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The genome-wide architecture of chromatin-associated proteins that maintains chromosome integrity and gene regulation is not well defined. Here we use chromatin immunoprecipitation, exonuclease digestion and DNA sequencing (ChIP–exo/seq) 1 , 2 to define this architecture in Saccharomyces cerevisiae . We identify 21 meta-assemblages consisting of roughly 400 different proteins that are related to DNA replication, centromeres, subtelomeres, transposons and transcription by RNA polymerase (Pol) I, II and III. Replication proteins engulf a nucleosome, centromeres lack a nucleosome, and repressive proteins encompass three nucleosomes at subtelomeric X-elements. We find that most promoters associated with Pol II evolved to lack a regulatory region, having only a core promoter. These constitutive promoters comprise a short nucleosome-free region (NFR) adjacent to a +1 nucleosome, which together bind the transcription-initiation factor TFIID to form a preinitiation complex. Positioned insulators protect core promoters from upstream events. A small fraction of promoters evolved an architecture for inducibility, whereby sequence-specific transcription factors (ssTFs) create a nucleosome-depleted region (NDR) that is distinct from an NFR. We describe structural interactions among ssTFs, their cognate cofactors and the genome. These interactions include the nucleosomal and transcriptional regulators RPD3-L, SAGA, NuA4, Tup1, Mediator and SWI–SNF. Surprisingly, we do not detect interactions between ssTFs and TFIID, suggesting that such interactions do not stably occur. Our model for gene induction involves ssTFs, cofactors and general factors such as TBP and TFIIB, but not TFIID. By contrast, constitutive transcription involves TFIID but not ssTFs engaged with their cofactors. From this, we define a highly integrated network of gene regulation by ssTFs. A ChIP–exo method is used to define the genome-wide positional organization of proteins associated with gene transcription, DNA replication, centromeres, subtelomeres and transposons, revealing distinct protein assemblies for constitutive and inducible gene expression.

TATA-box binding protein (TBP) is required for every single transcription event in archaea and eukaryotes. It binds DNA and harbors two repeats with an internal structural symmetry that show sequence asymmetry. At various times in evolution, TBP has acquired multiple interaction partners and different organisms have evolved TBP paralogs with additional protein regions. Together, these observations raise questions of what molecular determinants (i.e. key residues) led to the ability of TBP to acquire new interactions, resulting in an increasingly complex transcriptional system in eukaryotes. We present a comprehensive study of the evolutionary history of TBP and its interaction partners across all domains of life, including viruses. Our analysis reveals the molecular determinants and suggests a unified and multi-stage evolutionary model for the functional innovations of TBP. These findings highlight how concerted chemical changes on a conserved structural scaffold allow for the emergence of complexity in a fundamental biological process. The TATA-box binding protein (TBP) is required for transcription initiation in archaea and eukaryotes. Here the authors delineate how TBP’s function has evolved new functional features through context-dependent interactions with various protein partners.

In many repeat diseases, such as Huntington’s disease (HD), ongoing repeat expansions in affected tissues contribute to disease onset, progression and severity. Inducing contractions of expanded repeats by exogenous agents is not yet possible. Traditional approaches would target proteins driving repeat mutations. Here we report a compound, naphthyridine-azaquinolone (NA), that specifically binds slipped-CAG DNA intermediates of expansion mutations, a previously unsuspected target. NA efficiently induces repeat contractions in HD patient cells as well as en masse contractions in medium spiny neurons of HD mouse striatum. Contractions are specific for the expanded allele, independently of DNA replication, require transcription across the coding CTG strand and arise by blocking repair of CAG slip-outs. NA-induced contractions depend on active expansions driven by MutSβ. NA injections in HD mouse striatum reduce mutant HTT protein aggregates, a biomarker of HD pathogenesis and severity. Repeat-structure-specific DNA ligands are a novel avenue to contract expanded repeats. Naphthyridine-azaquinolone specifically binds slipped-CAG DNA intermediates, induces contractions of expanded repeats and reduces mutant HTT protein aggregates in cell and animal models of Huntington’s disease.

The recruitment of RNA polymerase to gene promoters is a critical step in gene expression. For RNA polymerase II, this process is initiated by TBP and TFIIB, with homologs of these TBP/TFIIB pairs found in all known multi-subunit RNA polymerase systems. Here, we describe a mode of promoter recognition by the poxviral intermediate transcription factor 3, VITF-3. This heterodimeric factor comprises an atypical TBP/TFIIB pair forming a stable ring structure inert towards DNA in the absence of viral RNA polymerase. Promoter recognition instead requires concerted VITF-3 and viral RNA polymerase binding, as shown by cryo-EM analysis of the intermediate pre-initiation complex. During the formation of this complex, viral RNA polymerase facilitates ring opening and loading of VITF-3 onto the promoter, anchoring the polymerase at the transcription start site. Our findings suggest viral RNA polymerase could act as a clamp loader for VITF-3 and identify VITF-3 as an unusual TBP/TFIIB pair. The recruitment of RNAPII to gene promoters is initiated by TBP and TFIIB. Here, the authors use cryo-EM to show how the RNAP from the Vaccinia virus recognizes promoters, identifying transcription factor VITF-3 as an atypical TBP/TFIIB pair.

Deletions within genes coding for subunits of the transcription coactivator SAGA caused strong genome-wide defects in transcription and SAGA-mediated chromatin modifications. In contrast, rapid SAGA depletion produced only modest transcription defects at 13% of protein-coding genes – genes that are generally more sensitive to rapid TFIID depletion. However, transcription of these ‘coactivator-redundant’ genes is strongly affected by rapid depletion of both factors, showing the overlapping functions of TFIID and SAGA at this gene set. We suggest that this overlapping function is linked to TBP-DNA recruitment. The remaining 87% of expressed genes that we term ‘TFIID-dependent’ are highly sensitive to rapid TFIID depletion and insensitive to rapid SAGA depletion. Genome-wide mapping of SAGA and TFIID found binding of both factors at many genes independent of gene class. Promoter analysis suggests that the distinction between the gene classes is due to multiple components rather than any single regulatory factor or promoter sequence motif.

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 .

Transcription regulation in cestodes has been little studied. Here, we characterize the Taenia solium TATA-binding protein (TBP) gene. We found binding sites for transcription factors such as NF1, YY1, and AP-1 in the proximal promoter. We also identified two TATA-like elements in the promoter; however, neither could bind TBP. Additionally, we mapped the transcription start site (A +1 ) within an initiator and identified a putative downstream promoter element (DPE) located at +27 bp relative to the transcription start site. These two elements are important and functional for gene expression. Moreover, we identified the genes encoding T . solium TBP-Associated Factor 6 (TsTAF6) and 9 (TsTAF9). A Western blot assay revealed that both factors are expressed in the parasite; electrophoretic mobility shift assays and super-shift assays revealed interactions between the DPE probe and TsTAF6-TsTAF9. Finally, we used molecular dynamics simulations to formulate an interaction model among TsTAF6, TsTAF9, and the DPE probe; we stabilized the model with interactions between the histone fold domain pair in TAFs and several pairs of nucleotides in the DPE probe. We discuss novel and interesting features of the TsTAF6-TsTAF9 complex for interaction with DPE on T . solium promoters.

The highly conserved multifunctional polymerase-associated factor 1 (Paf1) complex (PAF1C), composed of five core subunits Paf1, Leo1, Ctr9, Cdc73, and Rtf1, participates in all stages of transcription and is required for the Rad6/Bre1-mediated monoubiquitination of histone H2B (H2Bub). However, the molecular mechanisms underlying the contributions of the PAF1C subunits to H2Bub are not fully understood. Here, we report that Ctr9, acting as a hub, interacts with the carboxyl-terminal acidic tail of Rad6, which is required for PAF1C-induced stimulation of H2Bub. Importantly, we found that the Ras-like domain of Cdc73 has the potential to accelerate ubiquitin discharge from Rad6 and thus facilitates H2Bub, a process that might be conserved from yeast to humans. Moreover, we found that Rtf1 HMD stimulates H2Bub, probably through accelerating ubiquitin discharge from Rad6 alone or in cooperation with Cdc73 and Bre1, and that the Paf1/Leo1 heterodimer in PAF1C specifically recognizes the histone H3 tail of nucleosomal substrates, stimulating H2Bub. Collectively, our biochemical results indicate that intact PAF1C is required to efficiently stimulate Rad6/Bre1-mediated H2Bub.

RNA polymerase II (Pol II) carries out transcription of both protein-coding and non-coding genes. Whereas Pol II initiation at protein-coding genes has been studied in detail, Pol II initiation at non-coding genes, such as small nuclear RNA (snRNA) genes, is less well understood at the structural level. Here, we study Pol II initiation at snRNA gene promoters and show that the snRNA-activating protein complex (SNAPc) enables DNA opening and transcription initiation independent of TFIIE and TFIIH in vitro. We then resolve cryo-EM structures of the SNAPc-containing Pol IIpre-initiation complex (PIC) assembled on U1 and U5 snRNA promoters. The core of SNAPc binds two turns of DNA and recognizes the snRNA promoter-specific proximal sequence element (PSE), located upstream of the TATA box-binding protein TBP. Two extensions of SNAPc, called wing-1 and wing-2, bind TFIIA and TFIIB, respectively, explaining how SNAPc directs Pol II to snRNA promoters. Comparison of structures of closed and open promoter complexes elucidates TFIIH-independent DNA opening. These results provide the structural basis of Pol II initiation at non-coding RNA gene promoters. Rengachari et al. provide a structural investigation of Pol II initiation at snRNA gene promoters and find that the snRNA-activating protein complex enables DNA opening and transcription initiation independent of TFIIE and TFIIH in vitro.

Spinocerebellar ataxias 17 (SCA17) is caused by polyglutamine (polyQ) expansion in the TATA box-binding protein (TBP). The selective neurodegeneration in the cerebellum in SCA17 raises the question of why ubiquitously expressed polyQ proteins can cause neurodegeneration in distinct brain regions in different polyQ diseases. By expressing mutant TBP in different brain regions in adult wild-type mice via stereotaxic injection of adeno-associated virus, we found that adult cerebellar neurons are particularly vulnerable to mutant TBP. In SCA17 knock-in mice, mutant TBP inhibits SP1-mediated gene transcription to down-regulate INPP5A, a protein that is highly abundant in the cerebellum. CRISPR/Cas9-mediated deletion of Inpp5a in the cerebellum of wild-type mice leads to Purkinje cell degeneration, and Inpp5a overexpression decreases inositol 1,4,5-trisphosphate (IP 3 ) levels and ameliorates Purkinje cell degeneration in SCA17 knock-in mice. Our findings demonstrate the important contribution of a tissue-specific protein to the polyQ protein-mediated selective neuropathology. It is not yet clear how ubiquitously-expressed proteins can cause the selective degeneration of particular populations of neurons, such as in spinocerebellar ataxia type 17, SCA17, which results from a CAG trinucleotide repeat expansion in the ubiquitously expressed transcription factor TBP. Here, the authors show that mutant TBP suppresses the cerebellum-enriched transcription of Inpp5a and link altered levels of INPP5A to the selective degeneration of cerebellar neurons.