core promoter, defined as a region ±50 bp from the transcription start site (TSS) of a gene [1,2,3,4]. TATA box-containing promoters often produce cell typespecific or induced (e.g. by a hormone) transcripts, whereas housekeeping genes are often driven by CpG island promoters [5]. Both types of core promoters are bound by general TFs such as Pol and other components of the pre-initiation complex. Promoter activity can be increased by the action of site-specific, DNAbinding TFs that either bind proximal to the core promoter, stabilizing the recruitment of the transcriptional machinery, or to distal enhancer elements, bringing specific coregulators to the core promoter via long-range chromatin looping [6]
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