TATA Binding Research Articles

Spinocerebellar ataxia 17: full phenotype in a 41 CAG/CAA repeats carrier

BackgroundSpinocerebellar ataxia 17 (SCA17) is one of the most heterogeneous forms of autosomal dominant cerebellar ataxias with a large clinical spectrum which can mimic other movement disorders such as Huntington disease (HD), dystonia and parkinsonism. SCA17 is caused by an expansion of CAG/CAA repeat in the Tata binding protein (TBP) gene. Normal alleles contain 25 to 40 CAG/CAA repeats, alleles with 50 or greater CAG/CAA repeats are pathological with full penetrance. Alleles with 43 to 49 CAG/CAA repeats were also reported and their penetrance is estimated between 50 and 80%. Recently few symptomatic individuals having 41 and 42 repeats were reported but it is still unclear whether CAG/CAA repeats of 41 or 42 are low penetrance disease-causing alleles. Thus, phenotypic variability like the disease course in subject with SCA17 locus restricted expansions remains to be fully understood.Case presentationThe patients was a 63-year-old woman who, at 54 years, showed personality changes and increased frequency of falls. At 55 years of age neuropsychological tests showed executive attention and visuospatial deficit. At the age of 59 the patient developed dysarthria and a progressive cognitive deficit. The neurological examination showed moderate gait ataxia, dysdiadochokinesia and dysmetria, dysphagia, dysarthria and abnormal saccadic pursuit, severe axial asynergy during postural changes, choreiform dyskinesias. Molecular analysis of the TBP gene demonstrated an allele with 41 repeat suggesting that 41 CAG/CCG TBP repeats could be an allele associated with the full clinical spectrum of SCA17.ConclusionsThe described case with the other similar cases described in the literature suggests that 41 CAG/CAA trinucleotides should be considered as critical threshold in SCA17. We suggest that SCA17 diagnosis should be suspected in patients presenting with movement disorders associated with other neurodegenerative signs and symptoms.

Selection of reference genes for expression analysis in mouse models of acute alcoholic liver injury.

Investigations of hepatic gene expression are crucial for determining the molecular factors involved in acute alcoholic liver injury. The results of liver molecular investigations may reveal etiologically important genomic alterations. Therefore, it is necessary to normalize gene expression data to identify stable genes, which may be used as a reference under different experimental conditions. The aim of the present study was to apply reverse transcription‑quantitative polymerase chain reaction analysis and use analysis software to investigate the expression stability of candidate reference genes in hepatic tissues from mice with acute alcoholic liver injury. The acute alcoholic liver injury models were established by the intragastric administration of alcohol (5mg/kg) in Imprinting Control Region mice. Total RNA was isolated from the mouse livers, following which the expression levels of seven reference genes, β-actin, glyceraldehyde 3-phosphate dehydrogenase (Gadph), glucuronidase β, hypoxanthine phosphoribosyltransferase 1 (Hprt1), 18S ribosomal RNA, TATA binding protein and β‑2 microglobulin, were examined, and gene expression stability was assessed using the geNorm, NormFinder and BestKeeper tools. The geNorm analysis revealed that the gene with the lowest variability was Hprt1. Hprt1 and Gapdh were validated as the optimal reference gene pair in all samples from all groups. The NormFinder and BestKeeper results showed that Hprt1 was the most stable gene in all samples. Alcohol induces endoplasmic reticulum (ER) stress, causing changes in the expression levels of ER stress‑associated genes. The stability of Hprt1 was verified by the expression analysis of ER stress‑associated genes, and gene expression levels in the ethanol groups were upregulated, with a significant difference in expression, compared with those in the control group. Therefore, Hprt1 was selected as the most stable gene, and Hprt1 and Gapdh were determined to be the optimum gene pair in mouse models of acute alcoholic liver injury. The reliability of the Hprt1 gene was confirmed by expression analysis of ER stress‑associated genes.

Conformational Dynamics of the HIV-1 Trans-Activation Response Element RNA Hairpin Bound to a Lab-Evolved Peptide

We report microsecond-length classical molecular dynamics simulations of a lab-evolved peptide bound to the HIV-1 trans-activation response element (TAR) RNA hairpin. An essential 5′-structure in all HIV-1 mRNAs, TAR interacts with HIV-1 Tat to promote HIV-1 genome transcription, and is considered a promising therapeutic target. Using yeast display, a U1A-derived TAR binding protein (TBP) was selected that binds TAR with subnanomolar affinity and extraordinarily high specificity over the native U1A U1hpII target, thereby preventing Tat-dependent transcription in vitro. A co-crystal structure of TBP in complex with TAR reveals the basis for its unprecedented affinity and specificity. The complex exhibits a Watson-Crick C30-G34 base pair in the six-nucleotide apical loop. From chemical modification experiments and sequence conservation, this base pair has been posited as a prerequisite for Tat binding. We investigated the role of a short TBP peptide - responsible for the preponderance of TAR readout - in stabilizing the observed TAR structure using molecular dynamics simulations of TAR in the apo state and in complex with the full TBP protein and the short TBP peptide. To define discrete conformational states, we performed principal component analysis and clustered trajectories based on the resulting principal components. We observed that the apical loop C30-G34 base pair and a major groove U23-A27-U38 base triple are stable features of peptide-bound TAR on the microsecond timescale. In agreement with NMR studies of free TAR, dissolution of the major groove base triple occurs in trajectories of the apo state. The occupancy of contacts between the protein and the TAR major groove is greater for the arginine residues whose mutation to alanine has a greater impact on the binding affinity, as measured by isothermal calorimetry.

Mechanistic Differences in Transcription Initiation at TATA-Less and TATA-Containing Promoters.

A yeast in vitro system was developed that is active for transcription at both TATA-containing and TATA-less promoters. Transcription with extracts made from cells depleted of TFIID subunit Taf1 demonstrated that promoters of both classes are TFIID dependent, in agreement with recent in vivo findings. TFIID depletion can be complemented in vitro by additional recombinant TATA binding protein (TBP) at only the TATA-containing promoters. In contrast, high levels of TBP did not complement Taf1 depletion in vivo and instead repressed transcription from both promoter types. We also demonstrate the importance of the TATA-like sequence found at many TATA-less promoters and describe how the presence or absence of the TATA element is likely not the only feature that distinguishes these two types of promoters.

The HIV-1 Tat Protein: Mechanism of Action and Target for HIV-1 Cure Strategies.

The general mechanism involved in Tat activation of RNA Polymerase II (RNAP II) elongation of the integrated HIV-1 was elucidated over 20 years ago. This mechanism involves Tat binding to the TAR RNA element that forms at the 5' end of viral transcripts and recruiting a general RNAP II elongation factor termed as PTEFb. This elongation factor consists of CDK9 and Cyclin T1, and when recruited by Tat to TAR RNA, CDK9 was proposed to phosphorylate the carboxyl terminal domain of RNAP II and thereby activate elongation. Research in the past two decades has shown that the mechanism of Tat action is considerably more complicated than this simple model. In metabolically active cells, CDK9 and Cyclin T1 are now known to be largely sequestered in a RNA-protein complex termed the 7SK RNP. CDK9 and Cyclin T1 are released from the 7SK RNP by mechanisms not yet fully elucidated and along with Tat, bind to TAR RNA and orchestrate the assembly of a Super Elongation Complex (SEC) containing several additional proteins. CDK9 in the SEC then phosphorylates multiple substrates in the RNAP II complex to activate elongation. Importantly for therapeutic strategies, CDK9 and Cyclin T1 functions are down-regulated in resting CD4+ T cells that harbor latent HIV-1, and their up-regulation is required for reactivation of latent virus. Current strategies for a functional cure of HIV-1 infection therefore are likely to require development of latency reversal agents that up-regulate CDK9 and Cyclin T1 function in resting CD4+ T cells.

Allosteric modulatory effects of SRI-20041 and SRI-30827 on cocaine and HIV-1 Tat protein binding to human dopamine transporter

Dopamine transporter (DAT) is the target of cocaine and HIV-1 transactivator of transcription (Tat) protein. Identifying allosteric modulatory molecules with potential attenuation of cocaine and Tat binding to DAT are of great scientific and clinical interest. We demonstrated that tyrosine 470 and 88 act as functional recognition residues in human DAT (hDAT) for Tat-induced inhibition of DA transport and transporter conformational transitions. Here we investigated the allosteric modulatory effects of two allosteric ligands, SRI-20041 and SRI-30827 on cocaine binding on wild type (WT) hDAT, Y470 H and Y88 F mutants. Effect of SRI-30827 on Tat-induced inhibition of [3H]WIN35,428 binding was also determined. Compared to a competitive DAT inhibitor indatraline, both SRI-compounds displayed a similar decrease (30%) in IC50 for inhibition of [3H]DA uptake by cocaine in WT hDAT. The addition of SRI-20041 or SRI-30827 following cocaine slowed the dissociation rate of [3H]WIN35,428 binding in WT hDAT relative to cocaine alone. Moreover, Y470H and Y88F hDAT potentiate the inhibitory effect of cocaine on DA uptake and attenuate the effects of SRI-compounds on cocaine-mediated dissociation rate. SRI-30827 attenuated Tat-induced inhibition of [3H]WIN35,428 binding. These observations demonstrate that tyrosine 470 and 88 are critical for allosteric modulatory effects of SRI-compounds on the interaction of cocaine with hDAT.

Isolation and Characterization of plant TAF9, an Orthologous Gene for TATA Binding Protein Associated Factor 9, from Wintersweet (Chimonanthus praecox)

TAF9 is one of the TATA-binding protein-associated factors (TAFs) that constitute the TFIID complex. We isolated a plant TAF9 ortholog from the Chimonanthus praecox (L.) Link cDNA library and named it CpTAF9. The CpTAF9 protein contained the conserved TAF9 and H2A superfamily domain, which is highly conserved among the TAF9s of other organisms. It also showed a specific tissue expression pattern that the transcript level of CpTAF9 was higher in mature leaves than in other tissues. In addition, CpTAF9 expression in wintersweet leaves could be induced by treatment with salt or high temperatures, or by exogenous abscisic acid application. Overexpression of CpTAF9 in Arabidopsis thaliana (L.) Heynh. under the cauliflower mosaic virus 35S promoter improved salt and high-temperature stress tolerance to some extent. These results demonstrated that CpTAF9 may have a role in gene regulation associated with salt and heat stress responses in wintersweet, providing a foundation for further elucidating the molecular mechanisms that underlined TAF-mediated control of the stress response processes in plant.

HIV Tat protein and amyloid-β peptide form multifibrillar structures that cause neurotoxicity

We investigated direct interactions between the human immunodeficiency virus (HIV)-trans-activator of transcription (Tat) protein and amyloid β peptide. Amyloid β-Tat complexes are readily formed extracellularly in the brain. In vitro studies showed that in the presence of Tat, the uniform amyloid fibrils turned into double twisted fibrils followed by populations with thick unstructured filaments and aggregated large patches in a dose-dependent manner. The fibers became more rigid and mechanically resistant. Tat attached externally to fibrils, causing their lateral aggregation into thick multifibrilar structures. These present growth in β sheet and enhanced adhesion. The neurotoxic properties of Tat and amyloid β aggregates were strongly synergistic when complexed together in vitro and in animal models. These data suggest that the increased rigidity and mechanical resistance of the amyloid β-Tat complexes coupled with stronger adhesion due to the presence of Tat in the fibrils accounted for the increased damage, likely through pore formation in membranes.

MD simulation of the Tat/Cyclin T1/CDK9 complex revealing the hidden catalytic cavity within the CDK9 molecule upon Tat binding.

In this study, we applied molecular dynamics (MD) simulation to analyze the dynamic behavior of the Tat/CycT1/CDK9 tri-molecular complex and revealed the structural changes of P-TEFb upon Tat binding. We found that Tat could deliberately change the local flexibility of CycT1. Although the structural coordinates of the H1 and H2 helices did not substantially change, H1ʹ, H2ʹ, and H3ʹ exhibited significant changes en masse. Consequently, the CycT1 residues involved in Tat binding, namely Tat-recognition residues (TRRs), lost their flexibility with the addition of Tat to P-TEFb. In addition, we clarified the structural variation of CDK9 in complex with CycT1 in the presence or absence of Tat. Interestingly, Tat addition significantly reduced the structural variability of the T-loop, thus consolidating the structural integrity of P-TEFb. Finally, we deciphered the formation of the hidden catalytic cavity of CDK9 upon Tat binding. MD simulation revealed that the PITALRE signature sequence of CDK9 flips the inactive kinase cavity of CDK9 into the active form by connecting with Thr186, which is crucial for its activity, thus presumably recruiting the substrate peptide such as the C-terminal domain of RNA pol II. These findings provide vital information for the development of effective novel anti-HIV drugs with CDK9 catalytic activity as the target.

Allosteric DNA nanoswitches for controlled release of a molecular cargo triggered by biological inputs.

Here we demonstrate the rational design of a new class of DNA-based nanoswitches which are allosterically regulated by specific biological targets, antibodies and transcription factors, and are able to load and release a molecular cargo (i.e. doxorubicin) in a controlled fashion. In our first model system we rationally designed a stem-loop DNA-nanoswitch that adopts two mutually exclusive conformations: a "Load" conformation containing a doxorubicin-intercalating domain and a "Release" conformation containing a duplex portion recognized by a specific transcription-factor (here Tata Binding Protein). The binding of the transcription factor pushes this conformational equilibrium towards the "Release" state thus leading to doxorubicin release from the nanoswitch. In our second model system we designed a similar stem-loop DNA-nanoswitch for which conformational change and subsequent doxorubicin release can be triggered by a specific antibody. Our approach augments the current tool kit of smart drug release mechanisms regulated by different biological inputs.

Pretata: predicting TATA binding proteins with novel features and dimensionality reduction strategy.

BackgroundIt is necessary and essential to discovery protein function from the novel primary sequences. Wet lab experimental procedures are not only time-consuming, but also costly, so predicting protein structure and function reliably based only on amino acid sequence has significant value. TATA-binding protein (TBP) is a kind of DNA binding protein, which plays a key role in the transcription regulation. Our study proposed an automatic approach for identifying TATA-binding proteins efficiently, accurately, and conveniently. This method would guide for the special protein identification with computational intelligence strategies.ResultsFirstly, we proposed novel fingerprint features for TBP based on pseudo amino acid composition, physicochemical properties, and secondary structure. Secondly, hierarchical features dimensionality reduction strategies were employed to improve the performance furthermore. Currently, Pretata achieves 92.92% TATA-binding protein prediction accuracy, which is better than all other existing methods.ConclusionsThe experiments demonstrate that our method could greatly improve the prediction accuracy and speed, thus allowing large-scale NGS data prediction to be practical. A web server is developed to facilitate the other researchers, which can be accessed at http://server.malab.cn/preTata/.

Molecular mechanism: the human dopamine transporter histidine 547 regulates basal and HIV-1 Tat protein-inhibited dopamine transport.

Abnormal dopaminergic transmission has been implicated as a risk determinant of HIV-1-associated neurocognitive disorders. HIV-1 Tat protein increases synaptic dopamine (DA) levels by directly inhibiting DA transporter (DAT) activity, ultimately leading to dopaminergic neuron damage. Through integrated computational modeling prediction and experimental validation, we identified that histidine547 on human DAT (hDAT) is critical for regulation of basal DA uptake and Tat-induced inhibition of DA transport. Compared to wild type hDAT (WT hDAT), mutation of histidine547 (H547A) displayed a 196% increase in DA uptake. Other substitutions of histidine547 showed that DA uptake was not altered in H547R but decreased by 99% in H547P and 60% in H547D, respectively. These mutants did not alter DAT surface expression or surface DAT binding sites. H547 mutants attenuated Tat-induced inhibition of DA transport observed in WT hDAT. H547A displays a differential sensitivity to PMA- or BIM-induced activation or inhibition of DAT function relative to WT hDAT, indicating a change in basal PKC activity in H547A. These findings demonstrate that histidine547 on hDAT plays a crucial role in stabilizing basal DA transport and Tat-DAT interaction. This study provides mechanistic insights into identifying targets on DAT for Tat binding and improving DAT-mediated dysfunction of DA transmission.

Probing interaction of a fluorescent ligand with HIV TAR RNA

Trans-activator of Transcription (Tat) antagonists could block the interaction between Tat protein and its target, trans-activation responsive region (TAR) RNA, to inhibit Tat function and prevent human immunodeficiency virus type 1 (HIV-1) replication. For the first time, a small fluorescence ligand, ICR 191, was found to interact with TAR RNA at the Tat binding site and compete with Tat. It was also observed that the fluorescence of ICR 191 could be quenched when binding to TAR RNA and recovered when discharged via competition with Tat peptide or a well-known Tat inhibitor, neomycin B. The binding parameters of ICR 191 to TAR RNA were determined through theoretical calculations. Mass spectrometry, circular dichroism and molecular docking were used to further confirm the interaction of ICR 191 with TAR RNA. Inspired by these discoveries, a primary fluorescence model for the discovery of Tat antagonists was built using ICR 191 as a fluorescence indicator and the feasibility of this model was evaluated. This ligand-RNA interaction could provide a new strategy for research aimed at discovering Tat antagonists.

Characterization of transcription factor response kinetics in parallel.

BackgroundTranscription factors (TFs) are effectors of cell signaling pathways that regulate gene expression. TF networks are highly interconnected; one signal can lead to changes in many TF levels, and one TF level can be changed by many different signals. TF regulation is central to normal cell function, with altered TF function being implicated in many disease conditions. Thus, measuring TF levels in parallel, and over time, is crucial for understanding the impact of stimuli on regulatory networks and on diseases.ResultsHere, we report the parallel analysis of temporal TF level changes due to multiple stimuli in distinct cell types. We have analyzed short-term dynamic changes in the levels of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), signal transducer and activator of transcription 3 (Stat3), cAMP response element-binding protein (CREB), glucocorticoid receptor (GR), and TATA binding protein (TBP), in breast and liver cancer cells after tumor necrosis factor-alpha (TNF-α) and palmitic acid (PA) exposure. In response to both stimuli, NF-kB and CREB levels were increased, Stat3 decreased, and TBP was constant. GR levels were unchanged in response to TNF-α stimulation and increased in response to PA treatment.ConclusionsOur results show significant overlap in signaling initiated by TNF-α and by PA, with the exception that the events leading to PA-mediated cytotoxicity likely also include induction of GR signaling. These results further illuminate the dynamics of TF responses to cytokine and fatty acid exposure, while concomitantly demonstrating the utility of parallel TF measurement approaches in the analysis of biological phenomena.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0293-6) contains supplementary material, which is available to authorized users.

Conformational modulation mediated by polyglutamine expansion in CAG repeat expansion disease-associated proteins

We have previously reported TR-FRET based immunoassays to detect a conformational change imparted on huntingtin protein by the polyglutamine expansion, which we confirmed using biophysical methodologies. Using these immunoassays, we now report that polyglutamine expansion influences the conformational properties of other polyglutamine disease proteins, exemplified by the androgen receptor (associated with spinal bulbar muscular atrophy) and TATA binding protein (associated with spinocerebellar ataxia 17). Using artificial constructs bearing short or long polyglutamine expansions or a multimerized, unrelated epitope (mimicking the increase in anti-polyglutamine antibody epitopes present in polyglutamine repeats of increasing length) we confirmed that the conformational TR-FRET based immunoassay detects an intrinsic conformational property of polyglutamine repeats. The TR-FRET based conformational immunoassay may represent a rapid, scalable tool to identify modulators of polyglutamine-mediated conformational change in different proteins associated with CAG triplet repeat disorders.

A genetic screen for increasing metabolic flux in the isoprenoid pathway of Saccharomyces cerevisiae: Isolation of SPT15 mutants using the screen

A genetic screen to identify mutants that can increase flux in the isoprenoid pathway of yeast has been lacking. We describe a carotenoid-based visual screen built with the core carotenogenic enzymes from the red yeast Rhodosporidium toruloides. Enzymes from this yeast displayed the required, higher capacity in the carotenoid pathway. The development also included the identification of the metabolic bottlenecks, primarily phytoene dehydrogenase, that was subjected to a directed evolution strategy to yield more active mutants. To further limit phytoene pools, a less efficient version of GGPP synthase was employed. The screen was validated with a known flux increasing gene, tHMG1. New mutants in the TATA binding protein SPT15 were isolated using this screen that increased the yield of carotenoids, and an alternate isoprenoid, α-Farnesene confirming increase in overall flux. The findings indicate the presence of previously unknown links to the isoprenoid pathway that can be uncovered using this screen.

Protein Binding Bends the Gold Nanoparticle Capped DNA Sequence: Toward Novel Energy-Transfer-Based Photoelectrochemical Protein Detection.

In this work, we present a novel energy-transfer (ET)-based photoelectrochemical (PEC) probing of DNA-protein interactions, which associates intimately with many important intracellular processes in transcriptional regulatory networks. Specifically, Au nanoparticles (NPs) were confined onto the CdS quantum dots (QDs) functionalized PEC surface by the formation of duplex DNA, the subsequent binding of the TATA binding protein (TBP) and the resulting distortion of the Au NPs capped DNA sequence could adjust the interparticle distance and thereby modulate the PEC performance of CdS QDs through the ET process between the CdS QDs and Au NPs. Using the duplex DNA sequence as a rigid spacer, the relationship between the photocurrent quenching effect and the spacing distance was also studied and some experimental conditions were optimized, on the basis of which a novel ET-based PEC TBP biosensor was realized with high sensitivity and selectivity.

Targeting the prodromal stage of spinocerebellar ataxia type 17 mice: G-CSF in the prevention of motor deficits via upregulating chaperone and autophagy levels

Spinocerebellar ataxia type 17 (SCA17), an autosomal dominant cerebellar ataxia, is a devastating, incurable disease caused by the polyglutamine (polyQ) expansion of transcription factor TATA binding protein (TBP). The polyQ expansion causes misfolding and aggregation of the mutant TBP, further leading to cytotoxicity and cell death. The well-recognized prodromal phase in many forms of neurodegeneration suggests a prolonged period of partial neuronal dysfunction prior to cell loss that may be amenable to therapeutic intervention. The objective of this study was to assess the effects and molecular mechanisms of granulocyte-colony stimulating factor (G-CSF) therapy during the pre-symptomatic stage in SCA17 mice. Treatment with G-CSF at the pre-symptomatic stage improved the motor coordination of SCA17 mice and reduced the cell loss, insoluble mutant TBP protein, and vacuole formation in the Purkinje neurons of these mice. The neuroprotective effects of G-CSF may be produced by increases in Hsp70, Beclin-1, LC3-II and the p-ERK survival pathway. Upregulation of chaperone and autophagy levels further enhances the clearance of mutant protein aggregation, slowing the progression of pathology in SCA17 mice. Therefore, we showed that the early intervention of G-CSF has a neuroprotective effect, delaying the progression of SCA17 in mutant mice via increases in the levels of chaperone expression and autophagy.

Identification and Validation of Housekeeping Genes for Gene Expression Analysis of Cancer Stem Cells.

The characterization of cancer stem cell (CSC) subpopulation, through the comparison of the gene expression signature in respect to the native cancer cells, is particularly important for the identification of novel and more effective anticancer strategies. However, CSC have peculiar characteristics in terms of adhesion, growth, and metabolism that possibly implies a different modulation of the expression of the most commonly used housekeeping genes (HKG), like b-actin (ACTB). Although it is crucial to identify which are the most stable HKG genes to normalize the data derived from quantitative Real-Time PCR analysis to obtain robust and consistent results, an exhaustive validation of reference genes in CSC is still missing. Here, we isolated CSC spheres from different musculoskeletal sarcomas and carcinomas as a model to investigate on the stability of the mRNA expression of 15 commonly used HKG, in respect to the native cells. The selected genes were analysed for the variation coefficient and compared using the popular algorithms NormFinder and geNorm to evaluate stability ranking. As a result, we found that: 1) Tata Binding Protein (TBP), Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ), Peptidylprolyl isomerase A (PPIA), and Hydroxymethylbilane synthase (HMBS) are the most stable HKG for the comparison between CSC and native cells; 2) at least four reference genes should be considered for robust results; 3) the use of ACTB should not be recommended, 4) specific HKG should be considered for studies that are focused only on a specific tumor type, like sarcoma or carcinoma. Our results should be taken in consideration for all the studies of gene expression analysis of CSC, and will substantially contribute for future investigations aimed to identify novel anticancer therapy based on CSC targeting.

Activation of a T-box-Otx2-Gsc gene network independent of TBP and TBP-related factors.

Embryonic development relies on activating and repressing regulatory influences that are faithfully integrated at the core promoter of individual genes. In vertebrates, the basal machinery recognizing the core promoter includes TATA-binding protein (TBP) and two TBP-related factors. In Xenopus embryos, the three TBP family factors are all essential for development and are required for expression of distinct subsets of genes. Here, we report on a non-canonical TBP family-insensitive (TFI) mechanism of transcription initiation that involves mesoderm and organizer gene expression. Using TBP family single- and triple-knockdown experiments, α-amanitin treatment, transcriptome profiling and chromatin immunoprecipitation, we found that TFI gene expression cannot be explained by functional redundancy, is supported by active transcription and shows normal recruitment of the initiating form of RNA polymerase II to the promoter. Strikingly, recruitment of Gcn5 (also known as Kat2a), a co-activator that has been implicated in transcription initiation, to TFI gene promoters is increased upon depletion of TBP family factors. TFI genes are part of a densely connected TBP family-insensitive T-box-Otx2-Gsc interaction network. The results indicate that this network of genes bound by Vegt, Eomes, Otx2 and Gsc utilizes a novel, flexible and non-canonical mechanism of transcription that does not require TBP or TBP-related factors.