Synthetic Chromatin Epigenetics

Vision. Many people are familiar with “genetics,” the inheritance of visible traits like eye and hair color. Genetic traits are encoded by a molecular grammar (combinations of A,T,C, and G) within the iconic double helix structure DNA. Less familiar to non-specialists is chromatin, the network of protein particles and RNA that interact with DNA to control the physical organization chromosomes and the expression levels of genes. This regulatory process is epi-genetics (epi, EH-pee = upon or above). Our research group aims to engineer chromatin to control epigenetic states within human cells. Once this new technique is fully developed, scientists may be able to simultaneously control multiple genes and expression networks to stop cancer cells in their tracks, or to guide the growth of healthy tissues for limb and organ regeneration.

Technical approach. Signal sensor-actuator proteins (aka reader-effectors) are the central drivers of chromatin epigenetics. Combinations of chemical marks (histone modifications and DNA methylation) within chromatin act as a molecular brail upon which “reader” proteins dock, and then induce changes in the expression of nearby genes. We use protein engineering to design and build synthetic chromatin sensor-actuators that target chromatin marks that are associated with particular cellular states. These chromatin marks act as input signals that trigger the sensor-actuator to regulate genes within the surrounding DNA, and induce changes in cell behavior.

We designed the first synthetic chromatin sensor-actuator, the Polycomb Transcription Factor (PcTF) (Haynes & Silver 2011), to sense histone H3 trimethyl lysine 27 (H3K27me3), a signal that  accumulates near dozens of epigenetically silenced tumor-suppressor genes in cancer cells. When PcTF is introduced into cancer cells (bone, blood, brain, and breast) hundreds of silenced genes become activated. To determine if these changes are of practical use (e.g. for cancer treatment), our group uses bioinformatics to investigate  sensor-actuator-induced changes in gene expression and chromatin modifications that may affect important cellular pathways (immunomodulatory and epithelial-mesenchymal transition, EMT).

In our ongoing work, we are developing a variety of synthetic sensor-actuators to target disease-associated chromatin signals, defining design principles that determine the behavior of chromatin sensor-actuators (Tekel et al. 2017, Tekel et al. 2018), and exploring applications beyond cancer and disease treatment. We believe that designing and building the central drivers of chromatin epigenetics will generate a deeper understanding of coordinated gene regulation in all cells that share this mechanism.

PROJECT: Engineering Chromatin in Triple Negative Breast Cancer

Reprogramming cancer gene expression profiles, and therefore changing the production of proteins within cancer cells could be particularly useful for hard-to-treat cancers that lack sensitive drug targets. We are investigating how chromatin signals and sensor-actuators induce anti-cancer gene expression states in triple negative breast cancer.
Team: Dr. Natecia Williams (Emory), Cara Shields (Emory). Past members: Daniel Vargas (Biological Design MS, ASU), David Nyer (Research Tech, ASU). Support: Arizona Biomedical Research Commission ADHS14-082976 to K. Haynes; Wallace H. Coulter Department of Biomedical Engineering at Emory

Accomplishments & Products:

PROJECT: Rapid Design Pipeline for Chromatin Sensor-Actuators

Natural chromatin sensor-actuator proteins are extremely diverse in their primary sequences and peptide domain structures. To enable efficient exploration of the sensor-actuator design space, we have designed an efficient Golden gate assembly scheme, and a miniaturized, on-chip cell-free protein expression and histone modification-binding platform to quickly screen large libraries for functional histone-binding variants.
Team: J. Harrison Priode (Emory), Dr. Isioma Enwerem (Emory), Kierra Franklin, Dr. Christopher Plaisier (ASU). Past members: Stefan Tekel (Biological Design PhD, ASU). Support: NIH NCI R21CA232244 to K. Haynes.

Accomplishments & Products:

  • 2019 – Poster – “Controlling metastasis and immune signaling in breast cancer cells with epigenetic engineering” J.H. Priode, AfroBiotech Conference, Atlanta, GA

PROJECT: Chromatin Dynamics During Gene Delivery

Chromatin epigenetics influences the delivery of therapeutic genes into cells. We are designing genetically-encoded sensors to track the movement of synthetic DNA into the nuclei of human cells, and to observe the packaging of this DNA into chromatin complexes. We believe that our new molecular sensors will enable scientists to gather quantitative data on the fate of synthetic DNA in human cells, pinpoint and mitigate molecular barriers, and successfully use gene delivery for a broader spectrum of genetic diseases. Team: Dr. Natecia Baskin (Emory), Dr. Kaushal Rege (PI, ASU), Dr. Jacob Elmer (PI, Villanova).


Synthetic Chromatin for Cancer Research

We used the “PcTF” synthetic transcription activator that was first introduced in our earlier work (Haynes & Silver 2011) to regulate genes in cancer cells. By using powerful methods such as ChIP-seq and RNA-seq, we discovered genes that are controlled by PcTF. We also built and tested alternative versions of PcTF to determine how changes in protein design influenced the gene-activating function of PcTF. Team: Cassandra Barrett (Biological Design, PhD), Daniel Vargas (Biological Design MS), Stefan Tekel (Biological Design PhD). Collaborators: Dr. Melissa Wilson Sayres. Sponsor: NIH NCI K01CA188164 to K. Haynes.

Opening Silenced Chromatin

We are using synthetic chromatin proteins to build a permanently re-opened state at epigenetically silenced genes. We aim to achieve unprecedented reliability for the expression of synthetic genes and improvement of CRISPR/Cas9-mediated gene editing in mammalian cells. Team: René Daer (past, Biological Design, PhD), Cassandra Barrett (Biological Design, PhD). Collaborators: Dr. Kaushal Rege (PI, ASU), Dr. Jacob Elmer (PI, Villanova). Sponsor: NSF CBET 1403214 to K. Rege.

Microbial Communication With Synthetic Quorum Sensing

Bacterial engineering is highly accessible to beginners in synthetic biology. Our ongoing ‘engineered quorum sensing’ project (founded by Rene Davis) helps new trainees to learn the connection between gene engineering and cell behavior, and provides a pathway towards advanced projects in human cell synthetic biology. The quorum sensing project characterizes cross-talk between decoupled cell-cell communication systems from bacteria. Team: Rotating graduate mentors and undergraduate trainees. Sponsors: Women and Philanthropy, ASU Fulton Undergraduate Research Initiative (FURI), Kern Entrepreneurial Engineering Network (KEEN).


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