# Heat Stress Affects H3K9 me3 Level at Human Alpha Satellite DNA Repeats
## Results
### Influence of Suv39H1 Inhibition on Expression of Alpha Repeat-Associated Genes after HS  
To study the possible role of silent histone modification H3K9 me3 on suppression of alpha repeat-associated genes we used chaetocin, a specific inhibitor of the histone methyltransferase SU(VAR)3-9 of _Drosophila melanogaster_ and of its human orthologo Suv39H1 [27]. Suv39H1 is a key enzyme in establishing condensed heterochromatin by specifically di- and trimethylating Lys9 of histone H3 and the specificity of chaetocin for the enzyme makes this compound an excellent tool for the study of heterochromatin-mediated gene suppression. MJ90 hTERT cells were incubated in complete medium and chaetocin was added to a concentration of 150 uM [28] immediately before HS. In order to check if chaetocin under such conditions affects H3K9 me3 level at ARs, we examined H3K9 me3 levels at intronic ARs immediately after 2 h HS in the presence and in the absence of chaetocin (Figure 4A). As controls, we examined H3K9 me3 levels at regions positioned 2-6 kb from ARs. The results reveal a significant decrease of H3K9 me3 levels on all ARs ranging from 1.6\(\times\) for ARs No 14 and 18 to 3.9\(\times\) for AR No 25 (\(p<0.05\)). No significant change in the H3K9 me3 level was detected at regions 2-6 kb distant to ARs (Figure 4A; \(p>0.05\)). In the same way we tested the effect of chaetocin on H3K9 me3 level at ARs 10 and 29 in Cal 27, OV-90, and HepG2 cells, respectively, where these ARs exhibit insertion polymorphism. In the absence of the corresponding ARs (AR 10-, AR 29-) no change of H3K9 me3 level was observed (Figure 4A). We were interested to check if the observed decrease of H3K9 me3 at ARs reflects on the expression of their associated genes. Expression of six genes with intronic ARs (_SLC30A6_, _NR3C1_, _PRIM2 STAM_, _MYOIE_, and _ZNF675_) and eight genes associated with intergenic ARs (_SLC40A1_, _ASNSD1_, _ST6 GAL1_, _HTRA3_, _ACOX3_, _INTS1_, _PHF20L1_, and _DIP2C_) was measured by qRT-PCR after HS in the presence and in the absence of chaetocin (Figure 4B). An increase in the expression of all genes in MJ90 hTERT cells treated with chaetocin relative to the controls without chaetocin treatment was observed (Figure 4B; Table S4). Using the same conditions we monitored expression of genes _ZNF675_ and _NR3C1_ associated with polymorphic ARs 29 and 10 respectively, in cell lines Cal 27, OV-90, and HepG2. Expression of _ZNF675_ gene was not affected by chaetocin in cell lines Cal 27 and OV-90 where AR 29 is absent. _NR3C1_ expression from the allele with the AR 10 was increased, while _NR3C1_ expression from the allele without the alpha repeat was not affected by chaetocin in HepG2 cells (Figure 4B; Table S4). The results show that the decrease of H3K9 me3 level at ARs is coupled with the enhanced expression of alpha repeat-associated genes. In the absence of ARs, expression of two of these genes, _ZNF675_ and _NR3C1_, is not affected by chaetocin, indicating that H3K9 me3 enrichment at ARs might contribute to expression downregulation of these two genes after HS. Figure 4: **(A)** The level of H3K9 me3 at alpha satellite repeats and their neighboring regions after HS in the presence of chaetocin, inhibitor of histone methyltransferase Suv39H1. H3K9 me3 level at intronic alpha satellite repeats No 1, 10, 14, 18, 19, 25, 21, 28, 29, 31 and at the regions positioned 2–6 kb from each of the alpha repeat, either at 5\({}^{\prime}\) or 3\({}^{\prime}\) site is presented in MJ90 hTERT cells. In Cal 27, OV-90, and HepG2 cells, H3K9 me3 level was shown for polymorphic ARs 10 and 29. AR 29 is absent in Cal 27 and OV-90 cells (29-), while AR 10 is absent on a single allele in HepG2 cells (10-). **(B)** Expression of genes associated with dispersed alpha satellite repeats after HS in the presence of chaetocin. Genes associated with six intronic ARs: 1, 10, 14, 19, 25, and 29, and with eight intergenic ARs are expressed in MJ90 hTERT cells.