# Heat Stress Affects H3K9 me3 Level at Human Alpha Satellite DNA Repeats
## Results
### Expression of Alpha Repeat-Associated Genes after HS  
Among genes associated with 20 dispersed alpha satellite repeats previously tested for insertion polymorphism (Table S3), we selected those showing a reliable level of expression by qPCR in at least one cell line. Among genes that have alpha satellite elements located in introns, three genes (_PLA2G12B_, _DLG2_, _MAP7D2_) were expressed at a very low level while the seven remaining genes were suitable for expression study: _NR3C1_, _ZNF675_, _VAV1_, _SLC30A6_, _PRIM_, _STAM_, and _MYO1E_. Finally, we also tested expression of 14 genes closest to the intergenic alpha repeats, which are up to 50 kb away from the intergenic ARs (Tables S2 and S3), and eight of them were suitable for expression study (_SLC40A1_, _ASNSD1_, _ST6 GAL1_, _HTRA3_, _ACOX3_, _INTS1_, _PHF20L1_, and _DIP2C_). We first tested expression of genes associated with ARs that showed insertion polymorphism such as _ZNF675_ and _NR3C1_ genes. The dynamics of expression of the _ZNF675_ gene in MJ90 hTERT fibroblasts and in HepG2 cells is characterized by a downregulation of 1.7\(\times\) relative to the control immediately after 2 h of HS in both cell lines, while in Cal 27 and OV-90 cell lines where _ZNF675_ is not associated with an alpha satellite repeat, the expression was not affected by HS (Figure 3A). The maximal downregulation of _NR3C1_ expression relative to the control was observed immediately after 2 h of HS in MJ90 hTERT (3.5\(\times\)), Cal 27 (3.7\(\times\)), and OV-90 (2.7\(\times\)) cells and is followed by an increase in expression after recovery periods of 30 min and 1 h, respectively (Figure 3B). Since in HepG2 cells the alpha repeat within the intron of _NR3C1_ gene is heterozygous, we used allele-specific primers (Figure S1C) for the expression analysis. _NR3C1_ expression from the allele containing an alpha repeat was downregulated 2.7\(\times\) immediately after HS, followed by an increase in expression after 30 min of recovery, while _NR3C1_ expression from the allele without the alpha repeat was not affected by HS (Figure 3C). The results reveal transient downregulation of _NR3C1_ and _ZNF675_ genes associated with alpha repeats after HS in different cell lines, while in the absence of the alpha repeat no effect on the expression of these genes is observed. The dynamics of H3K9 me3 accumulation at ARs 10 and 29 after HS (Figure 2A,B,G,H) correlates with the suppression of AR 10 and AR 29-associated genes _NR3C1_ and _ZNF675_, respectively, after HS (Figure 3A-C), indicating a possible influence of H3K9 me3 enrichment at the ARs on gene expression downregulation. The expression analysis of genes _SLC30A6_, _PRIM2_, _STAM_, and _MYO1E_ with alpha satellite elements located with introns as well as of eight genes associated with intergenic alpha satellite elements was measured immediately after 2 h of HS at 42 \({}^{\circ}\)C in the MJ90 hTERT cell line (Figure 3D). The expression of the _VAV1_ gene was followed in the 697 cancer human Pre-B leukemia cell line because in MJ90 hTERT fibroblasts the gene is expressed at very low levels (Figure S6). HS was performed for 1 h since 697 cells do not completely survive longer HS. The decrease in gene expression after HS is observed for all tested genes (Figures 3D and S6). The level of downregulation with statistical significance expressed in P values, calculated by the unpaired t-test, is listed in Table 1 for all tested genes as well as the size and position of gene-associated alpha satellite elements, their distance from the transcription start site (TSS) and cell lines used in the expression analyses. The suppression level of alpha repeat-associated genes is within a range from 1.7\(\times\) for _ZNF675_ and _ACOX3_ to 5.6\(\times\) for _HTRA3_. The distance of dispersed alpha satellite elements from the TSS of genes as well as the distance of intergenic repeats from 5\({}^{\prime}\) or 3\({}^{\prime}\) gene ends, respectively, is not significantly correlated with the level of gene suppression (\(p\) values \(>\) 0.05).