How genome architecture controls Protocadherin gene choice at single-allele resolution

PROJECT SUMMARY
Neural self-avoidance is a fundamental yet poorly understood process that is essential for proper brain wiring.
Self-avoidance describes the tendency of neurites originating from the same cell to avoid each other while
innervating target brain regions and finding synaptic targets. This process is mediated by the clustered
Protocadherin (Pcdh) genes, which are differentially expressed between neurons and act as cell-surface
molecular barcodes: neurites presenting the same combination of Pcdh isoforms repel each other, while those
presenting different combinations can interact. Despite their central role in brain wiring, the regulation of Pcdh
genes remains an unsolved problem in neuroscience. Specifically, the mechanism by which neurons choose
specific Pcdh isoforms to express during development, and how this choice is maintained across the lifespan of
a neuron, are unclear. Recent work has demonstrated a role for cohesin-mediated DNA loop extrusion in tuning
Pcdh expression across cell types, suggesting that 3D genome architecture is a critical determinant of Pcdh
gene choice. This proposal aims to test this model in vivo by using optical reconstruction of chromatin
architecture (ORCA), a method for imaging single-allele genome structure in thousands of single cells, to study
Pcdh locus topology in neurons of the olfactory epithelium (OE) and their precursors. By combining ORCA with
RNA labelling of OE cell types and Pcdh isoforms, the first aim will establish a developmental clock of Pcdh gene
choice that links 3D genome folding to the onset of Pcdh expression during development. Genetic mouse lines
will be used to abolish cohesin activity in each cell type to determine how cohesin controls Pcdh choice across
development. The second aim will address how cells achieve transcriptional stability of Pcdh genes across their
lifetimes by considering a role for heterochromatin in the continued silencing of non-chosen Pcdh genes. Through
single-cell sequencing and ORCA experiments, the ability of heterochromatin to stabilize Pcdh gene expression
by sequestering non-chosen promoters away from Pcdh enhancers will be tested. Overall, these studies will
have implications across multiple fields, including brain wiring, translational neuroscience, and gene regulation.
First, they will reveal strategies by which neurons establish proper morphologies, an essential step in circuit
formation across the brain, as seen here through the formation of olfactory maps. Second, Pcdh dysregulation
is associated with multiple neuropsychiatric and neurodegenerative disorders, including schizophrenia and
autism. The principles of Pcdh gene regulation shown here will advance our understanding of these disorder
mechanisms and identify potential avenues for therapeutic intervention. And lastly, the functions of cohesin in
gene regulation have been difficult to determine. The protein complex has primarily been studied in dividing cells
in vitro, where it also plays a vital role in sister chromatid cohesion during cell division. These studies will reveal
new physiological activities of cohesin in single post-mitotic cells in vivo and therefore shed light on long-standing
debates over the ability of cohesin to activate and repress genes.

Grant Number: 5F31DC022526-02
NIH Institute/Center: NIH
Principal Investigator: Alexander Buckley