Search

TFIIH is an evolutionarily conserved complex that plays central roles in both RNA polymerase II (pol II) transcription and DNA repair. As an integral component of the pol II preinitiation complex, TFIIH regulates pol II enzyme activity in numerous ways. The TFIIH subunit XPB/Ssl2 is an ATP-dependent DNA translocase that stimulates promoter opening prior to transcription initiation. Crosslinking-mass spectrometry and cryo-EM results have shown a conserved interaction network involving XPB/Ssl2 and the C-terminal Hub region of the TFIIH p52/Tfb2 subunit, but the functional significance of specific residues is unclear. Here, we systematically mutagenized the HubA region of Tfb2 and screened for growth phenotypes in a TFB6 deletion background in Saccharomyces cerevisiae. We identified six lethal and 12 conditional mutants. Slow growth phenotypes of all but three conditional mutants were relieved in the presence of TFB6, thus identifying a functional interaction between Tfb2 HubA mutants and Tfb6, a protein that dissociates Ssl2 from TFIIH. Our biochemical analysis of Tfb2 mutants with severe growth phenotypes revealed defects in Ssl2 association, with similar results in human cells. Further characterization of these tfb2 mutant cells revealed defects in GAL gene induction, and reduced occupancy of TFIIH and pol II at GAL gene promoters, suggesting that functionally competent TFIIH is required for proper pol II recruitment to preinitiation complexes in vivo. Consistent with recent structural models of TFIIH, our results identify key residues in the p52/Tfb2 HubA domain that are required for stable incorporation of XPB/Ssl2 into TFIIH and for pol II transcription.

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.

Purpose of review Erythropoiesis is a hierarchical process by which hematopoietic stem cells give rise to red blood cells through gradual cell fate restriction and maturation. Deciphering this process requires the establishment of dynamic gene regulatory networks (GRNs) that predict the response of hematopoietic cells to signals from the environment. Although GRNs have historically been derived from transcriptomic data, recent proteomic studies have revealed a major role for posttranscriptional mechanisms in regulating gene expression during erythropoiesis. These new findings highlight the need to integrate proteomic data into GRNs for a refined understanding of erythropoiesis. Recent findings Here, we review recent proteomic studies that have furthered our understanding of erythropoiesis with a focus on quantitative mass spectrometry approaches to measure the abundance of transcription factors and cofactors during differentiation. Furthermore, we highlight challenges that remain in integrating transcriptomic, proteomic, and other omics data into a predictive model of erythropoiesis, and discuss the future prospect of single-cell proteomics. Summary Recent proteomic studies have considerably expanded our knowledge of erythropoiesis beyond the traditional transcriptomic-centric perspective. These findings have both opened up new avenues of research to increase our understanding of erythroid differentiation, while at the same time presenting new challenges in integrating multiple layers of information into a comprehensive gene regulatory model.

Unlike most other eukaryotes, Leishmania and other trypanosomatid protozoa have largely eschewed transcriptional control of gene expression; relying instead on post-transcriptional regulation of mRNAs derived from polycistronic transcription units (PTUs). In these parasites, a novel modified nucleotide base (β-D-glucopyranosyloxymethyluracil) known as J plays a critical role in ensuring that transcription termination occurs only at the end of each PTU, rather than at the polyadenylation sites of individual genes. To further understand the biology of J-associated processes, we used tandem affinity purification (TAP-tagging) and mass spectrometry to reveal proteins that interact with the glucosyltransferase performing the final step in J synthesis. These studies identified four proteins reminiscent of subunits in the PTW/PP1 complex that controls transcription termination in higher eukaryotes. Moreover, bioinformatic analyses identified the DNA-binding subunit of Leishmania PTW/PP1 as a novel J-binding protein (JBP3), which is also part of another complex containing proteins with domains suggestive of a role in chromatin modification/remodeling. Additionally, JBP3 associates (albeit transiently and/or indirectly) with the trypanosomatid equivalent of the PAF1 complex involved in regulation of transcription in other eukaryotes. Down-regulation of JBP3 expression levels in Leishmania resulted in a substantial increase in transcriptional read-through at the 3’ end of most PTUs. We propose that JBP3 recruits one or more of these complexes to the J-containing regions at the end of PTUs, where they halt progression of the RNA polymerase. This de-coupling of transcription termination from splicing of individual genes enables the parasites’ unique reliance on polycistronic transcription and post-transcriptional regulation of gene expression.ImportanceLeishmania parasites cause a variety of serious human diseases, with no effective vaccine and emerging resistance to current drug therapy. We have previously shown that a novel DNA base called J is critical for transcription termination at the ends of the polycistronic gene clusters that are a hallmark of Leishmania and related trypanosomatids. Here, we describe a new J-binding protein (JBP3) associated with three different protein complexes that are reminiscent to those involved in control of transcription in other eukaryotes. However, the parasite complexes have been reprogrammed to regulate transcription and gene expression in trypanosomatids differently than in the mammalian hosts, providing new opportunities to develop novel chemotherapeutic treatments against these important pathogens.

Significance Autism is a complex neurodevelopmental disorder whose causative mechanisms are unclear. Taking advantage of a unique cohort with recessively inherited autism, we identified six families with biallelic mutation of the neuronal-specific subunit of the BAF complex, ACTL6B (also known as BAF53b). Relative to all other genes, ACTL6B was the most statistically significant mutated gene in the recessive autism cohort. We describe autism-relevant phenotypes in human brain organoids and in mouse and fly models. We foresee the outcomes from this study will be the following: 1) a link between neuronal activity-dependent transcriptional repression and autism; 2) a characterization of mouse and fly models to study ACTL6B mutant autism; and 3) an understanding the role of ACTL6B and nBAF complexes in neuronal transcriptional regulation., Synaptic activity in neurons leads to the rapid activation of genes involved in mammalian behavior. ATP-dependent chromatin remodelers such as the BAF complex contribute to these responses and are generally thought to activate transcription. However, the mechanisms keeping such “early activation” genes silent have been a mystery. In the course of investigating Mendelian recessive autism, we identified six families with segregating loss-of-function mutations in the neuronal BAF (nBAF) subunit ACTL6B (originally named BAF53b). Accordingly, ACTL6B was the most significantly mutated gene in the Simons Recessive Autism Cohort. At least 14 subunits of the nBAF complex are mutated in autism, collectively making it a major contributor to autism spectrum disorder (ASD). Patient mutations destabilized ACTL6B protein in neurons and rerouted dendrites to the wrong glomerulus in the fly olfactory system. Humans and mice lacking ACTL6B showed corpus callosum hypoplasia, indicating a conserved role for ACTL6B in facilitating neural connectivity. Actl6b knockout mice on two genetic backgrounds exhibited ASD-related behaviors, including social and memory impairments, repetitive behaviors, and hyperactivity. Surprisingly, mutation of Actl6b relieved repression of early response genes including AP1 transcription factors (Fos, Fosl2, Fosb, and Junb), increased chromatin accessibility at AP1 binding sites, and transcriptional changes in late response genes associated with early response transcription factor activity. ACTL6B loss is thus an important cause of recessive ASD, with impaired neuron-specific chromatin repression indicated as a potential mechanism.

Summary Quantitative changes in transcription factor (TF) abundance regulate dynamic cellular processes, including cell fate decisions. Protein copy number provides information about the relative stoichiometry of TFs that can be used to determine how quantitative changes in TF abundance influence gene regulatory networks. In this protocol, we describe a targeted selected reaction monitoring (SRM)-based mass-spectrometry method to systematically measure the absolute protein concentration of nuclear TFs as human hematopoietic stem and progenitor cells differentiate along the erythropoietic lineage. For complete details on the use and execution of this protocol, please refer to Gillespie et al. (2020)., Graphical Abstract, Highlights • Protocol for absolute quantification of TFs in human erythropoiesis • Selected reaction monitoring mass-spectrometry parameters for each peptide • Validated SRM assays corresponding to >100 TFs • Copy number reveals the relative stoichiometries of TFs during erythropoiesis, Quantitative changes in transcription factor (TF) abundance regulate dynamic cellular processes, including cell fate decisions. Protein copy number provides information about the relative stoichiometry of TFs that can be used to determine how quantitative changes in TF abundance influence gene regulatory networks. In this protocol, we describe a targeted selected reaction monitoring (SRM)-based mass spectrometry method to systematically measure the absolute protein concentration of nuclear TFs as human hematopoietic stem and progenitor cells differentiate along the erythropoietic lineage.

Pyruvate dehydrogenase (PDH) is the gatekeeper enzyme into the tricarboxylic acid (TCA) cycle. Here we show that PARK7/DJ-1, a key familial Parkinson’s disease (PD) gene, is a pacemaker controlling PDH activity in CD4 regulatory T cells (Tregs). DJ-1 bound to PDH-E1 beta (PDHB), inhibiting the phosphorylation of PDH-E1 alpha (PDHA), thus promoting PDH activity and oxidative phosphorylation (OXPHOS). Dj-1 depletion impaired Treg proliferation and cellularity maintenance in older mice, increasing the severity during the remission phase of experimental autoimmune encephalomyelitis (EAE). The compromised proliferation and differentiation of Tregs in Dj-1 knockout mice were caused via regulating PDH activity. These findings provide novel insight into the already complicated regulatory machinery of the PDH complex and demonstrate that the DJ-1-PDHB axis represents a potent target to maintain Treg homeostasis, which is dysregulated in many complex diseases.

SummaryDynamic cellular processes such as differentiation are driven by changes in the abundances of transcription factors (TFs). Yet, despite years of studies we still do not know the protein copy number of TFs in the nucleus. Here, by determining the absolute abundances of 103 TFs and co-factors during the course of human erythropoiesis, we provide a dynamic and quantitative scale for TFs in the nucleus. Furthermore, we establish the first Gene Regulatory Network of cell fate commitment that integrates temporal protein stoichiometry data with mRNA measurements. The model revealed quantitative imbalances in TFs cross-antagonistic relationships that underlie lineage determination. Finally, we made the surprising discovery that in the nucleus, corepressors are dramatically more abundant than coactivators at the protein, but not at the RNA level, with profound implications for understanding transcriptional regulation. These analyses provide a unique quantitative framework to understand transcriptional regulation of cell differentiation in a dynamic context.

TFIIH is a 10-subunit RNA polymerase II basal transcription factor with a dual role in DNA repair. TFIIH contains three enzymatic functions and over 30 conserved subdomains and topological regions. We systematically tested the function of these regions in three TFIIH core module subunits, i.e., Ssl1, Tfb4, and Tfb2, in the DNA translocase subunit Ssl2, and in the kinase module subunit Tfb3. Our results are consistent with previously predicted roles for the Tfb2 Hub, Ssl2 Lock, and Tfb3 Latch regions, with mutations in these elements typically having severe defects in TFIIH subunit association. We also found unexpected roles for other domains whose function had not previously been defined. First, the Ssl1-Tfb4 Ring domains are important for TFIIH assembly. Second, the Tfb2 Hub and HEAT domains have an unexpected role in association with Tfb3. Third, the Tfb3 Ring domain is important for association with many other TFIIH subunits. Fourth, a partial deletion of the Ssl1 N-terminal extension (NTE) domain inhibits TFIIH function without affecting subunit association. Finally, we used site-specific cross-linking to localize the Tfb3-binding surface on the Rad3 Arch domain. Our cross-linking results suggest that Tfb3 and Rad3 have an unusual interface, with Tfb3 binding on two opposite faces of the Arch.

Summary Mammalian SWI/SNF complexes are ATP-dependent chromatin remodeling complexes that regulate genomic architecture. Here, we present a structural model of the endogenously purified human canonical BAF complex bound to the nucleosome, generated using cryoelectron microscopy (cryo-EM), cross-linking mass spectrometry, and homology modeling. BAF complexes bilaterally engage the nucleosome H2A/H2B acidic patch regions through the SMARCB1 C-terminal α-helix and the SMARCA4/2 C-terminal SnAc/post-SnAc regions, with disease-associated mutations in either causing attenuated chromatin remodeling activities. Further, we define changes in BAF complex architecture upon nucleosome engagement and compare the structural model of endogenous BAF to those of related SWI/SNF-family complexes. Finally, we assign and experimentally interrogate cancer-associated hot-spot mutations localizing within the endogenous human BAF complex, identifying those that disrupt BAF subunit-subunit and subunit-nucleosome interfaces in the nucleosome-bound conformation. Taken together, this integrative structural approach provides important biophysical foundations for understanding the mechanisms of BAF complex function in normal and disease states.

Dynamic cellular processes such as differentiation are driven by changes in the abundances of transcription factors (TFs). However, despite years of studies, our knowledge about the protein copy number of TFs in the nucleus is limited. Here, by determining the absolute abundances of 103 TFs and co-factors during the course of human erythropoiesis, we provide a dynamic and quantitative scale for TFs in the nucleus. Furthermore, we establish the first gene regulatory network of cell fate commitment that integrates temporal protein stoichiometry data with mRNA measurements. The model revealed quantitative imbalances in TFs' cross-antagonistic relationships that underlie lineage determination. Finally, we made the surprising discovery that, in the nucleus, co-repressors are dramatically more abundant than co-activators at the protein level, but not at the RNA level, with profound implications for understanding transcriptional regulation. These analyses provide a unique quantitative framework to understand transcriptional regulation of cell differentiation in a dynamic context.

Hematopoiesis provides an accessible system for studying the principles underlying cell-fate decisions in stem cells. Proposed models of hematopoiesis suggest that quantitative changes in lineage-specific transcription factors (LS-TFs) underlie cell-fate decisions. However, evidence for such models is lacking as TF levels are typically measured via RNA expression rather than by analyzing temporal changes in protein abundance. Here, we used single-cell mass cytometry and absolute quantification by mass spectrometry to capture the temporal dynamics of TF protein expression in individual cells during human erythropoiesis. We found that LS-TFs from alternate lineages are co-expressed, as proteins, in individual early progenitor cells and quantitative changes of LS-TFs occur gradually rather than abruptly to direct cell-fate decisions. Importantly, upregulation of a megakaryocytic TF in early progenitors is sufficient to deviate cells from an erythroid to a megakaryocyte trajectory, showing that quantitative changes in protein abundance of LS-TFs in progenitors can determine alternate cell fates.

Mammalian SWI/SNF (mSWI/SNF) ATP-dependent chromatin remodeling complexes are multi-subunit molecular machines that play vital roles in regulating genomic architecture and are frequently disrupted in human cancer and developmental disorders. To date, the modular organization and pathways of assembly of these chromatin regulators remain unknown, presenting a major barrier to structural and functional determination. Here, we elucidate the architecture and assembly pathway across three classes of mSWI/SNF complexes—canonical BRG1/BRM-associated factor (BAF), polybromo-associated BAF (PBAF), and newly defined ncBAF complexes—and define the requirement of each subunit for complex formation and stability. Using affinity purification of endogenous complexes from mammalian and Drosophila cells coupled with cross-linking mass spectrometry (CX-MS) and mutagenesis, we uncover three distinct and evolutionarily conserved modules, their organization, and the temporal incorporation of these modules into each complete mSWI/SNF complex class. Finally, we map human disease-associated mutations within subunits and modules, defining specific topological regions that are affected upon subunit perturbation.

Hematopoiesis provides an accessible system for studying the principles underlying cell-fate decisions in stem cells. Proposed models of hematopoiesis suggest that quantitative changes in lineage-specific transcription factors (LS-TFs) underlie cell-fate decisions. However, evidence for such models is lacking as TFs levels are typically measured via RNA expression rather than by analysing protein abundance. Here, we used single-cell mass-cytometry and absolute quantification by mass-spectrometry to capture the temporal dynamics of TF protein expression in individual cells during human erythropoiesis. We found that LS-TFs from alternate lineages are co-expressed, as proteins, in individual early progenitor cells and quantitative changes of LF-TFs occur gradually, rather than abruptly to direct cell-fate decisions. Importantly, upregulation of a megakaryocytic TF in early progenitors is sufficient to deviate cells from an erythroid to a megakaryocyte trajectory showing that quantitative changes in protein abundance of LS-TFs in progenitors can determine alternate cell-fates.

LXR–cofactor complexes activate the gene expression program responsible for cholesterol efflux in macrophages. Inflammation antagonizes this program, resulting in foam cell formation and atherosclerosis; however, the molecular mechanisms underlying this antagonism remain to be fully elucidated. We use promoter enrichment‐quantitative mass spectrometry (PE‐QMS) to characterize the composition of gene regulatory complexes assembled at the promoter of the lipid transporter Abca1 following downregulation of its expression. We identify a subset of proteins that show LXR ligand‐ and binding‐dependent association with the Abca1 promoter and demonstrate they differentially control Abca1 expression. We determine that NCOA5 is linked to inflammatory Toll‐like receptor (TLR) signaling and establish that NCOA5 functions as an LXR corepressor to attenuate Abca1 expression. Importantly, TLR3–LXR signal crosstalk promotes recruitment of NCOA5 to the Abca1 promoter together with loss of RNA polymerase II and reduced cholesterol efflux. Together, these data significantly expand our knowledge of regulatory inputs impinging on the Abca1 promoter and indicate a central role for NCOA5 in mediating crosstalk between pro‐inflammatory and anti‐inflammatory pathways that results in repression of macrophage cholesterol efflux.

Managing trade‐offs through gene regulation is believed to confer resilience to a microbial community in a fluctuating resource environment. To investigate this hypothesis, we imposed a fluctuating environment that required the sulfate‐reducer Desulfovibrio vulgaris to undergo repeated ecologically relevant shifts between retaining metabolic independence (active capacity for sulfate respiration) and becoming metabolically specialized to a mutualistic association with the hydrogen‐consuming Methanococcus maripaludis. Strikingly, the microbial community became progressively less proficient at restoring the environmentally relevant physiological state after each perturbation and most cultures collapsed within 3–7 shifts. Counterintuitively, the collapse phenomenon was prevented by a single regulatory mutation. We have characterized the mechanism for collapse by conducting RNA‐seq analysis, proteomics, microcalorimetry, and single‐cell transcriptome analysis. We demonstrate that the collapse was caused by conditional gene regulation, which drove precipitous decline in intracellular abundance of essential transcripts and proteins, imposing greater energetic burden of regulation to restore function in a fluctuating environment.

Faithful repair of DNA double-strand breaks by homologous recombination is crucial to maintain functional genomes. The major Escherichia coli pathway of DNA break repair requires RecBCD enzyme, a complex protein machine with multiple activities. Upon encountering a Chi recombination hotspot (5' GCTGGTGG 3') during DNA unwinding, RecBCD's unwinding, nuclease, and RecA-loading activities change dramatically, but the physical basis for these changes is unknown. Here, we identify, during RecBCD's DNA unwinding, two Chi-stimulated conformational changes involving RecC. One produced a marked, long-lasting, Chi-dependent increase in protease sensitivity of a small patch, near the Chi recognition domain, on the solvent-exposed RecC surface. The other change was identified by crosslinking of an artificial amino acid inserted in this RecC patch to RecB. Small-angle X-ray scattering analysis confirmed a major conformational change upon binding of DNA to the enzyme and is consistent with these two changes. We propose that, upon DNA binding, the RecB nuclease domain swings from one side of RecC to the other; when RecBCD encounters Chi, the nuclease domain returns to its initial position determined by crystallography, where it nicks DNA exiting from RecC and loads RecA onto the newly generated 3'-ended single-stranded DNA during continued unwinding; a crevice between RecB and RecC increasingly narrows during these steps. This model provides a physical basis for the intramolecular "signal transduction" from Chi to RecC to RecD to RecB inferred previously from genetic and enzymatic analyses, and it accounts for the enzymatic changes that accompany Chi's stimulation of recombination.

Blood is an ideal window for viewing our health and disease status. Because blood circulates throughout the entire body and carries secreted, shed, and excreted signature proteins from every organ and tissue type, it is thus possible to use the blood proteome to achieve a comprehensive assessment of multiple-organ physiology and pathology. To date, the blood proteome has been frequently examined for diseases of individual organs; studies on compound insults impacting multiple organs are, however, elusive. We believe that a characterization of peripheral blood for organ-specific proteins affords a powerful strategy to allow early detection, staging, and monitoring of diseases and their treatments at a whole-body level. In this paper we test this hypothesis by examining a mouse model of acetaminophen (APAP)-induced hepatic and extra-hepatic toxicity. We used a glycocapture-assisted global quantitative proteomics (gagQP) approach to study serum proteins and validated our results using Western blot. We discovered in mouse sera both hepatic and extra-hepatic organ-specific proteins. From our validation, it was determined that selected organ-specific proteins had changed their blood concentration during the course of toxicity development and recovery. Interestingly, the peak responding time of proteins specific to different organs varied in a time-course study. The collected molecular information shed light on a complex, dynamic, yet interweaving, multiorgan-enrolled APAP toxicity. The developed technique as well as the identified protein markers is translational to human studies. We hope our work can broaden the utility of blood proteomics in diagnosis and research of the whole-body response to pathogenic cues.

Regulation of gene expression involves the orchestrated interaction of a large number of proteins with transcriptional regulatory elements in the context of chromatin. Our understanding of gene regulation is limited by the lack of a protein measurement technology that can systematically detect and quantify the ensemble of proteins associated with the transcriptional regulatory elements of specific genes. Here, we introduce a set of selected reaction monitoring (SRM) assays for the systematic measurement of 464 proteins with known or suspected roles in transcriptional regulation at RNA polymerase II transcribed promoters in Saccharomyces cerevisiae . Measurement of these proteins in nuclear extracts by SRM permitted the reproducible quantification of 42% of the proteins over a wide range of abundances. By deploying the assay to systematically identify DNA binding transcriptional regulators that interact with the environmentally regulated FLO11 promoter in cell extracts, we identified 15 regulators that bound specifically to distinct regions along ∼600 bp of the regulatory sequence. Importantly, the dataset includes a number of regulators that have been shown to either control FLO11 expression or localize to these regulatory regions in vivo. We further validated the utility of the approach by demonstrating that two of the SRM-identified factors, Mot3 and Azf1, are required for proper FLO11 expression. These results demonstrate the utility of SRM-based targeted proteomics to guide the identification of gene-specific transcriptional regulators.

Perturbations to mammalian SWI/SNF (mSWI/SNF or BAF) complexes contribute to more than 20% of human cancers, with driving roles first identified in malignant rhabdoid tumor, an aggressive pediatric cancer characterized by biallelic inactivation of the core BAF complex subunit SMARCB1 (BAF47). However, the mechanism by which this alteration contributes to tumorigenesis remains poorly understood. We find that BAF47 loss destabilizes BAF complexes on chromatin, absent significant changes in complex assembly or integrity. Rescue of BAF47 in BAF47-deficient sarcoma cell lines results in increased genome-wide BAF complex occupancy, facilitating widespread enhancer activation and opposition of Polycomb-mediated repression at bivalent promoters. We demonstrate differential regulation by two distinct mSWI/SNF assemblies, BAF and PBAF complexes, enhancers and promoters, respectively, suggesting that each complex has distinct functions that are perturbed upon BAF47 loss. Our results demonstrate collaborative mechanisms of mSWI/SNF-mediated gene activation, identifying functions that are co-opted or abated to drive human cancers and developmental disorders.

The kinetochore is a macromolecular complex that controls chromosome segregation and cell cycle progression. When sister kinetochores make bioriented attachments to microtubules from opposite poles, the spindle checkpoint is silenced. Biorientation and the spindle checkpoint are regulated by a balance between the Ipl1/Aurora B protein kinase and the opposing activity of protein phosphatase I (PP1). However, little is known about the regulation of PP1 localization and activity at the kinetochore. Here, we developed a method to purify centromere-bound kinetochores and used quantitative proteomics to identify the Fin1 protein as a PP1 regulatory subunit. The Fin1/PP1 complex is regulated by phosphorylation and 14–3–3 protein binding. When Fin1 is mislocalized, bipolar spindles fail to assemble but the spindle checkpoint is inappropriately silenced due to PP1 activity. These data suggest that Fin1 is a PP1 regulatory subunit whose spatial and temporal activity must be precisely controlled to ensure genomic stability.

The kinetochore is a macromolecular complex that controls chromosome segregation and cell cycle progression. When sister kinetochores make bioriented attachments to microtubules from opposite poles, the spindle checkpoint is silenced. Biorientation and the spindle checkpoint are regulated by a balance between the Ipl1/Aurora B protein kinase and the opposing activity of protein phosphatase I (PP1). However, little is known about the regulation of PP1 localization and activity at the kinetochore. Here, we developed a method to purify centromere-bound kinetochores and used quantitative proteomics to identify the Fin1 protein as a PP1 regulatory subunit. The Fin1/PP1 complex is regulated by phosphorylation and 14–3–3 protein binding. When Fin1 is mislocalized, bipolar spindles fail to assemble but the spindle checkpoint is inappropriately silenced due to PP1 activity. These data suggest that Fin1 is a PP1 regulatory subunit whose spatial and temporal activity must be precisely controlled to ensure genomic stability.

SummaryKinetochores are the macromolecular complexes that interact with microtubules to mediate chromosome segregation [1]. Accurate segregation requires that kinetochores make bioriented attachments to microtubules from opposite poles. Attachments between kinetochores and microtubules are monitored by the spindle checkpoint, a surveillance system that prevents anaphase until every pair of chromosomes makes proper bioriented attachments [2]. Checkpoint activity is correlated with the recruitment of checkpoint proteins to the kinetochore [1]. Mps1 is a conserved protein kinase that regulates segregation and the spindle checkpoint, but few of the targets that mediate its functions have been identified. Here, we show that Mps1 is the major kinase activity that copurifies with budding yeast kinetochore particles and identify the conserved Spc105/KNL-1/blinkin kinetochore protein as a substrate. Phosphorylation of conserved MELT motifs within Spc105 recruits the Bub1 protein to kinetochores, and this is reversed by protein phosphatase I (PP1). Spc105 mutants lacking Mps1 phosphorylation sites are defective in the spindle checkpoint and exhibit growth defects. Together, these data identify Spc105 as a key target of the Mps1 kinase and show that the opposing activities of Mps1 and PP1 regulate the kinetochore localization of the Bub1 protein.

The indirect recruitment (tethering) of estrogen receptors (ERs) to DNA through other DNA-bound transcription factors (e.g. activator protein 1) is an important component of estrogen-signaling pathways, but our understanding of the mechanisms of ligand-dependent activation in this pathway is limited. Using proteomic, genomic, and gene-specific analyses, we demonstrate that a large repertoire of DNA-binding transcription factors contribute to estrogen signaling through the tethering pathway. In addition, we define a set of endogenous genes for which ERα tethering through activator protein 1 (e.g. c-Fos) and cAMP response element-binding protein family members mediates estrogen responsiveness. Finally, we show that functional interplay between c-Fos and cAMP response element-binding protein 1 contributes to estrogen-dependent regulation through the tethering pathway. Based on our results, we conclude that ERα recruitment in the tethering pathway is dependent on the ligand-induced formation of transcription factor complexes that involves interplay between the transcription factors from different protein families.

This study identifies KLF3 as a transcriptional regulator of muscle genes and reveals a novel synergistic interaction between KLF3 and serum response factor (SRF). Using quantitative proteomics, KLF3 was identified as one of several candidate factors that recognize the MPEX control element in the Muscle creatine kinase (MCK) promoter. Chromatin immunoprecipitation analysis indicated that KLF3 is enriched at many muscle gene promoters (MCK, Myosin heavy chain IIa, Six4, Calcium channel receptor alpha-1, and Skeletal alpha-actin), and two KLF3 isoforms are upregulated during muscle differentiation. KLF3 and SRF physically associate and synergize in transactivating the MCK promoter independently of SRF binding to CArG motifs. The zinc finger and repression domains of KLF3 plus the MADS box and transcription activation domain of SRF are implicated in this synergy. Our results provide the first evidence of a role for KLF3 in muscle gene regulation and reveal an alternate mechanism for transcriptional regulation by SRF via its recruitment to KLF binding sites. Since both factors are expressed in all muscle lineages, SRF may regulate many striated- and smooth-muscle genes that lack known SRF control elements, thus further expanding the breadth of the emerging CArGome.

The regenerative capacity of muscle is regulated by p38-γ, which phosphorylates MyoD and leads to formation of a complex that represses myogenin transcription., The mitogen-activated protein kinase p38-γ is highly expressed in skeletal muscle and is associated with the dystrophin glycoprotein complex; however, its function remains unclear. After induced damage, muscle in mice lacking p38-γ generated significantly fewer myofibers than wild-type muscle. Notably, p38-γ-deficient muscle contained 50% fewer satellite cells that exhibited premature Myogenin expression and markedly reduced proliferation. We determined that p38-γ directly phosphorylated MyoD on Ser199 and Ser200, which results in enhanced occupancy of MyoD on the promoter of myogenin together with markedly decreased transcriptional activity. This repression is associated with extensive methylation of histone H3K9 together with recruitment of the KMT1A methyltransferase to the myogenin promoter. Notably, a MyoD S199A/S200A mutant exhibits markedly reduced binding to KMT1A. Therefore, p38-γ signaling directly induces the assembly of a repressive MyoD transcriptional complex. Together, these results establish a hitherto unappreciated and essential role for p38-γ signaling in positively regulating the expansion of transient amplifying myogenic precursor cells during muscle growth and regeneration.

Phosphorylation of the Ndc80 kinetochore protein by the Ipl1/Aurora B kinase reduces its microtubule binding activity in vitro. We found that kinetochore-bound Ndc80 is phosphorylated on Ipl1 sites in vivo, but this phosphorylation is not essential. Instead, we show that additional Ipl1 targets contribute to segregation and the spindle checkpoint.

The Saccharomyces cerevisiae kinase Bur1 is involved in coupling transcription elongation to chromatin modification, but not all important Bur1 targets in the elongation complex are known. Using a chemical genetics strategy wherein Bur1 kinase was engineered to be regulated by a specific inhibitor, we found that Bur1 phosphorylates the Spt5 C-terminal repeat domain (CTD) both in vivo and in isolated elongation complexes in vitro. Deletion of the Spt5 CTD or mutation of the Spt5 serines targeted by Bur1 reduces recruitment of the PAF complex, which functions to recruit factors involved in chromatin modification and mRNA maturation to elongating polymerase II (Pol II). Deletion of the Spt5 CTD showed the same defect in PAF recruitment as rapid inhibition of Bur1 kinase activity, and this Spt5 mutation led to a decrease in histone H3K4 trimethylation. Brief inhibition of Bur1 kinase activity in vivo also led to a significant decrease in phosphorylation of the Pol II CTD at Ser-2, showing that Bur1 also contributes to Pol II Ser-2 phosphorylation. Genetic results suggest that Bur1 is essential for growth because it targets multiple factors that play distinct roles in transcription.

In Arabidopsis thaliana, oxidant-induced signalling has been shown to utilize the mitogen-activated protein kinase (MAPK), AtMPK6. To identify proteins whose accumulation is altered by ozone in an AtMPK6-dependent manner we employed isotope-coded affinity tagging (ICAT) technology to investigate the impact of AtMPK6-suppression on the protein profiles in Arabidopsis both before (air control) and during continuous ozone (O(3)) fumigation (500 nL L(-1) for 8 h). Among the 150 proteins positively identified and quantified in the O(3)-treated plants, we identified thirteen proteins whose abundance was greater in the AtMPK6-suppressed genotype than in wild-type (WT). These include the antioxidant proteins, monodehydroascorbate reductase, peroxiredoxin Q, and glutathione reductase. A further eighteen proteins were identified whose abundance was lower in the ozone-treated AtMPK6-suppressed line relative to ozone-exposed WT plants. These predominantly comprised proteins involved in carbohydrate-, energy-, and amino acid metabolism, and tetrapyrrole biosynthesis. In control plants, five proteins increased, and nine proteins decreased in abundance in the AtMPK6-suppressed genotype compared to that of the WT, reflecting changes in the protein composition of plants that have AtMPK6 constitutively suppressed. Since a number of these proteins are part of the redox response pathway, and loss of AtMPK6 renders Arabidopsis more susceptible to oxidative stress, we propose that AtMPK6 plays a key role in the plant's overall ability to manage oxidative stress.

We identified a conserved sequence within the Muscle creatine kinase (MCK) promoter that is critical for high-level activity in skeletal and cardiac myocytes (MCK Promoter Element X [MPEX]). After selectively enriching for MPEX-binding factor(s) (MPEX-BFs), ICAT-based quantitative proteomics was used to identify MPEX-BF candidates, one of which was MAZ (Myc-associated zinc finger protein). MAZ transactivates the MCK promoter and binds the MPEX site in vitro, and chromatin immunoprecipitation analysis demonstrates enrichment of MAZ at the endogenous MCK promoter and other muscle gene promoters (Skeletal alpha-actin, Desmin, and alpha-Myosin heavy chain) in skeletal and cardiac myocytes. Consistent with its role in muscle gene transcription, MAZ transcripts and DNA-binding activity are upregulated during skeletal myocyte differentiation. Furthermore, MAZ was shown to bind numerous sequences (e.g., CTCCTCCC and CTCCACCC) that diverge from the GA box binding motif. Alternate motifs were identified in many muscle promoters, including Myogenin and MEF2C, and one motif was shown to be critical for Six4 promoter activity in both skeletal and cardiac myocytes. Interestingly, MAZ occupies and is able to transactivate the Six4 promoter in skeletal but not cardiac myocytes. Taken together, these findings are consistent with a previously unrecognized role for MAZ in muscle gene regulation.

Cohesin is required to prevent premature dissociation of sister chromatids after DNA replication. Although its role in chromatid cohesion is well established, the functional significance of cohesin's association with interphase chromatin is not clear. Using a quantitative proteomics approach, we show that the STAG1 (Scc3/SA1) subunit of cohesin interacts with the CCTC-binding factor CTCF bound to the c-myc insulator element. Both allele-specific binding of CTCF and Scc3/SA1 at the imprinted IGF2/H19 gene locus and our analyses of human DM1 alleles containing base substitutions at CTCF-binding motifs indicate that cohesin recruitment to chromosomal sites depends on the presence of CTCF. A large-scale genomic survey using ChIP-Chip demonstrates that Scc3/SA1 binding strongly correlates with the CTCF-binding site distribution in chromosomal arms. However, some chromosomal sites interact exclusively with CTCF, whereas others interact with Scc3/SA1 only. Furthermore, immunofluorescence microscopy and ChIP-Chip experiments demonstrate that CTCF associates with both centromeres and chromosomal arms during metaphase. These results link cohesin to gene regulatory functions and suggest an essential role for CTCF during sister chromatid cohesion. These results have implications for the functional role of cohesin subunits in the pathogenesis of Cornelia de Lange syndrome and Roberts syndromes.

Enhancers have been functionally described for >35 years, but the molecular principles underlying the integration of regulatory inputs to alternate gene enhancers used during mammalian organogenesis remain incompletely understood. Using a combination ofin vivoenhancer mapping and proteomics approaches, we have established that two distant and distinct early enhancers, each requiring different transcription complexes, are required for full activation of the gene encoding the pituitary lineage determining factor, Pit1. A transcription factor belonging to the “giant, multiple-homeodomain and zinc finger family,” Atbf1, serves as a novel pituitary regulator for one of the two required enhancers as shown by genetic andin vitroanalysis.

MLL-containing complexes methylate histone H3 at lysine 4 (H3K4) and have been implicated in the regulation of transcription. However, it is unclear how MLL complexes are targeted to specific gene loci. Here, we show that the MLL2 complex associates with the hematopoietic activator NF-E2 in erythroid cells and is important for H3K4 trimethylation and maximal levels of transcription at the beta-globin locus. Furthermore, recruitment of the MLL2 complex to the beta-globin locus is dependent upon NF-E2 and coincides spatio-temporally with NF-E2 binding during erythroid differentiation. Thus, a DNA-bound activator is important initially for guiding MLL2 to a particular genomic location. Interestingly, while the MLL2-associated subunit ASH2L is restricted to the beta-globin locus control region 38 kb upstream of the beta(maj)-globin gene, the MLL2 protein spreads across the beta-globin locus, suggesting a previously undefined mechanism by which an activator influences transcription and H3K4 trimethylation at a distance.

The histone H4 N-terminal tail has long been regarded as a major regulator in chromatin structure and function. Although the underlying mechanism has not been unraveled, an emerging body of evidence supports that H4 tail and its post-translational modification function as a recruitment motif for key factors required for proper regulation of chromatin transcription. To investigate these aspects, we have generated HeLa cell lines that constitutively express ectopic H4 tail domain for biochemical purification of proteins associated with H4 tail. We found that expressed H4 tails stably associate with sets of transcription regulatory factors and histone methyltransferases distinct from those that associate with histone H3 tails. Importantly, point mutations of four major lysine substrates to block cellular acetylation of ectopic H4 tail significantly inhibited the association of histone methyltransferases and sets of transcription-activating factors, supporting a major role of acetylation on recruitmentbased action of H4 tail during transcription. Further, our transcription analysis revealed that the proteins associated with wild-type/acetylated H4 tail, but not with mutant/unacetylated H4 tail, can enhance p300-dependent chromatin transcription. Taken together, these findings demonstrate novel roles for H4 tail and its acetylation in mediating recruitment of multiple regulatory factors that can change chromatin states for transcription regulation.

We present a robust and general shotgun glycoproteomics approach to comprehensively profile glycoproteins in complex biological mixtures. In this approach, glycopeptides derived from glycoproteins are enriched by selective capture onto a solid support using hydrazide chemistry followed by enzymatic release of the peptides and subsequent analysis by tandem mass spectrometry. The approach was validated using standard protein mixtures that resulted in a close to 100% capture efficiency. Our capture approach was then applied to microsomal fractions of the cisplatin-resistant ovarian cancer cell line IGROV-1/CP. With a Protein Prophet probability value greater than 0.9, we identified a total of 302 proteins with an average protein identification rate of 136 +/- 19 (n = 4) in a single linear quadrupole ion trap (LTQ) mass spectrometer nano-LC-MS experiment and a selectivity of 91 +/- 1.6% (n = 4) for the N-linked glycoconsensus sequence. Our method has several advantages. 1) Digestion of proteins initially into peptides improves the solubility of large membrane proteins and exposes all of the glycosylation sites to ensure equal accessibility to capture reagents. 2) Capturing glycosylated peptides can effectively reduce sample complexity and at the same time increase the confidence of MS-based protein identifications (more potential peptide identifications per protein). 3) The utility of sodium sulfite as a quencher in our capture approach to replace the solid phase extraction step in an earlier glycoprotein chemical capture approach for removing excess sodium periodate allows the overall capture procedure to be completed in a single vessel. This improvement minimizes sample loss, increases sensitivity, and makes our protocol amenable for high throughput implementation, a feature that is essential for biomarker identification and validation of a large number of clinical samples. 4) The approach is demonstrated here on the analysis of N-linked glycopeptides; however, it can be applied equally well to O-glycoprotein analysis.

Mass spectrometry-based quantitative proteomics has become an important component of biological and clinical research. Although such analyses typically assume that a protein's peptide fragments are observed with equal likelihood, only a few so-called 'proteotypic' peptides are repeatedly and consistently identified for any given protein present in a mixture. Using >600,000 peptide identifications generated by four proteomic platforms, we empirically identified >16,000 proteotypic peptides for 4,030 distinct yeast proteins. Characteristic physicochemical properties of these peptides were used to develop a computational tool that can predict proteotypic peptides for any protein from any organism, for a given platform, with >85% cumulative accuracy. Possible applications of proteotypic peptides include validation of protein identifications, absolute quantification of proteins, annotation of coding sequences in genomes, and characterization of the physical principles governing key elements of mass spectrometric workflows (e.g., digestion, chromatography, ionization and fragmentation).

International audience; DNA repair-deficient trichothiodystrophy (TTD) results from mutations in the XPD and XPB subunits of the DNA repair and transcription factor TFIIH. In a third form of DNA repair-deficient TTD, called group A, none of the nine subunits encoding TFIIH carried mutations; instead, the steady-state level of the entire complex was severely reduced. A new, tenth TFIIH subunit (TFB5) was recently identified in yeast. Here, we describe the identification of the human TFB5 ortholog and its association with human TFIIH. Microinjection of cDNA encoding TFB5 (GTF2H5, also called TTDA) corrected the DNA-repair defect of TTD-A cells, and we identified three functional inactivating mutations in this gene in three unrelated families with TTD-A. The GTF2H5 gene product has a role in regulating the level of TFIIH. The identification of a new evolutionarily conserved subunit of TFIIH implicated in TTD-A provides insight into TFIIH function in transcription, DNA repair and human disease.

Transcriptional regulatory element X (Trex) is a positive control site within the Muscle creatine kinase (MCK) enhancer. Cell culture and transgenic studies indicate that the Trex site is important for MCK expression in skeletal and cardiac muscle. After selectively enriching for the Trex-binding factor (TrexBF) using magnetic beads coupled to oligonucleotides containing either wild-type or mutant Trex sites, quantitative proteomics was used to identify TrexBF as Six4, a homeodomain transcription factor of the Six/sine oculis family, from a background of approximately 900 copurifying proteins. Using gel shift assays and Six-specific antisera, we demonstrated that Six4 is TrexBF in mouse skeletal myocytes and embryonic day 10 chick skeletal and cardiac muscle, while Six5 is the major TrexBF in adult mouse heart. In cotransfection studies, Six4 transactivates the MCK enhancer as well as muscle-specific regulatory regions of Aldolase A and Cardiac troponin C via Trex/MEF3 sites. Our results are consistent with Six4 being a key regulator of muscle gene expression in adult skeletal muscle and in developing striated muscle. The Trex/MEF3 composite sequence ([C/A]ACC[C/T]GA) allowed us to identify novel putative Six-binding sites in six other muscle genes. Our proteomics strategy will be useful for identifying transcription factors from complex mixtures using only defined DNA fragments for purification.

We describe an algorithm for the automated statistical analysis of protein abundance ratios (ASAPRatio) of proteins contained in two samples. Proteins are labeled with distinct stable-isotope tags and fragmented, and the tagged peptide fragments are separated by liquid chromatography (LC) and analyzed by electrospray ionization (ESI) tandem mass spectrometry (MS/MS). The algorithm utilizes the signals recorded for the different isotopic forms of peptides of identical sequence and numerical and statistical methods, such as Savitzky-Golay smoothing filters, statistics for weighted samples, and Dixon's test for outliers, to evaluate protein abundance ratios and their associated errors. The algorithm also provides a statistical assessment to distinguish proteins of significant abundance changes from a population of proteins of unchanged abundance. To evaluate its performance, two sets of LC-ESI-MS/MS data were analyzed by the ASAPRatio algorithm without human intervention, and the data were related to the expected and manually validated values. The utility of the ASAPRatio program was clearly demonstrated by its speed and the accuracy of the generated protein abundance ratios and by its capability to identify specific core components of the RNA polymerase II transcription complex within a high background of copurifying proteins.

The goal of quantitative proteomics is to determine the identity and relative quantity of each protein present in two or more complex protein samples. Here we describe a novel approach to quantitative proteomics. It is based on a highly accurate algorithm for the automated quantification of chromatographically fractionated, isotope-coded affinity-tagged peptides and MALDI quadrupole time-of-flight tandem mass spectrometry for their identification. The method is capable of detecting and selectively identifying those proteins within a complex mixture that show a difference in relative abundance. We demonstrate the effectiveness and the versatility of this approach in the analysis of a standard protein mixture, protein expression profiling in a human prostate cancer cell line model, and identification of the specific components of the multiprotein transcriptional machinery in S. cerevisiae.

The adaptation of sequences of chemical reactions to a solid-phase format has been essential to the automation, reproducibility, and efficiency of a number of biotechnological processes including peptide and oligonucleotide synthesis and sequencing. Here we describe a method for the site-specific, stable isotopic labeling of cysteinyl peptides in complex peptide mixtures through a solid-phase capture and release process, and the concomitant isolation of the labeled peptides. The recovered peptides were analyzed by microcapillary liquid chromatography and tandem mass spectrometry (microLC-MS/MS) to determine their sequences and relative quantities. The method was used to detect galactose-induced changes in protein abundance in the yeast Saccharomyces cerevisiae. A side-by-side comparison with the isotope-coded affinity tag (ICAT) method demonstrated that the solid-phase method for stable isotope tagging of peptides is comparatively simpler, more efficient, and more sensitive.

The conserved transcription coactivator SAGA is comprised of several modules that are involved in activator binding, TBP binding, histone acetylation (HAT) and deubiquitination (DUB). Crosslinking and mass spectrometry, together with genetic and biochemical analyses, were used to determine the molecular architecture of the SAGA-TBP complex. We find that the SAGA Taf and Taf-like subunits form a TFIID-like core complex at the center of SAGA that makes extensive interactions with all other SAGA modules. SAGA-TBP binding involves a network of interactions between subunits Spt3, Spt8, Spt20, and Spt7. The HAT and DUB modules are in close proximity, and the DUB module modestly stimulates HAT function. The large activator-binding subunit Tra1 primarily connects to the TFIID-like core via its FAT domain. These combined results were used to derive a model for the arrangement of the SAGA subunits and its interactions with TBP. Our results provide new insight into SAGA function in gene regulation, its structural similarity with TFIID, and functional interactions between the SAGA modules.

Core Factor (CF) is a conserved RNA polymerase (Pol) I general transcription factor comprising Rrn6, Rrn11 and the TFIIB-related subunit Rrn7. CF binds TATA-binding protein (TBP), Pol I and the regulatory factors Rrn3 and upstream activation factor. We used chemical cross-linking-MS to determine the molecular architecture of CF and its interactions with TBP. The CF subunits assemble through an interconnected network of interactions between five structural domains that are conserved in orthologous subunits of the human Pol I factor SL1. We validated the cross-linking-derived model through a series of genetic and biochemical assays. Our combined results show the architecture of CF and the functions of the CF subunits in assembly of the complex. We extend these findings to model how CF assembles into the Pol I preinitiation complex, providing new insight into the roles of CF, TBP and Rrn3.

We demonstrate an integrated approach to build, test, and refine a model of a cellular pathway, in which perturbations to critical pathway components are analyzed using DNA microarrays, quantitative proteomics, and databases of known physical interactions. Using this approach, we identify 997 messenger RNAs responding to 20 systematic perturbations of the yeast galactose-utilization pathway, provide evidence that approximately 15 of 289 detected proteins are regulated posttranscriptionally, and identify explicit physical interactions governing the cellular response to each perturbation. We refine the model through further iterations of perturbation and global measurements, suggesting hypotheses about the regulation of galactose utilization and physical interactions between this and a variety of other metabolic pathways.

High levels of gene transcription by RNA polymerase II depend on high rates of transcription initiation and reinitiation. Initiation requires recruitment of the complete transcription machinery to a promoter, a process facilitated by activators and chromatin remodelling factors. Reinitiation probably occurs through a different pathway. After initiation, a subset of the transcription machinery remains at the promoter, forming a platform for assembly of a second transcription complex. Here we describe the isolation of a reinitiation intermediate that includes transcription factors TFIID, TFIIA, TFIIH, TFIIE and Mediator. This intermediate can act as a scaffold for formation of a functional reinitiation complex. Formation of this scaffold is dependent on ATP and TFIIH. The scaffold is stabilized in the presence of the activator Gal4-VP16, but not Gal4-AH, suggesting a new role for some activators and Mediator in promoting high levels of transcription.

Assembly and activity of yeast RNA polymerase II (Pol II) preinitiation complexes (PIC) was investigated with an immobilized promoter assay and extracts made from wild-type cells and from cells containing conditional mutations in components of the Pol II machinery. We describe the following findings: (1) In one step, TFIID and TFIIA assemble at the promoter independently of holoenzyme. In another step, holoenzyme is recruited to the promoter. Mutations in the CTD of Pol II, Srb2, Srb4, and Srb5, and two mutations in TFIIB disrupt recruitment of all holoenzyme components tested without affecting TFIID and TFIIA recruitment. These results indicate that the stepwise assembly pathway is blocked after TFIID/TFIIA binding. (2) Both the Gal4–AH and Gal4–VP16 activators stimulate formation of active PICs by increasing the extent of PIC formation. The Gal4–AH activator stimulated PIC formation by enhancing the binding of TFIID and TFIIA, whereas Gal4–VP16 could enhance the recruitment of TFIID, TFIIA, and holoenzyme. (3) Extracts deficient in TFIIA activity showed reduced assembly of all PIC components. These and other results suggest that TFIIA acts at an early step by enhancing the stable recruitment of TFIID. (4) An extract containing the TFIIB mutant E62G, had no defect in PIC formation, but had a severe defect in transcription. Similarly, mutation of the TATA box reduced PIC formation only two- to fourfold, but severely compromised transcription. These results demonstate an involvement of TFIIB and the TATA box in one or more steps after recruitment of factors to the promoter.