To investigate whether the interaction between STAT3 and CBP/p300 can result in the formation of a DNA loop between the promoter and coding region of GFAP, a 3C (chromosome conformation capture) assay [67] was performed. We also combined the 3C with ChIP (using p300 as a ChIP antibody) in a ChIP-loop assay [68] that presumably allowed us to determine which genomic sites would interact and how the candidate proteins mediate the interactions. Unfortunately, we failed to detect direct evidence of DNA looping at the GFAP promoter and thus conclusions must await further study. Although we had not yet establish a cause-and-effect relationship between DNA looping and GFAP gene transcription, our data of STAT3 and CBP/p300 binding suggest the possibility of DNA looping in the region surrounding the GFAP promoter. Alternatively, it was shown that during cell differentiation from a pluripotent to a committed state, there were global changes in the chromatin structure and interactions of chromatin-binding proteins [69] . Therefore, we cannot exclude the possibility that intrachromosomal interactions occurred in other regions where we did not examine in this study or that interactions between the promoter and coding region of GFAP may still occur in a more complicated manner [70] . Activation of GFAP without DNA demethylation It is known that binding of activated STAT3 to a consensus site in the GFAP promoter plays a role in the transcriptional activation of GFAP [53] . Our observations showed that GFAP expression was induced during astrocyte-like differentiation of NTera-2. ChIP experiments confirmed that there was a strong association of STAT3 with the GFAP promoter, suggesting the existence of mechanism that facilitates access of the STAT3 complex to the GFAP promoter. Methylation of CpGs in DNA constitutes one epigenetic mark that generally correlated with transcriptionally silent chromatin, and hypomethylated DNA in the promoter is a hallmark of vertebrate genes that are actively transcribed [71] . In mouse model, GFAP activation was associated with the loss of DNA methylation at the STAT3-binding site [22] . However, our investigation on the epigenetic regulation of GFAP expression in human NTera-2 cells did not observe changes of DNA methylation at the promoter (up to 4,500 bp upstream), indicating that regulation of the interaction of the STAT3 complex in the GFAP promoter was not mediated by DNA demethylation [21] . There seem to be several explanations that may account for the discrepancies between our results and previous data from the mouse study. First, since no enzyme has yet been identified that actively removes the methyl group from DNA, it is believed that DNA methylation is passively removed through multiple rounds of DNA replication [72] . In present study, the differentiated cells from NTera-2 underwent growth arrest on day 3 after induction. Therefore, it was not possible for these cells to have reduced the methylation level of DNA through cell division. Secondly, it was recently observed that DNA hypermethylation was completely maintained at the promoter region of the erythroid-specific carbonic anhydrase II upon hormone-induced activation [73] . Thus this finding challenges the paradigm that the methylation of promoter-containing CpG islands invariantly causes gene silencing. Finally, it was reported that parental allele-specific histone modifications in the promoter, rather than the differentiated methylated DNA, marked the imprinting status of imprinted genes [74] .