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.
BackgroundMounting evidence from genome-wide studies of cancer shows that chromatin-mediated epigenetic silencing at large cohorts of genes is strongly linked to a poor prognosis. This mechanism is thought to prevent cell differentiation and enable evasion of the immune system. Drugging the cancer epigenome with small molecule inhibitors to release silenced genes from the repressed state has emerged as a powerful approach for cancer research and drug development. Targets of these inhibitors include chromatin-modifying enzymes that can acquire drug-resistant mutations. In order to directly target a generally conserved feature, elevated trimethyl-lysine 27 on histone H3 (H3K27me3), we developed the Polycomb-based Transcription Factor (PcTF), a fusion activator that targets methyl-histone marks via its N-terminal H3K27me3-binding motif, and co-regulates sets of silenced genes.ResultsHere, we report transcriptome profiling analyses of PcTF-treated breast cancer model cell lines. We identified a set of 19 PcTF-upregulated genes, or PUGs, that were consistent across three distinct breast cancer cell lines. These genes are associated with the interferon response pathway.ConclusionsOur results demonstrate for the first time a chromatin-mediated interferon-related transcriptional response driven by an engineered fusion protein that physically links repressive histone marks with active transcription.Electronic supplementary materialThe online version of this article (10.1186/s12918-018-0608-4) contains supplementary material, which is available to authorized users.
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Disease states, such as breast cancer, arise from the disruption of chromatin, the central DNA-protein structures that package human genetic material. Mounting evidence from genome-wide studies across cancers show that Polycomb-mediated repression of sets of genes, called Polycomb modules, is strongly linked to a poor prognosis. We developed a synthetic transcriptional activator to release silenced genes from the repressed state. The Polycomb-based Transcription Factor (PcTF) is a synthetic effector that accumulates at methyl-histone marks and regulates hundreds of gene targets, including tumor suppressors. We recently reported the activity of PcTF in bone, blood, and brain sarcoma-derived model cell lines. Here, we expand our investigation of PcTF to three breast cancer-derived cell lines. We expressed PcTF in drug-responsive (MCF-7, BT-474) and nonresponsive triple negative (BT-549) breast cancer cell lines. RNA-seq showed that hundreds of genes were up-or down-regulated by PcTF as early as 24 hours after transfection. BT-549, the triple-negative cancer cell line, showed the highest number of PcTF-activated genes. We demonstrate the anti-cancer potential of PcTF by identifying 15 tumor suppressor genes that are upregulated across the three cell types. The data also provide new mechanistic insights into the relationship between chromatin organization and PcTF-mediated regulation of genes. Our results have exciting implications for cancer treatment with engineered biologics.
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 "Polycombbased transcription factor" (PcTF), that reads histone modifications through a proteinprotein interaction between the PCD motif and trimethylated lysine 27 of histone H3 (H3K27me3). Here, we describe the genomewide behavior of PcTF. Transcriptome and chromatin profiling revealed PcTFsensitive promoter regions marked by proximal PcTF and distal H3K27me3 binding. These results illuminate a mechanism in which PcTF interactions bridge epigenetic marks with the transcription initiation complex. In three cancerderived human cell lines tested here, many PcTFsensitive genes encode developmental regulators and tumor suppressors. Thus, PcTF represents a powerful new fusionproteinbased method for cancer research and treatment where silencing marks are translated into direct gene activation.
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