Various types of neurons, which are largely present in the brain and the spinal cord of the nervous system, control diverse functions in our body, such as recognition, movement, and behavior. Malfunction of neurons causes severe defects in our body functions, often associated with neurological disorders and neurodegenerative diseases, such as autism spectrum disorder (ASD) and ALS motor neuron disease. Several genome-wide association studies in patients with neurological diseases have identified genetic variations including single nucleotide polymorphisms (SNPs) in distal enhancer DNA, where transcription factors (TFs) and chromatin regulatory proteins bind to regulate gene expression. The gene regulatory mechanisms through which distal enhancers are bound by TFs and chromatin regulatory proteins in neural development and disease remain elusive. We established a rapid and efficient system to generate distinct types of neurons, such as cholinergic neurons and GABAergic neurons, via mouse embryonic stem (ES) cell-based differentiation protocol (Montanera and Rhee, 2020, J Vis Exp.; Jaura, Yeh, et al. and Rhee, 2022, Nature Commun). Despite the recent progress in genome-wide studies on TFs in cell-type-specific enhancers, the following essential questions are poorly understood.
1) How TFs select their functional binding sites out of thousands of similar DNA binding sequences on the genome via chromatin interactions between enhancers and neuron genes?
2) What are the molecular mechanisms of chromatin regulation in cell type-specific enhancers during neural development?
3) What are the genomic requirements for neuronal subtype specification regulated by distal enhancers?
To address these questions, the Rhee Lab uses an efficient mouse ES cell differentiation system and transgenic mouse models combined with CRISPR/Cas9 genome editing and genomic mapping approaches, such as chromatin accessibility, TF-binding sites, and long-distance chromatin interactions (Montanera et al. and Rhee, 2020, Epigenetics Met.; Yeh and Rhee, 2022, Methods Mol Biol). These cutting-edge approaches enabled us to generate detailed cell-fate programming molecular maps into distinct types of neurons, often associated with neurological disorders and diseases.
"WHAT MAKES A NEURON DIFFERENT FROM OTHER CELLS?"
In the mammalian body, there are thousands of cell types, which have the same genomic DNA in the nucleus of each cell. Genetically identical cells express a distinct set of genes during embryonic development, resulting in a variety of cell types. Our research focuses on understanding how genes are regulated and how a unique neuronal cell identity is established and maintained during mammalian development. This knowledge is key to understanding neurodevelopment and modeling neurodegenerative diseases. We study the molecular bases of global gene regulatory networks involved in neural development and disease.
"How are genes expressed from DNA to make a neuron?"
The acquisition of cell fate is driven by a cell type-specific gene expression program activated by DNA-binding proteins such as transcription factors. Nearly every gene is packaged into chromatin, which is a complex of DNA and protein in the nucleus of the cell. The recruitment of transcription factors to their target genes is accompanied by dynamic chromatin regulation. The genomic targets of some DNA-binding proteins have been recently identified. However, it is largely unknown how transcription factors are recruited to their target genes in the context of chromatin regulation during neural development. It results in challenges of stem cell differentiation into specific neuronal cell types for disease modeling and neurodevelopmental studies. We study mammalian gene regulation for more efficient stem cell differentiation methods to make specific types of neurons.
"How can we study gene regulation during neural development?"
To understand these processes, we utilize molecular, cellular, and epigenetics approaches combined with cutting-edge genomic techniques and stem cell differentiation methods. For example, we developed a novel genomic mapping method, called ChIP-exo, to detect the genomic binding locations of proteins at ultra-high resolution (Rhee et al., Cell, 2014 ). Furthermore, by using efficient embryonic stem cell differentiation and genome engineering systems (Rhee et al., Neuron, 2016 ), we are studying how cell type-specific transcription factors control gene expression (Rhee et al. Nature, 2012 ).
"Can we apply our expertise to translational research?”
Our expertise in these comprehensive approaches provides greater detail and insight into molecular mechanisms of cell type-specific gene expression programs in the mammalian central nervous system. We are now exploring whether this integration of multiple approaches is instrumental in identifying predictive rules for the progression and treatment of neurodegenerative diseases. We hope that our research on molecular mechanisms of gene regulation might be informative for diagnostic and therapeutic purposes.
CRISPR-mediated nBAF mutations in day 13 GABAergic neurons (courtesy of Alyssa)
RESEARCH FUNDS
The Rhee Lab is supported by the following research funds and federal funding agencies.
