Genetic Mechanisms of Regulated Stochastic Gene Expression

The adaptability and robustness of the central nervous system is partially explained by the vast diversity of neuronal identities. Molecular mechanisms generating such heterogeneity have evolved through multiple independent pathways. The olfactory sensory system provides a unique and tractable platform for investigating at least two orthogonal gene expression systems that generate neuronal diversity through stochastic promoter choice: olfactory receptor genes and clustered protocadherins. Olfactory sensory neuron identity is defined by the specific olfactory receptor (OR) gene chosen. Greater than 1300 OR genes are scattered throughout the mouse genome, and expression of an OR defines a unique sensory neuron class that responds to a selective set of odorants. This work further delineated an unprecedented network interchromosomal (trans) interactions indispensable for singular OR choice. In a largely orthogonal gene expression system, I sought to understand the molecular mechanisms governing stochastic protocadherin choice. Clustered protocadherins are an evolutionary- conserved system that is involved in cell-cell identification through a series of homo- and heterophilic interactions. This work uncovered a methylation-dependent mechanism for generating stochastic gene expression in the context of cis-regulatory elements. Overall, this work highlighted divergent cis and trans transcriptional regulatory mechanisms for generating stochastic gene expression and neuronal diversity

Antisense lncRNA Transcription Mediates DNA Demethylation to Drive Stochastic Protocadherin α Promoter Choice

Stochastic activation of clustered Protocadherin (Pcdh) α, β, and γ genes generates a cell-surface identity code in individual neurons that functions in neural circuit assembly. Here, we show that Pcdhα gene choice involves the activation of an antisense promoter located in the first exon of each Pcdhα alternate gene. Transcription of an antisense long noncoding RNA (lncRNA) from this antisense promoter extends through the sense promoter, leading to DNA demethylation of the CTCF binding sites proximal to each promoter. Demethylation-dependent CTCF binding to both promoters facilitates cohesin-mediated DNA looping with a distal enhancer (HS5-1), locking in the transcriptional state of the chosen Pcdhα gene. Uncoupling DNA demethylation from antisense transcription by Tet3 overexpression in mouse olfactory neurons promotes CTCF binding to all Pcdhα promoters, resulting in proximity-biased DNA looping of the HS5-1 enhancer. Thus, antisense transcription-mediated promoter demethylation functions as a mechanism for distance-independent enhancer/promoter DNA looping to ensure stochastic Pcdhα promoter choice