Promoter-like sequences regulating transcriptional activity in neurexin and neuroligin genes

Synapse function requires the cell-adhesion molecules neurexins (Nrxn) and neuroligins (Nlgn). Although these molecules are essential for neurotransmission and prefer distinct isoform combinations for interaction, little is known about their transcriptional regulation. Here, we started to explore this important aspect because expression of Nrxn1-3 and Nlgn1-3 genes is altered in mice lacking the transcriptional regulator methyl-CpG-binding protein2 (MeCP2). Since MeCP2 can bind to methylated CpG-dinucleotides and Nrxn/Nlgn contain CpG-islands, we tested genomic sequences for transcriptional activity in reporter gene assays. We found that their influence on transcription are differentially activating or inhibiting. As we observed an activity difference between heterologous and neuronal cell lines for distinct Nrxn1 and Nlgn2 sequences, we dissected their putative promoter regions. In both genes, we identify regions in exon1 that can induce transcription, in addition to the alternative transcriptional start points in exon2. While the 5′-regions of Nrxn1 and Nlgn2 contain two CpG-rich elements that show distinct methylation frequency and binding to MeCP2, other regions may act independently of this transcriptional regulator. These data provide first insights into regulatory sequences of Nrxn and Nlgn genes that may represent an important aspect of their function at synapses in health and disease.

. RT-qPCR analysis of Nrxn and Nlgn transcript levels Exon-spanning RT-qPCRs were performed between the indicated exons. Note that oligonucleotides for Nlgn1 and Nlgn2 only exist in the mRNA sequence. Values describe the relative expression of transcripts in wild-type (WT) vs. MeCP2-knockout (KO) samples from day P7 and P20. As described in Material and methods, values below 1 indicate that expression in the knockout was decreased, values above 1 indicate an increased expression in the knockout sample. Significance levels were tested with Student´s t-test, exact P values indicated, ns = not significant Quantitative immunoblots of brain lysates were used to determine the protein levels of Nlgn1-3 in WT and MeCP2 KO mice at age P7 and P20. a Expression of protein is calculated as relative luminescence signal in KO/signal in WT.
b Number (n) of blots quantified for each protein; significance was tested with Student´s t-test; ns = not significant. Several overlapping genomic fragments around exon1 and exon2 of the Nrxn1 gene were cloned in a luciferase vector, using the indicated primer pairs (No. refer to the order of fragment shown in Figure 4). The genomic localization of the sequences tested is indicated. Results of the luciferase assays in HEK293 cells, PC12 and differentiated PC12 cells (PC12 Diff) represent percent of vector control (= 100%). Each region was measured in 3-6 independent experiments. Significance levels were tested with Student´s ttest; and P values listed. Several overlapping genomic fragments in the vicinity of exon2 of the Nlgn2 gene were cloned in a luciferase vector, using the indicated primer pairs. The genomic localization of the sequences tested is shown. Results of the luciferase assays in HEK293 cells and PC12 cells represent percent of the vector control (= 100%). Each region was measured in 3-6 independent experiments. Significance levels were tested with Student´s t-test; and P values listed. Several overlapping genomic fragments between the first exons of the Nlgn2 and 18100RIK genes were cloned in a luciferase vector, using the indicated primer pairs (No. refer to the order of fragment shown in Fig. 7). The genomic localization of the sequences tested is shown. Results of the luciferase assays in HEK293 cells, PC12 and differentiated PC12 cells (PC12 Diff) represent percent of the vector control (= 100%). Each region was measured in 3-6 independent experiments. Significance levels were tested with Student´s t-test; and P values listed. Genomic fragments from the 5´-region of Nlgn2 were cloned in a luciferase vector, using the indicated primer pairs (No. refer to the fragments shown in Fig. 8). The sequences used are the same as in GS13 (MSP1) and GS14 (MSP2) displayed in Fig. 6. The DNA was methylated in vitro as described in

Supplementary
Material and methods before transfection into PC12 and differentiated PC12 (PC12 Diff) cells. The genomic localization of the sequences tested is indicated. Results of the luciferase assays represent percent of the methylated vector control. Each region was measured in 3 independent experiments.
Statistic and significance levels compared the results from both cells lines, and were tested with Student´s t-test; and P values listed.

Supporting Information
Supplementary Figures S1 to S3 Figure S1. Genomic organization and conservation of the transcriptional start site of Nrxn1.