Allelic Gene Regulation of Pcdh-α and Pcdh-γ Clusters Involving Both Monoallelic and Biallelic Expression in Single Purkinje Cells

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The closely linked human protocadherin (Pcdh) α, β, and γ gene clusters encode 53 distinct protein isoforms, which are expressed in a combinatorial manner to generate enormous diversity on the surface of individual neurons. This diversity is a consequence of stochastic promoter choice and alternative pre-mRNA processing. Here, we show that Pcdhα promoter choice is achieved by DNA looping between two downstream transcriptional enhancers and individual promoters driving the expression of alternate Pcdhα isoforms. In addition, we show that this DNA looping requires specific binding of the CTCF/cohesin complex to two symmetrically aligned binding sites in both the transcriptionally active promoters and in one of the enhancers. These findings have important implications regarding enhancer/promoter interactions in the generation of complex Pcdh cell surface codes for the establishment of neuronal identity and self-avoidance in individual neurons.T he clustered protocadherin (Pcdh) genes are expressed in the nervous system and organized into three closely linked clusters (α, β, and γ) (1-5). The human Pcdhα gene cluster contains 13 highly similar variable first exons (α1 to α13) arrayed in tandem and two more distantly related c-type variable first exons designated αc1 and αc2 (Fig. 1A). The variable first exons encode the extracellular, transmembrane, and juxtamembrane intracellular domains of the Pcdhα proteins. Each of these 15 variable first exons is cis-spliced to a single set of three downstream constant exons that encode a distal intracellular domain (1-3). The human β cluster is located downstream from the α cluster and contains a tandem array of 16 highly similar variable exons but with no constant exons, whereas the γ cluster contains 22 variable first exons arrayed in tandem and divided into three types (γa1 to γa12, γb1 to γb7, and γc3 to γc5) (Fig. 1D). As in the case of the α cluster, each of these 22 γ variable first exons is cis-spliced to a single set of three downstream constant exons, which are distinct from the α constant exons, to generate diverse γ mRNAs (1, 3, 6). Analyses of the α and γ transcripts have revealed that highly similar Pcdh alternate isoforms are expressed in a stochastic fashion, whereas all of the c-type divergent isoforms, αc1 and αc2 in the α cluster and γc3, γc4, and γc5 in the γ cluster, are expressed ubiquitously in all cells (1-3, 5, 7). Hereafter, we refer to the c-type genes as "ubiquitously expressed" in contrast to the "alternately expressed" Pcdh genes ( Fig. 1 A and D). A combination of stochastic activation of alternate promoters and constitutive activation of c-type ubiquitous promoters generates enormous single-cell diversity on the surface of individual neurons.Significant advances have been made in understanding the mechanisms by which individual neurons express distinct combinations of the clustered Pcdh genes (2, 3, 8-10). Two long-range cis-regulatory elements in the α cluster, HS5-1 and HS7 (hypersensitive sites 5-1 and 7), function as developmental and tissue...

Section: Resultsmentioning

confidence: 70%

“…However, we find that the human neuroblastoma cell line SK-N-SH is diploid (Fig. S1) (12) and that the pattern of Pcdh expression is what would be expected from a single neuron (6,7).…”

Section: Resultsmentioning

confidence: 99%

CTCF/cohesin-mediated DNA looping is required for protocadherin α promoter choice

Guo

Monahan

et al. 2012

236

329

“…As mentioned above, αc1 is expressed from both chromosomes in all neurons (5). The lower level of Rad21 binding at alternative isoforms is consistent with stochastic expression of these isoforms generating heterogeneity within the population of cells assayed, resulting in a reduced ChIP signal.…”

Section: Binding Of Rad21 To the Pcdhα Cse Correlates With Expressionmentioning

confidence: 74%

“…Over 50 clustered Pcdh genes are organized into three contiguous gene clusters: Pcdhα, Pcdhβ, and Pcdhγ. Single-cell analysis of Pcdhα and -γ gene expression in mouse Purkinje neurons revealed that "alternative" Pcdhα and -γ isoforms are stochastically expressed from each of the two chromosomes, whereas the five "c-type" isoforms are expressed biallelically in every cell (4,5). This pattern of expression could generate over 14,000 different combinations of alternative isoforms, comparable to that of the Drosophila Dscam gene.…”

mentioning

confidence: 99%

Role of CCCTC binding factor (CTCF) and cohesin in the generation of single-cell diversity of Protocadherin-α gene expression

Monahan

Rudnick

Kehayova

et al. 2012

143

126

Extraordinary single-cell diversity is generated in the vertebrate nervous system by the combinatorial expression of the clustered protocadherin genes (Pcdhα, -β, and -γ). This diversity is generated by a combination of stochastic promoter choice and alternative premRNA splicing. Here we show that both the insulator-binding protein CTCF and the cohesin complex subunit Rad21 bind to two highly conserved DNA sequences, the first within and the second downstream of transcriptionally active Pcdhα promoters. Both CTCF and Rad21 bind to these sites in vitro and in vivo, this binding directly correlates with alternative isoform expression, and knocking down CTCF expression reduces alternative isoform expression. Remarkably, a similarly spaced pair of CTCF/Rad21 binding sites was identified within a distant enhancer element (HS5-1), which is required for normal levels of alternative isoform expression. We also identify an additional, unique regulatory role for cohesin, as Rad21 binds to another enhancer (HS7) independently of CTCF, and knockdown of Rad21 reduces expression of the constitutive, biallelically expressed Pcdhα isoforms αc1 and αc2. We propose that CTCF and the cohesin complex initiate and maintain Pcdhα promoter choice by mediating interactions between Pcdhα promoters and enhancers.

“…There are >50 distinct Pcdh isoforms encoded by three closely linked gene clusters named Pcdhα, Pcdhβ, and Pcdhγ (2). Individual neurons stochastically express distinct combinations of Pcdh isoforms, thus generating enormous cell surface diversity that may contribute to the specificity of synaptic interactions (3,4).…”

mentioning

confidence: 99%

Proteolytic processing of protocadherin proteins requires endocytosis

Buchanan

Schalm

Maniatis

2010

The α-, β-, and γ-protocadherins (Pcdhα, Pcdhβ, and Pcdhγ) comprise a large family of single-pass transmembrane proteins predominantly expressed in the nervous system. These proteins contain six cadherin-like extracellular domains, and proteolysis of Pcdhα and Pcdhγ by the γ-secretase complex releases their intracellular domains into the cytoplasm where they may function locally and/ or enter the nucleus and affect gene expression. Thus, cleavage of Pcdhs may function to link intercellular contacts and intracellular signaling. Here we report that shedding of the Pcdhα extracellular domain and subsequent processing by γ-secretase require endocytosis and that Pcdhs interact with the regulator of vesicular sorting ESCRT-0 in undifferentiated cells. We also find that the accumulation of Pcdh cleavage products is regulated during development. Differentiation leads to an increase in the interactions between Pcdh proteins and a decrease in the accumulation of cleavage products. We conclude that Pcdh processing requires endocytosis and that the level of cleavage products is regulated during neuronal differentiation.γ-secretase | hepatocyte growth factor-regulated tyrosine kinase substrate | neurodevelopment | protein-protein interactions | proteolysis