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	Figure 1. NRSEs in fugu protocadherin clusters. (A) Fugu protocadherin clusters. Colored vertical bars represent individual variable exons, whereas the black vertical bars at the 3′-end of each subcluster represent the constant exons. Red and blue ovals in the intergenic regions represent canonical and non-canonical NRSEs, respectively. The dotted line in the fugu Pcdh2 cluster indicates an undetermined sequence gap. Fr: Fugu rubripes. (B) WebLogo display of the consensus sequence of canonical (left panel) and non-canonical (right panel) NRSEs. Note: the non-canonical NRSE consensus is a combination of both fugu and zebrafish elements.
	Figure 2. Functional assay of NRSEs in the fugu protocadherin cluster. EMSA for canonical (A) and non-canonical (B) NRSEs. The NRSE sequences are indicated by upper-case letters whereas the flanking sequences are indicated by lower-case letters. The asterisks indicate the critical guanine dinucleotide that was mutated to adenine nucleotides in NRSE mutants. Arrows in the autoradiography indicate the protein–oligonucleotide complex of the full-length fugu NRSF/REST (A: lanes 2 and 9; B: lanes 2, 4, 6 and 12), or the super-shifted complex in the presence of the anti-Myc antibodies (A: lanes 3 and 10; B: lanes 3, 5, 7 and 13). fN: nuclear extracts from pCMVmyc-fNRSF-transfected HEK293 cells; un: nuclear extracts from mock (pCMVmyc) transfected HEK293 cells. Fr2α33m, Fr2γ6m and Fr2γ34m represent the corresponding mutant NRSE oligonucleotides of Fr2α33, Fr2γ6 and Fr2γ34, respectively. HsαCX and MmαCX represent the NRSEs located in the constant regions of the human and mouse protocadherin α clusters. (C) Relative luciferase activity of Neuro2A stable cell lines harboring individual fugu protocadherin promoter-luciferase reporter cassettes. The solid bars represent the wild-type fugu protocadherin promoters, whereas the open boxes represent the mutant forms. The relative luciferase activity was calculated by the activity of the pCMVmyc-rNRSF-transfected cells over the mock plasmid transfected cells after normalization for the protein concentration. (D) ChIP assay showing that the ectopically-expressed NRSF/REST binds to the wild-type NRSE-containing promoters (Fr2α32, Fr2α33 and Fr2γ6), but not the mutant (Fr2α32mut, Fr2α33mut and Fr2γ6mut) or NRSE-lacking (Fr2α34 and Fr2α36) promoters. The expression of rat NRSF/REST was confirmed by western blot and immunostaining with anti-Myc antibody.
	Figure 3. NRSEs are responsible for the neuronal specificity of fugu protocadherin promoters. (A) The expression profile of fugu Pcdh2α and Pcdh2γ subcluster genes determined by real-time RT-PCR. The relative protocadherin expression level in each tissue is displayed as a percentage of the expression level in the brain after normalization by actin expression. (B) NRSE-containing protocadherin promoters: red ovals represent the wild-type NRSE, whereas blank ovals with a cross represent the mutant NRSEs. Images of transgenic tadpoles (9–10 days post-fertilization) harboring the wild-type (left panels) and mutant (right panels) protocadherin promoter-EGFP constructs. Upper panels: Fr2α33-EGFP; lower panels: Fr2γ6-EGFP. (C) Comparison of the NRSE-containing and NRSE-lacking protocadherin promoters: the whole body images of transgenic tadpoles (9–10 days post-fertilization) harboring a fugu NRSE-containing (Fr2α33, upper panel) or an NRSE-lacking (Fr2α36, lower panel) protocadherin promoter-EGFP showing that the NRSE-containing promoter directs EGFP expression specifically to the nervous system, whereas the NRSE-lacking promoter directs the transgene expression to both neuronal and non-neuronal tissues. (D) NRSE-lacking protocadherin promoters: images of transgenic tadpoles harboring NRSE-lacking Fr2α36-EGFP transgene in the absence (left panel) and presence (right panel) of an NRSE inserted at the 3′-end of EGFP coding sequence. (E) The transverse section of the brain/spinal cord region of transgenic tadpole harboring the Fr2α33-EGFP showing that the EGFP expression is predominantly neuronal. Left panel: EGFP florescence image; Middle panel: anti-neuron-specific tubulin βIII (TuJ1); Right panel: merged.
	Figure 4. NRSE regulates protocadherin promoter activity in the multi-gene cluster context. (A) Genomic organization of fugu BAC clone b245G6. Shaded ovals indicate the NRSEs, whereas open boxes and solid vertical bars indicate the variable and constant exons, respectively. The position of PCR primer pairs is shown by arrows. (B) ChIP assay of NRSE occupancy in b245G6-stably-transfected HeLa cells showing that NRSF/REST binds to NRSEs in fugu protocadherin cluster in vivo. Asterisks indicate NRSE-containing protocadherin genes. Input: genomic DNA of the b245G6-stably-transfected HeLa cells. (C) Characterization of the dominant-negative isoform of NRSF/REST (NRSF/RESTdn) in HeLa cells, showing that similar to the full-length NRSF/REST, the NRSF/RESTdn is predominantly expressed in nucleus. (D) Relative expression levels of fugu protocadherin genes in the NRSF/REST knock-down and NRSF/RESTdn-transfected cells. The data shown here (right panel) is average of three independent experiments. Asterisks indicate NRSE-containing protocadherin genes. Primers used for the ChIP assay and the RT-PCR analysis are listed in Supplementary Table S3. (E) Chromosomal spatial organization analysis by 3C assay showing that the promoter regions of Fr2γ1-3 are in proximity to the NRSE of Fr2γ4 in b245G6-stably-transfected HeLa cells. The AseI sites flanking the promoter region are indicated by ‘A’. DNA fragments corresponding to specific PCR products resulting from ligation of the sequence flanking the NRSE and the respective promoter sequences are indicated by arrows (panels 1–6). A fragment of the intergenic region of Fr2γ4 flanking the NRSE site (within the AseI fragment) was amplified by PCR with the Fr2γ4 primers (Supplementary Table S3) to confirm that equal amounts of DNA were used as template (last panel).
	Figure 5. NRSEs in the protocadherin clusters of zebrafish, human and mouse. (A) Zebrafish protocadherin clusters. The color vertical bars represent individual variable exons, whereas the black vertical bars at the 3′ end of each subcluster represent the constant exons. Red and blue ovals in the intergenic regions of the zebrafish clusters represent the canonical and non-canonical NRSEs, respectively. The dotted lines in the Pcdh2 cluster indicate undetermined sequence gaps. The consensus sequence of zebrafish canonical NRSEs is displayed as a WebLogo plot. Dr: Danio rerio. (B) EMSA for NRSEs located in the protein-coding sequence of human and mouse protocadherin clusters. The consensus sequences are displayed as WebLogo plots. Upper-case letters represent the NRSE sequence, and the lower-case letters represent the flanking sequences. The asterisks indicate the guanine dinucleotide which was mutated to adenine dinucleotide in NRSE mutants. Hsα5m represents the mutant NRSE in the protein-coding sequence of the Hsα5. Hs: Homo sapiens, Mm: Mus muscus. Autoradiography of EMSA: arrows indicate the protein–oligonucleotide complex of the full-length fugu NRSF/REST (lanes 1, 3, 5 and 7) or the super-shifted complex in the presence of the anti-Myc antibodies (lanes 2, 4, 6 and 8). fN: nuclear extracts from pCMVmyc-fNRSF-transfected HEK293 cells; un: nuclear extracts from mock (pCMVmyc) transfected HEK293 cells.

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