FIGURE 11.

HDAC3 regulates GFAP expression and interacts with the GFAP promoter in glial cells. A, glioblastoma U251 cells were transfected with HDAC3-FLAG or a GFP-encoding plasmid, and expression was analyzed 24 and 48 h later by Western blotting using a GFAP antibody. ERK1/2 was used as a loading control (n = 3). B, ChIP analysis examining the binding of HDAC3 to the GFAP promoter in U251 glioblastoma cells. Primers to an upstream region of the GFAP promoter (around −5000 bp) were used as a negative control. To show the specificity of HDAC3 binding at the GFAP promoter only, primer pairs targeted against nestin and Tubb2a gene promoters were used (n = 3). C, luciferase activity assay examining the change in GFAP promoter activity when HDAC3 is overexpressed. HEK293 cells were cotransfected with the pcDNA3.1 FLAG-HDAC3 and hGFAP-Luc vectors, which contain the human GFAP promoter, for 48 h (n = 4). Values are mean ± S.E. **, p < 0.01.