The New Face of Dynamic Mutation-The CAA [CAG]n CAA CAG Motif as a Mutable Unit in the TBP Gene Causative for Spino-Cerebellar Ataxia Type 17.

Since 1991, several genetic disorders caused by unstable trinucleotide repeats (TNRs) have been identified, collectively referred to as triplet repeat diseases (TREDs). They share a common mutation mechanism: the expansion of repeats (dynamic mutations) due to the propensity of repeated sequences to form unusual DNA structures during replication. TREDs are characterized as neurodegenerative diseases or complex syndromes with significant neurological components. Spinocerebellar ataxia type 17 (SCA17) falls into the former category and is caused by the expansion of mixed CAA/CAG repeats in the TBP gene. To date, a five-unit organization of this region [(CAG)3 (CAA)3] [(CAG)n] [CAA CAG CAA] [(CAG)n] [CAA CAG], with expansion in the second [(CAG)n] unit being the most common, has been proposed. In this study, we propose an alternative organization scheme for the repeats. A search of the PubMed database was conducted to identify articles reporting both the number and composition of GAC/CAA repeats in TBP alleles. Nineteen reports were selected. The sequences of all identified CAG/CAA repeats in the TBP locus, including 67 cases (probands and b relatives), were analyzed in terms of their repetition structure and stability in inheritance, if possible. Based on the analysis of three units [(CAG)3 (CAA)2] [CAA (CAG)n CAA CAG] [CAA (CAG)n CAA CAG], the organization of repeats is proposed. Detailed analysis of the CAG/CAA repeat structure, not just the number of repeats, in TBP-expanded alleles should be performed, as it may have a prognostic value in the prediction of stability/instability during transmission and the possible anticipation of the disease.

TBP facilitates RNA Polymerase I transcription following mitosis.

The TATA-box binding protein (TBP) is the sole transcription factor common in the initiation complexes of the three major eukaryotic RNA Polymerases (Pol I, II and III). Although TBP is central to transcription by the three RNA Pols in various species, the emergence of TBP paralogs throughout evolution has expanded the complexity in transcription initiation. Furthermore, recent studies have emerged that questioned the centrality of TBP in mammalian cells, particularly in Pol II transcription, but the role of TBP and its paralogs in Pol I transcription remains to be re-evaluated. In this report, we show that in murine embryonic stem cells TBP localizes onto Pol I promoters, whereas the TBP paralog TRF2 only weakly associates to the Spacer Promoter of rDNA, suggesting that it may not be able to replace TBP for Pol I transcription. Importantly, acute TBP depletion does not fully disrupt Pol I occupancy or activity on ribosomal RNA genes, but TBP binding in mitosis leads to efficient Pol I reactivation following cell division. These findings provide a more nuanced role for TBP in Pol I transcription in murine embryonic stem cells.

Novel Antennapedia and Ultrabithorax trimeric complexes with TBP and Exd regulate transcription.

BACKGROUND: Hox proteins interact with DNA and many other proteins, co-factors, transcriptional factors, chromatin remodeling components, non-coding RNAs and even the extracellular matrix that assembles the Hox complexes. The number of interacting partners continues to grow with diverse components and more transcriptional factors than initially thought. Hox complexes present many activities, but their molecular mechanisms to modulate their target genes remain unsolved. RESULTS: In this paper we showed the protein-protein interaction of Antp with Ubx through the homeodomain using BiFC in Drosophila. Analysis of Antp-deletional mutants showed that AntpHD helixes 1 and 2 are required for the interaction with Ubx. Also, we found a novel interaction of Ubx with TBP, in which the PolyQ domain of TBP is required for the interaction. Moreover, we also detected the formation of two new trimeric complexes of Antp with Ubx, TBP and Exd using BiFC-FRET; these proteins, however, do not form a trimeric interaction with BIP2 or TFIIEß. The novel trimeric complexes reduced Antp transcriptional activity, indicating that they could confer specificity for repression. CONCLUSIONS: Our results increase the number of transcriptional factors in the Antp and Ubx interactomes that form two novel trimeric complexes with TBP and Exd. We also report a new Ubx interaction with TBP. These novel interactions provide important clues of the dynamics of Hox-interacting complexes involved in transcriptional regulation, contributing to better understand Hox function.

Drosophila in the study of hTBP protein interactions in the development and modeling of SCA17.

BACKGROUND: Protein interactions participate in many molecular mechanisms involved in cellular processes. The human TATA box binding protein (hTBP) interacts with Antennapedia (Antp) through its N-terminal region, specifically via its glutamine homopeptides. This PolyQ region acts as a binding site for other transcription factors under normal conditions, but when it expands, it generates spinocerebellar ataxia 17 (SCA17), whose protein aggregates in the brain prevent its correct functioning. OBJECTIVE: To determine whether the hTBP glutamine-rich region is involved in its interaction with homeoproteins and the role it plays in the formation of protein aggregates in SCA17. MATERIAL AND METHODS: We characterized hTBP interaction with other homeoproteins using BiFC, and modeled SCA17 in Drosophila melanogaster by targeting hTBPQ80 to the fly brain using UAS/GAL4. RESULTS: There was hTBP interaction with homeoproteins through its glutamine-rich region, and hTBP protein aggregates with expanded glutamines were found to affect the locomotor capacity of flies. CONCLUSIONS: The study of hTBP interactions opens the possibility for the search for new therapeutic strategies in neurodegenerative pathologies such as SCA17.

ANTECEDENTES: Las interacciones proteicas participan en una gran cantidad de mecanismos moleculares que rigen los procesos celulares. La proteína de unión a la caja TATA humana (hTBP) interacciona con Antennapedia (Antp) a través de su extremo N-terminal, específicamente a través de sus homopéptidos de glutaminas. Esta región PolyQ sirve como sitio de unión a factores de transcripción en condiciones normales, pero cuando se expande genera la ataxia espinal cerebelosa 17 (SCA17), cuyos agregados proteicos en el cerebro impiden su funcionamiento correcto. OBJETIVO: Determinar si la región rica en glutaminas de hTBP interviene en su interacción con homeoproteínas y el papel que tiene en la formación de agregados proteicos en SCA17. MATERIAL Y MÉTODOS: Se caracterizó la interacción de hTBP con otras homeoproteínas usando BiFC y se modeló SCA17 en Drosophila melanogaster dirigiendo hTBPQ80 al cerebro de las moscas usando UAS/GAL4. RESULTADOS: Existió interacción de hTBP con homeoproteínas a través de su región rica en glutaminas. Los agregados proteicos de hTBP con las glutaminas expandidas afectaron la capacidad locomotriz de las moscas. CONCLUSIONES: El estudio de las interacciones de hTBP abre la posibilidad para la búsqueda de nuevas estrategias terapéuticas en patologías neurodegenerativas como SCA17.

Two more families supporting the existence of monogenic spinocerebellar ataxia 48.

The reduced penetrance of TBP intermediate alleles and the recently proposed possible digenic TBP/STUB1 inheritance raised questions on the possible mechanism involved opening a debate on the existence of SCA48 as a monogenic disorder. We here report clinical and genetic results of two apparently unrelated patients carrying the same STUB1 variant(c.244G > T;p.Asp82Tyr) with normal TBP alleles and a clinical picture fully resembling SCA48, including cerebellar ataxia, dysarthria and mild cognitive impairment. This report provides supportive evidence that this specific ataxia can also occur as a monogenic disease, considering classical TBP allelic ranges.

Three-step mechanism of promoter escape by RNA polymerase II.

The transition from transcription initiation to elongation is highly regulated in human cells but remains incompletely understood at the structural level. In particular, it is unclear how interactions between RNA polymerase II (RNA Pol II) and initiation factors are broken to enable promoter escape. Here, we reconstitute RNA Pol II promoter escape in vitro and determine high-resolution structures of initially transcribing complexes containing 8-, 10-, and 12-nt ordered RNAs and two elongation complexes containing 14-nt RNAs. We suggest that promoter escape occurs in three major steps. First, the growing RNA displaces the B-reader element of the initiation factor TFIIB without evicting TFIIB. Second, the rewinding of the transcription bubble coincides with the eviction of TFIIA, TFIIB, and TBP. Third, the binding of DSIF and NELF facilitates TFIIE and TFIIH dissociation, establishing the paused elongation complex. This three-step model for promoter escape fills a gap in our understanding of the initiation-elongation transition of RNA Pol II transcription.

Cognitive dysfunction, social behavior disorder, cerebellar ataxia, and atypical brain FDG-PET presentation in spinocerebellar ataxia 17: a case report.

BACKGROUND: Spinocerebellar ataxia 17 (SCA17) is a rare autosomal dominant form of inherited ataxia, caused by heterozygous trinucleotide repeat expansions encoding glutamine in the TATA box-binding protein (TBP) gene. CASE DESCRIPTION: We describe the clinical history, neuropsychological, and neuroimaging findings of a 42-year-old patient who presented for medical attention showing prevalent behavioral and cognitive problems along with progressively worsening gait disturbances. The patient's family history indicated the presence of SCA17 in the maternal lineage. Genetic analysis confirmed a heterozygous 52-CAG pathological expansion repeat in TBP (normal interval, 25-40 CAG. Brain 18-fluorodeoxyglucose positron emission tomography (FDG-PET) showed bilateral hypometabolism in the sensorimotor cortex, with a slight predominance on the right, as well as in the striatal nuclei and thalamic hypermetabolism, a finding similar to what is observed in Huntington's disease. The patient also underwent neuropsychological evaluation, which revealed mild cognitive impairment and difficulties in social interaction and understanding other's emotions (Faux Pas Test and Reading the Mind in the Eyes Test). CONCLUSION: Our report emphasizes the importance of considering SCA17 as a possible diagnosis in patients with a prevalent progressive cognitive and behavioral disorders, even with a pattern of FDG-PET hypometabolism not primarily indicative of this disease.

A Chinese Family with Digenic TBP/STUB1 Spinocerebellar Ataxia.

Spinocerebellar ataxias (SCAs) are inherited neurodegenerative diseases characterized by loss of balance, coordination, and slurred speech. Recently, a digenic mode of inheritance of TBP/STUB1 contributing to SCA was demonstrated. The clinical manifestations of SCATBP/STUB1 include not only ataxia but also obvious cognitive and behavioral impairment. Here, we describe a Chinese family with SCATBP/STUB1 and performed a literature search for similar cases. We identified a Chinese family with SCATBP/STUB1 and compare our clinical findings with other cases described in the literature so far. Four individuals in this family have been found to carry SCATBP/STUB1, of which three have clinical manifestations. A heterozygous deletion mutation in the STIP1-homologous and U-box containing protein 1 (STUB1) gene, NM_005861.4:c433_435del(p.K145del), was identified. The proband is a 34-year-old female with progressive dementia and dysarthria. The mother and uncle of the proband first presented with motor abnormalities and gradually developed cognitive impairment. The proband and her uncle showed cerebellar atrophy on MRI. The proband's brother carried digenic variants but was asymptomatic. SCATBP/STUB1 is a novel SCA subtype. The main clinical manifestations are motor, cognitive, and behavioral abnormalities. Brain MRI shows significant cerebellar atrophy and cortical thinning. The independent segregation of TBP and STUB1 alleles should be considered when evaluating patients with cognitive impairment and ataxia.

Participation of the TBP-associated factors (TAFs) in cell differentiation.

The understanding of the mechanisms that regulate gene expression to establish differentiation programs and determine cell lineages, is one of the major challenges in Developmental Biology. Besides the participation of tissue-specific transcription factors and epigenetic processes, the role of general transcription factors has been ignored. Only in recent years, there have been scarce studies that address this issue. Here, we review the studies on the biological activity of some TATA-box binding protein (TBP)-associated factors (TAFs) during the proliferation of stem/progenitor cells and their involvement in cell differentiation. Particularly, the accumulated evidence suggests that TAF4, TAF4b, TAF7L, TAF8, TAF9, and TAF10, among others, participate in nervous system development, adipogenesis, myogenesis, and epidermal differentiation; while TAF1, TAF7, TAF15 may be involved in the regulation of stem cell proliferative abilities and cell cycle progression. On the other hand, evidence suggests that TBP variants such as TBPL1 and TBPL2 might be regulating some developmental processes such as germ cell maturation and differentiation, myogenesis, or ventral specification during development. Our analysis shows that it is necessary to study in greater depth the biological function of these factors and its participation in the assembly of specific transcription complexes that contribute to the differential gene expression that gives rise to the great diversity of cell types existing in an organism. The understanding of TAFs' regulation might lead to the development of new therapies for patients which suffer from mutations, alterations, and dysregulation of these essential elements of the transcriptional machinery.

Ino2, activator of yeast phospholipid biosynthetic genes, interacts with basal transcription factors TFIIA and Bdf1.

Binding of general transcription factors TFIID and TFIIA to basal promoters is rate-limiting for transcriptional initiation of eukaryotic protein-coding genes. Consequently, activator proteins interacting with subunits of TFIID and/or TFIIA can drastically increase the rate of initiation events. Yeast transcriptional activator Ino2 interacts with several Taf subunits of TFIID, among them the multifunctional Taf1 protein. In contrast to mammalian Taf1, yeast Taf1 lacks bromodomains which are instead encoded by separate proteins Bdf1 and Bdf2. In this work, we show that Bdf1 not only binds to acetylated histone H4 but can also be recruited by Ino2 and unrelated activators such as Gal4, Rap1, Leu3 and Flo8. An activator-binding domain was mapped in the N-terminus of Bdf1. Subunits Toa1 and Toa2 of yeast TFIIA directly contact sequences of basal promoters and TFIID subunit TBP but may also mediate the influence of activators. Indeed, Ino2 efficiently binds to two separate structural domains of Toa1, specifically with its N-terminal four-helix bundle structure required for dimerization with Toa2 and its C-terminal ß-barrel domain contacting TBP and sequences of the TATA element. These findings complete the functional analysis of yeast general transcription factors Bdf1 and Toa1 and identify them as targets of activator proteins.

Structural convergence endows nuclear transport receptor Kap114p with a transcriptional repressor function toward TATA-binding protein.

The transcription factor TATA-box binding protein (TBP) modulates gene expression in nuclei. This process requires the involvement of nuclear transport receptors, collectively termed karyopherin-ß (Kap-ß) in yeast, and various regulatory factors. In previous studies we showed that Kap114p, a Kap-ß that mediates nuclear import of yeast TBP (yTBP), modulates yTBP-dependent transcription. However, how Kap114p associates with yTBP to exert its multifaceted functions has remained elusive. Here, we employ single-particle cryo-electron microscopy to determine the structure of Kap114p in complex with the core domain of yTBP (yTBPC). Remarkably, Kap114p wraps around the yTBPC N-terminal lobe, revealing a structure resembling transcriptional regulators in complex with TBP, suggesting convergent evolution of the two protein groups for a common function. We further demonstrate that Kap114p sequesters yTBP away from promoters, preventing a collapse of yTBP dynamics required for yeast responses to environmental stress. Hence, we demonstrate that nuclear transport receptors represent critical elements of the transcriptional regulatory network.

TATA-box-binding protein promotes hepatocellular carcinoma metastasis through epithelial-mesenchymal transition.

BACKGROUND: HCC characterizes malignant metastasis with high incidence and recurrence. Thus, it is pivotal to discover the mechanisms of HCC metastasis. TATA-box-binding protein (TBP), a general transcriptional factor (TF), couples with activators and chromatin remodelers to sustain the transcriptional activity of target genes. Here, we investigate the key role of TBP in HCC metastasis. METHODS: TBP expression was measured by PCR, western blot, and immunohistochemistry. RNA-sequencing was performed to identify downstream proteins. Functional assays of TBP and downstream targets were identified in HCC cell lines and xenograft models. Luciferase reporter and chromatin immunoprecipitation assays were used to demonstrate the mechanism mediated by TBP. RESULTS: HCC patients showed high expression of TBP, which correlated with poor prognosis. Upregulation of TBP increased HCC metastasis in vivo and in vitro, and muscleblind-like-3 (MBNL3) was the effective factor of TBP, positively related to TBP expression. Mechanically, TBP transactivated and enhanced MBNL3 expression to stimulate exon inclusion of lncRNA-paxillin (PXN)-alternative splicing (AS1) and, thus, activated epithelial-mesenchymal transition for HCC progression through upregulation of PXN. CONCLUSIONS: Our data revealed that TBP upregulation is an HCC enhancer mechanism that increases PXN expression to drive epithelial-mesenchymal transition.

Promoter-proximal nucleosomes attenuate RNA polymerase II transcription through TFIID.

A nucleosome is typically positioned with its proximal edge (NPE) ∼50 bp downstream from the transcription start site of metazoan RNA polymerase II promoters. This +1 nucleosome has distinctive characteristics, including the presence of variant histone types and trimethylation of histone H3 at lysine 4. To address the role of these features in transcription complex assembly, we generated templates with four different promoters and nucleosomes located at a variety of downstream positions, which were transcribed in vitro using HeLa nuclear extracts. Two promoters lacked TATA elements, but all supported strong initiation from a single transcription start site. In contrast to results with minimal in vitro systems based on the TATA-binding protein (TBP), TATA promoter templates with a +51 NPE were transcriptionally inhibited in extracts; activity continuously increased as the nucleosome was moved downstream to +100. Inhibition was much more pronounced for the TATA-less promoters: +51 NPE templates were inactive, and substantial activity was only seen with the +100 NPE templates. Substituting the histone variants H2A.Z, H3.3, or both did not eliminate the inhibition. However, addition of excess TBP restored activity on nucleosomal templates with TATA promoters, even with an NPE at +20. Remarkably, nucleosomal templates with histone H3 trimethylated at lysine 4 are active with an NPE at +51 for both TATA and TATA-less promoters. Our results strongly suggest that the +1 nucleosome interferes with promoter recognition by TFIID. This inhibition can be overcome with TBP alone at TATA promoters or through positive interactions with histone modifications and TFIID.

Paf1 complex subunit Rtf1 stimulates H2B ubiquitylation by interacting with the highly conserved N-terminal helix of Rad6.

Histone modifications coupled to transcription elongation play important roles in regulating the accuracy and efficiency of gene expression. The monoubiquitylation of a conserved lysine in H2B (K123 in Saccharomyces cerevisiae; K120 in humans) occurs cotranscriptionally and is required for initiating a histone modification cascade on active genes. H2BK123 ubiquitylation (H2BK123ub) requires the RNA polymerase II (RNAPII)-associated Paf1 transcription elongation complex (Paf1C). Through its histone modification domain (HMD), the Rtf1 subunit of Paf1C directly interacts with the ubiquitin conjugase Rad6, leading to the stimulation of H2BK123ub in vivo and in vitro. To understand the molecular mechanisms that target Rad6 to its histone substrate, we identified the site of interaction for the HMD on Rad6. Using in vitro cross-linking followed by mass spectrometry, we localized the primary contact surface for the HMD to the highly conserved N-terminal helix of Rad6. Using a combination of genetic, biochemical, and in vivo protein cross-linking experiments, we characterized separation-of-function mutations in S. cerevisiae RAD6 that greatly impair the Rad6-HMD interaction and H2BK123 ubiquitylation but not other Rad6 functions. By employing RNA-sequencing as a sensitive approach for comparing mutant phenotypes, we show that mutating either side of the proposed Rad6-HMD interface yields strikingly similar transcriptome profiles that extensively overlap with those of a mutant that lacks the site of ubiquitylation in H2B. Our results fit a model in which a specific interface between a transcription elongation factor and a ubiquitin conjugase guides substrate selection toward a highly conserved chromatin target during active gene expression.

Candidate SNP Markers Significantly Altering the Affinity of TATA-Binding Protein for the Promoters of Human Hub Genes for Atherogenesis, Atherosclerosis and Atheroprotection.

Atherosclerosis is a systemic disease in which focal lesions in arteries promote the build-up of lipoproteins and cholesterol they are transporting. The development of atheroma (atherogenesis) narrows blood vessels, reduces the blood supply and leads to cardiovascular diseases. According to the World Health Organization (WHO), cardiovascular diseases are the leading cause of death, which has been especially boosted since the COVID-19 pandemic. There is a variety of contributors to atherosclerosis, including lifestyle factors and genetic predisposition. Antioxidant diets and recreational exercises act as atheroprotectors and can retard atherogenesis. The search for molecular markers of atherogenesis and atheroprotection for predictive, preventive and personalized medicine appears to be the most promising direction for the study of atherosclerosis. In this work, we have analyzed 1068 human genes associated with atherogenesis, atherosclerosis and atheroprotection. The hub genes regulating these processes have been found to be the most ancient. In silico analysis of all 5112 SNPs in their promoters has revealed 330 candidate SNP markers, which statistically significantly change the affinity of the TATA-binding protein (TBP) for these promoters. These molecular markers have made us confident that natural selection acts against underexpression of the hub genes for atherogenesis, atherosclerosis and atheroprotection. At the same time, upregulation of the one for atheroprotection promotes human health.

Spt3 and Spt8 Are Involved in the Formation of a Silencing Boundary by Interacting with TATA-Binding Protein.

In Saccharomyces cerevisiae, a heterochromatin-like chromatin structure called the silencing region is present at the telomere as a complex of Sir2, Sir3, and Sir4. Although spreading of the silencing region is blocked by histone acetylase-mediated boundary formation, the details of the factors and mechanisms involved in the spread and formation of the boundary at each telomere are unknown. Here, we show that Spt3 and Spt8 block the spread of the silencing regions. Spt3 and Spt8 are members of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, which has histone acetyltransferase activity. We performed microarray analysis of the transcriptome of spt3Δ and spt8Δ strains and RT-qPCR analysis of the transcript levels of genes from the subtelomeric region in mutants in which the interaction of Spt3 with TATA-binding protein (TBP) is altered. The results not only indicated that both Spt3 and Spt8 are involved in TBP-mediated boundary formation on the right arm of chromosome III, but also that boundary formation in this region is DNA sequence independent. Although both Spt3 and Spt8 interact with TBP, Spt3 had a greater effect on genome-wide transcription. Mutant analysis showed that the interaction between Spt3 and TBP plays an important role in the boundary formation.

Functionally distinct promoter classes initiate transcription via different mechanisms reflected in focused versus dispersed initiation patterns.

Recruitment of RNA polymerase II (Pol II) to promoters is essential for transcription. Despite conflicting evidence, the Pol II preinitiation complex (PIC) is often thought to have a uniform composition and to assemble at all promoters via an identical mechanism. Here, using Drosophila melanogaster S2 cells as a model, we demonstrate that different promoter classes function via distinct PICs. Promoter DNA of developmentally regulated genes readily associates with the canonical Pol II PIC, whereas housekeeping promoters do not, and instead recruit other factors such as DREF. Consistently, TBP and DREF are differentially required by distinct promoter types. TBP and its paralog TRF2 also function at different promoter types in a partially redundant manner. In contrast, TFIIA is required at all promoters, and we identify factors that can recruit and/or stabilize TFIIA at housekeeping promoters and activate transcription. Promoter activation by tethering these factors is sufficient to induce the dispersed transcription initiation patterns characteristic of housekeeping promoters. Thus, different promoter classes utilize distinct mechanisms of transcription initiation, which translate into different focused versus dispersed initiation patterns.

RNA Polymerase II transcription independent of TBP in murine embryonic stem cells.

Transcription by RNA Polymerase II (Pol II) is initiated by the hierarchical assembly of the pre-initiation complex onto promoter DNA. Decades of research have shown that the TATA-box binding protein (TBP) is essential for Pol II loading and initiation. Here, we report instead that acute depletion of TBP in mouse embryonic stem cells has no global effect on ongoing Pol II transcription. In contrast, acute TBP depletion severely impairs RNA Polymerase III initiation. Furthermore, Pol II transcriptional induction occurs normally upon TBP depletion. This TBP-independent transcription mechanism is not due to a functional redundancy with the TBP paralog TRF2, though TRF2 also binds to promoters of transcribed genes. Rather, we show that the TFIID complex can form and, despite having reduced TAF4 and TFIIA binding when TBP is depleted, the Pol II machinery is sufficiently robust in sustaining TBP-independent transcription.

A DNA origami-based device for investigating DNA bending proteins by transmission electron microscopy.

The DNA origami technique offers precise positioning of nanoscale objects with high accuracy. This has facilitated the development of DNA origami-based functional nanomechanical devices that enable the investigation of DNA-protein interactions at the single particle level. Herein, we used the DNA origami technique to fabricate a nanoscale device for studying DNA bending proteins. For a proof of concept, we used TATA-box binding protein (TBP) to evaluate our approach. Upon binding to the TATA box, TBP causes a bend to DNA of ∼90°. Our device translates this bending into an angular change that is readily observable with a conventional transmission electron microscope (TEM). Furthermore, we investigated the roles of transcription factor II A (TF(II)A) and transcription factor II B (TF(II)B). Our results indicate that TF(II)A introduces additional bending, whereas TF(II)B does not significantly alter the TBP-DNA structure. Our approach can be readily adopted to a wide range of DNA-bending proteins and will aid the development of DNA-origami-based devices tailored for the investigation of DNA-protein interactions.

TFIID dependency of steady-state mRNA transcription altered epigenetically by simultaneous functional loss of Taf1 and Spt3 is Hsp104-dependent.

In Saccharomyces cerevisiae, class II gene promoters have been divided into two subclasses, TFIID- and SAGA-dominated promoters or TFIID-dependent and coactivator-redundant promoters, depending on the experimental methods used to measure mRNA levels. A prior study demonstrated that Spt3, a TBP-delivering subunit of SAGA, functionally regulates the PGK1 promoter via two mechanisms: by stimulating TATA box-dependent transcriptional activity and conferring Taf1/TFIID independence. However, only the former could be restored by plasmid-borne SPT3. In the present study, we sought to determine why ectopically expressed SPT3 is unable to restore Taf1/TFIID independence to the PGK1 promoter, identifying that this function was dependent on the construction protocol for the SPT3 taf1 strain. Specifically, simultaneous functional loss of Spt3 and Taf1 during strain construction was a prerequisite to render the PGK1 promoter Taf1/TFIID-dependent in this strain. Intriguingly, genetic approaches revealed that an as-yet unidentified trans-acting factor reprogrammed the transcriptional mode of the PGK1 promoter from the Taf1/TFIID-independent state to the Taf1/TFIID-dependent state. This factor was generated in the haploid SPT3 taf1 strain in an Hsp104-dependent manner and inherited meiotically in a non-Mendelian fashion. Furthermore, RNA-seq analyses demonstrated that this factor likely affects the transcription mode of not only the PGK1 promoter, but also of many other class II gene promoters. Collectively, these findings suggest that a prion or biomolecular condensate is generated in a Hsp104-dependent manner upon simultaneous functional loss of TFIID and SAGA, and could alter the roles of these transcription complexes on a wide variety of class II gene promoters without altering their primary sequences. Therefore, these findings could provide the first evidence that TFIID dependence of class II gene transcription can be altered epigenetically, at least in Saccharomyces cerevisiae.