# Heat Stress Affects H3K9 me3 Level at Human Alpha Satellite DNA Repeats
## Results
### Dynamics of H3K9 me3 at Alpha Satellite Repeats after HS  
The enrichment of repressive histone marks at dispersed repeats of a major satellite DNA and their neighboring regions after HS was previously demonstrated in the beetle _T. castaneum_[18]. Here we analyze distribution of the silent histone mark H3K9 me3 at dispersed alpha satellite repeats and flanking regions both at standard conditions and after heat stress. We performed chromatin immunoprecipitation (ChIP) coupled with quantitative real-time PCR, using specific primers (Table S1). H3K9 me3 levels were analyzed at 18 dispersed alpha satellite elements and their flanking regions, 10 of them located within introns and 8 at intergenic regions. Using the same method we also analyzed the level of H3K9 me3 at regions located within 0.5-6 kb distance to each of the AR. ChIP assay was performed on chromatin isolated from MJ90 hTERT, Cal 27, OV-90, or HepG2 cells subjected to a heat shock of 2 h at 42 \({}^{\circ}\)C. The level of H3K9 me3 was measured immediately after HS and at 30 min post-recovery at 37 \({}^{\circ}\)C, and was compared to the level of control (no HS) using the unpaired _t_-test. In addition, we followed the dynamics of binding of IgG to dispersed alpha satellite repeats and tandemly repeated satellite arrays and the amount of bound IgG was very low resulting in a signal below the qPCR threshold. The profile of H3K9 me3 distribution after HS at alpha repeats and their neighboring regions was first examined at intronic ARs No 10 (1.1 monomer), 19 (2.4 monomers), and 29 (0.6 monomer) in MJ90 hTERT cells using specific primers. The primers used amplified regions encompassing ARs as well as several regions, approximately 200 bp long, positioned 0.5-6 kb from the corresponding AR (indicated as red horizontal bars in Figure S3). The H3K9 me3 level was increased 2.1\(\times\) at the AR 10 immediately after HS and remains increased 1.5\(\times\) after 30 min of recovery (Figure 2A). At a region positioned 656 bp 3\({}^{\prime}\) to AR 10, 1.7\(\times\) (_p_ = 0.011) increase of H3K9 me3 was detected immediately after HS while no significant change in H3K9 me3 level was detected at regions located 1.8 kb 5\({}^{\prime}\)  
Figure 1: The dynamics of transcription of alpha satellite DNA in cell lines OV-90, MJ90 hTERT, HepG2 and Cal 27 under standard conditions (no HS), immediately after 2 h of heat stress at 42 \({}^{\circ}\)C (0 min), at 30 min and 1 h of recovery at 37 \({}^{\circ}\)C, respectively. Two experiments were performed on each cell line. No represents normalized average No value. Columns show average of two different RT-qPCR experiments performed in triplicate and error bars represent standard deviations. 1.9 kb 3', and 6 kb 5' to AR 10 (Figure 2A). Since a region 6 kb distant to AR 10 encompasses part of a L1 repeat while a 1.8 kb distant region at 5' end flanks an _Alu_ repeat (Figure S3), an unchanged H3K9 me3 level at these regions suggests no H3K93 enrichment on L1 and _Alu_ repeats. The profile of H3K9 me3 distribution at the AR 29 is characterized by an increase of 1.9\(\times\) and 1.3\(\times\) relative to the control immediately after HS and at 30 min of recovery, respectively (Figure 2B). At a region 625 bp 3' to AR 29, H3K9 me3 level was increased 1.6\(\times\) (\(p\) = 0.009) immediately after HS, while at more distant regions positioned 1.8 kb 5' and 1.9 kb 3' to AR a statistically insignificant increase of H3K9 me3 level was observed as well as no change at 6 kb distance (Figure 2B). At AR 19, the increase of H3K9 me3 immediately after HS was 3.6\(\times\), while at distances of 0.5 kb and 1.2 kb, the H3K9 me3 level increased 2.7\(\times\) and 2\(\times\) respectively, while at 2.4 kb distance the level was close to the control (Figure 2C). The results reveal that the H3K9 me3 profiles are characterized by a maximal increase at ARs and spreading of H3K9 me3 to nearby regions up to approx. 1-2 kb (Figure 2A-C).