# Heat Stress Affects H3K9 me3 Level at Human Alpha Satellite DNA Repeats
## 4 Discussion
For intergenic alpha repeats we assumed their association with the closest 5\({}^{\prime}\) and 3\({}^{\prime}\) positioned genes, however because of the 3-dimensional structure of the genome it might be possible that some intergenic repeats are associated with more distant genes and this can be assigned only based on high resolution chromatin interaction maps. The number of genes associated with dispersed alpha repeats is low (46) and gene ontology analysis revealed no significantly enriched pathways or molecular functions within this gene set. It is however interesting that some of the alpha repeat-associated genes are stress or disease-related: _NR3C1_ codes for the glucocorticoid receptor which upon binding of stress hormones participates in gene expression regulation [44], _HTRA3_ shows drastically downregulated expression in some cancers and it is considered to be a tumor suppressorgene [45; 46] while _VAV1_ and _PHF20L1_ have oncogenic potential [47; 48]. In addition, _ZNF675_ gene codes for a KRAB zinc-finger protein which specifically targets a subfamily of LTR retrotransposons and is responsible for their silencing [49]. The dispersion of satellite DNA repeats throughout the genome is most probably a consequence of the molecular mechanisms of satellite DNA evolution which still need deeper investigation to be fully elucidated [2; 50; 51]. We propose that some satellite repeat insertions could possibly cause a disease by affecting proper gene expression or by inducing gene mutation, as demonstrated for the insertion of human beta satellite repeats within the splice-acceptor site of a transmembrane serine protease gene [52]. Dispersed satellite repeats could also contribute to genomic instability by mutagenic recombination between these highly homologous elements as shown for recombination between _Alu_ repetitive elements which is responsible for human genetic diseases including cancer [53; 54]. In addition, euchromatic satellite repeats regulate gene expression, acting as modulators of local chromatin structure upon heat stress [18] or as a source of small RNAs which participate in degradation of maternally inherited transcripts during early embryonic development [55]. A relevant role of satellite DNA in the evolution of species has already been proposed [56; 57; 58], but further studies are necessary in order to clarify the role of dispersed, euchromatic satellite repeats in gene expression regulation and adaptive evolution, as well as their potential impact on disease. The following are available online at [[http://www.mdpi.com/2073-4425/11/6/663/s1](http://www.mdpi.com/2073-4425/11/6/663/s1)]([http://www.mdpi.com/2073-4425/11/6/663/s1](http://www.mdpi.com/2073-4425/11/6/663/s1)). Figure S1A: Polymorphism of AR 29 located within intron of _ZNF675_ gene among human cell lines; Figure S1B. Sequence of a short amplicon of AR No 29 within intron of _ZNF675_ gene; Figure S1C: Sequence of a part of intron of _NR3C1_ gene in HepG2 cells encompassing heterozygous AR No 10; Figure S2: Consensus sequence of 171 bp alpha satellite monomer and position of primers used in qPCR; Figure S3: Organization of repetitive sequences identified by RepeatMasker at intronic regions of genes _NR3C1_, _ZNF675_, and _PLA2G12_B; Figure S4: H3K9 me3 level at dispersed ARs in HepG2 cells; Figure S5: H3K4 me2/3 level at tandemly arranged and dispersed alpha satellite repeats; Figure S6: Dynamics of expression of _VAV1_ gene associated with dispersed AR 28 in cell line 697; Table S1: List of primers used for analysis of alpha repeat insertion polymorphism and in ChIP-qPCR experiments; Table S2: List of primers used for expression of alpha repeat-associated genes and of alpha satellite DNA; Table S3: List of alpha satellite repeats dispersed on human chromosomes; Table S4: Expression of genes associated with dispersed alpha satellite repeats after HS in the presence and in the absence of chaetocin.