Figure 7.

EGR family is a novel regulator of stimulus-dependent expression of Bdnf exon I and IV-containing transcripts. Rat cultured cortical neurons were treated at 8 DIV for the time indicated with 25 mM KCl (and 5 μM d-APV) or 50 ng/ml BDNF. A, ChIP-qPCR assay using anti-EGR1 antibody. Data is shown as enrichment to Bdnf promoter I and IV (pI and pIV, respectively) relative to the binding to the unrelated region (URR) in untreated cells. B, Western blots verifying the overexpression of EGR1 and dominant-negative proteins for EGR1 (Zn-EGR1) and EGR3 (Zn-EGR3). Coomassie staining is shown as a loading control. C, D, The neurons were transduced with lentiviral particles encoding (C) EGR1 or ZnEGR1 or EGFP as control; or (D) EGR3 or ZnEGR3 or EGFP as control. The expression levels of Bdnf transcripts containing either exon I or IV and total Bdnf mRNA levels were measured using RT-qPCR. E, On the left, schematic representation of Bdnf promoters I and IV with the described regulatory elements. Identical bases between the human and rat promoter regions are shown as dots. On the right, luciferase reporter assay with wild-type Bdnf promoter (pI WT or pIV WT) or with Bdnf promoter constructs where respective EGR-binding elements were mutated (EGR m1, m2, m3, shown in orange on the schematics on the left). The activity of Bdnf promoters was measured with luciferase reporter assay and data is shown relative to the activity of respective wild-type promoter in untreated neurons. The data of the wild-type promoter is the same as depicted in Figure 6. All data points are shown as dots and each biological replicate is denoted with the same color as shown in the legend. Numbers above the columns indicate the average of three to five independent experiments (A, n = 3; C, n = 5; D, E, n = 4). Error bars indicate SEM. Statistical significance in ChIP-qPCR assay was calculated relative to the binding to URR (black asterisks) or between Bdnf promoters (red asterisks) in respectively treated cells (A), in RT-qPCR experiments relative to the levels of respective transcripts in neurons transduced with EGFP-overexpressing cells (C, D), and in luciferase reporter assay relative to the activity of the respective wild-type promoter in respectively treated cells (E). ^#≤ 0.1, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 (paired two-tailed t test). AP1, AP1 family-binding element; BHLHB2-RE, BHLHB2 response element; CaRE, calcium response element; CRE, cAMP response element; NFkB-RE, NFkB response element; UBE, USF-binding element; PasRE, NPAS4-binding element.