Fig. 3

Neuronal depolarization evicts Yy1 and its interacting partner, the BET family member Brd4, from the Senp1 promoter. (a) Yy1 partners with Brd4 in cells. Co-immunoprecipitation of Yy1 and Brd4 in Neuro2A cells. Arrowhead indicates the Brd4 pulled down by Yy1. (b) Depletion of Yy1 using short interfering RNAs (siRNAs). Neuro2A cells were transfected with Scramble (scr) or siYy1. Knockdown was confirmed by immunoblotting whole cell extracts with anti-Yy1 and anti-Actin antibodies. (c) Yy1 recruits Brd4 to the Senp1 promoter. qChIP of Brd4 at − 1894 and − 80 following Yy1 siRNA. (d) Brd4 knockdown in Neuro2A cells. Western blot of Brd4 and scramble (Scr) control following knockdown in Neuro2A cells. Actin is used as a protein loading control. (e) Knockdown of Brd4 represses Senp1 transcription. (f) BET inhibition by JQ1 induces alterations of Senp1 mRNA levels. Cortical neurons were pretreated with 2 μM JQ1 for 22 hrs followed by 2 hr treatment with 60 mM KCl. The level of Senp1 relative mRNA levels were quantitatively analyzed by qRT-PCR. (g) The relative Yy1 mRNA levels after KCl treatment. Following 2 h KCl exposure, Yy1 mRNA levels increase. (h) The binding of Yy1 on the Senp1 promoter is inhibited by the neuronal depolarization. Chromatin was harvested from mouse cortical neurons with or without neuronal depolarization with 60 mM KCl for 2 hrs. The binding of Yy1 and IgG at Senp1 promoter was analyzed by qChIP with primers sets − 1894 and − 80. Relative enrichment was normalized with IgG. (i) Neuronal depolarization abolishes Brd4’s binding on the Senp1 promoter. Chromatin was harvested from unstimulated or stimulated mouse cortical neurons with 60 mM KCl for 2 hrs. Brd4 antibodies were qChIP with primers sets − 1894 and − 80. Relative enrichment was normalized with IgG. (j) Histone H4 acetylation (H4Ac) levels are also depleted at the Senp1 promoter following depolarization. The binding of H4Ac, and IgG at Senp1 promoter was analyzed by qChIP using PCR primers sets corresponding to − 1894 and − 80 Senp1 TSS. Relative enrichment was normalized with IgG. Graphs indicate three independent biological replicates. Error bars represent one standard deviation from the mean. *(p < 0.05). p value was determined using two-tailed unpaired t test