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Fig. 2 | Journal of Biomedical Science

Fig. 2

From: Yy1 regulates Senp1 contributing to AMPA receptor GluR1 expression following neuronal depolarization

Fig. 2

Yy1 binds to Senp1 promoter and activates its transcription in unpolarized neurons. (a) Scheme showing the 2.5-kilobases (kb) region upstream of the transcription start site (TSS) (designated by + 1) of mouse Senp1. In silico analysis shows numerous Yy1 binding motifs are present on Senp1 promoter. (b) Deduced Yy1 consensus at Senp1. (c) Yy1 is enriched on Senp1 promoter. Chromatin was harvested from mouse cortical neurons and qChIP was performed with primers sets − 1894 and − 80. Relative enrichment was normalized with 2% input. Graphs indicate three independent biological replicates. Error bars represent one standard deviation from the mean. Two different PCR amplicons situated 1894 bp and 80 bp upstream of the Senp1 TSS (− 1894 and − 80) were used for quantitative chromatin immunoprecipitation (qChIP). The PCR − 1894 amplicon spans the region of − 1894 to − 1741, whereas, the PCR − 80 amplicon spans the region between − 80 to + 7 relative to the Senp1 TSS. (d) Yy1 activates Senp1 promoter in luciferase assay. Upper panel verifies that Myc-Yy1 is over expressed in the cells subjected to the luciferase assay by immunoblot using anti-Myc antibodies. Lower panel shows that Yy1 transactivates the Senp1 promoter using a standard luciferase reporter assay. Luciferase activity was measured in Neuro2A cells co-transfected with luciferase reporter containing Senp1 promoter co-transfected with Myc-Yy1 or Myc alone (empty vector). Graphs indicate three independent biological replicates. Error bars represent one standard deviation from the mean. (e) Knockdown of Yy1 in Neuro2A cells by short hairpin RNAs (shRNAs). Whole cell extracts were prepared 46-48 hrs post transfection and immunoblotted with anti-Yy1 and anti-Actin antibodies. (f) Knock down of Yy1 reduces the expression of Senp1. 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

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