Regulatory elements required for the activation and repression of the protocadherin-alpha gene cluster

Abstract

The mouse protocadherin (Pcdh) -α, -β, and -γ gene clusters encode more than 50 protein isoforms, the combinatorial expression of which generates vast single-cell diversity in the brain. At present, the mechanisms by which this diversity is expressed are not understood. Here we show that two transcriptional enhancer elements, HS5-1 and HS7, play a critical role in Pcdhα gene expression in mice. We show that the HS5-1 element functions as an enhancer in neurons and a silencer in nonneuronal cells. The enhancer activity correlates with the binding of zinc finger DNA binding protein CTCF to the target promoters, and the silencer activity requires the binding of the REST/NRSF repressor complex in nonneuronal cells. Thus, the HS5-1 element functions as a neuron-specific enhancer and nonneuronal cell repressor. In contrast, the HS7 element functions as a Pcdhα cluster-wide transcription enhancer element. These studies reveal a complex organization of regulatory elements required for generating single cell Pcdh diversity.

Fig. 1.

HS5-1 deletion differentially affects Pcdhα isoform expression in neuronal tissues. Total mRNA was extracted from the whole brain, cortex, cerebellum, hippocampus, and olfactory bulb of HS5-1 KO homozygous mice and 129Sv/Jae wild-type mice at P9. Expression of individual Pcdhα isoforms was analyzed by qRT-PCR and normalized to Rps17. Each bar represents the average of at least three replicates and error bars represent SEM. Quantification of constant exon 3 (con3) represents total Pcdhα levels. *P < 0.05 comparing KO to wild-type expression using a two-tailed, unpaired Student t test.

Fig. 2.

CTCF binding at Pcdhα promoters correlates with expression. qPCR analysis of CTCF ChIP experiments performed on chromatin from whole brain of HS5-1 KO homozygous or 129Sv/Jae wild-type mice. Specific PCR primers were used to assay the quantity of DNA recovered in each immunoprecipitation and this quantity is expressed relative to the quantity detected in an input chromatin control. (A) Analysis of CTCF binding at the promoters of the C-type Pcdhα isoforms. Sites A and B are known intergenic CTCF sites (38). Neg A and Neg B are intergenic sites on chromosome 7 without CTCF motifs; −2.6kb αc1 is a site 2.6-kb upstream of the Pcdhαc1 transcription start site. Each bar represents the average of three or four experiments and error bars represent SEM. (B) Analysis of CTCF binding at the promoters of alternatively expressed Pcdhα isoforms. Negative control site data are the same as in A. *P < 0.05 and **P < 0.01 using a two-tailed, unpaired Student t test. N.D. indicates not determined.

Fig. 3.

HS7 deletion negatively affects the expression of Pcdhαs, including PcdhαC2, in a subset of brain tissues. Total mRNA was extracted from the whole brain, cerebellum, cortex, hippocampus, and olfactory bulb of HS7 KO homozygous mice and 129Sv/Jae wild-type mice. Data for cortex, hippocampus, and olfactory bulb are included in SI Materials and Methods. Expression of individual Pcdhα isoforms was analyzed by qRT-PCR and normalized to Rps17. Each bar represents the average of at least three replicates and error bars represent SEM. Quantification of constant exon 3 (con3) represents total Pcdhα levels. *P < 0.05 comparing KO to wild-type expression using a two-tailed, unpaired Student t test.

Fig. 4.

HS5-1 deletion leads to increase of Pcdhα expression in glia and kidneys. (A) Total mRNA was extracted from cultured glia and kidneys of HS5-1 KO homozygous or 129Sv/Jae wild-type mice. Expression of individual Pcdhα isoforms was analyzed by qRT-PCR and normalized to Rps17. Each bar represents the average of at least three replicates and error bars represent SEM. (B) HS7 deletion does not lead to substantial increase of Pcdhα expression in kidneys. Expression of individual Pcdhα isoforms was analyzed by qPCR as described in Fig. 4A. Each bar represents the average of at least three replicates and error bars represent SEM. Quantification of constant exon 3 (con3) represents total Pcdhα levels. *P < 0.05 comparing KO to wild-type expression using a two-tailed, unpaired Student t test.

Fig. 5.

NRSE site in HS5-1 represses nonneuronal Pcdhα4 promoter activity in Luciferase reporter assays. (A and B) Plot of NRSF ChIPseq signal at Pcdhα12 coding sequence NRSE (A) and HS5-1 NRSE (B) for eight human cell lines. Scaling of ChIPseq signal varies between cell lines but is the same at both sites. The position of the NRSE is indicated. The lower portion of each panel shows a plot of sequence conservation among mammals (Phastcons). (C) Diagram of the HS5-1 enhancer element indicating constructs used in reporter assays. The location of the NRSE sequence is highlighted. The plot below the figure indicates degree of sequence conservation among mammals. (D and E) Plot of luciferase reporter activity of Pcdhα4 promoter alone and in constructs bearing variants of HS5-1 constructs in TCMK1 mouse kidney cells (n = 3) (D) or undifferentiated CAD cells (n = 5) (E). Expression of each vector is compared with expression from the Pcdhα4 promoter alone, which is set to 1. Error bars represent SEM. *P < 0.05, **P < 0.01 in two-tailed, unpaired Student t test.