Yeast Transcription Factor Research Articles

Predicting with the quantify intensities of transcription factor-target genes binding using random forest technique

With the rapid development of technology, this development led to the emergence of microarray technology. It has the effect of studying the levels of gene expression in a way that makes it easier for researchers to observe the expression levels of millions of genes at the same time in a single experiment. Development also helped in the emergence of powerful tools to identify interactions between target genes and regulatory factors. The main aim of this study is to build models to predicate the relationship (Interaction) between Transcription Factors (TFs) proteins and target genes by selecting the subset of important genes (Relevant genes) from original dataset. The proposed methodology comprises into three major stages: the genes selection, merge datasets and the prediction stage. The process of reducing the computational space of gene data has been accomplished by using proposed mutual information method for genes selection based on the data of gene expression. In the prediction, the proposed prediction regression techniques are utilized to predict with binding rate between single TF-target gene. It has been compared the efficiency of two different proposed regression techniques including: Linear Regression and Random Forest Regression. Two available data sets have been utilized to achieve the objectives of this study: Gene’s expression data of Yeast Cell Cycle dataset and Transcription Factors dataset. The evaluation of predictions performance has been performed depending on two performance prediction measures (Root Mean Square Error (RMSE) and Mean Absolute Error (MAE) with (10) Folds-Cross Validation.

Abstract 2478: Elevating SOX2 inhibits the proliferation of tumor cells independently of DNA binding

Abstract Purpose: The purpose of these studies was to understand how elevated SOX2 affects tumor cell proliferation. The stem cell transcription factor SOX2 has been implicated in ~20 different tumor types. Importantly, several studies have demonstrated that the effects of SOX2 during development and malignancy are dependent on its dosage. High levels of SOX2 in both contexts are associated with a quiescent, stem-like phenotype. SOX2high quiescent tumor cells present a significant clinical challenge as these cells are largely resistant to conventional chemotherapies and, thus, provide a reservoir to drive disease recurrence. Therefore, understanding the mechanisms through which elevated SOX2 inhibits tumor cell proliferation will provide insights into the biology of drug-resistant, quiescent tumor cells. Methods: Tumor cell lines representative of medulloblastoma (MB) and colorectal (CRC) tumor cells were engineered for the doxycycline (Dox)-inducible expression of mutant forms of SOX2 that are unable to bind to DNA. One SOX2 mutant was created by substituting a proline for a glycine in the DNA binding domain of SOX2 [SOX2(G76P)]. A second SOX2 mutant replaces the DNA binding domain of SOX2 with the DNA binding domain of the yeast transcription factor Gal4 (Gal4-SOX2). The effects of elevating mutant forms of SOX2 on cell proliferation and the cell cycle distribution of the cells were compared to the effects of elevating wild-type SOX2. Results: Our studies demonstrate that elevating SOX2, SOX2(G76P) or Gal4-SOX2 decreases the proliferation of MB and CRC cells to similar extents. In contrast, inducible expression of the DNA binding domain of Gal4 had no effect on cell proliferation. We previously reported that while SOX2 elevation inhibits the proliferation of a diverse set of tumor cell lines, it does not significantly alter their cell cycle distribution. Similarly, elevating SOX2(G76P) or Gal4-SOX2 did not significantly alter cell cycle distribution. To extend these findings, we are engineering additional SOX2 mutants to map the location of the domain of SOX2 required for growth inhibition. We recently engineered MB and CRC cells to express a mutant form of SOX2 that lacks the c-terminal R1 transactivation domain [Gal4-SOX2(ΔR1)]. Significantly, removing the R1 transactivation domain eliminated the growth inhibitory effects of Gal4-SOX2 when elevated. Conclusion: Our findings demonstrate that elevating two mutant forms of SOX2 that cannot bind to DNA inhibit the proliferation of tumor cell lines that represent two different forms of cancer, similar to the effects of wild-type SOX2. Equally important, our studies indicate that the c-terminal R1 transactivation domain of SOX2 is necessary, but no sufficient, to inhibit proliferation when SOX2 levels are elevated. Collectively, these findings and our previously published work indicate that elevated levels of SOX2 inhibit cell proliferation independently of DNA binding. Citation Format: Ethan Patrick Metz, Phillip J. Wilder, Angie Rizzino. Elevating SOX2 inhibits the proliferation of tumor cells independently of DNA binding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2478.

Defining the role of the polyasparagine repeat domain of the S. cerevisiae transcription factor Azf1p.

Across eukaryotes, homopolymeric repeats of amino acids are enriched in regulatory proteins such as transcription factors and chromatin remodelers. These domains play important roles in signaling, binding, prion formation, and functional phase separation. Azf1p is a prion-forming yeast transcription factor that contains two homorepeat domains, a polyglutamine and a polyasparagine domain. In this work, we report a new phenotype for Azf1p and identify a large set of genes that are regulated by Azf1p during growth in glucose. We show that the polyasparagine (polyN) domain plays a subtle role in transcription but is dispensable for Azf1p localization and prion formation. Genes upregulated upon deletion of the polyN domain are enriched in functions related to carbon metabolism and storage. This domain may therefore be a useful target for engineering yeast strains for fermentation applications and small molecule production. We also report that both the polyasparagine and polyglutamine domains vary in length across strains of S. cerevisiae and propose a model for how this variation may impact protein function.

Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to Mediator

Gene activator proteins comprise distinct DNA-binding and transcriptional activation domains (ADs). Because few ADs have been described, we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity (“fuzzy” binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology.

Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to Mediator.

Gene activator proteins comprise distinct DNA-binding and transcriptional activation domains (ADs). Because few ADs have been described, we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity ('fuzzy' binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology.

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae.

The bipartite GAL4-UAS system is a versatile and powerful tool for functional genetic analysis. The essence of the system is to cross transgenic 'driver' lines that express the yeast transcription factor GAL4 in a tissue specific manner, with transgenic 'responder' lines carrying a candidate gene/RNA interference construct whose expression is controlled by Upstream Activation Sequences (UAS) that bind GAL4. In the ensuing progeny, the gene or silencing construct is thus expressed in a prescribed spatiotemporal manner, enabling the resultant phenotypes to be assayed and gene function inferred. The binary system enables flexibility in experimental approaches to screen phenotypes generated by transgene expression in multiple tissue-specific patterns, even if severe fitness costs are induced. We have adapted this system for Anopheles gambiae, the principal malaria vector in Africa. In this article, we provide some of the common procedures used during GAL4-UAS analysis. We describe the An. gambiae GAL4-UAS lines already generated, as well as the cloning of new responder constructs for upregulation and RNAi knockdown. We specify a step by step guide for sexing of mosquito pupae to establish genetic crosses, that also includes screening progeny to follow inheritance of fluorescent gene markers that tag the driver and responder insertions. We also present a protocol for clearing An. gambiae embryos to study embryonic development. Finally, we introduce potential adaptions of the method to generate driver lines through CRISPR/Cas9 insertion of GAL4 downstream of target genes.

Mediator subunit Med15 dictates the conserved \u201cfuzzy\u201d binding mechanism of yeast transcription activators Gal4 and Gcn4

The acidic activation domain (AD) of yeast transcription factor Gal4 plays a dual role in transcription repression and activation through binding to Gal80 repressor and Mediator subunit Med15. The activation function of Gal4 arises from two hydrophobic regions within the 40-residue AD. We show by NMR that each AD region binds the Mediator subunit Med15 using a “fuzzy” protein interface. Remarkably, comparison of chemical shift perturbations shows that Gal4 and Gcn4, two intrinsically disordered ADs of different sequence, interact nearly identically with Med15. The finding that two ADs of different sequence use an identical fuzzy binding mechanism shows a common sequence-independent mechanism for AD-Mediator binding, similar to interactions within a hydrophobic cloud. In contrast, the same region of Gal4 AD interacts strongly with Gal80 via a distinct structured complex, implying that the structured binding partner of an intrinsically disordered protein dictates the type of protein–protein interaction.

Duplex Telomere-Binding Proteins in Fungi With Canonical Telomere Repeats: New Lessons in the Rapid Evolution of Telomere Proteins.

The telomere protein assemblies in different fungal lineages manifest quite profound structural and functional divergence, implying a high degree of flexibility and adaptability. Previous comparative analyses of fungal telomeres have focused on the role of telomere sequence alterations in promoting the evolution of corresponding proteins, particularly in budding and fission yeast. However, emerging evidence suggests that even in fungi with the canonical 6-bp telomere repeat unit, there are significant remodeling of the telomere assembly. Indeed, a new protein family can be recruited to serve dedicated telomere functions, and then experience subsequent loss in sub-branches of the clade. An especially interesting example is the Tay1 family of proteins, which emerged in fungi prior to the divergence of basidiomycetes from ascomycetes. This relatively recent protein family appears to have acquired its telomere DNA-binding activity through the modification of another Myb-containing protein. Members of the Tay1 family evidently underwent rather dramatic functional diversification, serving, e.g., as transcription factors in fission yeast while acting to promote telomere maintenance in basidiomycetes and some hemi-ascomycetes. Remarkably, despite its distinct structural organization and evolutionary origin, a basidiomycete Tay1 appears to promote telomere replication using the same mechanism as mammalian TRF1, i.e., by recruiting and regulating Blm helicase activity. This apparent example of convergent evolution at the molecular level highlight the ability of telomere proteins to acquire new interaction targets. The remarkable evolutionary history of Tay1 illustrates the power of protein modularity and the facile acquisition of nucleic acid/protein-binding activity to promote telomere flexibility.

Inferring TF activities and activity regulators from gene expression data with constraints from TF perturbation data.

MotivationThe activity of a transcription factor (TF) in a sample of cells is the extent to which it is exerting its regulatory potential. Many methods of inferring TF activity from gene expression data have been described, but due to the lack of appropriate large-scale datasets, systematic and objective validation has not been possible until now.ResultsWe systematically evaluate and optimize the approach to TF activity inference in which a gene expression matrix is factored into a condition-independent matrix of control strengths and a condition-dependent matrix of TF activity levels. We find that expression data in which the activities of individual TFs have been perturbed are both necessary and sufficient for obtaining good performance. To a considerable extent, control strengths inferred using expression data from one growth condition carry over to other conditions, so the control strength matrices derived here can be used by others. Finally, we apply these methods to gain insight into the upstream factors that regulate the activities of yeast TFs Gcr2, Gln3, Gcn4 and Msn2.Availability and implementationEvaluation code and data are available at https://doi.org/10.5281/zenodo.4050573.Supplementary information Supplementary data are available at Bioinformatics online.

High-Throughput Affinity Measurements of Transcription Factor and DNA Mutations Reveal Affinity and Specificity Determinants.

Transcription factors (TFs) bind regulatory DNA to control gene expression, and mutations to either TFs or DNA can alter binding affinities to rewire regulatory networks and drive phenotypic variation. While studies have profiled energetic effects of DNA mutations extensively, we lack similar information for TF variants. Here, we present STAMMP (simultaneous transcription factor affinity measurements via microfluidic protein arrays), a high-throughput microfluidic platform enabling quantitative characterization of hundreds of TF variants simultaneously. Measured affinities for ∼210 mutants of a model yeast TF (Pho4) interacting with 9 oligonucleotides (>1,800 Kds) reveal that many combinations of mutations to poorly conserved TF residues and nucleotides flanking the core binding site alter but preserve physiological binding, providing a mechanism by which combinations of mutations in cis and trans could modulate TF binding to tune occupancies during evolution. Moreover, biochemical double-mutant cycles across the TF-DNA interface reveal molecular mechanisms driving recognition, linking sequence to function. A record of this paper's Transparent Peer Review process is included in the Supplemental Information.

Interaction Interface of Mason-Pfizer Monkey Virus Matrix and Envelope Proteins.

Retroviral envelope glycoprotein (Env) is essential for the specific recognition of the host cell and the initial phase of infection. As reported for human immunodeficiency virus (HIV), the recruitment of Env into a retroviral membrane envelope is mediated through its interaction with a Gag polyprotein precursor of structural proteins. This interaction, occurring between the matrix domain (MA) of Gag and the cytoplasmic tail (CT) of the transmembrane domain of Env, takes place at the host cell plasma membrane. To determine whether the MA of Mason-Pfizer monkey virus (M-PMV) also interacts directly with the CT of Env, we mimicked the in vivo conditions in an in vitro experiment by using a CT in its physiological trimeric conformation mediated by the trimerization motif of the GCN4 yeast transcription factor. The MA protein was used at the concentration shifting the equilibrium to its trimeric form. The direct interaction between MA and CT was confirmed by a pulldown assay. Through the combination of nuclear magnetic resonance (NMR) spectroscopy and protein cross-linking followed by mass spectrometry analysis, the residues involved in mutual interactions were determined. NMR has shown that the C terminus of the CT is bound to the C-terminal part of MA. In addition, protein cross-linking confirmed the close proximity of the N-terminal part of CT and the N terminus of MA, which is enabled in vivo by their location at the membrane. These results are in agreement with the previously determined orientation of MA on the membrane and support the already observed mechanisms of M-PMV virus-like particle transport and budding.IMPORTANCE By a combination of nuclear magnetic resonance (NMR) and mass spectroscopy of cross-linked peptides, we show that in contrast to human immunodeficiency virus type 1 (HIV-1), the C-terminal residues of the unstructured cytoplasmic tail of Mason-Pfizer monkey virus (M-PMV) Env interact with the matrix domain (MA). Based on biochemical data and molecular modeling, we propose that individual cytoplasmic tail (CT) monomers of a trimeric complex bind MA molecules belonging to different neighboring trimers, which may stabilize the MA orientation at the membrane by the formation of a membrane-bound net of interlinked Gag and CT trimers. This also corresponds with the concept that the membrane-bound MA of Gag recruits Env through interaction with the full-length CT, while CT truncation during maturation attenuates the interaction to facilitate uncoating. We propose a model suggesting different arrangements of MA-CT complexes between a D-type and C-type retroviruses with short and long CTs, respectively.

Mitochondrial HMG-Box Containing Proteins: From Biochemical Properties to the Roles in Human Diseases.

Mitochondrial DNA (mtDNA) molecules are packaged into compact nucleo-protein structures called mitochondrial nucleoids (mt-nucleoids). Their compaction is mediated in part by high-mobility group (HMG)-box containing proteins (mtHMG proteins), whose additional roles include the protection of mtDNA against damage, the regulation of gene expression and the segregation of mtDNA into daughter organelles. The molecular mechanisms underlying these functions have been identified through extensive biochemical, genetic, and structural studies, particularly on yeast (Abf2) and mammalian mitochondrial transcription factor A (TFAM) mtHMG proteins. The aim of this paper is to provide a comprehensive overview of the biochemical properties of mtHMG proteins, the structural basis of their interaction with DNA, their roles in various mtDNA transactions, and the evolutionary trajectories leading to their rapid diversification. We also describe how defects in the maintenance of mtDNA in cells with dysfunctional mtHMG proteins lead to different pathologies at the cellular and organismal level.

Generation and Characterization of a DNA-GCN4 Oligonucleotide-Peptide Conjugate: The Impact DNA/Protein Interactions on the Sensitization of DNA.

Radiotherapy, the most common therapy for the treatment of solid tumors, exerts its effects by inducing DNA damage. To fully understand the extent and nature of this damage, DNA models that mimic the in vivo situation should be utilized. In a cellular context, genomic DNA constantly interacts with proteins and these interactions could influence both the primary radical processes (triggered by ionizing radiation) and secondary reactions, ultimately leading to DNA damage. However, this is seldom addressed in the literature. In this work, we propose a general approach to tackle these shortcomings. We synthesized a protein-DNA complex that more closely represents DNA in the physiological environment than oligonucleotides solution itself, while being sufficiently simple to permit further chemical analyses. Using click chemistry, we obtained an oligonucleotide-peptide conjugate, which, if annealed with the complementary oligonucleotide strand, forms a complex that mimics the specific interactions between the GCN4 protein and DNA. The covalent bond connecting the oligonucleotide and peptide constitutes a part of substituted triazole, which forms due to the click reaction between the short peptide corresponding to the specific amino acid sequence of GCN4 protein (yeast transcription factor) and a DNA fragment that is recognized by the protein. DNAse footprinting demonstrated that the part of the DNA fragment that specifically interacts with the peptide in the complex is protected from DNAse activity. Moreover, the thermodynamic characteristics obtained using differential scanning calorimetry (DSC) are consistent with the interaction energies calculated at the level of metadynamics. Thus, we present an efficient approach to generate a well-defined DNA-peptide conjugate that mimics a real DNA-peptide complex. These complexes can be used to investigate DNA damage under conditions very similar to those present in the cell.

The Role of Variable Repetitive Protein Domainsin Azf1p Function and Prion Formation

Prions are naturally occurring, self‐propagating isoforms of proteins that represent a heritable mechanism for tuning protein function. The yeast transcription factor Azf1p was recently demonstrated to form a prion, [AZF1+], that confers resistance to the drug radicicol but reduces Azf1p target gene expression. Using the Prion‐Like Amino Acid Composition tool, we identified regions of Azf1p that are predicted to contribute to prion formation. Two notable candidates are the polyglutamine (polyQ) and polyasparagine (polyN) domains. Through a bioinformatic analysis of the genomes of 93 lab and wild isolates of S. cerevisiae, we determined that these domains vary in repeat copy number across strains of yeast. The polyQ domain has two alleles, while the polyN domain has six alleles ranging from 17 to 25 N residues. In this work, we are investigating the extent to which the variable polyQ and polyN domains of Azf1p play a role in its transcription factor activity and contribute to [AZF1+] prion formation. This research determines the effects of repeat‐length variation on protein localization and stability, prion activity, and target gene expression.Support or Funding InformationThis work is supported by the Army Research Office (W911NF‐19‐1‐0299)

Dual threshold optimization and network inference reveal convergent evidence from TF binding locations and TF perturbation responses.

A high-confidence map of the direct, functional targets of each transcription factor (TF) requires convergent evidence from independent sources. Two significant sources of evidence are TF binding locations and the transcriptional responses to direct TF perturbations. Systematic data sets of both types exist for yeast and human, but they rarely converge on a common set of direct, functional targets for a TF. Even the few genes that are both bound and responsive may not be direct functional targets. Our analysis shows that when there are many nonfunctional binding sites and many indirect targets, nonfunctional sites are expected to occur in the cis-regulatory DNA of indirect targets by chance. To address this problem, we introduce dual threshold optimization (DTO), a new method for setting significance thresholds on binding and perturbation-response data, and show that it improves convergence. It also enables comparison of binding data to perturbation-response data that have been processed by network inference algorithms, which further improves convergence. The combination of dual threshold optimization and network inference greatly expands the high-confidence TF network map in both yeast and human. Next, we analyze a comprehensive new data set measuring the transcriptional response shortly after inducing overexpression of a yeast TF. We also present a new yeast binding location data set obtained by transposon calling cards and compare it to recent ChIP-exo data. These new data sets improve convergence and expand the high-confidence network synergistically.

Binding and Regulation of Transcription by Yeast Ste12 Variants To Drive Mating and Invasion Phenotypes.

Amino acid substitutions are commonly found in human transcription factors, yet the functional consequences of much of this variation remain unknown, even in well-characterized DNA-binding domains. Here, we examine how six single-amino acid variants in the DNA-binding domain of Ste12-a yeast transcription factor regulating mating and invasion-alter Ste12 genome binding, motif recognition, and gene expression to yield markedly different phenotypes. Using a combination of the "calling-card" method, RNA sequencing, and HT-SELEX (high throughput systematic evolution of ligands by exponential enrichment), we find that variants with dissimilar binding and expression profiles can converge onto similar cellular behaviors. Mating-defective variants led to decreased expression of distinct subsets of genes necessary for mating. Hyper-invasive variants also decreased expression of subsets of genes involved in mating, but increased the expression of other subsets of genes associated with the cellular response to osmotic stress. While single-amino acid changes in the coding region of this transcription factor result in complex regulatory reconfiguration, the major phenotypic consequences for the cell appear to depend on changes in the expression of a small number of genes with related functions.

Identification of Highest-Affinity Binding Sites of Yeast Transcription Factor Families.

Transcription factors (TFs) play a crucial role in controlling key cellular processes and responding to the environment. Yeast is a single-cell fungal organism that is a vital biological model organism for studying transcription and translation in basic biology. The transcriptional control process of yeast cells has been extensively calculated and studied using traditional methods and high-throughput technologies. However, the identities of transcription factors that regulate major functional categories of genes remain unknown. Due to the avalanche of biological data in the post-genomic era, it is an urgent need to develop automated computational methods to enable accurate identification of efficient transcription factor binding sites from the large number of candidates. In this paper, we analyzed high-resolution DNA-binding profiles and motifs for TFs, covering all possible contiguous 8-mers. First, we divided all 8-mer motifs into 16 various categories and selected all sorts of samples from each category by setting the threshold of E-score. Then, we employed five feature representation methods. Also, we adopted a total of four feature selection methods to filter out useless features. Finally, we used Extreme Gradient Boosting (XGBoost) as our base classifier and then utilized the one-vs-rest tactics to build 16 binary classifiers to solve this multiclassification problem. In the experiment, our method achieved the best performance with an overall accuracy of 79.72% and Mathew's correlation coefficient of 0.77. We found the similarity relationship among each category from different TF families and obtained sequence motif schematic diagrams via multiple sequence alignment. The complexity of DNA recognition may act as an important role in the evolution of gene regulation. Source codes are available at https://github.com/guofei-tju/tfbs.

Transcription factor regulatory modules provide the molecular mechanisms for functional redundancy observed among transcription factors in yeast

BackgroundCurrent technologies for understanding the transcriptional reprogramming in cells include the transcription factor (TF) chromatin immunoprecipitation (ChIP) experiments and the TF knockout experiments. The ChIP experiments show the binding targets of TFs against which the antibody directs while the knockout techniques find the regulatory gene targets of the knocked-out TFs. However, it was shown that these two complementary results contain few common targets. Researchers have used the concept of TF functional redundancy to explain the low overlap between these two techniques. But the detailed molecular mechanisms behind TF functional redundancy remain unknown. Without knowing the possible molecular mechanisms, it is hard for biologists to fully unravel the cause of TF functional redundancy.ResultsTo mine out the molecular mechanisms, a novel algorithm to extract TF regulatory modules that help explain the observed TF functional redundancy effect was devised and proposed in this research. The method first searched for candidate TF sets from the TF binding data. Then based on these candidate sets the method utilized the modified Steiner Tree construction algorithm to construct the possible TF regulatory modules from protein-protein interaction data and finally filtered out the noise-induced results by using confidence tests. The mined-out regulatory modules were shown to correlate to the concept of functional redundancy and provided testable hypotheses of the molecular mechanisms behind functional redundancy. And the biological significance of the mined-out results was demonstrated in three different biological aspects: ontology enrichment, protein interaction prevalence and expression coherence. About 23.5% of the mined-out TF regulatory modules were literature-verified. Finally, the biological applicability of the proposed method was shown in one detailed example of a verified TF regulatory module for pheromone response and filamentous growth in yeast.ConclusionIn this research, a novel method that mined out the potential TF regulatory modules which elucidate the functional redundancy observed among TFs is proposed. The extracted TF regulatory modules not only correlate the molecular mechanisms to the observed functional redundancy among TFs, but also show biological significance in inferring TF functional binding target genes. The results provide testable hypotheses for biologists to further design subsequent research and experiments.

Chromatin Fiber Invasion and Nucleosome Displacement by the Rap1 Transcription Factor.

SummaryPioneer transcription factors (pTFs) bind to target sites within compact chromatin, initiating chromatin remodeling and controlling the recruitment of downstream factors. The mechanisms by which pTFs overcome the chromatin barrier are not well understood. Here, we reveal, using single-molecule fluorescence, how the yeast transcription factor Rap1 invades and remodels chromatin. Using a reconstituted chromatin system replicating yeast promoter architecture, we demonstrate that Rap1 can bind nucleosomal DNA within a chromatin fiber but with shortened dwell times compared to naked DNA. Moreover, we show that Rap1 binding opens chromatin fiber structure by inhibiting inter-nucleosome contacts. Finally, we reveal that Rap1 collaborates with the chromatin remodeler RSC to displace promoter nucleosomes, paving the way for long-lived bound states on newly exposed DNA. Together, our results provide a mechanistic view of how Rap1 gains access and opens chromatin, thereby establishing an active promoter architecture and controlling gene expression.

Structure of Fission Yeast Transcription Factor Pho7 Bound to pho1 Promoter DNA and Effect of Pho7 Mutations on DNA Binding and Phosphate Homeostasis.

Pho7 is the Schizosaccharomyces pombe fission yeast Zn2Cys6 transcriptional factor that drives a response to phosphate starvation in which phosphate acquisition genes are upregulated. Here we report a crystal structure at 1.6-Å resolution of the Pho7 DNA-binding domain (DBD) bound at its target site 2 in the pho1 promoter (5'-TCGGAAATTAAAAA). Comparison to the previously reported structure of Pho7 DBD in complex with its binding site in the tgp1 promoter (5'-TCGGACATTCAAAT) reveals shared determinants of target site specificity as well as variations in the protein-DNA interface that accommodate different promoter DNA sequences. Mutagenesis of Pho7 amino acids at the DNA interface identified nucleobase contacts at the periphery of the footprint that are essential for the induction of pho1 expression in response to phosphate starvation and for Pho7 binding to site 1 in the pho1 promoter.