Next, we sought to elucidate the mechanism by which TBPTALE in combination with VP64-TALEs robustly activated silent IL-2 gene expression in non-immune cells. We wanted to know if these artificially constructed TALE activators were able to activate the IL-2 gene expression by directly binding to the chromatin as reported by others or if chromatin remodeling was necessary. Within naı ¨ve T-cells, the IL-2 chromatin architecture is maintained in an inaccessible state, formed by nucleosome accumulation within the proximal promoter region, which masks TCR specific response elements. Consequently, nucleosome masking of these elements prevents TFs from binding to their cognate target sites and inhibits IL-2 gene transcription. However, upon T-cell stimulation, the IL-2 promoter is remodeled to an accessible state, which accommodates multiple TFs binding such as NFAT, AP-1, Oct-1 and NF-kB family members. Thus, chromatin remodeling accompanied by specific TFs is required to initiate IL-2 gene expression. Previous studies have shown that resting T-cells can position a distinct nucleosome between 60 to 200 bp upstream of the TSS and renders the IL-2 promoter inactive. During T-cell activation, this distinct nucleosome is subsequently displaced and accompanies increased DNase I hypersensitivity followed by ensuing IL-2 gene activation. To investigate the IL-2 promoter chromatin architecture in the presence of TBP-TALE and VP64-TALE activators in non-immune cells, we co-transfected 293FT cells with the most active combination of TALE activators or with empty vector control followed by CHART-PCR as previously described. CHART-PCR quantitatively measures the accessibility of a particular DNA region to DNase I cleavage as measured by real time qPCR. In concept, regions of open or relaxed chromatin DNA are more sensitive to DNase I cleavage, whereas regions of closed chromatin DNA are resistant to cleavage. Hence, we hypothesized that in the presence of TALE activators, the IL-2 promoter would be more sensitive to DNase I cleavage than that of empty vector control. As shown in Figure 7A, a series of primers were designed to probe the accessibility status of different loci within the IL-2 promoter region. The results from the CHART-PCR revealed that the combination of AD’CF TALEs but not empty vector control increased DNase I hypersensitivity across the IL-2 promoter with a significant increase observed in regions probed by primer sets 2 and 3. Interestingly, both primer sets amplified genomic regions either within the vicinity of a distinctly positioned nucleosome found in resting T-cells or within the TSS. Together, these data demonstrate that TBPTALE together with VP64-TALEs have either displaced or repositioned the nucleosome outside the detection range to allow for exclusive access of TALE activators to the IL-2 promoter regulatory regions, which controls gene activation.