Figure 2: Synthetic ZFP-based chromatin purification identified Ash1L as the transcriptional regulator of nrxn1α in mouse brain.

(A) Electrophoretic mobility shift assays were performed to test the binding capacity of the designed GST-ZFP-pnrxn1α. The PCR product of the nrxn1α promoter fragment (nrx1α promoter) was incubated with GST-ZFP-pnrxn1α or GST at the indicated concentrations. Lane 1, no protein; lane 2–6, 0.02, 0.04, 0.08, 0.12, 0.16 μM GST-ZFP-pnrxn1α, respectively; lane 7–10, 0.04, 0.08, 0.12, 0.16 μM GST, respectively (full-length gel in Fig. S2). (B,C) Competition assays for EMSA. The DNA probes with different paired-end tags were incubated with 0.08 μM GST-ZFP-pnrxn1α (B). The shift bands were subjected to semi-quantitative PCR to identify the ID of binding probes (C) (full-length gel in Fig. S2). (D) The experimental procedure of zinc finger protein-based chromatin purification. (E,F) PCR from purified chromatins showed specific enrichment of the promoter region of nrxn1α in GST-ZFP-pnrxn1α pull-down samples (E) (full-length gels in Fig. S2) and the enrichment near the TSS site of nrxn1α (F) (full-length gels in Fig. S2). (G) Proteins associated with the nrxn1α promoter were resolved by SDS-PAGE and silver staining (full-length gels in Fig. S2). (H,I) ChIP analysis showed the identified proteins specifically enriched at the nrxn1α promoter in the hippocampus of 3-month-old C57BL/6 mice were quantified according to the real-time PCR signal (full-length gels in Fig. S2). The relative enriched signal at the promoter region was normalized against the signal obtained at 3′UTR. (n > 3 biological samples for each group; two-way ANOVA with Bonferroni post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001).