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.

Protein interaction network revealed by quantitative proteomic analysis links TFIIB to multiple aspects of the transcription cycle.

Although TFIIB is widely regarded as an initiation factor, recent reports have implicated it in multiple aspects of eukaryotic transcription. To investigate the broader role of TFIIB in transcription, we performed quantitative proteomic analysis of yeast TFIIB. We purified two different populations of TFIIB; one from soluble cell lysate, which is not engaged in transcription, and the other from the chromatin fraction which yields the transcriptionally active form of the protein. TFIIB purified from the chromatin exhibits several interactions that explain its non-canonical roles in transcription. RNAPII, TFIIF and TFIIH were the only components of the preinitiation complex with a significant presence in chromatin TFIIB. A notable feature was enrichment of all subunits of CF1 and Rat1 3' end processing-termination complexes in chromatin-TFIIB preparation. Subunits of the CPF termination complex were also detected in both chromatin and soluble derived TFIIB preparations. These results may explain the presence of TFIIB at the 3' end of genes during transcription as well as its role in promoter-termination interaction.

TFIIB-Related Protein BRP5/PTF2 Is Required for Both Male and Female Gametogenesis and for Grain Formation in Rice.

Transcription factor IIB (TFIIB) is a general transcription factor for RNA polymerase II, exerting its influence across various biological contexts. In the majority of eukaryotes, TFIIB typically has two homologs, serving as general transcription factors for RNA polymerase I and III. In plants, however, the TFIIB-related protein family has expanded greatly, with 14 and 9 members in Arabidopsis and rice, respectively. BRP5/pollen-expressed transcription factor 2 (PTF2) proteins belong to a subfamily of TFIIB-related proteins found only in plants and algae. The prior analysis of an Arabidopsis atbrp5 mutant, characterized by a T-DNA insertion at the 5' untranslated region, demonstrated the essential role of BRP5/PTF2 during the process of pollen germination and embryogenesis in Arabidopsis. Using a rice transformation system based on CRISPR/Cas9 technology, we have generated transgenic rice plants containing loss-of-function frameshift mutations in the BRP5/PTF2 gene. Unlike in the Arabidopsis atbrp5 mutant, the brp5/ptf2 frameshift mutations were not transmitted to progeny in rice, indicating an essential role of BRP5/PTF2 in both male and female gamete development or viability. The silencing of rice BRP5/PTF2 expression through RNA interference (RNAi) had little effect on vegetative growth and panicle formation but strongly affected pollen development and grain formation. Genetic analysis revealed that strong RNAi silencing of rice BRP5/PTF2 was still transmissible to progeny almost exclusively through female gametes, as found in the Arabidopsis atbrp5 knockdown mutant. Thus, reduced rice BRP5/PTF2 expression impacted pollen preferentially by interfering with male gamete development or viability. Drawing upon these findings, we posit that BRP5/PTF2 assumes a distinct and imperative function in the realm of plant sexual reproduction.

TFIIB-related factor 1 is a nucleolar protein that promotes RNA polymerase I-directed transcription and tumour cell growth.

Eukaryotic RNA polymerase I (Pol I) products play fundamental roles in ribosomal assembly, protein synthesis, metabolism and cell growth. Abnormal expression of both Pol I transcription-related factors and Pol I products causes a range of diseases, including ribosomopathies and cancers. However, the factors and mechanisms governing Pol I-dependent transcription remain to be elucidated. Here, we report that transcription factor IIB-related factor 1 (BRF1), a subunit of transcription factor IIIB required for RNA polymerase III (Pol III)-mediated transcription, is a nucleolar protein and modulates Pol I-mediated transcription. We showed that BRF1 can be localized to the nucleolus in several human cell types. BRF1 expression correlates positively with Pol I product levels and tumour cell growth in vitro and in vivo. Pol III transcription inhibition assays confirmed that BRF1 modulates Pol I-directed transcription in an independent manner rather than through a Pol III product-to-45S pre-rRNA feedback mode. Mechanistically, BRF1 binds to the Pol I transcription machinery components and can be recruited to the rDNA promoter along with them. Additionally, alteration of BRF1 expression affects the recruitment of Pol I transcription machinery components to the rDNA promoter and the expression of TBP and TAF1A. These findings indicate that BRF1 modulates Pol I-directed transcription by controlling the expression of selective factor 1 subunits. In summary, we identified a novel role of BRF1 in Pol I-directed transcription, suggesting that BRF1 can independently regulate both Pol I- and Pol III-mediated transcription and act as a key coordinator of Pol I and Pol III.

A remodeled RNA polymerase II complex catalyzing viroid RNA-templated transcription.

Viroids, a fascinating group of plant pathogens, are subviral agents composed of single-stranded circular noncoding RNAs. It is well-known that nuclear-replicating viroids exploit host DNA-dependent RNA polymerase II (Pol II) activity for transcription from circular RNA genome to minus-strand intermediates, a classic example illustrating the intrinsic RNA-dependent RNA polymerase activity of Pol II. The mechanism for Pol II to accept single-stranded RNAs as templates remains poorly understood. Here, we reconstituted a robust in vitro transcription system and demonstrated that Pol II also accepts minus-strand viroid RNA template to generate plus-strand RNAs. Further, we purified the Pol II complex on RNA templates for nano-liquid chromatography-tandem mass spectrometry analysis and identified a remodeled Pol II missing Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9, contrasting to the canonical 12-subunit Pol II or the 10-subunit Pol II core on DNA templates. Interestingly, the absence of Rpb9, which is responsible for Pol II fidelity, explains the higher mutation rate of viroids in comparison to cellular transcripts. This remodeled Pol II is active for transcription with the aid of TFIIIA-7ZF and appears not to require other canonical general transcription factors (such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIS), suggesting a distinct mechanism/machinery for viroid RNA-templated transcription. Transcription elongation factors, such as FACT complex, PAF1 complex, and SPT6, were also absent in the reconstituted transcription complex. Further analyses of the critical zinc finger domains in TFIIIA-7ZF revealed the first three zinc finger domains pivotal for RNA template binding. Collectively, our data illustrated a distinct organization of Pol II complex on viroid RNA templates, providing new insights into viroid replication, the evolution of transcription machinery, as well as the mechanism of RNA-templated transcription.

Differential dependencies of human RNA polymerase II promoters on TBP, TAF1, TFIIB and XPB.

The effects of rapid acute depletion of components of RNA polymerase II (Pol II) general transcription factors (GTFs) that are thought to be critical for formation of preinitiation complexes (PICs) and initiation in vitro were quantified in HAP1 cells using precision nuclear run-on sequencing (PRO-Seq). The average dependencies for each factor across >70 000 promoters varied widely even though levels of depletions were similar. Some of the effects could be attributed to the presence or absence of core promoter elements such as the upstream TBP-specificity motif or downstream G-rich sequences, but some dependencies anti-correlated with such sequences. While depletion of TBP had a large effect on most Pol III promoters only a small fraction of Pol II promoters were similarly affected. TFIIB depletion had the largest general effect on Pol II and also correlated with apparent termination defects downstream of genes. Our results demonstrate that promoter activity is combinatorially influenced by recruitment of TFIID and sequence-specific transcription factors. They also suggest that interaction of the preinitiation complex (PIC) with nucleosomes can affect activity and that recruitment of TFIID containing TBP only plays a positive role at a subset of promoters.

RNA Polymerase II Transcription in Pneumocystis: TFIIB from Pneumocystis carinii Can Replace the Transcriptional Functions of Fission Yeast Schizosaccharomyces pombe TFIIB In Vivo and In Vitro.

The Pneumocystis genus is an opportunistic fungal pathogen that infects patients with AIDS and immunocompromised individuals. The study of this fungus has been hampered due to the inability to grow it in a (defined media/pure) culture. However, the use of modern molecular techniques and genomic analysis has helped researchers to understand its complex cell biology. The transcriptional process in the Pneumocystis genus has not been studied yet, although it is assumed that it has conventional transcriptional machinery. In this work, we have characterized the function of the RNA polymerase II (RNAPII) general transcription factor TFIIB from Pneumocystis carinii using the phylogenetically related biological model Schizosaccharomyces pombe. The results of this work show that Pneumocystis carinii TFIIB is able to replace the essential function of S. pombe TFIIB both in in vivo and in vitro assays. The S. pombe strain harboring the P carinii TFIIB grew slower than the parental wild-type S. pombe strain in complete media and in minimal media. The S. pombe cells carrying out the P. carinii TFIIB are larger than the wild-type cells, indicating that the TFIIB gene replacement confers a phenotype, most likely due to defects in transcription. P. carinii TFIIB forms very weak complexes with S. pombe TATA-binding protein on a TATA box promoter but it is able to form stable complexes in vitro when S. pombe TFIIF/RNAPII are added. P. carinii TFIIB can also replace the transcriptional function of S. pombe TFIIB in an in vitro assay. The transcription start sites (TSS) of the endogenous adh gene do not change when P. carinii TFIIB replaces S. pombe TFIIB, and neither does the TSS of the nmt1 gene, although this last gene is poorly transcribed in vivo in the presence of P. carinii TFIIB. Since transcription by RNA polymerase II in Pneumocystis is poorly understood, the results described in this study are promising and indicate that TFIIB from P. carinii can replace the transcriptional functions of S. pombe TFIIB, although the cells expressing the P. carini TFIIB show an altered phenotype. However, performing studies using a heterologous approach, like this one, could be relevant to understanding the basic molecular processes of Pneumocystis such as transcription and replication.

Initiator-Directed Transcription: Fission Yeast Nmtl Initiator Directs Preinitiation Complex Formation and Transcriptional Initiation.

The initiator element is a core promoter element encompassing the transcription start site, which is found in yeast, Drosophila, and human promoters. This element is observed in TATA-less promoters. Several studies have defined transcription factor requirements and additional cofactors that are needed for transcription initiation of initiator-containing promoters. However, those studies have been performed with additional core promoters in addition to the initiator. In this work, we have defined the pathway of preinitiation complex formation on the fission yeast nmt1 gene promoter, which contains a functional initiator with striking similarity to the initiator of the human dihydrofolate reductase (hDHFR) gene and to the factor requirement for transcription initiation of the nmt1 gene promoter. The results show that the nmt1 gene promoter possesses an initiator encompassing the transcription start site, and several conserved base positions are required for initiator function. A preinitiation complex formation on the nmt1 initiator can be started by TBP/TFIIA or TBP/TFIIB, but not TBP alone, and afterwards follows the same pathway as preinitiation complex formation on TATA-containing promoters. Transcription initiation is dependent on the general transcription factors TBP, TFIIB, TFIIE, TFIIF, TFIIH, RNA polymerase II, Mediator, and a cofactor identified as transcription cofactor for initiator function (TCIF), which is a high-molecular-weight protein complex of around 500 kDa. However, the TAF subunits of TFIID were not required for the nmt1 initiator transcription, as far as we tested. We also demonstrate that other initiators of the nmt1/hDHFR family can be transcribed in fission yeast whole-cell extracts.

Transcription factor GTF2B regulates AIP protein expression in growth hormone-secreting pituitary adenomas and influences tumor phenotypes.

BACKGROUND: Clinically, the low expression of wild-type aryl hydrocarbon receptor-interacting protein (AIP) in patients with sporadic growth hormone (GH)-secreting pituitary adenoma (GHPA) is associated with a more aggressive phenotype. However, the mechanism by which AIP expression is regulated in GHPA remains unclear. Herein, we investigated a transcription factor that regulates AIP expression and explored its role in tumor phenotypes. METHODS: General transcription factor IIB (GTF2B) was predicted by several bioinformatic tools to regulate AIP expression transcriptionally. Regulation by GTF2B was evaluated using chromatin immunoprecipitation (ChIP), reverse transcription PCR, luciferase reporter, and western blot experiments in SH-SY5Y cells. Furthermore, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, transwell invasive assay, ELISA, western blot, immunohistochemical staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling were performed to investigate the effects of GTF2B and AIP on tumor cell proliferation, apoptosis, growth hormone secretion, and invasiveness in GH3 cells and mouse xenograft models. Moreover, correlations between GTF2B and AIP expression were explored in GHPA cases. RESULTS: ChIP and luciferase reporter studies demonstrated that the regulation of AIP expression by GTF2B was dependent on the intergenic-5' untranslated region element of AIP and the initial residual S65 of GTF2B. In vitro and in vivo experiments indicated that GTF2B regulated AIP expression to impact the GHPA phenotype; this was confirmed by data from 33 GHPA cases. CONCLUSIONS: We determined the regulation by GTF2B of AIP transcription in GHPA and its impact on tumor phenotype. Our findings suggest that GTF2B may be a potential therapeutic target for GHPA with low AIP expression.

Beyond the canonical role of TFIIB in eukaryotic transcription.

The role of general transcription factor TFIIB in transcription extends well beyond its evolutionarily conserved function in initiation. Chromatin localization studies demonstrating binding of TFIIB to both the 5' and 3' ends of genes in a diverse set of eukaryotes strongly suggested a rather unexpected role of the factor in termination. TFIIB indeed plays a role in termination of transcription. TFIIB occupancy of the 3' end is possibly due to its interaction with the termination factors residing there. Interaction of the promoter-bound TFIIB with factors occupying the 3' end of a gene may be the basis of transcription-dependent gene looping. The proximity of the terminator-bound factors with the promoter in a gene loop has the potential to terminate promoter-initiated upstream anti-sense transcription thereby conferring promoter directionality. TFIIB, therefore, is emerging as a factor with pleiotropic roles in the transcription cycle. This could be the reason for preferential targeting of TFIIB by viruses. Further studies are needed to understand the critical role of TFIIB in viral pathogenesis in the context of its newly identified roles in termination, gene looping and promoter directionality.

Structures and implications of TBP-nucleosome complexes.

The TATA box-binding protein (TBP) is highly conserved throughout eukaryotes and plays a central role in the assembly of the transcription preinitiation complex (PIC) at gene promoters. TBP binds and bends DNA, and directs adjacent binding of the transcription factors TFIIA and TFIIB for PIC assembly. Here, we show that yeast TBP can bind to a nucleosome containing the Widom-601 sequence and that TBP-nucleosome binding is stabilized by TFIIA. We determine three cryo-electron microscopy (cryo-EM) structures of TBP-nucleosome complexes, two of them containing also TFIIA. TBP can bind to superhelical location (SHL) -6, which contains a TATA-like sequence, but also to SHL +2, which is GC-rich. Whereas binding to SHL -6 can occur in the absence of TFIIA, binding to SHL +2 is only observed in the presence of TFIIA and goes along with detachment of upstream terminal DNA from the histone octamer. TBP-nucleosome complexes are sterically incompatible with PIC assembly, explaining why a promoter nucleosome generally impairs transcription and must be moved before initiation can occur.

Structure of the human Mediator-RNA polymerase II pre-initiation complex.

Mediator is a conserved coactivator complex that enables the regulated initiation of transcription at eukaryotic genes1-3. Mediator is recruited by transcriptional activators and binds the pre-initiation complex (PIC) to stimulate the phosphorylation of RNA polymerase II (Pol II) and promoter escape1-6. Here we prepare a recombinant version of human Mediator, reconstitute a 50-subunit Mediator-PIC complex and determine the structure of the complex by cryo-electron microscopy. The head module of Mediator contacts the stalk of Pol II and the general transcription factors TFIIB and TFIIE, resembling the Mediator-PIC interactions observed in the corresponding complex in yeast7-9. The metazoan subunits MED27-MED30 associate with exposed regions in MED14 and MED17 to form the proximal part of the Mediator tail module that binds activators. Mediator positions the flexibly linked cyclin-dependent kinase (CDK)-activating kinase of the general transcription factor TFIIH near the linker to the C-terminal repeat domain of Pol II. The Mediator shoulder domain holds the CDK-activating kinase subunit CDK7, whereas the hook domain contacts a CDK7 element that flanks the kinase active site. The shoulder and hook domains reside in the Mediator head and middle modules, respectively, which can move relative to each other and may induce an active conformation of the CDK7 kinase to allosterically stimulate phosphorylation of the C-terminal domain.

A high-resolution protein architecture of the budding yeast genome.

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.

Expansion and Functional Diversification of TFIIB-Like Factors in Plants.

As sessile organisms, plants have evolved unique patterns of growth and development, elaborate metabolism and special perception and signaling mechanisms to environmental cues. Likewise, plants have complex and highly special programs for transcriptional control of gene expression. A case study for the special transcription control in plants is the expansion of general transcription factors, particularly the family of Transcription Factor IIB (TFIIB)-like factors with 15 members in Arabidopsis. For more than a decade, molecular and genetic analysis has revealed important functions of these TFIIB-like factors in specific biological processes including gametogenesis, pollen tube growth guidance, embryogenesis, endosperm development, and plant-microbe interactions. The redundant, specialized, and diversified roles of these TFIIB-like factors challenge the traditional definition of general transcription factors established in other eukaryotes. In this review, we discuss general transcription factors in plants with a focus on the expansion and functional analysis of plant TFIIB-like proteins to highlight unique aspects of plant transcription programs that can be highly valuable for understanding the molecular basis of plant growth, development and responses to stress conditions.

Disruption of the interaction between TFIIAαß and TFIIA recognition element inhibits RNA polymerase II gene transcription in a promoter context-dependent manner.

General transcription factors and core promoter elements play a pivotal role in RNA polymerase II (Pol II)-mediated transcription initiation. In the previous work, we have defined a TFIIA recognition element (IIARE) that modulates Pol II-directed gene transcription in a promoter context-dependent manner. However, how TFIIA interacts with the IIARE and whether the interaction between TFIIA and the IIARE is involved in the regulation of gene transcription by Pol II are not fully understood. In the present study, we confirm that both K348 and K350 residues in TFIIAαß are required for the interaction between TFIIAαß and the IIARE. Disruption of the interaction between them by gene mutations dampens TFIIAαß binding to the AdML-IIARE promoter and the transcriptional activation of the promoter containing a IIARE in vitro and in vivo. Stable expression of the TFIIAαß mutant containing both K348A and K350A in the cell line with endogenous TFIIAαß silence represses endogenous gene expression by reducing the occupancies of TFIIAαß, TBP, p300, and Pol II at the promoters containing a IIARE. The findings from this study provide a novel insight into the regulatory mechanism of gene transcription mediated by TFIIA and the IIARE.

Release of Human TFIIB from Actively Transcribing Complexes Is Triggered upon Synthesis of 7- and 9-nt RNAs.

RNA polymerase II (Pol II) and its general transcription factors assemble on the promoters of mRNA genes to form large macromolecular complexes that initiate transcription in a regulated manner. During early transcription, these complexes undergo dynamic rearrangement and disassembly as Pol II moves away from the start site of transcription and transitions into elongation. One step in disassembly is the release of the general transcription factor TFIIB, although the mechanism of release and its relationship to the activity of transcribing Pol II is not understood. We developed a single-molecule fluorescence transcription system to investigate TFIIB release in vitro. Leveraging our ability to distinguish active from inactive complexes, we found that nearly all transcriptionally active complexes release TFIIB during early transcription. Release is not dependent on the contacts TFIIB makes with its recognition element in promoter DNA. We identified two different points in early transcription at which release is triggered, reflecting heterogeneity across the population of actively transcribing complexes. TFIIB releases after both trigger points with similar kinetics, suggesting the rate of release is independent of the molecular transformations that prompt release. Together our data support the model that TFIIB release is important for Pol II to successfully escape the promoter as initiating complexes transition into elongation complexes.

Structural basis of RNA polymerase I pre-initiation complex formation and promoter melting.

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a prerequisite for the biosynthesis of ribosomes in eukaryotes. Compared to Pols II and III, the mechanisms underlying promoter recognition, initiation complex formation and DNA melting by Pol I substantially diverge. Here, we report the high-resolution cryo-EM reconstruction of a Pol I early initiation intermediate assembled on a double-stranded promoter scaffold that prevents the establishment of downstream DNA contacts. Our analyses demonstrate how efficient promoter-backbone interaction is achieved by combined re-arrangements of flexible regions in the 'core factor' subunits Rrn7 and Rrn11. Furthermore, structure-function analysis illustrates how destabilization of the melted DNA region correlates with contraction of the polymerase cleft upon transcription activation, thereby combining promoter recruitment with DNA-melting. This suggests that molecular mechanisms and structural features of Pol I initiation have co-evolved to support the efficient melting, initial transcription and promoter clearance required for high-level rRNA synthesis.

RNA decay during gammaherpesvirus infection reduces RNA polymerase II occupancy of host promoters but spares viral promoters.

In mammalian cells, widespread acceleration of cytoplasmic mRNA degradation is linked to impaired RNA polymerase II (Pol II) transcription. This mRNA decay-induced transcriptional repression occurs during infection with gammaherpesviruses including Kaposi's sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68), which encode an mRNA endonuclease that initiates widespread RNA decay. Here, we show that MHV68-induced mRNA decay leads to a genome-wide reduction of Pol II occupancy at mammalian promoters. This reduced Pol II occupancy is accompanied by down-regulation of multiple Pol II subunits and TFIIB in the nucleus of infected cells, as revealed by mass spectrometry-based global measurements of protein abundance. Viral genes, despite the fact that they require Pol II for transcription, escape transcriptional repression. Protection is not governed by viral promoter sequences; instead, location on the viral genome is both necessary and sufficient to escape the transcriptional repression effects of mRNA decay. We propose a model in which the ability to escape from transcriptional repression is linked to the localization of viral DNA within replication compartments, providing a means for these viruses to counteract decay-induced transcript loss.

PPARß/δ recruits NCOR and regulates transcription reinitiation of ANGPTL4.

In the absence of ligands, the nuclear receptor PPARß/δ recruits the NCOR and SMRT corepressors, which form complexes with HDAC3, to canonical target genes. Agonistic ligands cause dissociation of corepressors and enable enhanced transcription. Vice versa, synthetic inverse agonists augment corepressor recruitment and repression. Both basal repression of the target gene ANGPTL4 and reinforced repression elicited by inverse agonists are partially insensitive to HDAC inhibition. This raises the question how PPARß/δ represses transcription mechanistically. We show that the PPARß/δ inverse agonist PT-S264 impairs transcription initiation by decreasing recruitment of activating Mediator subunits, RNA polymerase II, and TFIIB, but not of TFIIA, to the ANGPTL4 promoter. Mass spectrometry identifies NCOR as the main PT-S264-dependent interactor of PPARß/δ. Reconstitution of knockout cells with PPARß/δ mutants deficient in basal repression results in diminished recruitment of NCOR, SMRT, and HDAC3 to PPAR target genes, while occupancy by RNA polymerase II is increased. PT-S264 restores binding of NCOR, SMRT, and HDAC3 to the mutants, resulting in reduced polymerase II occupancy. Our findings corroborate deacetylase-dependent and -independent repressive functions of HDAC3-containing complexes, which act in parallel to downregulate transcription.

RNA polymerase II plays an active role in the formation of gene loops through the Rpb4 subunit.

Gene loops are formed by the interaction of initiation and termination factors occupying the distal ends of a gene during transcription. RNAPII is believed to affect gene looping indirectly owing to its essential role in transcription. The results presented here, however, demonstrate a direct role of RNAPII in gene looping through the Rpb4 subunit. 3C analysis revealed that gene looping is abolished in the rpb4Δ mutant. In contrast to the other looping-defective mutants, rpb4Δ cells do not exhibit a transcription termination defect. RPB4 overexpression, however, rescued the transcription termination and gene looping defect of sua7-1, a mutant of TFIIB. Furthermore, RPB4 overexpression rescued the ssu72-2 gene looping defect, while SSU72 overexpression restored the formation of gene loops in rpb4Δ cells. Interestingly, the interaction of TFIIB with Ssu72 is compromised in rpb4Δ cells. These results suggest that the TFIIB-Ssu72 interaction, which is critical for gene loop formation, is facilitated by Rpb4. We propose that Rpb4 is promoting the transfer of RNAPII from the terminator to the promoter for reinitiation of transcription through TFIIB-Ssu72 mediated gene looping.