Many biomedically important processes such as oncogenesis, stem cell differentiation, and cellular reprogramming depend upon timely and appropriate changes in cellular memory and genomic regulation. Dysregulation, even transiently or in a small subpopulation of cells, may result in cancer or the loss of tissue homeostasis. Therefore, a mechanistic understanding of memory and spatial genomic regulation would benefit our understanding of normal developmental and cancerous processes. In addition, synthetic control of cellular memory would enable the development of in vivo diagnostic sensors of malignant or developmental events, enable biological researchers to stably regulate genes on demand, design targeted pharmaceuticals and biologics, as well as facilitate stem cell and cellular reprogramming studies towards cell replacement therapies. Chromatin, the structurally packaged proteins and DNA in the nucleus, has been implicated in regulating genomic architecture and memory-related processes. Chromatin structure is hypothesized to be regulated by a complex "histone code" in which modifications, including methylation and acetylation, placed on histone tails drives compact (silent) or open (active) chromatin formation. Interestingly, self-reinforcing mechanisms acting at these marks are hypothesized to confer stable chromatin-based memory and spatial spreading of chromatin over large genomic regions. However, current methods to study such mechanisms and the histone code rely largely on correlative observations between pan-genomic perturbations, such as gene knockouts and pharmacological agents, and genome-wide microarray expression analysis and chromatin- immunoprecipitation followed by genome-wide sequencing. This work proposes to address these limitations by building upon zinc-finger targeting capabilities to site-specifically recruit libraries of chromatin-regulating proteins to synthetic multi-reporter loci in yeast and human cells, thus directly assessing each protein's regulatory functions. The transcriptional regulation, memory, and spatial spreading properties of these proteins will be measured by flow cytometry and chromatin immunoprecipitation. In addition, de/reconstruction of non-native human chromatin machinery in budding yeast cells will present orthogonal interactions with which to study self-reinforcing human chromatin mechanisms in isolation from native protein interactions and also enable the design of novel functions. Hypothesized self- reinforcing behavior in chromatin-based memory will be tested using this synthetic system in conjunction with biochemistry, flow cytometry, and molecular biology techniques. Through these strategies, this proposal aims to identify chromatin proteins with novel memory and spatial regulatory properties as well as elucidate design principles underlying cellular memory and spatial genomic regulation, with implications for the design of chromatin-based approaches for biomedical applications and biological research.
Diverse biological processes including stem cell differentiation, cellular reprogramming, development, and tissue homeostasis depend on robust mechanisms of chromatin-based regulation with dysregulation leading to cancer and developmental disorders. This work aims to understand the mechanisms of chromatin- based cellular memory and spatial genomic regulation by site-specifically targeting native and non-native chromatin-regulating proteins to reporter loci in S.cerevisiae and human cells. A detailed understanding of cellular memory and spatial genomic regulatory mechanisms and the ability to implement epigenetic devices will be significant advances for our understanding of many classes of diseases and enable the design of powerful research tools as well as novel in vivo cellular sensors of malignant events.
|Keung, Albert J; Bashor, Caleb J; Kiriakov, Szilvia et al. (2014) Using targeted chromatin regulators to engineer combinatorial and spatial transcriptional regulation. Cell 158:110-20|