Biology subjects

Tekel, S. J.

Publications and source records attributed to Tekel, S. J..

3 recordsLinked to original sources

bioRxiv2020

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

The leading FDA-approved drugs for epigenetic cancer therapy are small molecule compounds that activate silenced tumor suppressors by inhibiting enzymes that generate aberrant repressive chromatin. Although promising, this approach is limited because chromatin-modifying enzymes often target non-chromatin proteins and can serve dual functions as gene repressors and activators. Previously, we have demonstrated that a transgenically expressed synthetic polycomb-derived transcription factor (PcTF) could activate genes in silenced chromatin via specific interactions with histone H3 trimethylated at lysine 27 (H3K27me3). Efficient non-viral intracellular delivery remains a challenge for protein-based biologics. Herein, we report the delivery of cell-penetrating PcTF (CP-PcTF) to cultured cells. We expressed and purified recombinant PcTF that was fused in frame with a nuclear localization signal and a cell penetrating peptide tag (TAT). We demonstrated rapid and efficient uptake of soluble CP-PcTF by osteosarcoma U-2 OS cells grown in 2-D monolayers and 3-D spheroids. However, CP-PcTF had a modest effect on gene expression and cell proliferation compared to transgenically-expressed PcTF from our previous work. Overall, these results suggest that TAT is a very effective delivery vehicle for the recombinant transcriptional regulator PcTF, and that further technical development is needed to deliver functional PcTF into cell nuclei.

synthetic biology↗

bioRxiv2018

Engineered orthogonal quorum sensing systems for synthetic gene regulation

Gene regulators that are controlled by membrane-permeable compounds called Homoserine lactones (HSLs) have become popular tools for building synthetic gene networks that coordinate behaviors across populations of engineered bacteria. Synthetic HSL-signaling systems are derived from natural DNA and protein elements from microbial quorum signaling pathways. Crosstalk, where a single HSL can activate multiple regulators, can lead to faults in networks composed of parallel signaling pathways. Here, we report an investigation of quorum sensing components to identify synthetic pathways that exhibit little to no crosstalk in liquid and solid cultures. In previous work, we characterized the response of a single regulator (LuxR) to ten distinct HSL-synthase enzymes. Our current study determined the responses of five different regulators (LuxR, LasR, TraR, BjaR, and AubR) to the same set of synthases. We identified two sets of orthogonal synthase-regulator pairs (BjaI/BjaR + EsaI/TraR and LasI/LasR + EsaI/TraR) that show little to no crosstalk when they are expressed in Escherichia coli BL21. These results expand the toolbox of characterized components for engineering microbial communities.

synthetic biology↗

bioRxiv2017

Tandem histone-binding domains enhance the activity of a synthetic chromatin effector

Fusion proteins that specifically interact with biochemical marks on chromosomes represent a new class of synthetic transcriptional regulators that decode cell state information rather than DNA sequences. In multicellular organisms, information relevant to cell state, tissue identity, and oncogenesis is often encoded as biochemical modifications of histones, which are bound to DNA in eukaryotic nuclei and regulate gene expression states. We have previously reported the development and validation of the \"Polycomb-based transcription factor\" (PcTF), a fusion protein that recognizes histone modifications through a protein-protein interaction between its polycomb chromodomain (PCD) motif and trimethylated lysine 27 of histone H3 (H3K27me3) at genomic sites. We demonstrated that PcTF activates genes at methyl-histone-enriched loci in cancer-derived cell lines. However, PcTF induces modest activation of a methyl-histone associated reporter compared to a DNA-binding activator. Therefore, we modified PcTF to enhance its target affinity. Here, we demonstrate the activity of a modified regulator called Pc2TF, which has two tandem copies of the H3K27me3-binding PCD at the N-terminus. Pc2TF shows higher affinity for H3K27me3 in vitro and shows enhanced gene activation in HEK293 cells compared to PcTF. These results provide compelling evidence that the intrinsic histone-binding activity of the PCD motif can be used to tune the activity of synthetic histone-binding transcriptional regulators.

synthetic biology↗