Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms

Dimerization plays a pivotal role in target-binding interactions among various transcription factors (TFs), including bZIPs, bHLHs, MYBs, and zinc finger TFs. The bZIP family is defined by an -helix class of TFs containing a basic region that binds to the major groove of double-stranded DNA (dsDNA), followed by a leucine zipper motif that mediates dimerization through coiled-coil structures, forming homo- or heterodimers. In polyploid bread wheat (Triticum aestivum cv.), bZIPs and their homeologs interact through the dimerization motifs to form specific bZIP pairs with distinct regulatory functions. The dimerization pattern of bZIPs is critical for gene regulation, however, the mechanisms underlying their specificity remain poorly understood. Here, we focus on the dimerization specificity of coiled-coil - helical bZIP proteins in bread wheat, highlighting the leucine zipper region as a key dimerization motif. We identified 265 bZIPs containing the bZIP motif, following dimerization criteria established in humans, Drosophila, and Arabidopsis based on the presence of specific amino acids at the a, d, e, and g positions in heptads. Eight key bZIP TFs were cloned, expressed, and purified for the structural analysis. This study not only provides the dimerization trend exhibited by bZIPs during thermal stability analysis but also provides the reversibility pattern followed by the thermal denaturation exposure at 85{degrees}C. Our study also points towards that how amino acid substitutions and the length of the leucine zipper influence the formation of coiled-coil structures among bZIP homeologs of polyploid wheat. Overall, the study advances our understanding of the role of leucine zippers, in determining the specificity and stability of bZIP dimers, which in turn governs DNA binding and gene regulation in plants.

Members of the R2R3-MYB4 subgroup are well-known negative regulatory transcription factors of phenylpropane and lignin pathways. In this study, we found that transgenic tobacco plants overexpressing a R2R3-MYB4 subgroup gene from Camellia sinensis (CsMYB4a) showed inhibited growth that was not regulated by phenylpropane and lignin pathways, and these plants exhibited altered sensitivity to synthetic auxin 1-naphthaleneacetic acid (-NAA) treatment. An auxin/indole-3-acetic acid 4 (AUX/IAA4) gene from Camellia sinensis (CsIAA4) participating in the regulation of the auxin signal transduction pathway was screened from the yeast two-hybrid library with CsMYB4a as the bait protein, and tobacco plants overexpressing this gene showed a series of auxin-deficiency phenotypes, such as dwarfism, small leaves, reduced lateral roots, and a shorter primary root. CsIAA4 transgenic tobacco plants were less sensitive to exogenous -NAA than control plants, which was consistent with the findings for CsMYB4a transgenic tobacco plants. The knockout of the endogenous NtIAA4 gene (a CsIAA4 homologous gene) in tobacco plants alleviated growth inhibition in CsMYB4a transgenic tobacco plants. Furthermore, protein-protein interaction experiments proved that domain II of CsIAA4 is the key motif for the interaction between CsIAA4 and CsMYB4a and that the degradation of CsIAA4 is prevented when CsMYB4a interacts with CsIAA4. In summary, our results suggest that CsMYB4a is a multifunctional transcription factor that regulates the auxin signaling pathway, phenylpropane and lignin pathways. This study provides new insights into the multiple functions of R2R3-MYB4 subgroup members as a group of well-known negative regulatory transcription factors. One-sentence summaryCsMYB4a act as multifunctional transcription factor that regulates the auxin signaling pathway, phenylpropane and lignin pathways.

BackgroundType II DNA topoisomerases (topo II) flip the spatial positions of two DNA duplexes, called G- and T-segments, by a cleavage-passage-resealing mechanism. In living cells, these DNA segments can be placed far from each other on the same chromosome. However, no direct evidence for this to occur has been described so far due to lack of proper methodology.\n\nResultsThe beta isoform of topo II (topo II{beta}) is essential for transcriptional regulation of genes expressed in the final stage of neuronal differentiation. To elucidate the enzymes role in the process, here we devise a genome-wide mapping technique for topo II{beta} target sites that can measure the genomic distance between G- and T-segments. It became clear that the enzyme operates in two distinctive modes, termed proximal strand passage (PSP) and distal strand passage (DSP). PSP sites are concentrated around transcription start sites, whereas DSP sites are heavily clustered in small number of hotspots. While PSP represent the conventional topo II targets that remove local torsional stresses, DSP sites have not been described previously. Most remarkably, DSP is driven by the pairing between homologous sequences or repeats located in a large distance. A model-building approach suggested that the DSP sites are intertwined or knotted and topo II{beta} is engaged in unknotting reaction that leads to chromatin decondensation and gene regulation.\n\nConclusionsWhen combined with categorized gene expression analysis, the model-based prediction of DSP sites reveals that DSP is one of the key factors for topo II{beta}-dependency of neuronal gene regulation.

Chromodomain helicase DNA binding protein 8 (CHD8) is a chromatin remodeler of the SNF2 family involved in gene transcription regulation. It has been shown that CHD8 is required for cell proliferation, cell differentiation and central nervous system development. In fact, CHD8 haploinsufficiency causes a human syndrome characterized by autism, macrocephaly, gastrointestinal complaints and some other clinical characteristics. However, the mechanism by which CHD8 controls transcription and how it is recruited to its targets in the chromatin is still unclear. We have previously shown that serum depletion causes that CHD8 detaches from chromatin. Here we demonstrate that serum-dependent recruitment of CHD8 to promoters requires the extracellular signal-regulated kinase (ERK)/ ETS-like (ELK) branch of the mitogen-activated protein kinase (MAPK) pathway. Our analysis of genomic occupancy data shows that CHD8 binding sites were strongly enriched in ELK1 and ELK4 DNA binding motifs and that CHD8 and ELK1 co-occupy multiple transcription start sites. We show that ELK1 and ELK4 are required for normal recruitment of CHD8 to the promoters of CCNA2, CDC6, CCNE2, BRCA2 and MYC genes. However, CHD8 is dispensable for ELK1 and ELK4 binding. Genome wide transcriptomic analysis evidenced that serum-dependent activation of a subset of immediate early genes, including the well-known ELK1 target gene FOS, was impaired upon depletion of CHD8. In summary, our results uncover the role of the ERK/ELK pathway in CHD8 recruitment to chromatin and provide evidences indicating a role of CHD8 in regulating serum-dependent transcription.

Transcription factors (TFs) regulate gene expression by specifically binding to DNA targets. Many factors have been revealed to influence TF-DNA binding specificity. Coevolution of residues in proteins occurs due to a common evolutionary history. However, it is unclear how coevolving residues in TFs contribute to DNA binding specificity. Here, we systematically analyzed TF-DNA interactions from high-throughput experiments for seven TF families, including Homeobox, HLH, bZIP_1, Ets, HMG_box, zf-C4 and Zn_clus TFs. Based on TF-DNA interactions, we detected TF subclass determining sites (TSDSs) defining the heterogeneity of DNA binding preference for each TF family. We showed that the TSDSs were more likely to be coevolving with TSDSs than with non-TSDSs, particularly for Homeobox, HLH, Ets, bZIP_1 and HMG_box TF families. Mutation of the highly coevolving residues could significantly reduce the stability of TF-DNA complex. The distant residues from the DNA interface also contributed to TF-DNA binding activity. Overall, our study gave evidence of the functional importance of coevolved residues in refining transcriptional regulation and provided clues to the application of engineered DNA-binding domains and protein.

The Spalt transcriptional regulators participate in a variety of cell fate decisions during multicellular development. Vertebrate Spalt proteins have been mostly associated to the organization of heterochromatic regions, but they also contribute regulatory functions through binding to A/T rich motives present in their target genes. The developmental processes in which the Drosophila spalt genes participate are well known through genetic analysis, but the mechanism by which the Spalt proteins regulate transcription are still unknown. Furthermore, despite the prominent changes in gene expression associated to mutations in the spalt genes, the specific DNA sequences they bind are also unknow. In this contribution we describe the analysis of a DNA fragment present in the regulatory region of the knirps gene. Spalt proteins are candidate repressors of knirps expression during the formation of the venation pattern in the wing disc, and here we identify a minimal conserved 30bp sequence that binds to Spalt major both in vivo and in vitro. This sequence mediates transcriptional repression in the central region of the wing blade, constituting the first confirmed case of a direct regulatory interaction between Spalt major and its target DNA in Drosophila.

The high mobility group N (HMGN) proteins, which were discovered over 50 years ago, are a multigene family of abundant nucleosome-specific binding factors that are present in all vertebrates. Despite their intriguing nucleosome-binding activity, the potential functions of the HMGN proteins in chromatin have not yet been assessed unambiguously due to the presence of several related HMGN genes in vertebrates and the lack of HMGN null cells. Here, we investigated the genome-wide activities of the human HMGN proteins by generating and analyzing an HMGN null cell line and isogenic HMGN rescue cell lines. These experiments revealed that the HMGN proteins function in the activation of gene expression at the level of transcription initiation at over a thousand specific sites that are mostly in promoters and enhancers. We additionally observed shared as well as unique functions of HMGN1 and HMGN2, which are likely to be the most abundant and ancient HMGN proteins. These findings thus indicate that the HMGN nucleosome-binding proteins are vertebrate-specific regulatory factors that primarily function in the activation of transcription initiation. Hence, any comprehensive model of vertebrate gene regulation should incorporate the contributions of the HMGN proteins, which are integral components of chromatin in all vertebrates.

Apart from its well-established role in initiation of transcription, the general transcription factor TFIIB has been implicated in the termination step as well. The ubiquity of TFIIB involvement in termination as well as mechanistic details of its termination function, however, remains largely unexplored. To determine the prevalence of TFIIBs role in termination, we performed GRO-seq analyses in sua7-1 mutant (TFIIBsua7-1) and the isogenic wild type (TFIIBWT) strains of yeast. Almost a three-fold increase in readthrough of the poly(A)-termination signal was observed in TFIIBsua7-1 mutant compared to the TFIIBWT cells. Of all genes analyzed in this study, nearly 74% genes exhibited a statistically significant increase in terminator readthrough in the mutant. To gain an understanding of the mechanistic basis of TFIIB involvement in termination, we performed mass spectrometry of TFIIB, affinity purified from chromatin and soluble cellular fractions, from TFIIBsua7-1 and TFIIBWT cells. TFIIB purified from the chromatin fraction of TFIIBWT cells exhibited significant enrichment of CF1A and Rat1 termination complexes. There was, however, a drastic decrease in TFIIB interaction with both CF1A and Rat1 termination complexes in TFIIBsua7-1 mutant. ChIP assay revealed that the recruitment of Pta1 subunit of CPF complex, Rna15 subunit of CF1 complex and Rat1 subunit of Rat1 complex registered nearly 90% decline in the mutant over wild type cells. The overall conclusion of these results is that TFIIB affects termination of transcription on a genome-wide scale, and TFIIB-termination factor interaction may play a crucial role in the process.

BackgroundChromatin interactions are essential in enhancer-promoter interactions (EPIs) and transcriptional regulation. CTCF and cohesin proteins located at chromatin interaction anchors and other DNA-binding proteins such as YY1, ZNF143, and SMARCA4 are involved in chromatin interactions. However, there is still no good overall understanding of proteins associated with chromatin interactions and insulator functions. ResultsHere, I describe a systematic and comprehensive approach for discovering DNA-binding motifs of transcription factors (TFs) that affect EPIs and gene expression. This analysis identified 96 biased orientations [64 forward-reverse (FR) and 52 reverse-forward (RF)] of motifs that significantly affected the expression level of putative transcriptional target genes in monocytes, T cells, HMEC, and NPC and included CTCF, cohesin (RAD21 and SMC3), YY1, and ZNF143; some TFs have more than one motif in databases; thus, the total number is smaller than the sum of FRs and RFs. KLF4, ERG, RFX, RFX2, HIF1, SP1, STAT3, and AP1 were associated with chromatin interactions. Many other TFs were also known to have chromatin-associated functions. The predicted biased orientations of motifs were compared with chromatin interaction data. Correlations in expression level of nearby genes separated by the motif sites were then examined among 53 tissues. ConclusionOne hundred FR and RF orientations associated with chromatin interactions and functions were discovered. Most TFs showed weak directional biases at chromatin interaction anchors and were difficult to identify using enrichment analysis of motifs. These findings contribute to the understanding of chromatin-associated motifs involved in transcriptional regulation, chromatin interactions/regulation, and histone modifications.

Hunchback (Hb) is considered a context-dependent transcription factor, able to activate or repress different enhancers during Drosophila embryo segmentation. The mechanism driving the contextdependent activity of Hb is however not well understood. Here we measure the activity of a large set of 20 synthetic enhancers that we design to elucidate the effect of Hb binding sites in Drosophila segmentation. We obtain quantitative data on the spatiotemporal dynamics of activity of all synthetic enhancers in-vivo, by using a quantitative and sensitive reporter system we recently developed. Our data reveal the dual role of Hb binding sites in segmentation enhancers: on the one hand, Hb act as a typical short range repressor by binding to its cognate sequences; on the other hand, we report a novel effect of a sequence containing multiple Hb binding sites, which is able to increase enhancer activity independently from Hb binding. This sequence, which contains multiple Poly-dA stretches, increases the activity of enhancers driven by different activators, possibly by disfavoring nucleosome occupancy. AUTHOR SUMMARYThe control of gene expression is a fundamental process that allows cells to respond to external stimuli and take on various identities in complex organisms. Enhancers are DNA sequences that play a key role in this process. In the simplest model of an enhancer, small parts of its sequence can be specifically bound by proteins called transcription factors and the occupancy pattern of these proteins on the enhancer determines the expression level of a specific gene. In this research work we have studied enhancers in the context of the development of a fruit fly embryo. We have built synthetic enhancer sequences containing binding sites for a few specific factors and measured their activity in living embryos using fluorescence microscopy. Our results revealed that binding sites for a particular protein, Hunchback, are able to influence the activity of the enhancer even independently from Hunchback binding to them. This discovery might help to explain the complex effects that have been observed when studying Hunchback binding sites in natural enhancers.

The chromatin remodeling protein, dIno80 (Drosophila Ino80) regulates homeotic genes. We show that Ino80, along with Trx and ETP (Enhancer of Trithorax and Polycomb) proteins, interacts with two Polycomb/Trithorax Responsive Elements (PRE/TRE), iab-7 and bxd PRE in flies and the larval imaginal discs. In S2 cells, dIno80 localizes to the endogenous iab-7 and bxd-PREs. The localization of Ino80 and Pleiohomeotic (Pho) at the PRE is sensitive to the cellular abundance of each other; when levels of Ino80 are limiting, there is increased Pho enrichment, and Pho knock-down leads to increased enrichment of Ino80. We demonstrate that over-expression of dIno80 rescues the pupal lethality in pleiohomeotic (pho) deficient flies, which suggests that dIno80 has a role in cellular memory. The apparent competition between Pho and Ino80 for binding at the PRE indicates that Ino80 may act as a potential recruiter of the regulatory complex in addition to being a chromatin remodeler.\n\nAuthor SummaryThe null mutants of Pho and dIno80 show lethality at different stages of development in the fly, implying that they may function independent of each other. The observation that Pho-lethality can be rescued by overexpression of dIno80 with significant penetrance and that Ino80 has its own DNA binding domain, led us to predict that Ino80 may have Pho-independent functions, perhaps through non-canonical complexes. In the current study, we show that dIno80 interacts with bxd and iab-7 PRE in cooperation with Polycomb and Trithorax proteins and regulate the homeotic genes. The effect of knock-down or mutation of dIno80 results in altered phenotype in adult flies and rescue of Lac-Z expression in imaginal discs, in parallel with similar effect of Pho mutation or knock-down. We provide evidence of direct interaction of dIno80 with iab7- and bxd-PRE using chromatin immunoprecipitation. The dIno80 localization in and around the PRE sequence was enhanced in the absence of Pho, indicating competition between Pho and dIno80 for binding at the PRE.

Breast Cancer Susceptibility Gene 1 (BRCA1) codes for a DNA repair protein that facilitates the repair of double-stranded DNA breaks (DSBs) in human cells through the homologous recombination (HR) pathway. Mutations of BRCA1 are highly associated with breast cancer; however, many variants remain unclassified with unknown cellular phenotypes. The DNA binding activity of BRCA1 is localized primarily to its central region, which can be divided into two distinct domains: DNA Binding Domain 1 (DBD1; amino acids (aa) 330-554) and 2 (DBD2; aa 894-1057). We previously proposed a model in which DBD1 targets BRCA1 to DSBs for the promotion of DNA end resection, while DBD2 targets BRCA1 to telomeres to function in chromatin remodeling and telomere regulation. In this study, we hypothesized that unknown DBD variants (T374I, K408E, N417S, N909I, M1008I, and R1028H) with similar properties to known disease-causing variants (Q356H, F461L, R496H, D940Y, S1027N, and E1038G) would also be pathogenic. The affinities of each variant for single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and a G-quadruplex (G4) sequence were measured via biolayer interferometry. The DNA repair phenotypes of each variant were analyzed by overexpression in HEK cells to determine correlation between binding activity and DNA damage response. Altogether, these results provide insight into how missense mutations affect the ability of BRCA1 DBDs to facilitate the DNA damage response.

The discovery of functional long non-coding RNAs (lncRNAs) changed their initial concept as transcriptional noise. LncRNAs have been found to participate in the regulation of multiple biological processes, including chromatin structure, gene expression, splicing, and mRNA degradation and translation. However, functional studies of lncRNAs are hindered by the usual lack of phenotypes upon deletion or inhibition. Here, we used Drosophila imaginal discs as a model system to identify lncRNAs involved in development and regeneration. We examined a subset of lncRNAs expressed in the wing, leg, and eye disc development. Additionally, we analyzed transcriptomic data from regenerating wing discs to profile the expression pattern of lncRNAs during tissue repair. We focused on the lncRNA CR40469, which is upregulated during regeneration. We generated CR40469 mutant flies that developed normally but showed impaired wing regeneration upon the induction of cell death. The ability of these mutants to regenerate was restored by the ectopic expression of CR40469. Furthermore, we found that the lncRNA CR34335 has a high degree of sequence similarity with CR40469 and can partially compensate for its function during regeneration in the absence of CR40469. Our findings point to a potential role of the lncRNA CR40469 in trans during the response to damage in the wing imaginal disc.

In Bacillus species, the interaction between the repressor AbrB and the antirepressor AbbA is vital for regulating gene expression. Our study reveals that AbrB binds DNA cooperatively the promoter of the phyC gene through multiple tetramers, forming a complex regulatory mechanism. AbbA disrupts AbrBs DNA binding by competing for its DNA-binding sites, as shown by surface plasmon resonance (SPR) and gel shift assays. Circular dichroism (CD) confirmed that AbbA does not bind directly to the phyC promoter but mimics DNA to interfere with AbrB. Small-angle X-ray scattering (SAXS) data suggest that AbbA resembles a deformed DNA double helix. Our results indicate that AbbA binds to AbrBs DNA-binding sites located with the N-terminal domain causing AbrB displacement. The interaction exhibits negative cooperativity, with both high- and low-affinity binding sites, as evidenced by Scatchard plots and kinetic studies. Our findings suggest that AbbA effectively mimics DNA to displace AbrB, activating transition-state genes. This research enhances our understanding of bacterial gene regulation and provides insights into the complex mechanisms controlling transcription in Bacillus species.

C2H2 zinc finger proteins (ZFPs) comprise of the largest group of DNA-binding proteins in human genome, and many of them contain long, tandem array of fingers, making the motif discovery, prediction of in vivo cis-regulatory elements (CREs), and understanding their functions particularly challenging. Previous work established that due to the dependent recognition between sub-motifs, the simple, additive recognition model impedes motif discovery and compromises our understanding about how ZFPs work. This work uses ZFP3, a 13-finger long ZFP with no known function, as case example to address the reverse question---given the full-length motif learned through in vitro experiments, like Spec-seq and HT-SELEX, how to reliably identify its in vivo cis-regulatory elements (CREs) and further predict this genes functions. Through sorting of all possible sites within the ChIP-seq peaks with similar predicted binding energy into groups and comparing the aggregate ChIP-seq signals between groups, it is evident that either its full-length or individual sub-motif alone fails to correctly identify all high-affinity specific sites without false-positives, thus it is necessary to revise current algorithm, and use both the core and upstream motifs as separate components to improve the prediction accuracy. Furthermore, significant number of regulatory elements of ZFP3 are found to be proximal to genes associated with microtubules organization and ciliogenesis, which coincides with the fact that ZFP3 is specifically upregulated in multiple ciliated cells. At last, local chromatin accessibility and active chromatin marks like H3K27ac are found to positively associate with the differential binding of ZFP3 between tested cell lines. Overall, this work establishes a novel "From motif to function" strategy for long ZFPs, and the data analysis workflows are implemented through R package TFCookbook for reuse onto other ZFPs.

Cells of many ectothermic species, including Drosophila melanogaster, maintain homeostatic function within a considerable temperature range. The cellular mechanisms enabling temperature acclimation are still poorly understood. At the transcriptional level, the heat shock response has been extensively analyzed. The opposite has received less attention. Here, using cultured Drosophila cells, we have identified genes with increased transcript levels at the lower end of the readily tolerated temperature range, as well as chromatin regions with increased DNA accessibility. Candidate cis-regulatory elements (CREs) for transcriptional upregulation at low temperature were selected and evaluated with a novel reporter assay for accurate assessment of their temperature-dependency. Robust transcriptional upregulation at low temperature could be demonstrated for a fragment from the pastrel gene, which expresses more transcript and protein at reduced temperatures. The CRE is controlled by the JAK/STAT signaling pathway and antagonizing activities of the transcription factors Pointed and Ets97D.

Histones bind directly to DNA and play a role in regulating gene expression mediated by chromatin structure. DNA sequences of these histone genes are quite similar, which has hindered individual analyses. The exact function of the 13 different isoforms of histone H2B remains unclear. In this report, a comprehensive gene expression analysis of the H2B isoforms, focusing on tissue specificity, was conducted. We generated mice lacking the H2bc27 gene, which exhibited brain-specific expression of E14.5, and proceeded to characterize the brain tissue. The phenotype of H2bc27 knockout brains was similar to that of wild-type brains, yet transcriptome analysis indicated that H2bc27 is associated with regulating the expression of several functional genes involved in mouse brain development. The methods used in this study may serve to facilitate comprehensive H2B isoform analysis.

The THAP (Thanatos-associated protein) domain is a DNA-binding domain which binds DNA via a zinc coordinating C2CH motif. Although THAP domains share a conserved structural fold, they bind different DNA sequences in different THAP proteins which in turn perform distinct cellular functions. In this study, we investigate (using multiple sequence alignment, in silico motif and secondary structure prediction) THAP domain conservation within the homologs of the human THAP (hTHAP) protein family. We report that there is significant variation in sequence and predicted secondary structure elements across hTHAP homologs. Interestingly, we report that the THAP domain can be either longer or shorter than the conventional 90 residues and the amino terminal C2CH motif within the THAP domain serves as a hotspot for insertion or deletion. Our results lay the foundation for future studies which will further our understanding of the evolution of THAP domain and regulation of its function.

G-quadruplexes (G4), stable four-stranded non-canonical DNA structures, are highly related to function of promoters and initiation of gene transcription. We found that G4 structures were also enriched in the enhancers across different cell lines. However, the relationship between G4 structures and enhancer activity remains unknown. Here, we proved that G4 structures on enhancers lead to the re-positioning of nucleosomes create nucleosome depleted regions (NDRs). Moreover, stable NDRs and special secondary structures of G4 help enhancers to recruit abundant TFs to co-bind, especially for architectural proteins including CTCF, RAD21, and SMC3. These architectural proteins, which play critical roles in the formation of higher-order chromatin organization, further influenced the chromatin interactions of G4 enhancers. Additionally, we revealed that G4 enhancers harbored significantly higher enrichment of eQTLs than typical enhancers, suggesting G4 enhancers displayed more enhancer regulatory activity. We found that most super enhancers (SEs) contain G4 structures. Even though the enrichment of chromatin accessibility and histone modifications around G4-containing SEs are not significantly higher than those around other SEs, G4-containing SEs still possess much more TFs across different cell lines. According to these results, we proposed a model in which the formation of G4 structures on enhancer exclude nucleosome occupancy and recruit abundant TFs which lead to the stable chromatin interaction between G4 enhancers and their target genes. Because of the relevance between G4 structures and enhancers, we hypothesized that G4 structures may be a potential markers indicating enhancer regulatory activity.

One of the main replicative enzymes in most eukaryotes, DNA polymerase {varepsilon} (POLE), is composed of four subunits, namely a single catalytic and three regulatory subunits. In Arabidopsis, the catalytic subunit of POLE is encoded by two genes: Arabidopsis thaliana DNA POLYMERASE EPSILON CATALYTIC SUBUNIT A (AtPOL2A) and B (AtPOL2B). Although studies have shown AtPOL2A to be involved in various biological processes, the role of AtPOL2B is unclear. Here, we investigated the transcriptomes of both atpol2a and atpol2b mutants, and the promoter sequences to provide a better insight into the targets of AtPOL2s at the molecular level. In the present study, leaf cDNA libraries of four AtPOL2 mutants (atpol2a-1 and atpol2b-1, -2 and - 3) were sequenced using the Illumina platform. Analysis of gene expression profiles identified a total of 198, 76, 141 and 67 differentially expressed genes in atpol2a-1, atpol2b-1, atpol2b-2 and atpol2b-3, respectively; the majority of pericentromeric transposable elements were transcriptionally active in atpol2a-1 as compared to atpol2b mutants and wild type. Protein-protein interaction network analysis and molecular docking identified three (CER1, RPA1E and AT5G60250) and two (PR1 and AT5G48490) proteins as potential interactors (cluster size > 60 and balanced score < -900) of AtPOL2A and AtPOL2B, respectively; Interestingly, these five proteins also showed a significant interaction between POLE catalytic subunit of Saccharomyces cerevisiae. Our in silico promoter analysis showed that the AtPOL2A promoter sequence is overrepresented with cis-acting regulatory elements (CREs) associate with cell cycle regulation, meristematic/reproductive tissue-specific pattern of expression and MYB protein recognition, whereas the AtPOL2B promoter sequence was mainly enriched with stress-responsive elements. The information provided here has led to the identification of targets of AtPOL2s at the molecular level and CREs putatively associated with the regulation of AtPOL2s. To our knowledge, this study provides the first comparative transcriptome profiling of single-gene mutants of AtPOL2s.