RP58 controls neuron and astrocyte differentiation by downregulating the expression of Id1–4 genes in the developing cortex RP58 directly bound to the.

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RP58 controls neuron and astrocyte differentiation by downregulating the expression of Id1–4 genes in the developing cortex RP58 directly bound to the regulatory regions of Id genes and repressed their activity. (A) Schematic shows two E‐boxes (CANNTG) with specific sequences bound by RP58. The mutation was introduced into the latter E‐box. Besides the typical E‐boxes (CANNTG), there is only one pair of Rp58‐binding E‐boxes (ACANCTG) situated in close proximity to the id‐encoding genomic sequences. (B) Luciferase assay representing the effects of RP58 on the putative RP58‐binding region of each Id. Cos7 cells were transiently transfected with 0.2 μg pGL4P‐Id1, ‐Id2, ‐Id3, or ‐Id4 regulatory region‐luc as a reporter. The luciferase activity of cells co‐transfected with Rp58 expression vector or pCDNA3.1 (+) empty vector (0.2 μg) was measured. The mean Photinus pyralis firefly activities were normalized to the mean activities of Renilla luciferase vector (0.1 μg) (t‐test: **P<0.01). Data were represented as mean values±s.d. (n=3). (C) ChIP analysis of the putative RP58‐binding region of each Id gene. Mouse E16.5 homogenized cerebral cortex was immunoprecipitated with an antibody specific for RP58 or rabbit‐IgG as a negative control. As a positive control, 0.2% total input chromatin DNA was used, and the amplification products of the β‐actin promoter region were used as a negative control. (D) The agarose gel image presented in (C) was quantified using Fiji software available online (http://pacific.mpi-cbg.de/wiki/index.php/Fiji) (t‐test: *P<0.05, **P<0.01). Data were represented as mean±s.d. (n=3). IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. EMBO J, Volume: 31, Issue: 5, Pages: 1190-1202, First published: 10 January 2012, DOI: (10.1038/emboj.2011.486)