Fig. 8.

Inverse relationship between MYCN and CDKL5 expression in CGPs.

A,B: Quantification by RT-qPCR of MYCN (A) and CDKL5 (B) expression in freshly dissociated cerebellar granular cell precursors (DIV0) and after 7 days in vitro (DIV7). Data, in A and B are given as percentage of DIV0 condition and are expressed as the mean ± SE of 3 independent experiments. ***P < 0.001; two-tailed t-test. C: Labeling index (LI) was determined for cerebellar granular cells 3 h after plating (indicated as DIV0) and at DIV7. Cultures were treated with BrdU for 2 h, fixed and processed for immunofluorescence with an anti-BrdU antibody. Data are expressed as mean ± SE. ***P < 0.001, two-tailed t-test. D: Dual cross-linking ChIP and quantitative RT-PCR were applied in either freshly dissociated cerebellar granular precursors (DIV0) or after 7 days in vitro (DIV7). Real-time PCR with primers targeting the negative control region (Amplicon mA) or four regions overlapping MYCN-canonical or non canonical binding site (amplicons mB–mD) or SP1-binding sites (amplicons mB,mE) were performed. Fold enrichment of mouse Cdkl5 promoter regions immunoprecipitated by pre-immune serum (IgG), anti-SP1 and anti-MYCN antibodies was calculated as the logarithm of the difference between the cycle-threshold obtained with pre-immune serum and the cycle-threshold obtained with the specific antibody. Schematic representation of the mouse Cdkl5 gene promoter containing the SP1 binding sites.