Eukaryotic Transcription Factors Research Articles

Using Restriction Endonuclease, Protection, Selection, and Amplification to Identify Preferred DNA-Binding Sequences of Microbial Transcription Factors.

Regulation of gene expression is a vital component of cellular biology. Transcription factor proteins often bind regulatory DNA sequences upstream of transcription start sites to facilitate the activation or repression of RNA polymerase. Research laboratories have devoted many projects to understanding the transcription regulatory networks for transcription factors, as these regulated genes provide critical insight into the biology of the host organism. Various in vivo and in vitro assays have been developed to elucidate transcription regulatory networks. Several assays, including SELEX-seq and ChIP-seq, capture DNA-bound transcription factors to determine the preferred DNA-binding sequences, which can then be mapped to the host organism's genome to identify candidate regulatory genes. In this protocol, we describe an alternative in vitro, iterative selection approach to ascertaining DNA-binding sequences of a transcription factor of interest using restriction endonuclease, protection, selection, and amplification (REPSA). Contrary to traditional antibody-based capture methods, REPSA selects for transcription factor-bound DNA sequences by challenging binding reactions with a type IIS restriction endonuclease. Cleavage-resistant DNA species are amplified by PCR and then used as inputs for the next round of REPSA. This process is repeated until a protected DNA species is observed by gel electrophoresis, which is an indication of a successful REPSA experiment. Subsequent high-throughput sequencing of REPSA-selected DNAs accompanied by motif discovery and scanning analyses can be used for determining transcription factor consensus binding sequences and potential regulated genes, providing critical first steps in determining organisms' transcription regulatory networks. IMPORTANCE Transcription regulatory proteins are an essential class of proteins that help maintain cellular homeostasis by adapting the transcriptome based on environmental cues. Dysregulation of transcription factors can lead to diseases such as cancer, and many eukaryotic and prokaryotic transcription factors have become enticing therapeutic targets. Additionally, in many understudied organisms, the transcription regulatory networks for uncharacterized transcription factors remain unknown. As such, the need for experimental techniques to establish transcription regulatory networks is paramount. Here, we describe a step-by-step protocol for REPSA, an inexpensive, iterative selection technique to identify transcription factor-binding sequences without the need for antibody-based capture methods.

Expression Pattern and Clinical Significance of E2F Transcription Factors in Skin Cutaneous Melanoma

Introduction: E2F is a group of genes that encodes a family of transcription factors in higher eukaryotes. Although emerging evidence indicates that E2Fs are implicated in various cancer types, the diverse expression patterns and prognostic values of E2F transcription factors in SKCM have yet to be elucidated. The purpose of this study was to enhance our knowledge concerning the role of E2Fs in SKCM patients.. Methods: GEPIA, The Human Protein Atlas, TIMER, cBioPortal, GeneMANIA, DAVID 6.8 and KEGG PATHWAY Database were utilized in this study.

Genome-Wide Characterization and Analysis of R2R3-MYB Genes Related to Fruit Ripening and Stress Response in Banana (Musa acuminata L. AAA Group, cv. 'Cavendish').

MYB is an important type of transcription factor in eukaryotes. It is widely involved in a variety of biological processes and plays a role in plant morphogenesis, growth and development, primary and secondary metabolite synthesis, and other life processes. In this study, bioinformatics methods were used to identify the R2R3-MYB transcription factor family members in the whole Musa acuminata (DH-Pahang) genome, one of the wild ancestors of banana. A total of 280 MaMYBs were obtained, and phylogenetic analysis indicated that these MaMYBs could be classified into 33 clades with MYBs from Arabidopsis thaliana. The amino acid sequences of the R2 and R3 Myb-DNA binding in all MaMYB protein sequences were quite conserved, especially Arg-12, Arg-13, Leu-23, and Leu-79. Distribution mapping results showed that 277 MaMYBs were localized on the 11 chromosomes in the Musa acuminata genome. The MaMYBs were distributed unevenly across the 11 chromosomes. More than 40.0% of the MaMYBs were located in collinear fragments, and segmental duplications likely played a key role in the expansion of the MaMYBs. Moreover, the expression profiles of MaMYBs in different fruit development and ripening stages and under various abiotic and biotic stresses were investigated using available RNA-sequencing data to obtain fruit development, ripening-specific, and stress-responsive candidate genes. Weighted gene co-expression network analysis (WGCNA) was used to analyze transcriptome data of banana from the above 11 samples. We found MaMYBs participating in important metabolic biosynthesis pathways in banana. Collectively, our results represent a comprehensive genome-wide study of the MaMYB gene family, which should be helpful in further detailed studies on MaMYBs functions related to fruit development, postharvest ripening, and the seedling response to stress in an important banana cultivar.

Quantifying the Binding and Target-Search Kinetics of Transcriptional Regulatory Factors by Live-Cell Single-Molecule Tracking.

Eukaryotic transcriptional regulatory factors, such as transcription factors and epigenetic regulatory factors, must locate, bind, and assemble at specific genomic regions to execute functions within the complex and crowded environment of the nucleus. These dynamic processes are typically at nonequilibrium, so quantifying their binding and target-search processes within the native environment is essential for understanding transcriptional mechanisms. Live-cell single-molecule tracking (SMT) is an emerging technique that can be utilized to observe molecular trajectories of individual transcriptional regulatory complexes within the nucleus. Here, we describe the use of live-cell SMT to observe trajectories of individual transcriptional regulatory complexes. We delineate the imaging analysis to obtain chromatin-bound fraction and residence time. Finally, we elaborate on the kinetic modeling to estimate target-search parameters. These binding and target-search parameters facilitate the understanding of how transcription is spatially and temporally regulated under physiological and pathological conditions.

Molecular biology of the cell

Molecular biology of the cell

Transcriptome-wide characterization, evolutionary analysis, and expression pattern analysis of the NF-Y transcription factor gene family and salt stress response in Panax ginseng

Jilin ginseng (Panax ginseng C. A. Meyer) has a long history of medicinal use worldwide. The quality of ginseng is governed by a variety of internal and external factors. Nuclear factor Y (NF-Y), an important transcription factor in eukaryotes, plays a crucial role in the plant response to abiotic stresses by binding to a specific promoter, the CCAAT box. However, the NF-Y gene family has not been reported in Panax ginseng. In this study, 115 PgNF-Y transcripts with 40 gene IDs were identified from the Jilin ginseng transcriptome database. These genes were classified into the PgNF-YA (13), PgNF-YB (14), and PgNF-YC (13) subgroups according to their subunit types, and their nucleotide sequence lengths, structural domain information, and amino acid sequence lengths were analyzed. The phylogenetic analysis showed that the 79 PgNF-Y transcripts with complete ORFs were divided into three subfamilies, NF-YA, NF-YB, and NF-YC. PgNF-Y was annotated to eight subclasses under three major functions (BP, MF, and CC) by GO annotation, indicating that these transcripts perform different functions in ginseng growth and development. Expression pattern analysis of the roots of 42 farm cultivars, 14 different tissues of 4-year-old ginseng plants, and the roots of 4 different-ages of ginseng plants showed that PgNF-Y gene expression differed across lineages and had spatiotemporal specificity. Coexpression network analysis showed that PgNF-Ys acted synergistically with each other in Jilin ginseng. In addition, the analysis of the response of PgNF-YB09, PgNF-YC02, and PgNF-YC07-04 genes to salt stress treatment was investigated by fluorescence quantitative PCR. The expression of these genes increased after salt stress treatment, indicating that they may be involved in the regulation of the response to salt stresses in ginseng. These results provide important functional genetic resources for the improvement and gene breeding of ginseng in the future.Conclusions: This study fills a knowledge gap regarding the NF-Y gene family in ginseng, provides systematic theoretical support for subsequent research on PgNF-Y genes, and provides data resources for resistance to salt stress in ginseng.

CREB1 promotes proliferation and differentiation by mediating the transcription of CCNA2 and MYOG in bovine myoblasts

The cAMP response element binding protein 1 (CREB1) is an important nuclear transcription factor in eukaryotes. To explore the potential role of CREB1 on Qinchuan bovine skeletal myoblasts, we investigated the function of CREB1 on proliferation and differentiation. In this study, we found that CREB1 promoted cell proliferation by promoting DNA synthesis in S phase and cell division in G2 phase and promoted myogenic differentiation process in bovine myoblasts. Through dual luciferase experiments, we found that CREB1 can bind to the proximal promoter regions of CCNA2 and MyoG, indicating that CREB1 can play a positive regulatory role in the proliferation and differentiation of myoblasts by mediating the transcription of CCNA2 and MyoG. In addition, through downstream target gene analysis and transcriptome sequencing, we found that CREB1 plays a role in cell proliferation, myogenic differentiation, skeletal muscle repair and other related pathways.

Structural insights into cooperative DNA recognition by the CCAAT-binding complex and its bZIP transcription factor HapX

The heterotrimeric CCAAT-binding complex (CBC) is a fundamental eukaryotic transcription factor recognizing the CCAAT box. In certain fungi, like Aspergilli, the CBC cooperates with the basic leucine zipper HapX to control iron metabolism. HapX functionally depends on the CBC, and the stable interaction of both requires DNA. To study this cooperative effect, X-ray structures of the CBC-HapX-DNA complex were determined. Downstream of the CCAAT box, occupied by the CBC, a HapX dimer binds to the major groove. The leash-like N terminus of the distal HapX subunit contacts the CBC, and via a flexible polyproline type II helix mediates minor groove interactions that stimulate sequence promiscuity. Invitro and invivo mutagenesis suggest that the structural and functional plasticity of HapX results from local asymmetry and its ability to target major and minor grooves simultaneously. The latter feature may also apply to related transcription factors such as yeast Hap4 and distinct Yap family members.

Independent Evolution of the MYB Family in Brown Algae.

Myeloblastosis (MYB) proteins represent one of the largest families of eukaryotic transcription factors and regulate important processes in growth and development. Studies on MYBs have mainly focused on animals and plants; however, comprehensive analysis across other supergroups such as SAR (stramenopiles, alveolates, and rhizarians) is lacking. This study characterized the structure, evolution, and expression of MYBs in four brown algae, which comprise the biggest multicellular lineage of SAR. Subfamily 1R-MYB comprised heterogeneous proteins, with fewer conserved motifs found outside the MYB domain. Unlike the SHAQKY subgroup of plant 1R-MYB, THAQKY comprised the largest subgroup of brown algal 1R-MYBs. Unlike the expansion of 2R-MYBs in plants, brown algae harbored more 3R-MYBs than 2R-MYBs. At least ten 2R-MYBs, fifteen 3R-MYBs, and one 6R-MYB orthologs existed in the common ancestor of brown algae. Phylogenetic analysis showed that brown algal MYBs had ancient origins and a diverged evolution. They showed strong affinity with stramenopile species, while not with red algae, green algae, or animals, suggesting that brown algal MYBs did not come from the secondary endosymbiosis of red and green plastids. Sequence comparison among all repeats of the three types of MYB subfamilies revealed that the repeat of 1R-MYBs showed higher sequence identity with the R3 of 2R-MYBs and 3R-MYBs, which supports the idea that 1R-MYB was derived from loss of the first and second repeats of the ancestor MYB. Compared with other species of SAR, brown algal MYB proteins exhibited a higher proportion of intrinsic disordered regions, which might contribute to multicellular evolution. Expression analysis showed that many MYB genes are responsive to different stress conditions and developmental stages. The evolution and expression analyses provided a comprehensive analysis of the phylogeny and functions of MYBs in brown algae.

Directed mutational scanning reveals a balance between acidic and hydrophobic residues in strong human activation domains.

SUMMARYAcidic activation domains are intrinsically disordered regions of the transcription factors that bind coactivators. The intrinsic disorder and low evolutionary conservation of activation domains have made it difficult to identify the sequence features that control activity. To address this problem, we designed thousands of variants in seven acidic activation domains and measured their activities with a high-throughput assay in human cell culture. We found that strong activation domain activity requires a balance between the number of acidic residues and aromatic and leucine residues. These findings motivated a predictor of acidic activation domains that scans the human proteome for clusters of aromatic and leucine residues embedded in regions of high acidity. This predictor identifies known activation domains and accurately predicts previously unidentified ones. Our results support a flexible acidic exposure model of activation domains in which the acidic residues solubilize hydrophobic motifs so that they can interact with coactivators. A record of this paper’s transparent peer review process is included in the supplemental information.

The diversity of heme sensor systems - heme-responsive transcriptional regulation mediated by transient heme protein interactions.

Heme is a versatile molecule that is vital for nearly all cellular life by serving as prosthetic group for various enzymes or as nutritional iron source for diverse microbial species. However, elevated levels of heme is toxic to cells. The complexity of this stimulus has shaped the evolution of diverse heme sensor systems, which are involved in heme-dependent transcriptional regulation in eukaryotes and prokaryotes. The functions of these systems are manifold-ranging from the specific control of heme detoxification or uptake systems to the global integration of heme and iron homeostasis. This review focuses on heme sensor systems, regulating heme homeostasis by transient heme protein interaction. We provide an overview of known heme-binding motifs in prokaryotic and eukaryotic transcription factors. Besides the central ligands, the surrounding amino acid environment was shown to play a pivotal role in heme binding. The diversity of heme-regulatory systems, therefore, illustrates that prediction based on pure sequence information is hardly possible and requires careful experimental validation. Comprehensive understanding of heme-regulated processes is not only important for our understanding of cellular physiology, but also provides a basis for the development of novel antibacterial drugs and metabolic engineering strategies.

The auto-inhibition mechanism of transcription factor Ets-1 induced by phosphorylation on the intrinsically disordered region

As the most abundant post-translation modifications (PTMs), the phosphorylation usually occurred on the intrinsically disordered regions (IDRs). The regulation on the structures and interactions of IDRs induced by phosphorylation is critical to the function performing. The eukaryotic transcription factor 1 (Ets-1) is a member of transcription factor family, which participates in many important biological processes. The DNA-binding ability of Ets-1 is auto-inhibited by a disordered serine-rich region (SRR) on the Ets-1. The inhibition ability of SRR is greatly enhanced by the phosphorylation of the serine on the SRR. Nevertheless, the molecular mechanisms of the phosphorylation regulation on the structure and activity of Ets-1 are still unclear and under debates. By using both of the molecular simulations and biochemical experiments, we studied the molecule mechanism of phosphorylation regulation on the auto-inhibition of the Ets-1. The reasons of stabilization of Ets-1 core by phosphorylation on SRR region were elucidated. More important, the free energy landscapes (FEL) show that both of the steric hindrance and allosteric regulation are responsible for the DNA-binding inhibitory induced by phosphorylation, but the steric effects contribute greater than the allosteric regulation. The phosphorylation not only enhances the electrostatic interactions to facilitate the steric impedance, but also promotes the formation of hydrophobic residue clusters, which provide major driven force for the allosteric regulation. The structural basis of auto-inhibition of Ets-1 induced by the phosphorylation revealed in this study would great help the developing of inhibitor for the cancer therapy.

The Piragua Gene Is not Essential for Drosophila Development.

Proteins with clusters of C2H2 zinc finger domains (C2H2-proteins) constitute the most abundant class of transcription factors in higher eukaryotes. N-terminal ZAD (zinc finger-associated domain) dimerization domain has been identified in a large group of C2H2-proteins mostly in insects. The piragua gene encodes one of these proteins, Fu2. We have generated CRISPR/Cas9-mediated deletion of the piragua gene that has no phenotype. We have used φC31-mediated attP/attB recombination to generate a transgenic line expressing Fu2 protein fused with HA epitope. This line will be useful for analysis of DNA binding profile and functions of Fu2 protein.

Genome-Wide Identification and Analysis of the NF-Y Gene Family in Potato (Solanum tuberosum L.).

Nuclear factor Y (NF-Y) is a ubiquitous transcription factor in eukaryotes, which is composed of three subunits (NF-YA, NF-YB, and NF-YC). NF-Y has been identified as a key regulator of multiple pathways in plants. Although the NF-Y gene family has been identified in many plants, it has not been reported in potato (Solanum tuberosum). In the present study, a total of 41 NF-Y proteins in potato (StNF-Ys) were identified, including 10 StNF-YA, 22 StNF-YB, and nine StNF-YC subunits, and their distribution on chromosomes, gene structure, and conserved motif was analyzed. A synteny analysis indicated that 14 and 38 pairs of StNF-Y genes were orthologous to Arabidopsis and tomato (Solanum lycopersicum), respectively, and these gene pairs evolved under strong purifying selection. In addition, we analyzed the expression profiles of NF-Y genes in different tissues of double haploid (DM) potato, as well as under abiotic stresses and hormone treatments by RNA-seq downloaded from the Potato Genome Sequencing Consortium (PGSC) database. Furthermore, we performed RNA-seq on white, red, and purple tuber skin and flesh of three potato cultivars at the tuber maturation stage to identify genes that might be involved in anthocyanin biosynthesis. These results provide valuable information for improved understanding of StNF-Y gene family and further functional analysis of StNF-Y genes in fruit development, abiotic stress tolerance, and anthocyanin biosynthesis in potato.

A heat-shock response regulated by the PfAP2-HS transcription factor protects human malaria parasites from febrile temperatures.

Periodic fever is a characteristic clinical feature of human malaria, but how parasites survive febrile episodes is not known. Although Plasmodium spp. genomes encode a full set of chaperones, they lack the conserved eukaryotic transcription factor HSF1, which activates the expression of chaperones upon heat-shock. Here, we show that PfAP2-HS, a transcription factor in the ApiAP2 family, regulates the protective heat-shock response in Plasmodium falciparum. PfAP2-HS activates transcription of hsp70–1 and hsp90 at elevated temperatures. The main binding site of PfAP2-HS in the entire genome coincides with a tandem G-box DNA motif in the hsp70–1 promoter. Engineered parasites lacking PfAP2-HS have reduced heat-shock survival and severe growth defects at 37°C, but not at 35°C. Parasites lacking PfAP2-HS also have increased sensitivity to imbalances in protein homeostasis (proteostasis) produced by artemisinin, the frontline antimalarial drug, or by the proteasome inhibitor epoxomicin. We propose that PfAP2-HS contributes to maintenance of proteostasis under basal conditions and upregulates specific chaperone-encoding genes at febrile temperatures to protect the parasite against protein damage.

Sequence-specific dynamics of DNA response elements and their flanking sites regulate the recognition by AP-1 transcription factors.

Activator proteins 1 (AP-1) comprise one of the largest families of eukaryotic basic leucine zipper transcription factors. Despite advances in the characterization of AP-1 DNA-binding sites, our ability to predict new binding sites and explain how the proteins achieve different gene expression levels remains limited. Here we address the role of sequence-specific DNA flexibility for stability and specific binding of AP-1 factors, using microsecond-long molecular dynamics simulations. As a model system, we employ yeast AP-1 factor Yap1 binding to three different response elements from two genetic environments. Our data show that Yap1 actively exploits the sequence-specific flexibility of DNA within the response element to form stable protein–DNA complexes. The stability also depends on the four to six flanking nucleotides, adjacent to the response elements. The flanking sequences modulate the conformational adaptability of the response element, making it more shape-efficient to form specific contacts with the protein. Bioinformatics analysis of differential expression of the studied genes supports our conclusions: the stability of Yap1–DNA complexes, modulated by the flanking environment, influences the gene expression levels. Our results provide new insights into mechanisms of protein–DNA recognition and the biological regulation of gene expression levels in eukaryotes.

Enhanced regulation of prokaryotic gene expression by a eukaryotic transcriptional activator

Expanding the genetic toolbox for prokaryotic synthetic biology is a promising strategy for enhancing the dynamic range of gene expression and enabling new engineered applications for research and biomedicine. Here, we reverse the current trend of moving genetic parts from prokaryotes to eukaryotes and demonstrate that the activating eukaryotic transcription factor QF and its corresponding DNA-binding sequence can be moved to E. coli to introduce transcriptional activation, in addition to tight off states. We further demonstrate that the QF transcription factor can be used in genetic devices that respond to low input levels with robust and sustained output signals. Collectively, we show that eukaryotic gene regulator elements are functional in prokaryotes and establish a versatile and broadly applicable approach for constructing genetic circuits with complex functions. These genetic tools hold the potential to improve biotechnology applications for medical science and research.

LEAFY, a Pioneer Transcription Factor in Plants: A Mini-Review.

A subset of eukaryotic transcription factors (TFs) possess the ability to reprogram one cell type into another. Genes important for cellular reprograming are typically located in closed chromatin, which is covered by nucleosomes. Pioneer factors are a special class of TFs that can initially engage their target sites in closed chromatin prior to the engagement with, opening of, or modification of the sites by other factors. Although many pioneer factors are known in animals, a few have been characterized in plants. The TF LEAFY (LFY) acts as a pioneer factor specifying floral fate in Arabidopsis. In response to endogenous and environmental cues, plants produce appropriate floral inducers (florigens). During the vegetative phase, LFY is repressed by the TERMINAL FLOWER 1 (TFL1)–FD complex, which functions as a floral inhibitor, or anti-florigen. The florigen FLOWERING LOCUS T (FT) competes with TFL1 to prevent the binding of the FD TF to the LFY locus. The resulting FT–FD complex functions as a transient stimulus to activate its targets. Once LFY has been transcribed in the appropriate spatiotemporal manner, LFY binds to nucleosomes in closed chromatin regions. Subsequently, LFY opens the chromatin by displacing H1 linker histones and recruiting the SWI/SNF chromatin-remodeling complex. Such local changes permit the binding of other TFs, leading to the expression of the floral meristem identity gene APETALA1. This mini-review describes the latest advances in our understanding of the pioneer TF LFY, providing insight into the establishment of gene expression competence through the shaping of the plant epigenetic landscape.

Competition for DNA binding between paralogous transcription factors determines their genomic occupancy and regulatory functions.

Most eukaryotic transcription factors (TFs) are part of large protein families, with members of the same family (i.e., paralogous TFs) recognizing similar DNA-binding motifs but performing different regulatory functions. Many TF paralogs are coexpressed in the cell and thus can compete for target sites across the genome. However, this competition is rarely taken into account when studying the in vivo binding patterns of eukaryotic TFs. Here, we show that direct competition for DNA binding between TF paralogs is a major determinant of their genomic binding patterns. Using yeast proteins Cbf1 and Pho4 as our model system, we designed a high-throughput quantitative assay to capture the genomic binding profiles of competing TFs in a cell-free system. Our data show that Cbf1 and Pho4 greatly influence each other's occupancy by competing for their common putative genomic binding sites. The competition is different at different genomic sites, as dictated by the TFs’ expression levels and their divergence in DNA-binding specificity and affinity. Analyses of ChIP-seq data show that the biophysical rules that dictate the competitive TF binding patterns in vitro are also followed in vivo, in the complex cellular environment. Furthermore, the Cbf1-Pho4 competition for genomic sites, as characterized in vitro using our new assay, plays a critical role in the specific activation of their target genes in the cell. Overall, our study highlights the importance of direct TF-TF competition for genomic binding and gene regulation by TF paralogs, and proposes an approach for studying this competition in a quantitative and high-throughput manner.

DNA‐Binding Specificity of Cardiac Transcription Factor Complex Formed by NKX2‐5 and TBX5

Transcription factors (TFs) are sequence-specific DNA-binding proteins responsible for cellular differentiation and development. Eukaryotic TFs are known for binding DNA as multimeric complexes to regulate gene expression. Transcription factors NKX2-5 and TBX5 are fundamental elements of the gene regulatory networks that control mammalian heart development. More importantly, NKX2-5 and TBX5 can bind to DNA as a cooperative complex. Our aim was to identify the intrinsic DNA-binding preferences and the motif spacing and orientation pattern of the NKX2-5:TBX5 heterodimer complex. In this study, we expressed full-length GST-tagged NKX2-5 and TBX5 using a wheat germ cell-free system. We successfully determined the in vitro DNA-binding specificity of the NKX2-5:TBX5 complex using Systematic Evolution of Ligands by Exponential Enrichment (SELEX-seq). Our preliminary results shows NKX2-5:TBX5 binding in a head-to-tail orientation with 0bp and 3bps spacing between core binding sites: GGTGT for TBX5 and CACTT for NKX2-5. The spacing preference is consistent with ChIP-exo data from cardiomyocytes and cardiac precursors. Our results highlight the utility of using in vitro techniques to determined DNA-binding specificity to understand genomic binding. The findings of our study will help understand the spatial and temporal gene regulation rules involved in normal heart development.