Regulation of cancer epigenomes with a histone-binding synthetic transcription factor

Abstract

Chromatin proteins have expanded the mammalian synthetic biology toolbox by enabling control of active and silenced states at endogenous genes. Others have reported synthetic proteins that bind DNA and regulate genes by altering chromatin marks, such as histone modifications. Previously, we reported the first synthetic transcriptional activator, the “Polycomb-based transcription factor” (PcTF) that reads histone modifications through a protein–protein interaction between the polycomb chromodomain motif and trimethylated lysine 27 of histone H3 (H3K27me3). Here, we describe the genome-wide behavior of the polycomb-based transcription factor fusion protein. Transcriptome and chromatin profiling revealed several polycomb-based transcription factor-sensitive promoter regions marked by distal H3K27me3 and proximal fusion protein binding. These results illuminate a mechanism in which polycomb-based transcription factor interactions bridge epigenomic marks with the transcription initiation complex at target genes. In three cancer-derived human cell lines tested here, some target genes encode developmental regulators and tumor suppressors. Thus, the polycomb-based transcription factor represents a powerful new fusion protein-based method for cancer research and treatment where silencing marks are translated into direct gene activation.

Delivery of cell-penetrating chromatin sensor-actuators to human osteosarcoma cells

“…However, the RFP signal from the fusion protein was granular and not entirely within the nucleus. In our previous work, transgenically-expressed PcTF showed a homogeneous distribution of RFP signal entirely within the nucleus [18,28] (Figure 5A). These differences in subcellular distribution might affect the gene-regulating activity of the PcTF protein.…”

“…Three of the CP-PcTF-responsive genes, including AC002398. 9 (a locus that contains PSENEN and LIN37 ), ADORA2A , and PALD1 were upregulated 3.7, 1.6, and 1.7-fold respectively by transgenic PcTF (TG-PcTF) and not by the TG-mut negative control in our previous RNA-seq study [18] .…”

“…We identified 103 genes that were upregulated specifically by CP-PcTF and not by the nonbinding control protein (CP-mut). Three of these genes were also specifically activated by transgene-expressed PcTF (TG-PcTF) in our previous study [18] , which is just a small fraction of the 3807 genes that responded TG-PcTF. Although we observed nearly 100% uptake of CP-PcTF via flow cytometry at this concentration, high-resolution fluorescence microscopy suggested that inefficient nuclear import might hinder gene-regulating activity of the protein.…”

“…Gene expression analysis revealed a modest level of gene activation in 2-D cultured cells that were treated with CP-PcTF. Based on our previous results from overexpression of PcTF from a transgene in U-2 OS cells [18,28] , we expected CP-PcTF to enter the cell nucleus, bind multiple H3K27me3-enriched sites, and co-activate many epigenetically-repressed genes. We identified 103 genes that were upregulated specifically by CP-PcTF and not by the nonbinding control protein (CP-mut).…”

“…We have previously designed chromatin sensor-actuator proteins that bind histone post-translational modifications and co-activate groups of repressed genes. We demonstrated that a H3K27me3-specific sensor-actuator called PcTF activates epigenetically repressed tumor suppressor genes in several cancer cell lines [18,19] . However, experiments with epigenome editors and sensor-actuators have relied heavily on the delivery and overexpression of fusion-encoding transgenes.…”