Sequence-specific Transcription Factors Research Articles

Long range regulation of transcription scales with genomic distance in a gene specific manner.

While critical for tuning the timing and level of transcription, enhancer communication with distal promoters is not well understood. Here we bypass the need for sequence-specific transcription factors and recruit activators directly using CARGO-VPR, an approach for targeting dCas9-VPR using a multiplexed array of RNA guides. We show that this approach achieves effective activator recruitment to arbitrary genomic sites, even those inaccessible by single dCas9. We utilize CARGO-VPR across the Prdm8-Fgf5 locus in mESCs, where neither gene is expressed. We demonstrate that while activator recruitment to any tested region results in transcriptional induction of at least one gene, the expression level strongly depends on the genomic distance between the promoter and activator recruitment site. However, the expression-distance relationship for each gene scales distinctly in a manner not attributable to differences in 3D contact frequency, promoter DNA sequence or presence of the repressive chromatin marks at the locus.

Position-dependent function of human sequence-specific transcription factors.

Patterns of transcriptional activity are encoded in our genome through regulatory elements such as promoters or enhancers that, paradoxically, contain similar assortments of sequence-specific transcription factor (TF)binding sites1-3. Knowledge of how these sequence motifs encode multiple, often overlapping, gene expression programs is central to understanding gene regulation and how mutations in non-coding DNA manifest in disease4,5. Here, by studying gene regulation from the perspective of individual transcription start sites (TSSs), using natural genetic variation, perturbation of endogenous TF protein levels and massively parallel analysis of natural and synthetic regulatory elements, we show that the effect of TF binding on transcription initiation is position dependent. Analysing TF-binding-site occurrences relative to the TSS, we identified several motifs with highly preferential positioning. We show that these patterns are a combination of a TF's distinct functional profiles-many TFs, including canonical activators such as NRF1, NFY and Sp1, activate or repress transcription initiation depending on their precise position relative to the TSS. As such, TFs and their spacing collectively guide the site and frequency of transcription initiation. More broadly, these findings reveal how similar assortments of TFbinding sites can generate distinct gene regulatory outcomes depending on their spatial configuration and how DNA sequence polymorphisms may contribute to transcription variation and disease and underscore a critical role for TSS data in decoding the regulatory information of our genome.

DNA sequence and chromatin differentiate sequence-specific transcription factor binding in the human malaria parasite Plasmodium falciparum.

Development of the malaria parasite, Plasmodium falciparum, is regulated by a limited number of sequence-specific transcription factors (TFs). However, the mechanisms by which these TFs recognize genome-wide binding sites is largely unknown. To address TF specificity, we investigated the binding of two TF subsets that either bind CACACA or GTGCAC DNA sequence motifs and further characterized two additional ApiAP2 TFs, PfAP2-G and PfAP2-EXP, which bind unique DNA motifs (GTAC and TGCATGCA). We also interrogated the impact of DNA sequence and chromatin context on P. falciparum TF binding by integrating high-throughput in vitro and in vivo binding assays, DNA shape predictions, epigenetic post-translational modifications, and chromatin accessibility. We found that DNA sequence context minimally impacts binding site selection for paralogous CACACA-binding TFs, while chromatin accessibility, epigenetic patterns, co-factor recruitment, and dimerization correlate with differential binding. In contrast, GTGCAC-binding TFs prefer different DNA sequence context in addition to chromatin dynamics. Finally, we determined that TFs that preferentially bind divergent DNA motifs may bind overlapping genomic regions due to low-affinity binding to other sequence motifs. Our results demonstrate that TF binding site selection relies on a combination of DNA sequence and chromatin features, thereby contributing to the complexity of P. falciparum gene regulatory mechanisms.

Planarian LDB and SSDP proteins scaffold transcriptional complexes for regeneration and patterning

Sequence-specific transcription factors often function as components of large regulatory complexes. LIM-domain binding protein (LDB) and single-stranded DNA-binding protein (SSDP) function as core scaffolds of transcriptional complexes in animals and plants. Little is known about potential partners and functions for LDB/SSDP complexes in the context of tissue regeneration. In this work, we find that planarian LDB1 and SSDP2 promote tissue regeneration, with a particular function in anterior regeneration and mediolateral polarity reestablishment. We find that LDB1 and SSDP2 interact with one another and with characterized planarian LIM-HD proteins Arrowhead, Islet1, and Lhx1/5–1. We also show that SSDP2 and LDB1 function with islet1 in polarity reestablishment and with lhx1/5–1 in serotonergic neuron maturation. Finally, we find new roles for LDB1 and SSDP2 in regulating gene expression in the planarian intestine and parenchyma; these functions are likely LIM-HD-independent. Together, our work provides insight into LDB/SSDP complexes in a highly regenerative organism. Further, our work provides a strong starting point for identifying and characterizing potential binding partners of LDB1 and SSDP2 and for exploring roles for these proteins in diverse aspects of planarian physiology.

Nuclear factor Y, a key player in neuronal gene regulation.

Establishing a functional nervous system is a complex process requiring tightly controlled gene expression programs to achieve the correct differentiation of distinct neuronal subtypes. The molecular programs required for neurons to acquire neuron-type-specific, and core pan-neuronal features mostly rely on sequence-specific transcription factors (TFs), which recognize and bind to cis-regulatory motifs present in the promoters of target genes. Recently, we investigated the role and mode of action of the NF-Y complex, a ubiquitously expressed transcriptional master regulator, in the Caenorhabditis elegans nervous system. We found that NFYA-1 is a pervasive regulator of neuron-specific and pan-neuronal gene batteries that are essential for neuronal development and function. Furthermore, we concluded that NFYA-1 acts cell autonomously by either directly binding to conserved motifs in target gene promoter regions or indirectly by regulating other transcriptional regulators to fine-tune gene expression. However, further studies are required to fully define the impact of the NF-Y complex on nervous system regulatory networks and how NF-Y coordinates with other TFs in this regard.

Dynamic modulation of enhancer-promoter and promoter-promoter connectivity in gene regulation.

Enhancers are short segments of regulatory DNA that control when and in which cell-type genes should be turned on in response to a variety of extrinsic and intrinsic signals. At the molecular level, enhancers serve as a genomic scaffold that recruits sequence-specific transcription factors and co-activators to facilitate transcription from linked promoters. However, it remains largely unclear how enhancers communicate with appropriate target promoters in the context of higher-order genome topology. In this review, we discuss recent progress in our understanding of the functional interplay between enhancers, genome topology, and the molecular properties of transcription machineries in gene regulation. We suggest that the activities of transcription hubs are highly regulated through the dynamic rearrangement of enhancer-promoter and promoter-promoter connectivity during animal development.

Basis of gene-specific transcription regulation by the Integrator complex

The Integrator complex attenuates gene expression via the premature termination of RNA polymerase II (RNAP2) at promoter-proximal pausing sites. It is required for stimulus response, cell differentiation, and neurodevelopment, but how gene-specific and adaptive regulation by Integrator is achieved remains unclear. Here, we identify two sites on human Integrator subunits 13/14 that serve as binding hubs for sequence-specific transcription factors (TFs) and other transcription effector complexes. When Integrator is attached to paused RNAP2, these hubs are positioned upstream of the transcription bubble, consistent with simultaneous TF-promoter tethering. The TFs co-localize with Integrator genome-wide, increase Integrator abundance on target genes, and co-regulate responsive transcriptional programs. For instance, sensory cilia formation induced by glucose starvation depends on Integrator-TF contacts. Our data suggest TF-mediated promoter recruitment of Integrator as a widespread mechanism for targeted transcription regulation.

An updated compendium and reevaluation of the evidence for nuclear transcription factor occupancy over the mitochondrial genome.

In most eukaryotes, mitochondrial organelles contain their own genome, usually circular, which is the remnant of the genome of the ancestral bacterial endosymbiont that gave rise to modern mitochondria. Mitochondrial genomes are dramatically reduced in their gene content due to the process of endosymbiotic gene transfer to the nucleus; as a result most mitochondrial proteins are encoded in the nucleus and imported into mitochondria. This includes the components of the dedicated mitochondrial transcription and replication systems and regulatory factors, which are entirely distinct from the information processing systems in the nucleus. However, since the 1990s several nuclear transcription factors have been reported to act in mitochondria, and previously we identified 8 human and 3 mouse transcription factors (TFs) with strong localized enrichment over the mitochondrial genome using ChIP-seq (Chromatin Immunoprecipitation) datasets from the second phase of the ENCODE (Encyclopedia of DNA Elements) Project Consortium. Here, we analyze the greatly expanded in the intervening decade ENCODE compendium of TF ChIP-seq datasets (a total of 6,153 ChIP experiments for 942 proteins, of which 763 are sequence-specific TFs) combined with interpretative deep learning models of TF occupancy to create a comprehensive compendium of nuclear TFs that show evidence of association with the mitochondrial genome. We find some evidence for chrM occupancy for 50 nuclear TFs and two other proteins, with bZIP TFs emerging as most likely to be playing a role in mitochondria. However, we also observe that in cases where the same TF has been assayed with multiple antibodies and ChIP protocols, evidence for its chrM occupancy is not always reproducible. In the light of these findings, we discuss the evidential criteria for establishing chrM occupancy and reevaluate the overall compendium of putative mitochondrial-acting nuclear TFs.

GAGA zinc finger transcription factor searches chromatin by 1D-3D facilitated diffusion.

To elucidate how eukaryotic sequence-specific transcription factors (TFs) search for gene targets on chromatin, we used multi-color smFRET and single-particle imaging to track the diffusion of purified GAGA-Associated Factor (GAF) on DNA and nucleosomes. Monomeric GAF DNA-binding domain (DBD) bearing one zinc finger finds its cognate site by 1D or 3D diffusion on bare DNA and rapidly slides back-and-forth between naturally clustered motifs for seconds before escape. Multimeric, full-length GAF also finds clustered motifs on DNA by 1D-3D diffusion, but remains locked on target for longer periods. Nucleosome architecture effectively blocks GAF-DBD 1D-sliding into the histone core but favors retention of GAF-DBD when targeting solvent-exposed sites by 3D-diffusion. Despite the occlusive power of nucleosomes, 1D-3D facilitated diffusion enables GAF to effectively search for clustered cognate motifs in chromatin, providing a mechanism for navigation to nucleosome and nucleosome-free sites by a member of the largest TF family.

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.

Individual transcription factors modulate both the micromovement of chromatin and its long-range structure

The control of eukaryotic gene expression is intimately connected to highly dynamic chromatin structures. Gene regulation relies on activator and repressor transcription factors (TFs) that induce local chromatin opening and closing. However, it is unclear how nucleus-wide chromatin organization responds dynamically to the activity of specific TFs. Here, we examined how two TFs with opposite effects on local chromatin accessibility modulate chromatin dynamics nucleus-wide. We combine high-resolution diffusion mapping and dense flow reconstruction and correlation in living cells to obtain an imaging-based, nanometer-scale analysis of local diffusion processes and long-range coordinated movements of both chromatin and TFs. We show that the expression of either an individual transcriptional activator (CDX2) or repressor (SIX6) with large numbers of binding sites increases chromatin mobility nucleus-wide, yet they induce opposite coherent chromatin motions at the micron scale. Hi-C analysis of higher-order chromatin structures shows that induction of the pioneer factor CDX2 leads both to changes in local chromatin interactions and the distribution of A and B compartments, thus relating the micromovement of chromatin with changes in compartmental structures. Given that inhibition of transcription initiation and elongation by RNA Pol II has a partial impact on the global chromatin dynamics induced by CDX2, we suggest that CDX2 overexpression alters chromatin structure dynamics both dependently and independently of transcription. Our biophysical analysis shows that sequence-specific TFs can influence chromatin structure on multiple architectural levels, arguing that local chromatin changes brought by TFs alter long-range chromatin mobility and its organization.

DNA binding analysis of rare variants in homeodomains reveals homeodomain specificity-determining residues.

Homeodomains (HDs) are the second largest class of DNA binding domains (DBDs) among eukaryotic sequence-specific transcription factors (TFs) and are the TF structural class with the largest number of disease-associated mutations in the Human Gene Mutation Database (HGMD). Despite numerous structural studies and large-scale analyses of HD DNA binding specificity, HD-DNA recognition is still not fully understood. Here, we analyze 92 human HD mutants, including disease-associated variants and variants of uncertain significance (VUS), for their effects on DNA binding activity. Many of the variants alter DNA binding affinity and/or specificity. Detailed biochemical analysis and structural modeling identifies 14 previously unknown specificity-determining positions, 5 of which do not contact DNA. The same missense substitution at analogous positions within different HDs often exhibits different effects on DNA binding activity. Variant effect prediction tools perform moderately well in distinguishing variants with altered DNA binding affinity, but poorly in identifying those with altered binding specificity. Our results highlight the need for biochemical assays of TF coding variants and prioritize dozens of variants for further investigations into their pathogenicity and the development of clinical diagnostics and precision therapies.

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.

Epigenetics of Genes Preferentially Expressed in Dissimilar Cell Populations: Myoblasts and Cerebellum.

While studying myoblast methylomes and transcriptomes, we found that CDH15 had a remarkable preference for expression in both myoblasts and cerebellum. To understand how widespread such a relationship was and its epigenetic and biological correlates, we systematically looked for genes with similar transcription profiles and analyzed their DNA methylation and chromatin state and accessibility profiles in many different cell populations. Twenty genes were expressed preferentially in myoblasts and cerebellum (Myob/Cbl genes). Some shared DNA hypo- or hypermethylated regions in myoblasts and cerebellum. Particularly striking was ZNF556, whose promoter is hypomethylated in expressing cells but highly methylated in the many cell populations that do not express the gene. In reporter gene assays, we demonstrated that its promoter's activity is methylation sensitive. The atypical epigenetics of ZNF556 may have originated from its promoter's hypomethylation and selective activation in sperm progenitors and oocytes. Five of the Myob/Cbl genes (KCNJ12, ST8SIA5, ZIC1, VAX2, and EN2) have much higher RNA levels in cerebellum than in myoblasts and displayed myoblast-specific hypermethylation upstream and/or downstream of their promoters that may downmodulate expression. Differential DNA methylation was associated with alternative promoter usage for Myob/Cbl genes MCF2L, DOK7, CNPY1, and ANK1. Myob/Cbl genes PAX3, LBX1, ZNF556, ZIC1, EN2, and VAX2 encode sequence-specific transcription factors, which likely help drive the myoblast and cerebellum specificity of other Myob/Cbl genes. This study extends our understanding of epigenetic/transcription associations related to differentiation and may help elucidate relationships between epigenetic signatures and muscular dystrophies or cerebellar-linked neuropathologies.

Purification of 3×Myc PKG-Puro-Poly A Gene for Bacterial Transformation

Dysregulation of the oncoprotein c-Myc (Myc) is involved in many types of cancer. Myc is a sequence-specific transcription factor that regulates the transcription of genes involved in the control of cell proliferation and apoptosis through mechanisms that are not well understood. The method of this research is experimental. The experiment result will be described. Some processes will be done by polymerase chain reaction (PCR) to amplify the gene and then it will be extracted to pure the targeted gene. This research has been succesful to purify 3×Myc PKG-Puro-Poly A gene. c-MYC (hereinafter MYC) is an oncoprotein consisting of 439 amino acids contains a well-characterized C-terminal DNA compound and an N-terminal transactivation domain (TAD). The C-terminal region »100 residues comprises a basic leucine zipper-helix-loop-helix (bHLH-LZ) segment that regulates heterodimerization between MYC and its partner bHLH-LZ MAX mediate in their binding to gene promoters.

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.

TAF1, the Largest Subunit of Tfiid, Is Dispensable for Adult Hematopoiesis

Temporal and spatial control of gene expression is central to normal hematopoietic stem cell (HSC) biology. Many sequence-specific transcription factors have been identified as key regulators of HSC fate decision, while the function of general transcription factors in HSC behavior is poorly understood. We previously reported that TAF1, the largest subunit of the TFIID complex, plays a critical role in AML1-ETO driven acute myeloid leukemogenesis. To evaluate the function of TAF1 in normal fetal and adult hematopoiesis, we generated TAF1 conditional knockout (CKO) mice and identified an essential role of TAF1 in fetal erythropoiesis. Surprisingly, deletion of TAF1 in adult mice was not lethal to hematopoiesis; rather, we observed a marked expansion of hematopoietic stem and progenitor cell (HSPC) populations, with increased self-renewal and impaired differentiation capacity in these TAF1- null cells. Consistently, we found that TAF1-null HSPCs failed to up-regulate key differentiation-associated genes when induced to differentiate in vitro. Using a variety of genome-wide assays and biochemical approaches, we found that TAF1 loss not only disrupted TFIID complex formation and chromatin recruitment, but also reduced promoter accessibility, impaired RNA Polymerase II promoter recruitment and activity, as well as its promoter-proximal pausing mechanism. Thus, the effect of TAF1 loss on normal adult hematopoiesis primarily relates to the inability to upregulate genes involved in differentiation, while HSC self-renewal and the expression of self-renewal genes proceeds relatively normally.

Transcriptional responses of cancer cells to heat shock-inducing stimuli involve amplification of robust HSF1 binding

Responses of cells to stimuli are increasingly discovered to involve the binding of sequence-specific transcription factors outside of known target genes. We wanted to determine to what extent the genome-wide binding and function of a transcription factor are shaped by the cell type versus the stimulus. To do so, we induced the Heat Shock Response pathway in two different cancer cell lines with two different stimuli and related the binding of its master regulator HSF1 to nascent RNA and chromatin accessibility. Here, we show that HSF1 binding patterns retain their identity between basal conditions and under different magnitudes of activation, so that common HSF1 binding is globally associated with distinct transcription outcomes. HSF1-induced increase in DNA accessibility was modest in scale, but occurred predominantly at remote genomic sites. Apart from regulating transcription at existing elements including promoters and enhancers, HSF1 binding amplified during responses to stimuli may engage inactive chromatin.

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.

BENDing with Polycomb in pluripotency and cancer.

Three recent publications on BEND3 firmly establish its role as a novel sequence-specific transcription factor that is essential for PRC2 recruitment and maintenance of pluripotency. Here, we briefly review our current understanding of the BEND3-PRC2 axis in the regulation of pluripotency and also explore the possibility of a similar connection in cancer.