RNA-seq data of mammalian cells treated with artificial transcription factors

Interferon (IFN)-γ is a central regulator of cell-mediated immunity in human health and disease, but reduced expression of the target receptors impairs signaling activity and leads to immunotherapy resistance. Although intracellular expression of IFN-γ restores signaling and downstream functions, we lack tools to activate the IFNG gene instead of cell surface receptors. This work describes the design and characterization of an artificial transcription factor (ATF) protein that recognizes the IFNG gene with six zinc finger domains, which are dovetailed to a VP64 signaling domain that promotes gene transcription and translation. Biological studies with human Jurkat T cells reveal that the ATF treatment induces potent IFN-γ expression and amplifies the gene transcriptome profile for multiple class I HLA alleles and interferon-stimulated genes (ISGs). Biophysical characterization showed the recombinant ATF protein recognizes the human IFNG gene with nanomolar affinity (KD = 5.27 ± 0.3 nM), adopts a protein secondary structure associated with the ββα-fold of zinc finger domains, and is resistant to thermal denaturation. These studies demonstrate that transcriptional targeting of cytokine genes, rather than surface receptors, can activate cytokine expression and show potential for directing immune function. We profiled the gene expresion of human Jurkat T cells after treatment with an anti-IFNG artificial transciption factor (ATF), isotype control (Aart6), and vehicle (media). The cells were transfected with plasmid by electroportation , and supplemented after 2 hours with zinc chloride, PMA/Ionomycin, and Brefeldin A. After 48 hours, the cells were collected and processed to extract the total RNA content with an RNeasy Minikit (Qiagen, 74106) and RNase-free DNase (Qiagen, 79254). The RNA quality was verified by electrophoresis. The sample isolates that passed quality control were sequenced on an Illumina Genome Analyser (Azenta).