Sequence-specific DNA-binding Transcription Factors Research Articles

Study on the active components and mechanism of Atractylodis Macrocephalae Rhizoma for invigorating the spleen and tonifying qi based on spectrum-effect relationship and network pharmacology.

Spleen deficiency can lead to various abnormal physiological functions of the spleen. Atractylodis Macrocephalae Rhizoma (AMR) is a traditional Chinese medicine used to invigorate the spleen and tonify qi. The study aimed to identify the primary active components influencing the efficacy of AMR in strengthening the spleen and replenishing qi through spectrum-effect relationship and chemometrics. Network pharmacology was used to investigate the mechanism by which AMR strengthens the spleen and replenishes qi, with molecular docking utilized for validation purposes. The findings indicated that bran-fried AMR exhibited superior efficacy, with atractylenolides and atractylone identified as the primary active constituents. Atractylenolide II emerged as the most influential component impacting the effectiveness of AMR, while the key target was androgen receptor. Furthermore, crucial pathways implicated included the mitogen-activated protein cascade (MAPK) cascade, RNA polymerase II transcription factor activity, ligand-activated sequence-specific DNA binding, and RNA polymerase II sequence-specific DNA-binding transcription factor binding. In summary, our study has identified the primary active components associated with the efficacy of AMR and has provided an initial exploration of its mechanism of action. This offers a theoretical foundation for future investigations into the material basis and molecular mechanisms underlying the pharmacodynamics of AMR.

A function of spalt major as a sequence-specific DNA binding transcription factor mediates repression of knirps in the Drosophila wing imaginal disc

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 unknow. Here, we analyze 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 we identified 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. Interestingly, we also find similar sequences in a set of eight novel candidate Spalt target genes, pointing to a common mechanism of transcriptional repression mediated by Spalt proteins.

Combining network pharmacology and molecular docking to decipher molecular mechanism of Wugen decoction in influenza A prevention

Wugen decoction, a classic traditional Chinese medical diet, is widely used to prevent influenza A virus infection in China. However, the principal effective ingredients and potential mechanism of Wugen decoction remain unclear. Therefore, a systemic analysis-based approach combined with molecular docking and network pharmacology is used to elucidate the molecular mechanism of Wugen decoction in prevention of influenza A. The efficient bioactive and potential targets of Wugen decoction are initially retrieved from public databases and relevant references. To further analyze the core targets, a protein-protein interaction network is investigated through cluster screening, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, and Gene Ontology analysis. Then the binding behaviors between crucial bioactive ingredients and core targets are obtained via molecular docking. Quercetin, stigmasterol, beta-sitosterol, genistein, oxalic acid, and daidzein with higher degrees are collected and screened from the “Herb-Ingredients” network. Meanwhile, AKT1, IL1B, IL6, INS, TNF, and VEGFA are identified from cluster screening and topological investigation. The coincident targets are primarily concentrated to biological processes including hydrogen peroxide, cellular response to synapse, and molecular function to cysteine-type endopeptidase activity involved in tumor necrosis factor receptor binding. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis manifests that the signaling pathways related to influenza A prevention are closely associated with tumor necrosis factor receptor binding, RNA polymerase II sequence-specific DNA binding transcription factor binding, and protein phosphatase binding. Molecular docking results highlight that the effective ingredients can spontaneously bind with the core targets. In all, the present research comprehensively illustrates the potential mechanism of Wugen decoction against influenza A virus infection with the ‘multicomponent-multitarget-multipathway’ mode. This research also provides an effective approach to uncover the scientific basis of Wugen decoction within food and health care industries.

Functional Characteristics of Diverse PAX6 Mutations Associated with Isolated Foveal Hypoplasia.

The human fovea is a specialized pit structure in the central retina. Foveal hypoplasia is a condition where the foveal pit does not fully develop, and it is associated with poor vision. Autosomal dominant isolated foveal hypoplasia (FVH1) is a rare condition of foveal hypoplasia (FH) that lacks any other ocular manifestations. FVH1 is associated with hypomorphic mutations in the PAX6 gene that encodes a sequence-specific DNA-binding transcription factor for morphogenesis and evolution of the eye. We report our findings in 17 patients with PAX6 mutations associated with FVH1 or FH with aniridia and corneal opacities. Patients with three mutations, p.V78E, p.V83F and p.R128H, in the C-terminal subdomain of the paired domain (CTS) consistently have severe FH. Luciferase assays for a single reporter containing a representative PAX6 binding site indicated that the transcriptional activities of these mutations were significantly reduced, comparable to that of the truncation mutation of p.G65Rfs*5. Patients with p.P20S in the N-terminal subdomain of the paired domain, and a patient with p.N365K in the proline-serine-threonine-rich domain (PSTD) had mild FH. A patient with p.Q255L in the homeodomain had severe FH. The P20S and Q255L mutants did not affect the transcriptional activity. Mutant N365K has a retained DNA-binding activity but a reduced transcriptional activity, due to a low PSTD transactivation. These findings demonstrated that mutations associated with FVH1 underlie a functional divergence between DNA-binding ability and transcriptional activity. We conclude that a wide range of mutations in the PAX6 gene is not limited to the CST region and are responsible for FVH1.

Identification and Characterization of Development-Related microRNAs in the Red Flour Beetle, Tribolium castaneum

MicroRNAs (miRNAs) play important roles in insect growth and development, but they were poorly studied in insects. In this study, a total of 883 miRNAs were detected from the early embryo (EE), late larva (LL), early pupa (EP), late pupa (LP), and early adult (EA) of Tribolium castaneum by microarray assay. Further analysis identified 179 differentially expressed unique miRNAs (DEmiRNAs) during these developmental stages. Of the DEmiRNAs, 102 DEmiRNAs exhibited stage-specific expression patterns during development, including 53 specifically highly expressed miRNAs and 20 lowly expressed miRNAs in EE, 19 highly expressed miRNAs in LL, 5 weakly expressed miRNAs in EP, and 5 abundantly expressed miRNAs in EA. These miRNAs were predicted to target 747, 265, 472, 234, and 121 genes, respectively. GO enrichment analysis indicates that the targets were enriched by protein phosphorylation, calcium ion binding, sequence-specific DNA binding transcription factor activity, and cytoplasm. An RNA interference-mediated knockdown of the DEmiRNAs tca-miR-6-3p, tca-miR-9a-3p, tca-miR-9d-3p, tca-miR-11-3p, and tca-miR-13a-3p led to defects in metamorphosis and wing development of T. castaneum. This study has completed the identification and characterization of development-related miRNAs in T. castaneum, and will enable us to investigate their roles in the growth and development of insect.

The Mediator complex as a master regulator of transcription by RNA polymerase II.

The Mediator complex, which in humans is 1.4 MDa in size and includes 26 subunits, controls many aspects of RNA polymerase II (Pol II) function. Apart from its size, a defining feature of Mediator is its intrinsic disorder and conformational flexibility, which contributes to its ability to undergo phase separation and to interact with a myriad of regulatory factors. In this Review, we discuss Mediator structure and function, with emphasis on recent cryogenic electron microscopy data of the 4.0-MDa transcription preinitiation complex. We further discuss how Mediator and sequence-specific DNA-binding transcription factors enable enhancer-dependent regulation of Pol II function at distal gene promoters, through the formation of molecular condensates (or transcription hubs) and chromatin loops. Mediator regulation of Pol II reinitiation is also discussed, in the context of transcription bursting. We propose a working model for Mediator function that combines experimental results and theoretical considerations related to enhancer–promoter interactions, which reconciles contradictory data regarding whether enhancer–promoter communication is direct or indirect. We conclude with a discussion of Mediator’s potential as a therapeutic target and of future research directions.

Thirty Years' History since the Discovery of Pax6: From Central Nervous System Development to Neurodevelopmental Disorders.

Pax6 is a sequence-specific DNA binding transcription factor that positively and negatively regulates transcription and is expressed in multiple cell types in the developing and adult central nervous system (CNS). As indicated by the morphological and functional abnormalities in spontaneous Pax6 mutant rodents, Pax6 plays pivotal roles in various biological processes in the CNS. At the initial stage of CNS development, Pax6 is responsible for brain patterning along the anteroposterior and dorsoventral axes of the telencephalon. Regarding the anteroposterior axis, Pax6 is expressed inversely to Emx2 and Coup-TF1, and Pax6 mutant mice exhibit a rostral shift, resulting in an alteration of the size of certain cortical areas. Pax6 and its downstream genes play important roles in balancing the proliferation and differentiation of neural stem cells. The Pax6 gene was originally identified in mice and humans 30 years ago via genetic analyses of the eye phenotypes. The human PAX6 gene was discovered in patients who suffer from WAGR syndrome (i.e., Wilms tumor, aniridia, genital ridge defects, mental retardation). Mutations of the human PAX6 gene have also been reported to be associated with autism spectrum disorder (ASD) and intellectual disability. Rodents that lack the Pax6 gene exhibit diverse neural phenotypes, which might lead to a better understanding of human pathology and neurodevelopmental disorders. This review describes the expression and function of Pax6 during brain development, and their implications for neuropathology.

Advanced Strategies for Therapeutic Targeting of Wild-Type and Mutant p53 in Cancer.

TP53 is a tumor suppressor gene that encodes a sequence-specific DNA-binding transcription factor activated by stressful stimuli; it upregulates target genes involved in growth suppression, cell death, DNA repair, metabolism, among others. TP53 is the most frequently mutated gene in tumors, with mutations not only leading to loss-of-function (LOF), but also gain-of-function (GOF) that promotes tumor progression, and metastasis. The tumor-specific status of mutant p53 protein has suggested it is a promising target for cancer therapy. We summarize the current progress of targeting wild-type and mutant p53 for cancer therapy through biotherapeutic and biopharmaceutical methods for (1) boosting p53 activity in cancer, (2) p53-dependent and p53-independent strategies for targeting p53 pathway functional restoration in p53-mutated cancer, (3) targeting p53 in immunotherapy, and (4) combination therapies targeting p53, p53 checkpoints, or mutant p53 for cancer therapy.

A systematic genome-wide account of binding sites for the model transcription factor Gcn4

Sequence-specific DNA-binding transcription factors are central to gene regulation. They are often associated with consensus binding sites that predict far more genomic sites than are bound in vivo. One explanation is that most sites are blocked by nucleosomes, such that only sites in nucleosome-depleted regulatory regions are bound. We compared the binding of the yeast transcription factor Gcn4 in vivo using published ChIP-seq data (546 sites) and in vitro, using a modified SELEX method (“G-SELEX”), which utilizes short genomic DNA fragments to quantify binding at all sites. We confirm that Gcn4 binds strongly to an AP-1-like sequence (TGACTCA) and weakly to half-sites. However, Gcn4 binds only some of the 1078 exact matches to this sequence, even in vitro. We show that there are only 166 copies of the high-affinity RTGACTCAY site (exact match) in the yeast genome, all occupied in vivo, largely independently of whether they are located in nucleosome-depleted or nucleosomal regions. Generally, RTGACTCAR/YTGACTCAY sites are bound much more weakly and YTGACTCAR sites are unbound, with biological implications for determining induction levels. We conclude that, to a first approximation, Gcn4 binding can be predicted using the high-affinity site, without reference to chromatin structure. We propose that transcription factor binding sites should be defined more precisely using quantitative data, allowing more accurate genome-wide prediction of binding sites and greater insight into gene regulation.

Integration of transcription coregulator complexes with sequence-specific DNA-binding factor interactomes

The domain of transcription regulation has been notoriously difficult to annotate in the Gene Ontology, partly because of the intricacies of gene regulation which involve molecular interactions with DNA as well as amongst protein complexes. The molecular function ‘transcription coregulator activity’ is a part of the biological process ‘regulation of transcription, DNA-templated’ that occurs in the cellular component ‘chromatin’. It can mechanistically link sequence-specific DNA-binding transcription factor (dbTF) regulatory DNA target sites to coactivator and corepressor target sites through the molecular function ‘cis-regulatory region sequence-specific DNA binding’. Many questions arise about transcription coregulators (coTF). Here, we asked how many unannotated, putative coregulators can be identified in protein complexes? Therefore, we mined the CORUM and hu.MAP protein complex databases with known and strongly presumed human transcription coregulators. In addition, we trawled the BioGRID and IntAct molecular interaction databases for interactors of the known 1457 human dbTFs annotated by the GREEKC and GO consortia. This yielded 1093 putative transcription factor coregulator complex subunits, of which 954 interact directly with a dbTF. This substantially expands the set of coTFs that could be annotated to ‘transcription coregulator activity’ and sets the stage for renewed annotation and wet-lab research efforts. To this end, we devised a prioritisation score based on existing GO annotations of already curated transcription coregulators as well as interactome representation. Since all the proteins that we mined are parts of protein complexes, we propose to concomitantly engage in annotation of the putative transcription coregulator-containing complexes in the Complex Portal database.

Transcription Factor-Based Genetic Engineering in Microalgae.

Sequence-specific DNA-binding transcription factors (TFs) are key components of gene regulatory networks. Advances in high-throughput sequencing have facilitated the rapid acquisition of whole genome assembly and TF repertoires in microalgal species. In this review, we summarize recent advances in gene discovery and functional analyses, especially for transcription factors in microalgal species. Specifically, we provide examples of the genome-scale identification of transcription factors in genome-sequenced microalgal species and showcase their application in the discovery of regulators involved in various cellular functions. Herein, we highlight TF-based genetic engineering as a promising framework for designing microalgal strains for microalgal-based bioproduction.

Physiological, biochemical, and transcriptional regulation in a leguminous forage Trifolium pratense L. responding to silver ions

Trifolium pratense L. (red clover) is an important leguminous crop with great potential for Ag-contaminated environment remediation. Whereas, the molecular mechanisms of Ag tolerance in red clover are largely unknown. Red clover seedlings were used for physiological and transcriptomic investigation under 0, 20, 50, and 100 mg/L Ag+ stress in our research to reveal potential molecular resistance mechanism. Research showed that red clover possessed fairly strong Ag absorbance capacity, the Ag level reached 0.14 and 2.35 mg/g·FW in the leaves and roots under 100 mg/L AgNO3 stress condition. Root fresh weight, root dry weight, root water content, and photosynthetic pigments contents were significantly decreased with elevating AgNO3 concentration. Obvious withered plant tissue, microstructure disorder, and disrupted organelles were observed. In vitro evaluations (e.g., PI and DCFH-DA staining) represented that AgNO3 at high concentration (100 mg/L) exhibited obvious inhibition on cell viability, which was due possibly to the induction of reactive oxygen species (ROS) accumulation. A total of 44643 differentially expressed genes (DEGs) were identified under Ag stress, covering 27155 upregulated and 17488 downregulated genes. 12 stress-responsive DEGs was authenticated utilizing real-time quantitative PCR (qRT-PCR). Gene ontology (GO) analysis revealed that the DEGs were mostly related to metal ion binding (molecular function), nucleus (cellular component), and defense response (biological process). Involved DEGs in sequence-specific DNA binding transcription factor activity, response to various hormones (e.g., abscisic acid, IAA/Auxin, salicylic acid, and etc), calcium signal transduction, and protein ubiquitination were concluded to play crucial roles in Ag tolerance of red clover. On the other hand, Kyoto Encyclopedia of Genes and Genomes (KEGG) database annotated several stress responsive pathways such as plant-pathogen interaction, phenylpropanoid biosynthesis, ubiquitin mediated proteolysis, hormone signal transduction, and autophagy. Several down-regulated genes (e.g., RSF2, RCD1, DOX1, and etc) were identified indicating possible metabolic disturbance. Besides, protein-protein interaction network (PPI) identified several pivotal genes such as ribosomal proteins, TIR, and ZAT.

Stable Cells with NF-κB-ZsGreen Fused Genes Created by TALEN Editing and Homology Directed Repair for Screening Anti-inflammation Drugs.

BackgroundNF-κB is a sequence-specific DNA-binding transcription factor that plays key roles in inflammation and cancer. It is well known that NF-κB is over-activated in these diseases. NF-κB inhibitors are therefore developed as promising drugs for these diseases. However, finding NF-κB inhibitors is dependent on effective screening platforms.MethodsFor providing an easy and visualizable tool for screening NF-κB inhibitors, and other NF-κB-related studies, this study edited all five genes of NF-κB family (RELA, RELB, CREL, NF-κB1, NF-κB2) in three different cell lines (293T, HepG2, and PANC1) with both TALEN and CRISPR. The edited NF-κB genes were repaired by homology-dependent repair using a linear homologous donor containing ZsGreen coding sequence. The edit efficiency was thus directly evaluated by detecting cellular fluorescence. The editing efficiency was also confirmed by PCR detection of NF-κB-ZsGreen fused genes.ResultsIt was found that all genes were more efficiently edited by TALEN in all cells than CRISPR. The positive cells were then isolated from the TALEN-edited cell pool by flow cytometry. The purified positive cells were finally evaluated by regulating NF-κB activity with a known NF-κB inhibitor, BAY 11–7082, and an NF-κB-targeting artificial microRNA, miR533. The results revealed that all the labeled NF-κB genes responded well to the two kinds of NF-κB activity regulators in all cell lines.ConclusionThis study thus obtained 15 cell lines with NF-κB-ZsGreen fused genes, which provide an easy and visualizable tool for screening NF-κB inhibitors and other NF-κB-related studies.

Genome-Wide DNA Methylation Analysis of Soybean Curled-Cotyledons Mutant and Functional Evaluation of a Homeodomain-Leucine Zipper (HD-Zip) I Gene GmHDZ20.

DNA methylation is a major, conserved epigenetic modification that influences many biological processes. Cotyledons are specialized tissues that provide nutrition for seedlings at the early developmental stage. To investigate the patterns of genomic DNA methylation of germinated cotyledons in soybean (Glycine max) and its effect on cotyledon development, we performed a genome-wide comparative analysis of DNA methylation between the soybean curled-cotyledons (cco) mutant, which has abnormal cotyledons, and its corresponding wild type (WT) by whole-genome bisulfite sequencing. The cco mutant was methylated at more sites but at a slightly lower level overall than the WT on the whole-genome level. A total of 46 CG-, 92 CHG-, and 9723 CHH- (H = A, C, or T) differentially methylated genes (DMGs) were identified in cotyledons. Notably, hypomethylated CHH-DMGs were enriched in the gene ontology term “sequence-specific DNA binding transcription factor activity.” We selected a DMG encoding a homeodomain-leucine zipper (HD-Zip) I subgroup transcription factor (GmHDZ20) for further functional characterization. GmHDZ20 localized to the nucleus and was highly expressed in leaf and cotyledon tissues. Constitutive expression of GmHDZ20 in Arabidopsis thaliana led to serrated rosette leaves, shorter siliques, and reduced seed number per silique. A yeast two-hybrid assay revealed that GmHDZ20 physically interacted with three proteins associated with multiple aspects of plant growth. Collectively, our results provide a comprehensive study of soybean DNA methylation in normal and aberrant cotyledons, which will be useful for the identification of specific DMGs that participate in cotyledon development, and also provide a foundation for future in-depth functional study of GmHDZ20 in soybean.

Proteomic Profiling of Fast Neutron-Induced Soybean Mutant Unveiled Pathways Associated with Increased Seed Protein Content.

Mutagenesis through fast neutron (FN) radiation of soybean resulted in a mutant with a 15% increase in seed protein content. A comparative genomic hybridization analysis confirmed that the mutant is lacking 24 genes located at chromosomes 5 and 10. A tandem mass tag-based proteomic profiling of the wild type and the FN mutant revealed 3,502 proteins, of which 206 proteins exhibited increased abundance and 214 proteins showed decreased abundance. Among the abundant proteins, basic 7S globulin increased fourfold, followed by vacuolar-sorting receptor and protein transporters. The differentially expressed proteins were mapped on the global metabolic pathways. It was observed that there was an enrichment of 29 ribosomal proteins, 16 endoplasmic reticular proteins, and several proteins in export metabolic pathways. The deletion of the sequence-specific DNA binding transcription factor along with 23 other genes may have altered the negative regulation of protein syntheses processes, resulting in an increase in the overall protein content of the mutant seed. This mutant is a valuable resource for researchers to understand the metabolic pathways that may affect an increase in seed protein content (the mass spectrometry data files were submitted to massive.ucsd.edu # MassIVE MSV000084228).

Interactome of vertebrate GAF/ThPOK reveals its diverse functions in gene regulation and DNA repair

GAGA associated factor (GAF) is a sequence-specific DNA binding transcription factor that is evolutionarily conserved from flies to humans. Emerging evidence shows a context-dependent function of vertebrate GAF (vGAF, a.k.a. ThPOK) in multiple processes like gene activation, repression, and enhancer-blocking. We hypothesize that context-dependent interaction of vGAF with a diverse set of proteins forms the basis for the multifunctional nature of vGAF. To this end, we deciphered the protein-protein interactome of vGAF and show that vGAF interacts with chromatin remodelers, RNA metabolic machinery, transcriptional activators/ repressors, and components of DNA repair machinery. We further validated the biological significance of our protein-protein interaction data with functional studies and established a novel role of vGAF in DNA repair and cell-survival after UV-induced DNA damage. One of the major risk factors for skin cutaneous melanoma is prolonged exposure of UV and subsequent DNA damage. vGAF is highly expressed in normal skin tissue. Interestingly, our analysis of high-throughput RNA-sequencing data shows that vGAF is heavily downregulated across all major stages of skin cutaneous melanoma suggesting its potential as a diagnostic biomarker. Taken together, our study provides a plausible explanation for the diverse gene regulatory functions of vGAF and unravels its novel role in DNA repair.

Identification and characterization of circular RNAs in atrial appendage of patients with atrial fibrillation

Circular RNAs (circRNAs) have emerged as a novel type of non-coding RNA (ncRNA) of interest in gene regulation, especially for its vital function underlying many diseases. Atrial fibrillation is the most common sustained arrythmia. However, the expression spectrum and function of circRNAs in atrial appendage of patients with atrial fibrillation (AF) has seldomly been investigated. Human atrial appendage tissues were acquired during cardiac surgery, which were divided into the AF group and the Sinus rhythm (SR) group. The expression characterization of circRNAs of two groups was revealed by high-throughput sequencing. The dysregulated circRNAs were identified and analyzed by bioinformatics methods, and further validated by realtime PCR. A total 18109 circRNAs in human atrial appendage tissues were targeted. Among them, 147 differentially expressed circRNAs (102 up-regulated and 45 down-regulated) were found between AF group and SR group. Gene ontology (GO) and KEGG pathway analysis indicated that many mRNAs transcribed from the host genes of altered circRNAs were implicated in regulation of sequence-specific DNA binding transcription factor activity, as well as nicotinate and nicotinamide metabolism pathways. Analysis of the association between differently expressed circRNA and miRNA were explored, which revealed an ample interaction. Our study firstly revealed the expression spectrum of circRNAs in both left and right atrial appendage of patients with or without AF. Differentially expressed circRNAs in the atrial appendage were also identified, analyzed and validated. The results of this study may provide novel biomarkers and potential therapeutic targets for AF.

Overexpression of VIRE2-INTERACTING PROTEIN2 in Arabidopsis regulates genes involved in Agrobacterium-mediated plant transformation and abiotic stresses

Arabidopsis VIRE2-INTERACTING PROTEIN2 (VIP2) was previously described as a protein with a NOT domain, and Arabidopsis vip2 mutants are recalcitrant to Agrobacterium-mediated root transformation. Here we show that VIP2 is a transcription regulator and the C-terminal NOT2 domain of VIP2 interacts with VirE2. Interestingly, AtVIP2 overexpressor lines in Arabidopsis did not show an improvement in Agrobacterium-mediated stable root transformation, but the transcriptome analysis identified 1,634 differentially expressed genes compared to wild-type. These differentially expressed genes belonged to various functional categories such as membrane proteins, circadian rhythm, signaling, response to stimulus, regulation of plant hypersensitive response, sequence-specific DNA binding transcription factor activity and transcription regulatory region binding. In addition to regulating genes involved in Agrobacterium-mediated plant transformation, AtVIP2 overexpressor line showed differential expression of genes involved in abiotic stresses. The majority of the genes involved in abscisic acid (ABA) response pathway, containing the Abscisic Acid Responsive Element (ABRE) element within their promoters, were down-regulated in AtVIP2 overexpressor lines. Consistent with this observation, AtVIP2 overexpressor lines were more susceptible to ABA and other abiotic stresses. Based on the above findings, we hypothesize that VIP2 not only plays a role in Agrobacterium-mediated plant transformation but also acts as a general transcriptional regulator in plants.

Profiling of chromatin accessibility and identification of general cis-regulatory mechanisms that control two ocular lens differentiation pathways

BackgroundPromoters and enhancers are cis-regulatory DNA sequences that control specificity and quantity of transcription. Both are rich on clusters of cis-acting sites that interact with sequence-specific DNA-binding transcription factors (TFs). At the level of chromatin, these regions display increased nuclease sensitivity, reduced nucleosome density, including nucleosome-free regions, and specific combinations of posttranslational modifications of core histone proteins. Together, “open” and “closed” chromatins represent transcriptionally active and repressed states of individual genes, respectively. Cellular differentiation is marked by changes in local chromatin structure. Lens morphogenesis, regulated by TF Pax6, includes differentiation of epithelial precursor cells into lens fibers in parallel with differentiation of epithelial precursors into the mature lens epithelium.ResultsUsing ATAC-seq, we investigated dynamics of chromatin changes during mouse lens fibers and epithelium differentiation. Tissue-specific features of these processes are demonstrated via comparative studies of embryonic stem cells, forebrain, and liver chromatins. Unbiased analysis reveals cis-regulatory logic of lens differentiation through known (e.g., AP-1, Ets, Hsf4, Maf, and Pax6 sites) and novel (e.g., CTCF, Tead, and NF1) motifs. Twenty-six DNA-binding TFs, recognizing these cis-motifs, are markedly up-regulated in differentiating lens fibers. As specific examples, our ATAC-seq data uncovered both the regulatory regions and TF binding motifs in Foxe3, Prox1, and Mip loci that are consistent with previous, though incomplete, experimental data. A cross-examination of Pax6 binding with ATAC-seq data demonstrated that Pax6 bound to both open (H3K27ac and P300-enriched) and closed chromatin domains in lens and forebrain.ConclusionsOur study has generated the first lens chromatin accessibility maps that support a general model of stage-specific chromatin changes associated with transcriptional activities of batteries of genes required for lens fiber cell formation. Analysis of active (or open) promoters and enhancers reveals important cis-DNA motifs that establish the molecular foundation for temporally and spatially regulated gene expression in lens. Together, our data and models open new avenues for the field to conduct mechanistic studies of transcriptional control regions, reconstruction of gene regulatory networks that govern lens morphogenesis, and identification of cataract-causing mutations in noncoding sequences.

Transcriptomic analyses identify key differentially expressed genes and clinical outcomes between triple-negative and non-triple-negative breast cancer.

PurposeThere are significant differences in the biological behavior between triple-negative breast cancer (TNBC) and non-triple-negative breast cancer (non-TNBC). In the present study, we identify key differential genes and clinical outcomes between TNBC and non-TNBC.Materials and methodsTranscriptomic analyses used GEO datasets (GSE76275), gene ontology, KEGG pathway analysis and cBioPortal. Quantitative RT-PCR analysis (qRT-PCR) was used to validate the differentially expressed genes. We used the KM Plotter Online Tool and 240 patients with TNBC tissue microarray to assay the prognostic value of HORMAD1.ResultsThe upregulated differentially expressed genes were enriched in transcription factor activity, sequence-specific DNA binding and nucleic acid binding transcription factor activity. Only 16 genes were upregulated when further screened for fold change >4-fold change. HORMAD1 and SOX8 exhibited high frequencies of change of greater than 10% (HORMAD1 was close to 20%). qRT-PCR results indicated that HORMAD1 and SOX8 mRNA levels were significantly upregulated in TNBC samples. In KM Plotter Online Tool, high HORMAD1 was associated with worse outcome. In our tissue microarray (including 240 TNBC tissues), IHC analysis revealed that 29.7% (55/240) of the tumor samples exhibited high HORMAD1 expression and 70.3% (185/240) of the tumor samples exhibited low HORMAD1 expression levels. Meanwhile, high HORMAD1 group has a bad prognosis.ConclusionThe status of transcriptional activation is an important difference between TNBC and non-TNBC. HORMAD1 is a key differential gene associated with poor outcome in TNBC. Epigenetic therapy and agents targeting cancer/testis antigens might potentially help to customize therapies of TNBC.