RUI: Investigating enhancer grammar that underlies naive-state pluripotency

Despite decades of research, it remains a challenge to predict enhancer functions from genetic information. Enhancers are sequences in genomes that control when and where genes are expressed and have profound roles during development of multicellular organisms. While they do not code for proteins, the information that enhancers contain is encoded as binding sites for transcription factors (TFs), which are key proteins in gene regulation.

It is thought that a key contributor to enhancer function is grammar, or the characteristics of TF binding sites, such as type, number, binding affinities, and arrangement. However, there are still many questions about the principles of enhancer grammar and how those underlie mechanisms for gene regulation. A deeper understanding of enhancers will help us predict phenotypes from genetic information and can be applied in other areas, such as medicine and agriculture.

This project will study enhancer grammar in naïve-state pluripotent stem cells, which represent the earliest stages of mammalian development. In addition to advancing knowledge about how gene regulation is encoded into the genome, this work will engage 25-50 undergraduate students per year in mentored, investigative, and original research in preparation for science careers.

To investigate grammatical constraints that underlie naïve-state specific enhancer function, this project will focus on the enhancers for the naïve-state gene Klf4, which contain binding sites for the TFs OCT4, SOX2, ESRRB and STAT3. Preliminary work has indicated that low-affinity TF binding sites are critical for the naïve-state specific function of one Klf4 enhancer.

To illuminate the role of this binding affinity constraint in transcription activation mechanisms, this project will utilize molecular approaches to measure the effects of optimizing TF binding sites in this enhancer. Prior studies also have shown that TFs bind to the Klf4 enhancers in a hierarchical order, suggesting that the arrangement of TF binding sites is key to facilitate enhanceosome assembly, particularly interactions among TFs at the Klf4 enhancers.

To address these questions, this research will utilize a combination of reporter assays, gene editing, and molecular techniques. Lastly, this project will apply knowledge from these and prior studies of the Klf4 enhancers to other naïve-state specific enhancers. A combination of bioinformatics and molecular biology techniques will be employed to identify and analyze putative naïve-state specific enhancers.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.