HAT assay development using GCN5

The histone acetyltransferase GCN5 acetylates the histones H3 and H4 in non-nucleosomal context with preference for H3K4me3 modified histones^[138]. GCN5 within the SAGA

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Figure 3.19: NCP-H3 library with additional acetylation on H3K4, H3K9 and H3K27 (A) 96 well plate organization of randomized Library #1 and #2, with wells for calibrating Cy5-fluorescence (calibr. Cy5). (B) PTM patterns of combinatorial NCP-H3 library with 36 members.

3 RESULTS

plex augments HAT activity and acetylates histones within the NCP^[59]. In addition, recom-binant GCN5 was found to acetylate nucleosomal substrates in vitro under certain condi-tions^[58]. With respect to the different acetylation behavior towards histones within the NCP, we wanted to see whether the experimental set up was able to observe the incorporation of radioisotope labeled acetyl groups onto immobilized NCP by the HAT GCN5 in Flash plates (figure 3.20 (A)). To this end, NCPs containing wild type histones (WT-NCP, for octamer and NCP reconstitution see Appendix figure 5.2) were immobilized with a coating volume of 200µL and the HAT assay was performed (figure 3.20 (B)). We observed GCN5 depen-dent incorporation of 14C-labeled acetyl-groups into the NCP in the absence of either GCN5 (– GNC5) or NCP substrate (BG), only little background signal was observed. Concluding, we were able to detect GCN5 dependent acetylation of WT-NCP immobilized on Flash plates.

Streptavidin coated Flash plates had a biotin binding capacity of≈40 pmol/well with a strep-tavidin coating volume of 200µL. Thus, the streptavidin coating density was lower compared to the BA-plates (biotin binding capacity: 125 pmol/well in 100µL coating volume). As the coating volume defined the amount of immobilized NCP, we were interested in how much the NCP immobilization degree would decrease by the reduction of the NCP coating sur-face. To this end, WT-NCP were immobilized with 50, 100 and 200µL coating volume into 96-well Flash plates while NCP coverage was observed by in-well Cy5-fluorescence (see figure 3.20 (C)). With fluorescence readings normalized with respect to 200µL, the fluores-cence intensities of Cy5-NCP coverage within the well showed 55 and 62 % signal intensity for 50 and 100µL Cy5-NCP coating volume. While the difference of NCP coverage for 50 and 100µL correlated with the theoretical available surface of 50 and 100µL, there was a loss in signal intensity of 40 % between 100 and 200µL Cy5-NCP coating volume. Thus, the reduction of the coating volume by a factor of 0.5 and 0.25 resulted in 62 and 55 % of NCP coverage. Therefore, it seemed more efficient to use either 50 or 100µL coating volume for NCP immobilization.

The main interest however, was to find the minimal NCP coating volume where a stable and good acetylation signal could be obtained. To this end, GCN5 dependent acetylation with 14C-labeled AcCoA was performed on WT-NCP in 50, 100 and 200µL reaction volume (fig-ure 3.20 (D)). Signal intensities were corrected for background and counts were normalized with respect to the acetylation counts in 200µL reaction volume. The observed NCP

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Figure 3.20: The acetylation of immobilized WT-NCP by GCN5 was observed using Flash plates

(A) Schematic representation of HAT assay with NCP immobilized on Flash plates, which have the scintillant incorporated into the well. Scintillant con-verted energy of β-particle emitted by incorporated isotope (14C) into pho-tons (λ) which were detected. (B) Acetylation of immobilized WT-NCP in the presence (+) or absence (-) of GCN5 with NCP-coverage shown in the inset.

BG: + GCN5 in empty wells. (C) Comparison of theoretical available coating surface in 50, 100, and 200µL coating volume (blue) and effectively covered surface by NCP relative to 200µL coverage. Values are plotted as mean±s.d.

of 3 replicates. (D) background corrected CPM of acetylated NCPs in 50, 100, and 200µL reaction volume relative to acetylation in 200µL. Values are plotted as mean±s.d. of 3 replicates.

3 RESULTS

lation in correlation with the reaction volume displayed a linear behavior. This was contrary the expectation, that would suggest a correlation between NCP coverage and acetylation signal. Nevertheless, for 100µL both NCP coverage and NCP acetylation displayed 60 % signal intensity relative to 200µL reaction per coating volume. Whereas the acetylation sig-nal observed within 50µL coating surface did not correlate with NCP-coverage. 100µL NCP or larger NCP coating and GCN5 reaction volume led to good correlation of NCP coverage and detected acetylation degree. Thus it was chosen to work with an NCP coating volume per GCN5 reaction volume of 100µL.

Replicate #1 Replicate #2

Figure 3.21: Acetylation of WT-NCP by GCN5 over 2 hours time course

Two replicates of HAT assay on immobilized WT-NCP by GCN5 over 120 min-utes. Raw data was corrected for background and normalized to NCP coverage.

Next, we were interested in the time dependent acetylation of immobilized NCP by GCN5. To this end, the GCN5 reaction mix was incubated with immobilized WT-NCP and was removed from the plate at indicated time points. Wells were washed and the 14C incorporation and Cy5-NCP coverage was detected. In figure 3.21 the acetylation degree per NCP is plotted in dependence of incubation time. Acetylation of NCPs by GCN5 displayed a linear correlation over 120 min. Thus, enzymatic acetylation of WT-NCP by GCN5 had not reached saturation.

As the acetylation degree of WT-NCP progressed the most within the observed time frame at 120 min without saturation, the HAT assays on the H3-NCP libraries were incubated for 120 min. This way we reasoned, we would obtain the largest range for the observation of GCN5 activity in dependence of the pre-modification of H3-NCP.

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