Dr. Natecia Williams (Senior Research Specialist) has been working on producing batches of epigenetic sensor-actuator proteins that can penetrate human cells, which we hope to use to treat cancer. First recombinant DNA (plasmid pTXB1), constructed by Harrison Priode (Research Specialist I), that encodes a cell-penetrating polycomb transcription factor (CP-PcTF) is transformed into E. coli cells (illustration, left). The transformed cells are grown as a large culture (200 mL) and show a characteristic beige color when they are concentrated at the bottom of a flask (left photo). When an inducer chemical is added (IPTG), this activates production of the CP-PcTF protein. CP-PcTF includes a red fluorescent protein, which looks magenta under white light (right photo).
Next, Dr. Williams will gently break open the cells to release the proteins, use a special resin to capture the CP-PcTF proteins by a “handle” on the end of each protein (column-binding tag), and purify the proteins to eliminate cell residue. She will add the purified proteins to triple negative breast cancer cells in culture plates to determine how effectively the proteins enter nuclei and alter the expression of genes. If these experiments are successful we will test CP-PcTF in mice that carry breast cancer tumors. We will continue to post updates on this and other exciting projects!
Research – bioRxiv – Delivery of cell-penetrating chromatin sensor-actuators to human osteosarcoma cells
Delivery of cell-penetrating chromatin sensor-actuators to human osteosarcoma cells
Tekel SJ, Brookhouser N, Haynes KA. (2020) bioRxiv. https://doi.org/10.1101/2020.02.28.969907
Recent research has revealed that a key vulnerability in hard-to-treat cancer cells (e.g. triple negative breast cancer) may be chromatin, a system of proteins and nucleic acids that controls chromosome organization and gene expression. Small molecule drugs called epigenetic inhibitors can easily get into the nucleus to disrupt chromatin in cancer, but these molecules do not carry enough biological information to direct specific changes in gene expression. This unmet need inspired us to build artificial transcription factors that could be delivered to cells in a similar manner as soluble drugs, bind to aberrant chromatin, and induce gene activation. In this report, we describe how the addition of short cell-penetrating signal to the chromatin sensor-actuator PcTF enabled 100% uptake in cultured cells (monolayers), and up to 50% uptake by cells grown as spheroids (used to represent tumors). For gene activation, these cell-penetrating PcTF proteins did not appear to be as effective as the PcTF protein we had expressed from synthetic DNA in past experiments. Further technical development is needed to deliver functional PcTF regulators into cancer cell nuclei.
Dr. Haynes was invited to Davidson College to present a seminar and to teach a lecture, both related to her work in chromatin epigenetic engineering. On Monday morning, February 10, 202 she visited the Genomics course, taught by Debbie Thurtle-Schmidt. Dr. Haynes’ lecture focused on how evolutionary variations of Polycomb proteins across species were used to identify histone-binding modules that have recently been used as molecular tools (fusion proteins) (Tekel 2018). That evening, Dr. Haynes presented a seminar “Genomic analysis to achieve multi-gene regulation by chromatin design,” as part of the Genomics Program seminar series. Her seminar explained how the fusion proteins she discussed in the Genomics class were used in cancer cells to co-regulate sets of genes, and how bioinformatics methods were used to evaluate changes in gene expression.
The visit was also a reunion with past mentors and colleagues including Malcolm Campbell, and Laurie Heyer. Dr. Haynes was an HHMI supported postdoc/ lecturer at Davidson College from 2006-2008, where she began her training in synthetic biology and inquiry-driven college classroom teaching.
Dr. Haynes has been invited to give a talk, “Chromatin epigenetic engineering: combining synthetic biology with molecular bioinformatics,” at the Quantitative Biology Seminar Series hosted by the Quantitative Biology (qBio) program at the University of California San Diego (UCSD) on Monday, January 27, 2020. The qBio PhD track is designed to equip quantitatively-inclined students with the knowledge and skills necessary to carry out quantitative, multi-faceted investigations of living systems.
Dr. Haynes will present an invited talk, “A Nuclear Genetic Sensor to Measure and Optimize Delivery of Non-Viral DNA into Human Cells,” at the 2020 PepTalk Conference on Monday, January 20, 2020 in the session “Vector Design and Development for Gene and Cell Therapies.” Her talk will focus on a new project to develop genetically-encoded sensors that produce visible signals when therapeutic genes reach their targets (cell nuclei) and become associated with chromatin proteins in living cells.
Research – IJMS – Components from the human c-myb transcriptional regulation system reactivate epigenetically repressed transgenes
Components from the human c-myb transcriptional regulation system reactivate epigenetically repressed transgenes
Barrett CM, McCracken R, Elmer J, Haynes KA. (2020) Int. J. Mol. Sci. 21: 530. PMID: 31947658. PMCID: PMC7014047.
This work was inspired by a problem faced by many bioengineers who are interested in adding new, synthetic genes to human cells to fight disease, produce therapeutic proteins, and regenerate tissues and organs. New genetic material that is delivered into cells generally starts out working quite well: it is transcribed and translated like, or even better than, a “normal” gene. But eventually the synthetic gene is often shut down (silenced) by a DNA packaging and regulation system called chromatin. In our paper, we report the results of a project where we tested different DNA and protein components for their ability to protect synthetic genes from silencing. We use a priori knowledge of chromatin features at the synthetic gene to understand the mechanism of reactivation.
- Pre-print: Use of MYB as a new synthetic activator to enhance transgene expression from within repressed Polycomb chromatin. Barrett CM, McCracken R, Elmer J, Haynes KA. (2018) bioRxiv. https://doi.org/10.1101/487736
Research – APL BioE – Site-directed targeting of transcriptional activation-associated proteins to repressed chromatin restores CRISPR activity
Site-directed targeting of transcriptional activation-associated proteins to repressed chromatin restores CRISPR activity
Daer R, Hamna F, Barrett CM, and Haynes KA. (2020) APL Bioengineering. 4: 016102. PMID: 31967103. PMCID: PMC6960031.
CRISPR is a powerful and popular tool for editing DNA in living cells. Scientists are becoming more interested in using CRISPR to correct mistakes in DNA that lead to diseases, to artificially generate mutations to research the origins of diseases, and for other important applications. However, CRISPR originated in bacteria and has probably not evolved to function very well in genomes that are packed in configurations (open and closed chromatin) as complex as those found in human cells. In a recent report (Daer et al. 2017), we demonstrated that CRISPR activity was inhibited at a DNA sequence that became artificially condensed into closed chromatin. Our new study shows that targeted re-opening of closed chromatin leads to enhanced CRISPR activity in the same region. The epigenetic drug we tested (UNC1999) was not sufficient to generate a transcriptionally active or CRISPR-accessible state. In contrast, strong direct transcriptional activation of the target gene with a DNA-binding p65 protein did enhance CRISPR accessibility. We also identified four other fusion proteins that did not activate transcription but still enhanced CRISPR efficiency, showing that CRISPR can be improved without activating expression of the editing target.
- Histone modifications and active gene expression are associated with enhanced CRISPR activity in de-silenced chromatin. Daer R, Barrett C, Haynes KA. (2017) bioRxiv. https://www.biorxiv.org/content/early/2018/03/11/228601
- Press release – Opening up DNA to delete disease: Custom-built molecules enable editing of genes previously obscured by DNA’s innately protective structure. EurekAlert! https://www.eurekalert.org/pub_releases/2020-01/aiop-oud011320.php