The generation of a protocadherin cell-surface recognition code for neural circuit assembly

Abstract

The assembly of functional neural circuits in vertebrate organisms requires complex mechanisms of self-recognition and self-avoidance. Neurites (axons and dendrites) from the same neuron recognize and avoid self, but engage in synaptic interactions with other neurons. Vertebrate neural self-avoidance requires the expression of distinct repertoires of clustered Protocadherin (Pcdh) cell-surface protein isoforms, which act as cell-surface molecular barcodes that mediate highly specific homophilic self-recognition, followed by repulsion. The generation of sufficiently diverse cell-surface barcodes is achieved by the stochastic and combinatorial activation of a subset of clustered Pcdh promoters in individual neurons. This remarkable mechanism leads to the generation of enormous molecular diversity at the cell surface. Here we review recent studies showing that stochastic expression of individual Pcdhα isoforms is accomplished through an extraordinary mechanism involving the activation of 'antisense strand' promoter within Pcdhα 'variable' exons, antisense transcription of a long non-coding RNA through the upstream 'sense strand' promoter, demethylation of this promoter, binding of the CTCF/cohesin complex and DNA looping to a distant enhancer through a mechanism of chromatin 'extrusion'.

Figure 1:. Neuron-type specific expression of cluster Pcdh genes (stochastic vs determinative)

(A) Organization of the murine Pcdh gene clusters. White: Pcdhα alternate genes; Aqua: Pcdhβ genes; Grey: Pcdhγ alternate genes; Purple: c-type genes from the Pcdh α and γ clusters. Arrows indicate all the promoters of the Pcdh genes. (B) Example of a stochastic and combinatorial expression of Pcdh genes from all three clusters in olfactory sensory neurons (OSNs). (C) Example of a stochastic and combinatorial expression of Pcdh alternate genes from all three clusters in Purkinje neurons. In these neurons, the c-type are biallelically and constitutively expressed in every cell. (D) Example of a determinative expression of the sole cluster Pcdhαc2 in serotonergic neurons. The expression of Pcdh genes shown in (B-C) is meant to summarize expression from both chromosomes, as the two chromosomes behave independently with regards to Pcdh promoter choice.

Figure 2:. Functional role of a Pcdh diversity code in OSNs

Left: Schematics of how a Pcdh cell-surface diversity code provides: (1) self-recognition of sister neurites (axons and dendrites) to allow them to innervate their territory and (2) attraction of non-sister neurites (axons and dendrites) to allow different neurons to converge to form a glomerulus. Right: Schematics of the phenotypes observed by deletion of the Pcdh gene tri-cluster (Pcdh Δ) and overexpression of a specific subset of Pcdh α, β and γ isoforms in every OSN (Pcdh UNI). The data presented here are from Mountoufaris et al. Science 2017.

Figure 3:. Differential regulation of cluster Pcdhα alternate and c-type generates distinct transcriptional programs

(A) Differential regulation of expression of alternate and c-type promoters in the Pcdhα gene cluster. Pcdhα alternate genes are expressed by long-range DNA contacts between their promoters and the HS5–1 enhancer. These contacts are mediated by the CTCF and Cohesin protein complexes. The choice of Pcdhα alternate promoters is also regulated by histone and DNA methylation (H3K9me3 and 5mC, respectively) and the activity of the Smcdh1 protein. (B) Relation between DNA methylation and promoter activation in the Pcdhα cluster: DNA demethylation of CTCF binding sites and, the DNA sequence between them, correlates with CTCF occupancy of its CBS sites, engagement with the HS5–1 enhancer and transcriptional activation of the unmethylated promoter.

Figure 4:. Model of stochastic Pcdhα alternate promoter choice

Coupling antisense lncRNA transcription to DNA demethylation mediates stochastic promoter choice of clustered Pcdhα alternate promoters by CTCF and Cohesin through DNA loop extrusion. This mechanism ensures that the choice of a Pcdhα alternate promoter occurs independently from the distance between the stochastically chosen promoter and the HS5–1 enhancer.