# Heat Stress Affects H3K9 me3 Level at Human Alpha Satellite DNA Repeats
## Results
### Dynamics of H3K9 me3 at Alpha Satellite Repeats after HS
The level of H3K9 me3 after HS was also examined at the seven remaining alpha satellite repeats located within introns of genes: ARs No 1, 14, 18, 21, 25, 28, and 31 (Figure 2D). The H3K9 me3 mark was also examined at the intronic regions positioned 2-6 kb from each of the AR (Figure 2D). It was expected that other ARs also exhibit spreading of H3K9 me3 up to 1-2 kb to the neighboring regions making therefore 2-6 kb ARs' distant regions suitable as negative controls. In the same way we examined H3K9 me3 level at intergenic ARs No 3, 4, 15, 16, 17, 20, and 30 as well as at the regions positioned 2-6 kb from each of the intergenic AR (Figure 2E). For intergenic AR 5 which is composed of a 3.4 mer (Table S3) and due to its size cannot be efficiently amplified by qPCR, we examined H3K9 me3 level at the regions positioned 1 kb 5' and 2.4 kb 3' to AR, respectively (Figure 2E). The H3K9 me3 level at all ARs, either intergenic or intronic, is characterized by an increase ranging from 1.7\(\times\) for the intergenic AR No 30 to 4.3\(\times\) for the intronic AR No 18 relative to the control, immediately after HS (\(p<0.05\); Figure 2D,E; Table 1), while after 30 min of recovery the level of H3K9 me3 decreases at all ARs. The H3K9 me3 level at the region positioned 1 kb 5' to AR No 5 is increased 1.8\(\times\) immediately after HS (Figure 2E). No significant correlation between size of either intergenic or intronic ARs and their H3K9 me3 enrichment upon heat stress (Table 1) is found. At regions 2-6 kb distant to ARs no significant change in H3K9 me3 level was observed upon heat stress (Figure 2D,E), confirming similar spreading of H3K9 me3 from all ARs after HS which is up to maximally 2 kb to the nearby region. We also followed the level of H3K9 me3 at tandemly arranged alpha satellite repeats characteristic for heterochromatin (Figure 2F). In MJ90 hTERT cells H3K9 me3 levels increased 3.2\(\times\) (\(p\) = 0.009) immediately after HS while at 30 min of recovery the level decreases and drops to the level of the control at 1h of recovery (Figure 2F). In addition to MJ90 hTERT cells, we also examined the H3K9 me3 level at intronic ARs in a HepG2 cell line and the results revealed a significant increase of H3K9 me3 of 2-3.4\(\times\) (\(p<0.05\)) after HS on all ARs while no significant change was observed at the regions 2-6 kb distant to ARs which served as negative controls (Figure S4). Figure 2: The level of H3K9 me3 at alpha satellite repeats (ARs) and their proximal regions. **(A)** The profiles of H3K9 me3 distribution in MJ90 hTERT cells at alpha repeat 10, **(B)** alpha repeat 29, **(C)** alpha repeat 19, and at their neighboring regions positioned 0.5–6 kb to each of the AR. **(D)** H3K9 me3 level at intronic alpha satellite repeats No 1, 14, 18, 25, 21, 28, 31, and **(E)** intergenic alpha satellite repeats No 3, 4, 15, 16, 17, 20, and 30 as well as at the region 1 kb 5\({}^{\prime}\) to intergenic AR No 5. In addition, H3K9 me3 level at the regions positioned 2–6 kb from each of the alpha repeat, either at 5\({}^{\prime}\) or 3\({}^{\prime}\) site is shown. **(F)** H3K9 me3 level at tandemly arranged alpha satellite repeats characteristic for heterochromatin and at **(G)** AR 10 in intron of _N_R3 Cl gene in Cal 27 and OV-90 cells as well as in HepG2 cells on allele with (HepG2+) and without AR (HepG2-), **(H)** AR 29 in intron of _ZNF_675 gene in HepG2 cells as well as in the corresponding intron region without alpha satellite repeat in Cal 27 and OV-90 cells. H3K9 me3 levels were measured by ChIP coupled by quantitative real-time PCR at standard conditions (no HS), immediately after 2 h of HS (0 min), at 30 min (30 min) and 1 h of recovery. No value was normalized using No value of input fraction and represents the H3K9 me3 level. Columns show average of three independent experiments and error bars indicate the standard deviations.