Satellite Non-Coding Transcription as a Response Mechanism: Comparison
Please note this is a comparison between Version 1 by Sandra Louzada and Version 2 by Jason Zhu.

Organisms are often subjected to conditions that promote cellular stress. Cell responses to stress include the activation of pathways to defend against and recover from the stress, or the initiation of programmed cell death to eliminate the damaged cells. One of the processes that can be triggered under stress is the transcription and variation in the number of copies of satellite DNA sequences (satDNA), which are involved in response mechanisms. Satellite DNAs are highly repetitive tandem sequences, mainly located in the centromeric and pericentromeric regions of eukaryotic chromosomes, where they form the constitutive heterochromatin. Satellite non-coding RNAs (satncRNAs) are important regulators of cell processes, and their deregulation has been associated with disease.

  • cellular stress
  • satellite DNA
  • satellite non-coding RNAs
  • stress-response mechanisms

1. Introduction

Cells can be subject to numerous internal and external factors that can trigger signaling mechanisms, namely environmental conditions (such as radiation or temperature), and responses to chemical or pathogenic agents. These conditions cause stress to the cells and promote a cellular response which is dependent on the type of stress, and also the type of cell, tissue and organism. The stress response and the consequent activation of cellular pathways have been related to mechanisms that encompass the deregulation of a series of genes [1][2][3][1,2,3]. Studies demonstrate that non-coding RNAs (ncRNAs), including those originating from satellite DNA sequences, are also important players in various mechanisms of cellular response to stress conditions [4].
Satellite DNAs (satDNAs) are highly repetitive sequences present in the eukaryotic genomes. These sequences were initially identified as bands with distinct densities concerning the rest of the genome [5][6][5,6] and were later shown to be organized in tandem arrays accounting for a significant amount of the total DNA content of some genomes [7]. These sequences are the main component of constitutive heterochromatin and are located primarily in the centromeric and pericentromeric regions of eukaryotic chromosomes [8][9][10][8,9,10]. However, they have been also found in euchromatin in shorter arrays dispersed in the genome [10][11][10,11].
The transcription of satDNAs into satellite non-coding RNAs (satncRNAs) has gathered increasing interest due to their association with important functions in the organization and regulation of the genome of several organisms: vertebrate [4][10][12][13][14][4,10,12,13,14] and invertebrate [15][16][15,16]. A growing number of studies report that satncRNAs are important regulators of cell processes and their deregulation has been associated with disease, namely the tumor process [16][17][18][19][16,17,18,19]. In particular, these transcripts integrate a significant fraction of differentially expressed sequences in response to various stress stimuli and their stability seems to be adjusted according to their regulatory functions, mechanisms of action and the physiological state of the cell [3].

2. Heat Shock Response Mechanisms as a Way to Counterpart Thermal and Oxidative Stress

Eukaryotic cells developed a highly conserved protective response, the heat shock response (HSR), that allows them to cope with stressful conditions, such as exposure to high temperatures or the presence of oxidants, that can cause protein misfolding and denaturation [20]. The HSR is regulated by a family of transcription factors called heat shock factors (HSFs) [21]. Upon exposure to heat or other stressors, heat shock transcription factor 1 (HSF1) becomes activated and translocates to the nucleus, where it binds to heat shock response elements (HSEs) in the promoter regions of heat shock genes [1,22–24]. These genes are rapidly upregulated in response to stress, resulting in the production of heat shock proteins (HSPs) [23,25].

Heat shock stress leads to the increased expression of satDNAs. This increased transcription seems to depend on the localization of the original sequence. Heterochromatic tandemly repeated satellites are particularly sensitive to heat shock, which appears to be a shared characteristic of upregulated satDNAs between different species.  SatDNA upregulation has been analyzed in several species: mammals, such as Homo Sapiens [17,31– several species: mammals, such as Homo Sapiens [17,31–33] and Mus musculus [34,35]; but also insects, such as Tribolium castaneum [16,36,37] and but also insects, such as Tribolium castaneum [16,36,37] and Drosophila melanogaster [38–40].  The involvement of satncRNAs, namely human satellite 3 (Hsat3) transcripts, in the  human heat stress response mechanism is the best-characterized relation between these  types of transcripts and cellular stress conditions. Heat shock (HS) has already been  been shown to induce the transcription of HSat3 repeat arrays located at the pericentromeric  heterochromatin of specific human chromosomes [41].  Human pericentromeric GC-rich satellite DNA 2 (HSat2) sequence, was also demon- strated to be overexpressed in heat shock stressed cells (Table 1) [53,54]. The overexpression of Hsat2 transcripts can reflect global changes in heterochromatin silencing [53], namely causing the overexpression of other satDNA sequences.  SatDNA overexpression under heat shock cellular stress was also described in rodents. The highly conserved mouse Cassini ncRNA (murine -satellite) was revealed to be upreg- ulated in acute lymphoblastic leukemia (ALL) cells under conditions of heat shock, even though researcherswe still have no indication if these transcripts are strategically involved in the stress response or have adverse consequences [55].  Some insect species have also shown increased transcription of satDNAs when cells undergo heat stress. The major satellite of Tribolium castaneum (TCAST1) is demethylated and overexpressed upon heat shock [56].  Heat stress is one of the environmental stressors that have been extensively studied in relation to its impact on satDNA transcription. Regardless of the species or the specific satDNA sequences being considered, there is a consistent trend of increased transcription of satDNA sequences under conditions of heat stress. As already proposed for humans and mice, satDNAs might share inducibility [55], which suggests a potentially shared functional role.

Oxidative stress is characterized by an imbalance between reactive oxygen species (ROS) production and antioxidant defense [18,62], which can cause damage to cellular components such as proteins, lipids, and DNA, potentially leading to cellular dysfunc- tion and disease [63,64]. Eukaryotic cells are particularly vulnerable to oxidative stress because they contain organelles such as mitochondria and peroxisomes that produce ROS as part of their normal function [63,66,67]. Human satellite 3 RNA has been shown to be induced by multiple stress agents, such as oxidative stress when cells were treated with H2O2 (Table 1) [12]. This treatment has been shown to moderately increase the expression of HSat3 as well as trigger the formation of nSBs in a small number of cells [12]. The activation of HSF1 via ROS results from an indirect pathway involving the activation of transcription factors like nuclear factor erythroid 2 related factor 2 (Nrf2) [18,72]. Nrf2 orchestrates the transcription of genes encoding antioxidant enzymes and detoxification proteins, bolstering the cellular defense against oxidative stress-induced harm [72,73].

3. The Relation between Osmotic Stress and Satellite DNA Transcription

Osmotic stress occurs when there is an excessive amount of salt in the surrounding environment, which promotes changes in external osmolarity resulting in the disruption of ions and water balance within the cell [20][77]. When eukaryotic cells are under osmotic stress, ROS production is triggered as a consequence of altered metabolic processes, particularly at the mitochondria electron transport chain [21][78]. Notably, ROS function as secondary messengers, instigating the activation of crucial signaling pathways [22][79]. For instance, osmotic stress-associated ROS are known to influence mitogen-activated protein kinases (MAPK) and calcium signaling, both important in the cell’s adaptive response to osmotic challenges [23][80]. As osmotic stress activates ROS production, cells mobilize their antioxidant defense mechanisms to avert potential damage [22][79]. Antioxidant enzymes like superoxide dismutase, catalase, and glutathione peroxidase are deployed to neutralize ROS and reinstate redox equilibrium [24][73]. Moreover, ROS are not merely passive participants; they actively shape gene expression patterns by engaging transcription factors [25][81]. These factors, such as nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and protein 53 (P53) are pivotal in initiating the expression of genes that contribute to the cell’s stress response and adaptation [22][24][73,79].
It has been described that hyperosmotic stress also induces the transcriptional activation of HSat3, involving a transcription factor tonicity enhancer binding protein, the transcription factor tone enhancer (TonEBP) [12]. The TonEBP transcripts also accumulate at the 9q12 locus (location of HSat3) [26][35], just as with HSF during heat-shock. In addition, TonEBP is also known for the activation of T-cell nuclear factor 5 (NFAT5), described to regulate gene expression in response to osmotic stress and to be vital in kidney function and protection against high levels of salinity [27][28][29][82,83,84].

43. The Pathways of DNA Damage Response (DDR) Encompass Satellite DNA Transcription

Cellular exposure to the molecules that are produced by physical-chemical or environmental agents, such as ultraviolet radiation, ionizing radiation, and chemotherapy drugs, can promote genome instability. This cellular outcome is related to the development of several diseases and to avoid them, cells developed DNA repair pathways [30][85]. Despite the lack of research regarding the function of satncRNAs in these DNA damage response (DDR) mechanisms, some works suggest that they can be key players in protecting the cells against these factors [31][32][86,87].
Ultraviolet radiation (UV-A, UV-B, UV-C) can cause double-stranded DNA breaks (DSBs) [33][34][88,89], which results in cell cycle arrest through the recruitment of ATM (ataxia-telangiectasia mutated) protein kinase that mobilizes one of the most extensive signaling networks as an attempt to repair them, and when it is not possible, the apoptosis pathway is triggered by P53 protein [35][36][90,91]. Although studies are still very limited, there is already evidence that cellular exposure to this type of radiation can influence the transcription of satDNA sequences. Valgardsdottir and colleagues (2008) described the increased transcription of HSat3 after UV-C radiation treatment. They proposed that UV-C stress also triggers the heat-shock response and the formation of structures similar to nSBs [12]. Some authors suggest that these nSBs may protect the large heterochromatic block at 9q12 from chromosomal rearrangements induced by stress [17]. Another study that focused on the functional characterization of human HSat2 in DDR showed that the UV-C stress induction in arising retinal pigment epithelia (ARPE-19) cells does not result in a significant alteration of its transcription levels, possibly as a consequence of the type of DNA damage and its cellular outcome [37][58].
DDR mechanisms can be also triggered by cytotoxic agents, i.e., the association of satncRNAs aberrant expression with chromosomal instability (CIN) and DDR [38][39][40][92,93,94]. In 2018, Ichida and colleagues demonstrated that the overexpression of αSat led to CIN and copy number changes at specific chromosomes [41][95]. Despite the growing number of studies that highlight the relation between drug response and satncRNA levels, the mechanisms are still poorly understood [40][94]. Human cells treated with zeocin (an antibiotic that mimics the effect of ionizing radiation on cells) or etoposide (an anticancer agent that causes DSBs and prevents their repair by Topoisomerase II) showed an increased expression of HSat2 RNA of about 500 and 700 times, respectively. This accumulation of transcripts is regulated by DDR mechanisms and it is not dependent on P53 pathways [37][58]. It has been also demonstrated that HSat3 transcripts can promote resistance to etoposide and that the epigenetic modification of the HSat3 locus and its expression can be used to predict the response to this treatment. The same authors suggest that HSat3 transcripts are capable of recruiting topoisomerase II alpha (TOP2A) to the nSBs in response to stress. This will result in resistance to etoposide treatment because it prevents the formation of the etoposide-TOP2A complex, and consequently, the decrease in DNA damage. However, it is possible to restore etoposide sensitivity when HSat3 expression is reduced [42][96]. Another study supports these data and relates HSat3 overexpression to resistance to chemotherapeutic drugs such as staurosporine, fluorouracil (5-FU), and cisplatin (interferes with the cell cycle and target caspase-specific cell death) [43][33]. HSat3 knockdown restores the P53 function, promoting cell death, and sensitizing cells to these agents. Also, P53 regulates the levels of HSat3 ncRNAs to induce cell death. These authors suggest that HSat3 is an important regulator of human cancers, facilitating cancer progression by a different pathway from the heat stress pathway [43][33]. Some works on mouse satellite RNAs also showed the importance of satncRNA in DDR. First, the Cassini satellite (from the mouse γ-satellite family) was shown to be upregulated in acute lymphoblastic leukemia cells when they are treated with cytostatic drugs [44][55]. More recently, a study demonstrated the relation between the overexpression of the mouse major satellite RNA (MaSat) and DDR features, namely abnormal segregation (involving micronuclei formation and anaphase bridging) and increased levels of the DNA damage marker γ phosphorylated form of the histone H2AX (γH2AX). The authors also showed a relation between overexpression of MaSat and sensitivity to camptothecin (CPT) (topoisomerase I inhibitor) via CIN induction [40][94]. After etoposide or zeomicin treatment in murine cells, it was also observed the increased expression of MaSat and minor satellite repeats (MiSat), being this last more evident. However, this increase in MiSat is dependent on P53 presence, suggesting that a stabilized form of P53 can bind to non-canonical sites in MiSat repeats, activating its transcription in the DDR signaling pathway [45][34].

54. Beyond Satellite Transcription: Satellite DNA Copy Number Variation in Response to Emotional Stress during Aging and Disease

Although most work focuses on the analysis of satDNA transcription, the number of copies of these satDNAs can also be associated with several biological processes and diseases. Recently, Ershova and colleagues (2019) associated the HSat3 copy number variation with aging. The sub-fraction of HSat3 located at the 1q12 region (characterized for being a rather unstable region of the human genome) was studied in a group of healthy individuals with ages ranging from two to ninety-one years, separated into different age groups. HSat3 was quantified using a specific probe by means of a non-radioactive quantitative hybridization method, and it was observed that in young people the number of copies of HSat3 was much lower than in the group of elderly people aged between 77 and 91 years [46][97]. The authors associated the increase in the number of copies with emotional stress caused by environmental changes (social conditions and radiation exposure) to which the studied population was subject. The population born between 1912 and 1925, experienced the First World War, Russian Revolution and Russian Civil War (living in unfavorable social conditions), and according to the data obtained, it was the population group that had a greater number of copies. The latter finding is similar to the results obtained in the analysis of individuals who were born between 1975 and 2000 and exposed to high levels of radiation (Chernobyl incident), and to the stressful conditions caused by the former Soviet Union, and to individuals from the group 1926 to 1975, that worked with ionizing radiation sources. Realizing that variations in the number of copies of satDNA sequences are related to environmental factors is an interesting path that can help in the deeper knowledge of different pathologies and psychiatric illnesses, such as schizophrenia.
Schizophrenia is a serious, disabling and chronic psychiatric illness characterized essentially by auditory, visual and olfactory hallucinations [47][98]. It was recently demonstrated that patients with schizophrenia have a lower number of copies of HSat3 when compared to healthy individuals (quantification by non-radioactive hybridization) [48][99]. HSat3 content in patients diagnosed with schizophrenia varies across different brain areas [49][100]. Moreover, it has been reported that patients submitted to antipsychotic therapy show an increase or decrease in the number of copies of HSat3, depending if they have initially a low or high HSat3 content, respectively [49][100]. It is known that the number of copies of HSat3 is influenced by both endogenous (variable number of copies between individuals in the same population) and exogenous (environmental stresses such as ionizing radiation and oxidative stress conditions) [46][50][97,101]. This may indicate two possible scenarios, either the low amount of HSat3 is a general feature of schizophrenia patients, or these patients’ genomes react to chronic oxidative stress (caused by this disease) by reducing the number of HSat3 copies [49][100]. This realization opens a new path to investigate the role of satellite DNAs in psychiatric diseases, accounting that they may share common features. In this sense, more of this type of illness has to be studied regarding these repetitive sequences state in patients versus healthy individuals, not only in terms of the number of copies but also in terms of its transcription levels and possible regulatory functions.
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