PROJECT SUMMARY Cellular differentiation is controlled by epigenetic regulatory systems that integrate multiple sensory inputs over time to direct long-term cell fate decisions. Harnessing epigenetic regulation in the design of synthetic gene circuits would greatly augment synthetic biology. Synthetic epigenetic circuits based on controlling chromatin state present many attractive advantages over current forms of artificial cellular memory (e.g. recombinase or Cas9-based switches/cascades) which can be limited in scalability, stability, and temporal control. Natural epigenetic systems support stable memory states without altering genetic information, can induce state changes in either deterministic or stochastic fashion, and still maintain reversibility. We propose to generate a synthetic toolbox to regulate chromatin state in response to user-specified inputs, thereby allowing construction of circuits with key epigenetic properties, such as memory, fate bifurcation and temporally controlled gene expression. Our proposed circuits will be designed to a) respond to a variety of extracellular input cues through synthetic Notch receptors, b) discriminate inputs by duration to specifically induce cell fate changes only in response to persistent environmental stimuli, c) temporally control gene expression programs to promote sequences of cellular behaviors, and finally d) establish divergent differentiation states to allow functionally advantageous specialization within a cell population. Our work will be developed in the testbed of CAR T cell immunotherapy, a major application area for mammalian cellular engineering that could greatly benefit from synthetic circuits that incorporate epigenetic memory and temporal control capabilities. The resulting epigenetic toolkit and circuits, however, will be applicable to a much larger range of engineered cells, including applications in regenerative medicine and cell therapies more broadly.