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Knowledge of how Mediator and TFIID cross-talk contributes to promoter–enhancer (P-E) communication is important for elucidating the mechanism of enhancer function. We conducted an shRNA knockdown screen in murine embryonic stem cells to identify the functional overlap between Mediator and TFIID subunits on gene expression. Auxin-inducible degrons were constructed for TAF12 and MED4, the subunits eliciting the greatest overlap. Degradation of TAF12 led to a dramatic genome-wide decrease in gene expression accompanied by destruction of TFIID, loss of Pol II preinitiation complex (PIC) at promoters, and significantly decreased Mediator binding to promoters and enhancers. Interestingly, loss of the PIC elicited only a mild effect on P-E looping by promoter capture Hi-C (PCHi-C). Degradation of MED4 had a minor effect on Mediator integrity but led to a consistent twofold loss in gene expression, decreased binding of Pol II to Mediator, and decreased recruitment of Pol II to the promoters, but had no effect on the other PIC components. PCHi-C revealed no consistent effect of MED4 degradation on P-E looping. Collectively, our data show that TAF12 and MED4 contribute mechanistically in different ways to P-E communication but neither factor appears to directly control P-E looping, thereby dissociating P-E communication from physical looping.

After years of only low-resolution and partial assemblies, the entire human preinitiation complex (PIC), including the large and flexible Mediator and TFIID complexes, has come into focus. Five recent papers from three different research groups have transformed our understanding of transcription initiation by RNA polymerase II.

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.

Saccharomyces cerevisiae TBP associated factor 14 (Taf14) is a well-studied transcriptional regulator that controls diverse physiological processes and that physically interacts with at least seven nuclear complexes in yeast. Despite multiple previous Taf14 structural studies, the nature of its disparate transcriptional regulatory functions remains opaque. Here, we demonstrate that the extra-terminal (ET) domain of Taf14 (Taf14ET) recognizes a common motif in multiple transcriptional coactivator proteins from several nuclear complexes, including RSC, SWI/SNF, INO80, NuA3, TFIID, and TFIIF. Moreover, we show that such partner binding promotes liquid-liquid phase separation (LLPS) of Taf14ET, in a mechanism common to YEATS-associated ET domains (e.g., AF9ET) but not Bromo-associated ET domains from BET-family proteins. Thus, beyond identifying the molecular mechanism by which Taf14ET associates with many transcriptional regulators, our study suggests that Taf14 may function as a versatile nuclear hub that orchestrates transcriptional machineries to spatiotemporally regulate diverse cellular pathways., S. cerevisiae TBP associated factor 14 (Taf14) is a transcriptional regulator that interacts with multiple nuclear complexes. Here, the authors report that the extra-terminal domain of Taf14 (Taf14ET) recognizes a common motif in various transcriptional coactivator proteins and they solve the NMR structure of Taf14ET bound the ET-binding motif of Sth1, the catalytic subunit of the RSC (Remodel the Structure of Chromatin) complex, and furthermore show that Taf14ET undergoes liquid-liquid phase separation, which is enhanced by Taf14 interaction partners.

To better understand human RNA polymerase II (Pol II) promoters in the context of promoter-proximal pausing and local chromatin organization, 5′ and 3′ ends of nascent capped transcripts and the locations of nearby nucleosomes were accurately identified through sequencing at exceptional depth. High-quality visualization tools revealed a preferred sequence that defines over 177 000 core promoters with strengths varying by >10 000-fold. This sequence signature encompasses and better defines the binding site for TFIID and is surprisingly invariant over a wide range of promoter strength. We identified a sequence motif associated with promoter-proximal pausing and demonstrated that cap methylation only begins once transcripts are about 30 nt long. Mapping also revealed a ∼150 bp periodic downstream sequence element (PDE) following the typical pause location, strongly suggestive of a +1 nucleosome positioning element. A nuclear run-off assay utilizing the unique properties of the DNA fragmentation factor (DFF) coupled with sequencing of DFF protected fragments demonstrated that a +1 nucleosome is present downstream of paused Pol II. Our data more clearly define the human Pol II promoter: a TFIID binding site with built-in downstream information directing ubiquitous promoter-proximal pausing and downstream nucleosome location.

In Saccharomyces cerevisiae, class II gene promoters contain two classes of TATA elements: the TATA box and the TATA-like element. Functional loss of TFIID and SAGA transcription complexes selectively impacts steady-state mRNA levels expressed from TATA-like element-containing (i.e., TATA-less) and TATA box-containing promoters, respectively. While nascent RNA analysis has revealed that TFIID and SAGA are required for both types of promoters, the division of their roles remains unclear. We show here that transcription from the PGK1 promoter decreased in some cases by more than half after disruption of the TATA box or SPT3 (spt3Δ), whereas spt3Δ did not affect transcription from the TATA-less promoter, consistent with the prevailing view that Spt3 functions specifically in a TATA box-dependent manner. Transcription from this promoter was abolished in the spt3Δ taf1-N568Δ strain but unaffected in the taf1-N568Δ strain, regardless of TATA box presence, suggesting that TFIID was functionally dispensable for PGK1 transcription at least in the SPT3 strain. Furthermore, transcription from the TATA box-containing PGK1 promoter was slightly reduced in the taf1 strain lacking TAND (taf1-ΔTAND) upon temperature shift from 25 to 37 ℃, with restoration to normal levels within 2 h, in an Spt3-dependent manner. Interestingly, when SPT3 was reintroduced into the spt3Δ TAF1, spt3Δ taf1-N568Δ or spt3Δ taf1-ΔTAND strain, TATA box-dependent transcription from this promoter was largely restored, but TFIID independence in transcription was not restored, especially from the TATA-less promoter, and transient TAND/Spt3-dependent fluctuations of transcription after the temperature shift were also not recapitulated. Collectively, these observations suggest that Spt3 has multiple functions in PGK1 transcription, some of which may be intimately connected to Taf1 function and may therefore become unrestorable when the TFIID and SAGA functions are simultaneously compromised.

Mittal and colleagues have raised questions about mapping transcription factor locations on DNA using the MNase-based ChEC-seq method (Mittal et al., 2021). Partly due to this concern, we modified the experimental conditions of the MNase cleavage step and subsequent computational analyses, resulting in more stringent conditions for mapping protein-DNA interactions (Donczew et al., 2020). The revised method (dx.doi.org/10.17504/protocols.io.bizgkf3w) answers questions raised by Mittal et al. and, without changing earlier conclusions, identified widespread promoter binding of the transcription coactivators TFIID and SAGA at active genes. The revised method is also suitable for accurately mapping the genome-wide locations of DNA sequence-specific transcription factors.

International audience; The human general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). In eukaryotic cells, TFIID is thought to nucleate RNA polymerase II (Pol II) preinitiation complex formation on all protein coding gene promoters and thus, be crucial for Pol II transcription. TFIID is composed of three lobes, named A, B and C. A 5TAF core complex can be assembled in vitro constituting a building block for the further assembly of either lobe A or B in TFIID. Structural studies showed that TAF8 forms a histone fold pair with TAF10 in lobe B and participates in connecting lobe B to lobe C. To better understand the role of TAF8 in TFIID, we have investigated the requirement of the different regions of TAF8 for the in vitro assembly of lobe B and C, and the importance of certain TAF8 regions for mouse embryonic stem cell (ESC) viability. We have identified a region of TAF8 distinct from the histone fold domain important for assembling with the 5TAF core complex in lobe B. We also delineated four more regions of TAF8 each individually required for interacting with TAF2 in lobe C. Moreover, CRISPR/Cas9-mediated gene editing indicated that the 5TAF core-interacting TAF8 domain and the proline-rich domain of TAF8 that interacts with TAF2 are both required for mouse embryonic stem cell survival. Thus, our study defines distinct TAF8 regions involved in connecting TFIID lobe B to lobe C that appear crucial for TFIID function and consequent ESC survival.

The preinitiation complex (PIC) for transcriptional initiation by RNA polymerase (Pol) II is composed of general transcription factors that are highly conserved. However, analysis of ChIP-seq datasets reveals kinetic and compositional differences in the transcriptional initiation process among eukaryotic species. In yeast, Mediator associates strongly with activator proteins bound to enhancers, but it transiently associates with promoters in a form that lacks the kinase module. In contrast, in human, mouse, and fly cells, Mediator with its kinase module stably associates with promoters, but not with activator-binding sites. This suggests that yeast and metazoans differ in the nature of the dynamic bridge of Mediator between activators and Pol II and the composition of a stable inactive PIC-like entity. As in yeast, occupancies of TATA-binding protein (TBP) and TBP-associated factors (Tafs) at mammalian promoters are not strictly correlated. This suggests that within PICs, TFIID is not a monolithic entity, and multiple forms of TBP affect initiation at different classes of genes. TFIID in flies, but not yeast and mammals, interacts strongly at regions downstream of the initiation site, consistent with the importance of downstream promoter elements in that species. Lastly, Taf7 and the mammalian-specific Med26 subunit of Mediator also interact near the Pol II pause region downstream of the PIC, but only in subsets of genes and often not together. Species-specific differences in PIC structure and function are likely to affect how activators and repressors affect transcriptional activity.

International audience; In somatic cells, RNA polymerase II (Pol II) transcription initiation starts by the binding of the general transcription factor TFIID, containing the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs), to core promoters. However, in growing oocytes active Pol II transcription is TFIID/TBP-independent, as during oocyte growth TBP is replaced by its vertebrate-specific paralog TBPL2. TBPL2 does not interact with TAFs, but stably associates with TFIIA. The maternal transcriptome is the population of mRNAs produced and stored in the cytoplasm of growing oocytes. After fertilization, maternal mRNAs are inherited by the zygote from the oocyte. As transcription becomes silent after oocyte growth, these mRNAs are the sole source for active protein translation. They will participate to complete the protein pool required for oocyte terminal differentiation, fertilization and initiation of early development, until reactivation of transcription in the embryo, called zygotic genome activation (ZGA). All these events are controlled by an important reshaping of the maternal transcriptome. This procedure combines cytoplasmic readenylation of stored transcripts, allowing their translation, and different waves of mRNA degradation by deadenylation coupled to decapping, to eliminate transcripts coding for proteins that are no longer required. The reshaping ends after ZGA with an almost total clearance of the maternal transcripts. In the past, the murine maternal transcriptome has received little attention but recent progresses have brought new insights into the regulation of maternal mRNA dynamics in the mouse. This review will address past and recent data on the mechanisms associated with maternal transcriptome dynamic in the mouse.

The Mediator coactivator complex is divided into four modules: head, middle, tail, and kinase. Deletion of the architectural subunit Med16 separates core Mediator (cMed), comprising the head, middle, and scaffold (Med14), from the tail. However, the direct global effects of tail/cMed disconnection are unclear. We find that rapid depletion of Med16 downregulates genes that require the SAGA complex for full expression, consistent with their reported tail dependence, but also moderately overactivates TFIID-dependent genes in a manner partly dependent on the separated tail, which remains associated with upstream activating sequences. Suppression of TBP dynamics via removal of the Mot1 ATPase partially restores normal transcriptional activity to Med16-depleted cells, suggesting that cMed/tail separation results in an imbalance in the levels of PIC formation at SAGA-requiring and TFIID-dependent genes. We propose that the preferential regulation of SAGA-requiring genes by tailed Mediator helps maintain a proper balance of transcription between these genes and those more dependent on TFIID., Author summary Composed of over two dozen subunits, the Mediator complex plays several roles in RNA polymerase II (RNAPII) transcription in eukaryotes. In yeast, deletion of Med16, which splits Mediator into two stable subcomplexes, both increases and decreases transcript levels, suggesting that Med16 might play a repressive role. However, the direct effects of Med16 removal on RNAPII transcription have not been assessed, owing to the use of deletion mutants and measurement of steady-state RNA levels in prior studies. Here, using a combination of inducible protein depletion and analysis of nascent RNA, we find that Med16 removal 1) downregulates a small group of genes reported to be highly dependent on the SAGA complex and 2) upregulates a larger set of genes reported to be more dependent on the TFIID complex in a manner dependent on another component of Mediator. We find that artificially altering the balance of transcription pre-initiation complex (PIC) formation toward SAGA-requiring promoters and away from TFIID-dependent promoters partially restores normal transcription, indicating a contribution of altered PIC formation to the transcriptional alterations observed with Med16 loss. Taken together, our results indicate that the structural integrity of Mediator is important for maintaining balanced transcription between different gene classes.

The human general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). In eukaryotic cells, TFIID is thought to nucleate RNA polymerase II (Pol II) preinitiation complex formation on all protein coding gene promoters and thus, be crucial for Pol II transcription. TFIID is composed of three lobes, named A, B and C. Structural studies showed that TAF8 forms a histone fold pair with TAF10 in lobe B and participates in connecting lobe B to lobe C. In the present study, we have investigated the requirement of the different regions of TAF8 for in vitro TFIID assembly, and the importance of certain TAF8 regions for mouse embryonic stem cell (ESC) viability. We have identified a TAF8 region, different from the histone fold domain of TAF8, important for assembling with the 5TAF core complex in lobe B, and four regions of TAF8 each individually required for interacting with TAF2 in lobe C. Moreover, we show that the 5TAF coreinteracting TAF8 domain, and the proline rich domain of TAF8 that interacts with TAF2, are both required for mouse embryonic stem cell survival. Thus, our study demonstrates that distinct TAF8 regions involved in connecting lobe B to lobe C are crucial for TFIID function and consequent ESC survival.

DOT1L, the only H3K79 methyltransferase in human cells and a homolog of the yeast Dot1, normally forms a complex with AF10, AF17, and ENL or AF9, is dysregulated in most cases of mixed-lineage leukemia (MLLr), and has been believed to regulate transcriptional elongation on the basis of its colocalization with RNA polymerase II (Pol II), the sharing of subunits (AF9 and ENL) between the DOT1L and super elongation complexes, and the distribution of H3K79 methylation on both promoters and transcribed regions of active genes. Here we show that DOT1L depletion in erythroleukemic cells reduces its global occupancy without affecting the traveling ratio or the elongation rate (assessed by 4sUDRB-seq) of Pol II, suggesting that DOT1L does not play a major role in elongation in these cells. In contrast, analyses of transcription initiation factor binding reveal that DOT1L and ENL depletions each result in reduced TATA binding protein (TBP) occupancies on thousands of genes. More importantly, DOT1L and ENL depletions concomitantly reduce TBP and Pol II occupancies on a significant fraction of direct (DOT1L-bound) target genes, indicating a role for the DOT1L complex in transcription initiation. Mechanistically, proteomic and biochemical studies suggest that the DOT1L complex may regulate transcriptional initiation by facilitating the recruitment or stabilization of transcription factor IID, likely in a monoubiquitinated H2B (H2Bub1)-enhanced manner. Additional studies show that DOT1L enhances H2Bub1 levels by limiting recruitment of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex. These results advance our understanding of roles of the DOT1L complex in transcriptional regulation and have important implications for MLLr leukemias.

Human bromodomain and extra-terminal domain (BET) family members are promising targets for therapy of cancer and immunoinflammatory diseases, but their mechanisms of action and functional redundancies are poorly understood. Bdf1/2, yeast homologues of the human BET factors, were previously proposed to target transcription factor TFIID to acetylated histone H4, analogous to bromodomains that are present within the largest subunit of metazoan TFIID. We investigated the genome-wide roles of Bdf1/2 and found that their important contributions to transcription extend beyond TFIID function as transcription of many genes is more sensitive to Bdf1/2 than to TFIID depletion. Bdf1/2 co-occupy the majority of yeast promoters and affect preinitiation complex formation through recruitment of TFIID, Mediator, and basal transcription factors to chromatin. Surprisingly, we discovered that hypersensitivity of genes to Bdf1/2 depletion results from combined defects in transcription initiation and early elongation, a striking functional similarity to human BET proteins, most notably Brd4. Our results establish Bdf1/2 as critical for yeast transcription and provide important mechanistic insights into the function of BET proteins in all eukaryotes., eLife digest When a healthy cell creates new proteins, it activates a standard two-step biological manufacturing process. Firstly, DNA is transcribed from a specific gene to generate a strand of messenger RNA, or mRNA. Next, this mRNA molecule is translated to create the final protein product. This process of converting DNA into mRNA is supported by a series of helper proteins, including proteins from the bromodomain and extra-terminal domain (BET) family. Cancer cells can become ‘addicted’ to the process of converting DNA into RNA, leading to the overproduction of mRNA molecules, uncontrolled cell growth and tumor formation. Knocking out BET helper proteins could potentially bring cancer cells under control by halting transcription and preventing tumor growth. However, the precise ways in which BET helper proteins regulate transcription are currently poorly understood, and therefore developing rational ways to target them is a challenge. Building on their previous work, Donczew and Hahn have investigated how two BET helper proteins, Bdf1 and Bdf2, help to regulate transcription in budding yeast. Using a range of genomic techniques, Donczew and Hahn found that Bdf1 and Bdf2 had important roles for initiating transcription and elongating mRNA molecules. Both BET proteins were also involved in recruiting other protein factors to help with the transcription process, including TFIID and Mediator. Based on these findings, it is likely that cooperation between BET proteins, TFIID and Mediator represents a common pathway through which gene expression is regulated across all eukaryotic organisms. Both Bdf1 and Bdf2 were also found to provide the same functions in yeast as similar BET proteins in humans. Using this robust yeast model system to perform further detailed studies of BET factors could therefore provide highly relevant information to expand our understanding of human biology and disease. Ultimately, this research provides important insights into how two members of the BET family of helper proteins contribute to the control of transcription in yeast. This information could be used to guide the design of new drugs for cancer therapy that target not only BET proteins themselves but also other proteins they recruit, including TFIID and Mediator. Such targeted drugs would be expected to be more harmful for cancer cells than for healthy cells, which could reduce unwanted side effects.

Transcription factor c-MYC is a potent oncoprotein; however, the mechanism of transcriptional regulation via MYC-protein interactions remains poorly understood. The TATA-binding protein (TBP) is an essential component of the transcription initiation complex TFIID and is required for gene expression. We identify two discrete regions mediating MYC-TBP interactions using structural, biochemical and cellular approaches. A 2.4 -A resolution crystal structure reveals that human MYC amino acids 98–111 interact with TBP in the presence of the amino-terminal domain 1 of TBP-associated factor 1 (TAF1TAND1). Using biochemical approaches, we have shown that MYC amino acids 115–124 also interact with TBP independently of TAF1TAND1. Modeling reveals that this region of MYC resembles a TBP anchor motif found in factors that regulate TBP promoter loading. Site-specific MYC mutants that abrogate MYC-TBP interaction compromise MYC activity. We propose that MYC-TBP interactions propagate transcription by modulating the energetic landscape of transcription initiation complex assembly. Structural, biophysical and modeling approaches, combined with cell-based assays, reveal how the oncogenic transcription factor MYC interacts with subunits of the general transcription factor TFIID to modulate gene expression.

Background Intermittent dehydration caused by tidal changes is one of the most important abiotic factors that intertidal seaweeds must cope with in order to retain normal growth and reproduction. However, the underlying molecular mechanisms for the adaptation of red seaweeds to repeated dehydration-rehydration cycles remain poorly understood. Results We chose the red seaweed Gloiopeltis furcata as a model and simulated natural tidal changes with two consecutive dehydration-rehydration cycles occurring over 24 h in order to gain insight into key molecular pathways and regulation of genes which are associated with dehydration tolerance. Transcription sequencing assembled 32,681 uni-genes (GC content = 55.32%), of which 12,813 were annotated. Weighted gene co-expression network analysis (WGCNA) divided all transcripts into 20 modules, with Coral2 identified as the key module anchoring dehydration-induced genes. Pathways enriched analysis indicated that the ubiquitin-mediated proteolysis pathway (UPP) and phosphatidylinositol (PI) signaling system were crucial for a successful response in G. furcata. Network-establishing and quantitative reverse transcription PCR (qRT-PCR) suggested that genes encoding ubiquitin-protein ligase E3 (E3–1), SUMO-activating enzyme sub-unit 2 (SAE2), calmodulin (CaM) and inositol-1,3,4-trisphosphate 5/6-kinase (ITPK) were the hub genes which responded positively to two successive dehydration treatments. Network-based interactions with hub genes indicated that transcription factor (e.g. TFIID), RNA modification (e.g. DEAH) and osmotic adjustment (e.g. MIP, ABC1, Bam1) were related to these two pathways. Conclusions RNA sequencing-based evidence from G. furcata enriched the informational database for intertidal red seaweeds which face periodic dehydration stress during the low tide period. This provided insights into an increased understanding of how ubiquitin-mediated proteolysis and the phosphatidylinositol signaling system help seaweeds responding to dehydration-rehydration cycles.

Basal transcription factor TFIID connects transcription activation to the assembly of the RNA polymerase II preinitiation complex at the core promoter of genes. The mechanistic understanding of TFIID function and dynamics has been limited by the lack of high-resolution structures of the holo-TFIID complex. Recent cryo-electron microscopy studies of yeast and human TFIID complexes provide insight into the molecular organization and structural dynamics of this highly conserved transcription factor. Here, we discuss how these TFIID structures provide new paradigms for: (i) the dynamic recruitment of TFIID; (ii) the binding of TATA-binding protein (TBP) to promoter DNA; (iii) the multivalency of TFIID interactions with (co)activators, nucleosomes, or promoter DNA; and (iv) the opportunities for regulation of TBP turnover and promoter dynamics.

The advent of quantitative approaches that enable interrogation of transcription at single nucleotide resolution has allowed a novel understanding of transcriptional regulation previously undefined. However, little is known, at such high resolution, how transcription factors directly influence RNA Pol II pausing and directionality. To map the impact of transcription/elongation factors on transcription dynamics genome-wide at base pair resolution, we developed an adapted NET-seq protocol called NET-prism (Native Elongating Transcription by Polymerase-Regulated Immunoprecipitants in the Mammalian genome). Application of NET-prism on elongation factors (Spt6, Ssrp1), splicing factors (Sf1), and components of the pre-initiation complex (PIC) (TFIID, and Mediator) reveals their inherent command on transcription dynamics, with regards to directionality and pausing over promoters, splice sites, and enhancers/super-enhancers. NET-prism will be broadly applicable as it exposes transcription factor/Pol II dependent topographic specificity and thus, a new degree of regulatory complexity during gene expression.

Transcription factor II D (TFIID), a multiprotein complex consisting of TATA-binding protein (TBP) and 13-14 TBP-associated factors (Tafs), plays a central role in transcription and regulates nearly all class II genes. The N-terminal domain of Taf1p (TAND) can be divided into two subdomains, TAND1 and TAND2, which bind to the concave and convex surfaces of TBP, respectively. The interaction between TAND and TBP is thought to be regulated by TFIIA, activators and/or DNA during transcriptional activation, as the TAND1-bound form of TBP cannot bind to the TATA box. We previously demonstrated that Drosophila TAND1 binds to TBP with a much stronger affinity than yeast TAND1 and that the expression levels of full-length chimeric Taf1p, whose TAND1 is replaced with the Drosophila counterpart, can be varied in vivo by substituting several methionine residues downstream of TAND2 with alanine residues in various combinations. In this study, we examined the transcriptional activation of the GAL1-lacZ reporter or endogenous genes such as RNR3 or GAL1 in yeast cells expressing various levels of full-length chimeric Taf1p. The results showed that the substitution of TAND1 with the Drosophila counterpart in yeast TFIID weakened the transcriptional activation of GAL1-lacZ and RNR3 but not that of GAL1. These findings strongly support a model in which TBP must be released efficiently from TAND1 within TFIID upon transcriptional activation.

Summary: Despite substantial progress in our understanding of the players involved and the regulatory mechanisms controlling the initiation and elongation steps of transcription, little is known about the recruitment of elongation factors at promoter-proximal regions for the initiation-to-elongation transition. Here, we show evidence that human TFIID, which initiates pre-initiation complex (PIC) assembly, contributes to regulating the recruitment of super-elongation complex (SEC) components at the promoter-proximal region through interactions among selective TAF and SEC components. In vitro direct interactions, coupled with cell-based assays, identified an important poly-Ser domain within SEC components that are involved in their interaction with TFIID. DNA template-based recruitment assays, using purified components, further show a direct role for poly-Ser domain-dependent TFIID interaction in recruiting SEC components on target DNA. Consistently, ChIP and RNA analyses have shown the importance of this mechanism in TFIID-dependent SEC recruitment and target gene expression within mammalian cells. : Yadav et al. show a role for human TFIID in recruiting SEC at the promoter-proximal region through poly-Ser domain-dependent interaction between selective SEC components and TFIID subunits for the transcriptional activation of target genes. Keywords: transcription, TFIID, super elongation complex, P-TEFb, AF9, poly-Ser, TAF6, pause, elongation

International audience; In eukaryotes, transcription of protein encoding genes is initiated by the controlled deposition of the TATA-box binding protein TBP onto gene promoters, followed by the ordered assembly of a pre-initiation complex. The SAGA co-activator is a 19-subunit complex that stimulates transcription by the action of two chromatin-modifying enzymatic modules, a transcription activator binding module, and by delivering TBP. Recent cryo electron microscopy structures of yeast SAGA with bound nucleosome or TBP reveal the architecture of the different functional domains of the co-activator. An octamer of histone fold domains is found at the core of SAGA. This octamer, which deviates considerably from the symmetrical analogue forming the nucleosome, establishes a peripheral site for TBP binding where steric hindrance represses interaction with spurious DNA. The structures point to a mechanism for TBP delivery and release from SAGA that requires TFIIA and whose efficiency correlates with the affinity of DNA to TBP. These results provide a structural basis for understanding specific TBP delivery onto gene promoters and the role played by SAGA in regulating gene expression. The properties of the TBP delivery machine harboured by SAGA are compared with the TBP loading device present in the TFIID complex and show multiple similitudes.

Transcription of eukaryotic protein-coding genes involves co-activator complexes, including TFIID and SAGA. A new study has determined the first high-resolution cryo-EM structure of the human SAGA complex, and has implications for defining SAGA function during multiple stages of eukaryotic transcription.

Regulation of transcription is a tightly choreographed process. The establishment of RNA polymerase II promoter proximal pausing soon after transcription initiation and the release of Pol II into productive elongation are key regulatory processes that occur in early elongation. We describe the techniques and tools that have become available for the study of promoter proximal pausing and their utility for future experiments. We then provide an overview of the factors and interactions that govern a multipartite pausing process and address emerging questions surrounding the mechanism of RNA polymerase II's subsequent advancement into the gene body. Finally, we address remaining controversies and future areas of study.

SUMMARYTranscription initiation by RNA polymerase II (Pol II) requires preinitiation complex (PIC) assembly at gene promoters. In the dynamic nucleus where thousands of promoters are broadly distributed in chromatin, it is unclear how ten individual components converge on any target to establish the PIC. Here, we use live-cell, single-molecule tracking in S. cerevisiae to document subdiffusive, constrained exploration of the nucleoplasm by PIC components and Mediator’s key functions in guiding this process. On chromatin, TBP, Mediator, and Pol II instruct assembly of a short-lived PIC, which occurs infrequently but efficiently at an average promoter where initiation-coupled disassembly may occur within a few seconds. Moreover, PIC exclusion by nucleosome encroachment underscores regulated promoter accessibility by chromatin remodeling. Thus, coordinated nuclear exploration and recruitment to accessible targets underlies dynamic PIC establishment in yeast. Collectively, our study provides a global spatio-temporal model for transcription initiation in live cells.

ChEC-seq is a method used to identify protein-DNA interactions across a genome. It involves fusing micrococcal nuclease (MNase) to a protein of interest. In principle, specific genome-wide interactions of the fusion protein with chromatin result in local DNA cleavages that can be mapped by DNA sequencing. ChEC-seq has been used to draw conclusions about broad gene-specificities of certain protein-DNA interactions. In particular, the transcriptional regulators SAGA, TFIID, and Mediator are reported to generally occupy the promoter/UAS of genes transcribed by RNA polymerase II in yeast. Here we compare published yeast ChEC-seq data performed with a variety of protein fusions across essentially all genes, and find high similarities with negative controls. We conclude that ChEC-seq patterning for SAGA, TFIID, and Mediator differ little from background at most promoter regions, and thus cannot be used to draw conclusions about broad gene specificity of these factors.

During oocyte growth, transcription is required to create RNA and protein reserves to achieve maternal competence. During this period, the general transcription factor TATA binding protein (TBP) is replaced by its paralogue, TBPL2 (TBP2 or TRF3), which is essential for RNA polymerase II transcription. We show that in oocytes TBPL2 does not assemble into a canonical TFIID complex. Our transcript analyses demonstrate that TBPL2 mediates transcription of oocyte-expressed genes, including mRNA survey genes, as well as specific endogenous retroviral elements. Transcription start site (TSS) mapping indicates that TBPL2 has a strong preference for TATA-like motif in core promoters driving sharp TSS selection, in contrast with canonical TBP/TFIID-driven TATA-less promoters that have broader TSS architecture. Thus, we show a role for the TBPL2/TFIIA complex in the establishment of the oocyte transcriptome by using a specific TSS recognition code., The vertebrate TATA-binding protein (TBP) paralogue (TBPL2) is only expressed in growing oocytes, where TBP is absent. Here the authors highlight a unique role for the TBPL2/TFIIA complex in the establishment of the oocyte transcriptome by using a specific TSS recognition code.

SUMMARY DOT1L, the only H3K79 methyltransferase in human cells and a homolog of the yeast Dot1, normally forms a complex with AF10, AF17 and ENL/AF9, is dysregulated in most of the cases of mixed lineage leukemia (MLL) and is believed to regulate transcriptional elongation without much evidence. Here we show that the depletion of DOT1L reduced the global occupancy without affecting the traveling ratio or the elongation rate of Pol II, suggesting it not a major elongation factor. An examination of general transcription factors (GTFs) binding revealed globally reduced TBP and TFIIA occupancies near promoters after DOT1L loss, pointing to a role in transcriptional initiation. Proteomic studies uncovered that DOT1L regulates transcriptional initiation likely by facilitating the recruitment of TFIID. Moreover, ENL, a DOT1L complex subunit with a known role in DOT1L recruitment, also regulates transcriptional initiation. Furthermore, DOT1L stimulates H2B monoubiquitination by limiting the recruitment of human SAGA complex, and the connection between Dot1/DOT1L and SAGA complex is conserved between yeast and human. These results advanced current understanding of roles of DOT1L complex in transcriptional regulation and MLL.

Although we have made significant progress, we still possess a limited understanding of how genomic and epigenomic information directs gene expression programs through sequence-specific transcription factors (TFs). Extensive research has settled on three general classes of TF targets in metazoans: promoter accessibility via chromatin regulation (e.g., SAGA), assembly of the general transcription factors on promoter DNA (e.g., TFIID), and recruitment of RNA polymerase (Pol) II (e.g., Mediator) to establish a transcription pre-initiation complex (PIC). Here we discuss TFs and their targets. We also place this in the context of our current work with Saccharomyces (yeast), where we find that promoters typically lack an architecture that supports TF function. Moreover, yeast promoters that support TF binding also display interactions with cofactors like SAGA and Mediator, but not TFIID. It is unknown to what extent all genes in metazoans require TFs and their cofactors.

Numerous transcription regulatory players have emerged in order to meet the increasing demand for transcriptional complexity in the eukaryotic branch of life. Being the first member of the pre-initiation complex to engage genes at their core promoters and accurately stabilize the RNA polymerase II (RNAPII) enzyme at the transcription start site (TSS), TFIID protein complex is key player in eukaryotic transcription initiation. The work described in this thesis examines comprehensively the evolutionary history of TFIID and places its origins in a common with the SAGA complex ancestor, dating prior to the last eukaryotic common ancestor (LECA). Through numerous duplication and sub-functionalization events, a gradual divergence of the two complexes is observed. Notably, while the core subunits of TFIID remain mostly conserved, dynamic gains and losses of epigenetic reader domains accompany the expansion of the eukaryotic branch, indicating continuous readjustment of the complex to the transcriptional demands and rules of different organisms. The structure of human TFIID is extensively analysed through a cell-based expression system coupled with affinity enrichment and label-free quantitative mass spectrometry analyses. Key mechanistic steps in the formation and dynamics of the complex were identified, including: 1) nuclear holo-TFIID assembly from discrete cytoplasmic submodules, 2) checkpoint role for the chaperonin complex, TRiC/CCT, during the formation of a central for TFIID integrity submodule, TAF5/TAF6/TAF9, 3) the atomic resolution of TAF5/TAF6/TAF9, revealing a novel TAF5/TAF9 interface with a crucial role in the release of newly-synthesized TAF5 from the TRiC/CCT, 4) disintegration of holo-TFIID once chromatin-bound to a primarily core-TFIID structure and a specific loss of TBP, as the protein engages other transcription players onto the DNA, and 5) a canonical holo-TFIID formation by its non-canonical TAF1L paralog, with integration efficiency dependent on the presence or absence of the canonical TAF1 subunit. Altogether, the work described in the thesis exemplifies comprehensive experimental and computational methods for the characterization of evolutionary and structural dynamics of large molecular machineries. Being central for the initiation of accurate eukaryotic transcription events, such an extensive picture of TFIID gives an opportunity to rationally place its structure and function in the context of the immense molecular network of eukaryotic transcription.

Assembling for transcription initiation Eukaryotic transcription initiation by RNA polymerase II (Pol II) requires the assembly of a preinitiation complex (PIC) on core promoters. The binding of TATA box–binding protein (TBP) to the TATA box promoter has been thought to be a general rule in PIC assembly and transcription initiation. However, most coding genes lack a TATA box, and nearly all Pol II–mediated gene transcription requires the TBP-containing multisubunit complex transcription factor IID (TFIID). Chen et al. determined the structures of human TFIID-based PIC in sequential assembly states and revealed that TFIID supports distinct PIC assembly on TATA-containing and TATA-lacking promoters. The finding resolves the long-standing mystery of how one set of general transcription machinery initiates transcription on diverse promoters. Science , this issue p. eaba8490

Basic helix-loop-helix (bHLH) proteins belong to a large family of transcription factors that are known to play important roles in cell proliferation, differentiation and oncogenesis. These proteins are structurally featured by a bHLH motif, which is responsible for protein dimerization and sequence-specific DNA binding (e.g., E-box). Recently we isolated a cDNA from a human liver library by a gene trapping method. Based on the Kozak rule, this cDNA encodes a protein with 415 amino acids, which is hereafter designated as CCAF. The objective of this thesis is to establish the molecular mass of this protein and to test the hypothesis that CCAF is a transcriptional modulator involving the regulation of cell cycle events. To establish the molecular mass, CCAF was in vitro translated with TNT reticulocyte lysate and analyzed by autoradiography. Addition of the CCAF cDNA to the reaction mixture yielded a single product with a molecular weight of 52 kDa. This mass is consistent with the estimated weight and suggests that the Kozak favorable sequence indeed harbors the codon for translation initiation. In order to determine whether CCAF undergoes posttranslational modifications, immunochemical experiments were performed. An antibody was raised against a peptide derived from CCAF and subjected to affinity chromatography. This antibody detected a 52-kDa protein in the CCAF cDNA transfected cells but not in the control cells. These results further support the notion that the functional CCAF is a 52-kDa protein and undergoes little post-translational modifications.

During oocyte growth, transcription is required to create RNA and protein reserves to achieve maternal competence. During this period, the general transcription factor TATA binding protein (TBP) is replaced by its paralogue, TBPL2 (TBP2 or TRF3), which is essential for RNA polymerase II transcription. We show that in oocytes TBPL2 does not assemble into a canonical TFIID complex. Our transcript analyses demonstrate that TBPL2 mediates transcription of oocyte-expressed genes, including mRNA survey genes, as well as specific endogenous retroviral elements. Transcription start site (TSS) mapping indicates that TBPL2 has a strong preference for TATA-like motif in core promoters driving sharp TSS selection, in contrast with canonical TBP/TFIID-driven TATA-less promoters that have broader TSS architecture. Thus, we show a role for the TBPL2/TFIIA complex in the establishment of the oocyte transcriptome by using a specific TSS recognition code.

Background The YEATS domain is a highly conserved protein structure that interacts with acetylated and crotonylated lysine residues in N-terminal tails of histones. The budding yeast genome encodes three YEATS domain proteins (Taf14, Yaf9, and Sas5) that are all the subunits of different complexes involved in histone acetylation, gene transcription, and chromatin remodeling. As the strains deficient in all these three genes are inviable, it has been proposed that the YEATS domain is essential in yeast. In this study we investigate in more detail the requirement of YEATS domain proteins for yeast survival and the possible roles of Taf14 YEATS domain in the regulation of gene transcription. Results We found that YEATS domains are not essential for the survival of Saccharomyces cerevisiae cells. Although the full deletion of all YEATS proteins is lethal in yeast, we show that the viability of cells can be restored by the expression of the YEATS-less version of Taf14 protein. We also explore the in vivo functions of Taf14 protein and show that the primary role of its YEATS domain is to stabilize the transcription pre-initiation complex (PIC). Our results indicate that Taf14-mediated interactions become crucial for PIC formation in rpb9Δ cells, where the recruitment of TFIIF to the PIC is hampered. Although H3 K9 residue has been identified as the interaction site of the Taf14 YEATS domain in vitro, we found that it is not the only interaction target in vivo. Conclusions Lethality of YEATS-deficient cells can be rescued by the expression of truncated Taf14 protein lacking the entire YEATS domain, indicating that the YEATS domains are not required for cell survival. The YEATS domain of Taf14 participates in PIC stabilization and acetylated/crotonylated H3K9 is not the critical target of the Taf14 YEATS domain in vivo.

RNA polymerase II (Pol II) transcribes all protein-coding genes and many noncoding RNAs in eukaryotic genomes. Although Pol II is a complex, 12-subunit enzyme, it lacks the ability to initiate transcription and cannot consistently transcribe through long DNA sequences. To execute these essential functions, an array of proteins and protein complexes interact with Pol II to regulate its activity. In this review, we detail the structure and mechanism of over a dozen factors that govern Pol II initiation (e.g., TFIID, TFIIH, and Mediator), pausing, and elongation (e.g., DSIF, NELF, PAF, and P-TEFb). The structural basis for Pol II transcription regulation has advanced rapidly in the past decade, largely due to technological innovations in cryoelectron microscopy. Here, we summarize a wealth of structural and functional data that have enabled a deeper understanding of Pol II transcription mechanisms; we also highlight mechanistic questions that remain unanswered or controversial.

TAF4b is a subunit of the TFIID complex that is highly expressed in the ovary and testis and required for mouse fertility. TAF4b-deficient male mice undergo a complex series of developmental defects that result in the inability to maintain long-term spermatogenesis. To decipher the transcriptional mechanisms upon which TAF4b functions in spermatogenesis, we used two-hybrid screening to identify a novel TAF4b-interacting transcriptional cofactor, ZFP628. Deletion analysis of both proteins reveals discrete and novel domains of ZFP628 and TAF4b protein that function to bridge their direct interaction in vitro. Moreover, coimmunoprecipitation of ZFP628 and TAF4b proteins in testis-derived protein extracts supports their endogenous association. Using CRISPR-Cas9, we disrupted the expression of ZFP628 in the mouse and uncovered a postmeiotic germ cell arrest at the round spermatid stage in the seminiferous tubules of the testis in ZFP628-deficient mice that results in male infertility. Coincident with round spermatid arrest, we find reduced mRNA expression of transition protein (Tnp1 and Tnp2) and protamine (Prm1 and Prm2) genes, which are critical for the specialized maturation of haploid male germ cells called spermiogenesis. These data delineate a novel association of two transcription factors, TAF4b and ZFP628, and identify ZFP628 as a novel transcriptional regulator of stage-specific spermiogenesis.

Transcription is a highly regulated process that supplies living cells with coding and non-coding RNA molecules. Failure to properly regulate transcription is associated with human pathologies, including cancers. RNA polymerase II is the enzyme complex that synthesizes messenger RNAs that are then translated into proteins. In spite of its complexity, RNA polymerase requires a plethora of general transcription factors to be recruited to the transcription start site as part of a large transcription pre-initiation complex, and to help it gain access to the transcribed strand of the DNA. This chapter reviews the structure and function of these eukaryotic transcription pre-initiation complexes, with a particular emphasis on two of its constituents, the multisubunit complexes TFIID and TFIIH. We also compare the overall architecture of the RNA polymerase II pre-initiation complex with those of RNA polymerases I and III, involved in transcription of ribosomal RNA and non-coding RNAs such as tRNAs and snRNAs, and discuss the general, conserved features that are applicable to all eukaryotic RNA polymerase systems.

The TSS region, which is also called core promoter, is a pre-requisite for the understanding how transcription by Pol II is controlled. Pol II is the core of the basal transcriptional machinery that contains a large number of general transcription factors, such as the TATA box binding protein (TBP), many of which are summarized as the TFIID complex. The TATA box is the prototype of a site-specific transcription factor binding site determining the position of Pol II on the TSS. However, genome-wide analysis showed that the majority of human genes use alternative binding sites for general transcription factors. Diversity and complexity of the transcriptome are based on that most genes have multiple TSS regions and that the TSS of many genes is not a single defined nucleotide. The basal transcriptional machinery interacts via another multi-protein complex of co-activators, termed the Mediator complex, with a large variation of transcription factors. In parallel, the Mediator complex coordinates the action of co-activators and co-repressors, some of which are chromatin modifiers.

International audience; Transcript buffering involves reciprocal adjustments between overall rates in mRNA synthesis and degradation to maintain similar cellular concentrations of mRNAs. This phenomenon was first discovered in yeast and encompasses coordination between the nuclear and cytoplasmic compartments. Transcript buffering was revealed by novel methods for pulse labeling of RNA to determine in vivo synthesis and degradation rates. In this Perspective, we discuss the current knowledge of transcript buffering. Emphasis is placed on the future challenges to determine the nature and directionality of the buffering signals, the generality of transcript buffering beyond yeast, and the molecular mechanisms responsible for this balancing.

Human T-cell lymphotropic virus type 1 (HTLV-1) Tax oncoprotein is required for viral gene expression. Tax transactivates the viral promoter by recruiting specific transcription factors but also by interfering with general transcription factors involved in the preinitiation step, such as TFIIA and TFIID. However, data are lacking regarding Tax interplay with TFIIH, which intervenes during the last step of preinitiation. We previously reported that XPB, the TFIIH subunit responsible for promoter opening and promoter escape, is required for Tat-induced human-immunodeficiency virus promoter transactivation. Here, we investigated whether XPB may also play a role in HTLV-1 transcription. We report that Tax and XPB directly interact in vitro and that endogenous XPB produced by HTLV-1-infected T cells binds to Tax and is recruited on proviral LTRs. In contrast, XPB recruitment at the LTR is not detected in Tax-negative HTLV-1-infected T cells and is strongly reduced when Tax-induced HTLV-1 LTR transactivation is blocked. XPB overexpression does not affect basal HTLV-1 promoter activation but enhances Tax-mediated transactivation in T cells. Conversely, downregulating XPB strongly reduces Tax-mediated transactivation. Importantly, spironolactone (SP)-mediated inhibition of LTR activation can be rescued by overexpressing XPB but not XPD, another TFIIH subunit. Furthermore, an XPB mutant defective for the ATPase activity responsible for promoter opening does not show rescue of the effect of SP. Finally, XPB downregulation reduces viability of Tax-positive but not Tax-negative HTLV-1-transformed T cell lines. These findings reveal that XPB is a novel cellular cofactor hijacked by Tax to facilitate HTLV-1 transcription., IMPORTANCE HTLV-1 is considered the most potent human oncovirus and is also responsible for severe inflammatory disorders. HTLV-1 transcription is undertaken by RNA polymerase II and is controlled by the viral oncoprotein Tax. Tax transactivates the viral promoter first via the recruitment of CREB and its cofactors to the long terminal repeat (LTR). However, how Tax controls subsequent steps of the transcription process remains unclear. In this study, we explore the link between Tax and the XPB subunit of TFIIH that governs, via its ATPase activity, the promoter-opening step of transcription. We demonstrate that XPB is a novel physical and functional partner of Tax, recruited on HTLV-1 LTR, and required for viral transcription. These findings extend the mechanism of Tax transactivation to the recruitment of TFIIH and reinforce the link between XPB and transactivator-induced viral transcription.

In eukaryotes, gene expression is performed by three RNA polymerases that are targeted to promoters by molecular complexes. A unique common factor, the TATA-box binding protein (TBP), is thought to serve as a platform to assemble pre-initiation complexes competent for transcription. Herein, we describe a novel molecular mechanism of nutrient regulation of gene transcription by dynamic O-GlcNAcylation of TBP. We show that O-GlcNAcylation at T114 of TBP blocks its interaction with BTAF1, hence the formation of the B-TFIID complex, and its dynamic cycling on and off of DNA. Transcriptomic and metabolomic analyses of TBP(T114A) CRISPR/Cas9 edited cells showed that loss of O-GlcNAcylation at T114 increases TBP binding to BTAF1 and directly impacts expression of 408 genes. Lack of O-GlcNAcylation at T114 is associated with a striking reprogramming of cellular metabolism induced by a profound modification of the transcriptome, leading to gross alterations in lipid storage.

TAF6, bearing the histone H4-like histone-fold domain (HFD), is a subunit of the core TAF module in TFIID and SAGA transcriptional regulatory complexes. We isolated and characterized several yeast TAF6 mutants bearing amino acid substitutions in the HFD, the middle region or the HEAT repeat domain. The TAF6 mutants were highly defective for transcriptional activation by the Gcn4 and Gal4 activators. CHIP assays showed that the TAF6-HFD and the TAF6-HEAT domain mutations independently abrogated the promoter occupancy of TFIID and SAGA complex in vivo. We employed genetic and biochemical assays to identify the relative contributions of the TAF6 HFD and HEAT domains. First, the temperature-sensitive phenotype of the HEAT domain mutant was suppressed by overexpression of the core TAF subunits TAF9 and TAF12, as well as TBP. The HFD mutant defect, however, was suppressed by TAF5 but not by TAF9, TAF12 or TBP. Second, the HEAT mutant but not the HFD mutant was defective for growth in the presence of transcription elongation inhibitors. Third, coimmunoprecipitation assays using yeast cell extracts indicated that the specific TAF6 HEAT domain residues are critical for the interaction of core TAF subunits with the SAGA complex but not with TFIID. The specific HFD residues in TAF6, although required for heterodimerization between TAF6 and TAF9 recombinant proteins, were dispensable for association of the core TAF subunits with TFIID and SAGA in yeast cell extracts. Taken together, the results of our studies have uncovered the non-overlapping requirement of the evolutionarily conserved HEAT domain and the HFD in TAF6 for transcriptional activation.

RNA polymerase (Pol) III transcribes small untranslated RNAs that are essential for cellular homeostasis and growth. Its activity is regulated by inactivation of tumor suppressor proteins and overexpression of the oncogene c-MYC, but the concerted action of these tumor-promoting factors on Pol III transcription has not yet been assessed. In order to comprehensively analyse the regulation of Pol III transcription during tumorigenesis we employ a model system that relies on the expression of five genetic elements to achieve cellular transformation. Expression of these elements in six distinct transformation intermediate cell lines leads to the inactivation of TP53, RB1, and protein phosphatase 2A, as well as the activation of RAS and the protection of telomeres by TERT, thereby conducting to full tumoral transformation of IMR90 fibroblasts. Transformation is accompanied by moderately enhanced levels of a subset of Pol III-transcribed RNAs (7SK; MRP; H1). In addition, mRNA and/or protein levels of several Pol III subunits and transcription factors are upregulated, including increased protein levels of TFIIIB and TFIIIC subunits, of SNAPC1 and of Pol III subunits. Strikingly, the expression of POLR3G and of SNAPC1 is strongly enhanced during transformation in this cellular transformation model. Collectively, our data indicate that increased expression of several components of the Pol III transcription system accompanied by a 2-fold increase in steady state levels of a subset of Pol III RNAs is sufficient for sustaining tumor formation.

In previous studies, we showed that the insulator protein Su(Hw) containing zinc finger domains interacts with the ENY2 protein and recruits the ENY2-containing complexes on Su(Hw)-dependent insulators, participating in the regulation of transcription and in the positioning of replication origins. Here, we found interaction between ENY2 and CG9890 protein, which also contains zinc finger domains. The interaction between ENY2 and CG9890 was confirmed. It was established that CG9890 protein is localized in the nucleus and interacts with the SAGA, ORC, dSWI/SNF, TFIID, and THO protein complexes.

TFIID is a critical component of the eukaryotic transcription pre-initiation complex (PIC) required for the recruitment of RNA Pol II to the start site of protein-coding genes. Within the PIC, TFIID’s role is to recognize and bind core promoter sequences and recruit the rest of the PIC components. Due to its size and its conformational complexity, TFIID poses a serious challenge for structural characterization. The limited amounts of purified TFIID that can be obtained by present methods of purification from endogenous sources has limited structural studies to cryo-EM visualization, which requires very small amounts of sample. Previous cryo-EM studies have shed light on how the extreme conformational flexibility of TFIID is involved in core promoter DNA binding. Recent progress in cryo-EM methodology has facilitated a parallel progress in the study of human TFIID, leading to an improvement in resolution and the identification of the structural elements in the complex directly involved in DNA interaction. While many questions remain unanswered, the present structural knowledge of human TFIID suggests a mechanism for the sequential engagement with different core promoter sequences and how it could be influenced by regulatory factors.

The general transcription factor TFIID is a multiprotein complex that is essential for specific transcription initiation by RNA polymerase II. It is composed of the TATA box-binding protein (TBP) and ~13 different TBP-associated factors (TAFs). Purification of TFIID free of other general transcription factors and coactivators is essential to analyze the transcription regulatory mechanisms in reconstituted systems in vitro. A breakthrough in TFIID purification was the generation of HeLa cell lines that express a FLAG epitope-tagged TBP subunit and immunopurification protocols with monoclonal anti-FLAG antibodies. Purification of TFIID from HeLa nuclear extracts generally required a two-step purification procedure involving phosphocellulose P11 chromatography followed by anti-flag M2 affinity purification (Chiang et al., 1993; Ge et al., 1996) [1,2]. Here we show first that the MED26 (CRSP70) coactivator subunit of Mediator co-purifies with TFIID in the above two-step protocol and interacts strongly with TFIID under high salt conditions. We further show that a MED26-free TFIID complex can be obtained by including a simple additional DE52 chromatography step following P11 fractionation. Thus, we demonstrate that MED26 strongly interacts with TFIID and recommend the use of a P11-DE52-M2 resin affinity three-step purification procedure to obtain MED26-free TFIID for analyzing Mediator-dependent transcription regulatory mechanisms in purified transcription systems in vitro.

Transcription As a critical transcription coactivator, the multisubunit Mediator complex binds RNA polymerase II (Pol II), facilitates preinitiation complex (PIC) assembly, and stimulates transcription and phosphorylation of the Pol II C-terminal domain (CTD). However, how these critical transcriptional events are coordinated by Mediator is not fully understood. Chen et al. determined the structures of human Mediator and Mediator-bound PIC in distinct conformational states, the latter of which represents a complete PIC-Mediator complex assembled on the 14-subunit transcription factor IID (TFIID). The structures show that Mediator undergoes reorganization during PIC-Mediator assembly, sandwiches and facilitates phosphorylation of Pol II CTD, and works with TFIID to organize TFIIH in PIC for transcription initiation. Science , abg0635, this issue p. [eabg0635][1] [1]: /lookup/doi/10.1126/science.abg0635

TFIID binds promoter DNA to recruit RNA polymerase II and other basal factors for transcription. Although the TATA-Binding Protein (TBP) subunit of TFIID is necessary and sufficient for in vitro transcription, the TBP-Associated Factor (TAF) subunits recognize downstream promoter elements, act as co-activators, and interact with nucleosomes. Here we show that transcription induces stable TAF binding to downstream promoter DNA, independent of upstream contacts, TBP, or other basal transcription factors. This transcription-dependent TAF complex promotes subsequent activator-independent transcription, and promoter response to TAF mutations in vivo correlates with the level of downstream, rather than overall, Taf1 crosslinking. We propose a new model in which TAFs function as reinitiation factors, accounting for the differential responses of promoters to various transcription factor mutations.