# Heat Stress Affects H3K9 me3 Level at Human Alpha Satellite DNA Repeats
## 4 Discussion  
Transcription of heterochromatic satellite DNAs is activated by heat stress in different organisms, from plants [29; 30] and insects [14; 31] to humans [11; 12]. In particular, transcription of human pericentromeric satellite III which binds HSF1 is drastically increased during and after HS, exceeding controls by 10-40\(\times\)[32]. On the other hand, induction of transcription of alpha satellite DNA by HS is much lower relative to satellite III, as observed previously in some cell lines [32]. In agreement with this observation, we detected 2\(\times\) overexpression of alpha satellite DNA exceeding control, immediately after heat stress in different cell lines. What is the potential function of stress induced transcription of satellite DNA? It was shown that human satellite III transcripts mediate the recruitment of a number of RNA binding proteins involved in pre-mRNA processing and participate in the control of gene expression upon heat stress, at the level of splicing regulation [33]. Transcripts of many satellite DNAs are necessary for heterochromatin establishment [34] and increased satellite transcription after heat stress correlates with H3K9 me3 enrichment at tandemly arranged satellite repeats, as shown previously for a major TCAST1 satellite DNA [13] and here for a major human alpha satellite DNA. It was proposed that increased TCAST1 satellite transcription reinforces \"heterochromatinization\" and helps heterochromatin recovery upon heat stress [14]. Since heterochromatin is important for genome stability and integrity, satellite DNA transcripts might have protective effects in stressed cells/organisms. Centromeric satellite DNA transcripts also play a structural role in centromere or kinetochore integrity in many species including humans and beetles [35; 36] and their accumulation under stress conditions seems to be a conserved feature of the cellular stress response [37]. In addition to H3K9 me3 enrichment within heterochromatin, the collateral \"heterochromatinization\" at satellite repeats dispersed within euchromatin occurs, as shown previously for TCAST1 satellite and here for human alpha satellite DNA. Aside from satellite DNA, some transposons in plants [38], _Drosophila_[39; 40] and mammals [41; 42] are enriched for repressive epigenetic marks and were shown to spread the heterochromatin mark H3K9 me2/3 from the insertion sites. Considering the influence of dispersed, euchromatic alpha repeats on the expression of neighboring genes, the observed downregulation of all ARs-associated genes upon heat stress as well as differences in the expression of two genes associated with polymorphic alpha satellite repeats suggest a possible contribution of H3K9 me3 enriched alpha repeats in gene silencing. It is known that the genome-wide transcriptional response to heat shock in mammals is modulated at promoter-proximal pause, either by increasing or reducing RNA polymerase release, resulting in up or downregulation of many genes [43]. We can exclude the possible influence of this gene regulatory mechanism of heat stress response on _NR3C1_ and _ZNF675_ genes whose expression is not affected by HS in the absence of associated alpha repeats. However, for other tested genes it is possible that their downregulation listed in Table 1 results not only from H3K9 me3 enriched alpha repeats but also from the general transcriptional response to heat shock. Further studies are necessary to resolve contribution of H3K9 me3 enrichment at alpha repeats on downregulation of each of these genes. Comparison of expression of a gene with and without particular intronic alpha repeat in the same genetic background (the same cell line) can be helpful in resolving the potential of alpha repeats to modulate gene expression upon heat stress. However, abundant repetitive elements (_Alu_, L1, etc.) adjacent to intronic ARs and to most of intergenic ARs prevented elimination of alpha repeats using CRISPR/Cas9 and creation of modified cell lines suitable for further studies.