Architecture of the Human and Yeast General Transcription and DNA Repair Factor TFIIH.

TFIIH is essential for both RNA polymerase II transcription and DNA repair, and mutations in TFIIH can result in human disease. Here, we determine the molecular architecture of human and yeast TFIIH by an integrative approach using chemical crosslinking/mass spectrometry (CXMS) data, biochemical analyses, and previously published electron microscopy maps. We identified four new conserved "topological regions" that function as hubs for TFIIH assembly and more than 35 conserved topological features within TFIIH, illuminating a network of interactions involved in TFIIH assembly and regulation of its activities. We show that one of these conserved regions, the p62/Tfb1 Anchor region, directly interacts with the DNA helicase subunit XPD/Rad3 in native TFIIH and is required for the integrity and function of TFIIH. We also reveal the structural basis for defects in patients with xeroderma pigmentosum and trichothiodystrophy, with mutations found at the interface between the p62 Anchor region and the XPD subunit.

Regulation of Mec1 kinase activity by the SWI/SNF chromatin remodeling complex.

ATP-dependent chromatin remodeling complexes alter chromatin structure through interactions with chromatin substrates such as DNA, histones, and nucleosomes. However, whether chromatin remodeling complexes have the ability to regulate nonchromatin substrates remains unclear. Saccharomyces cerevisiae checkpoint kinase Mec1 (ATR in mammals) is an essential master regulator of genomic integrity. Here we found that the SWI/SNF chromatin remodeling complex is capable of regulating Mec1 kinase activity. In vivo, Mec1 activity is reduced by the deletion of Snf2, the core ATPase subunit of the SWI/SNF complex. SWI/SNF interacts with Mec1, and cross-linking studies revealed that the Snf2 ATPase is the main interaction partner for Mec1. In vitro, SWI/SNF can activate Mec1 kinase activity in the absence of chromatin or known activators such as Dpb11. The subunit requirement of SWI/SNF-mediated Mec1 regulation differs from that of SWI/SNF-mediated chromatin remodeling. Functionally, SWI/SNF-mediated Mec1 regulation specifically occurs in S phase of the cell cycle. Together, these findings identify a novel regulator of Mec1 kinase activity and suggest that ATP-dependent chromatin remodeling complexes can regulate nonchromatin substrates such as a checkpoint kinase.

TAF8 regions important for TFIID lobe B assembly or for TAF2 interactions are required for embryonic stem cell survival.

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 Architecture of Trypanosoma brucei editosomes.

Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei Editing is catalyzed by three distinct ∼20S editosomes that have a common set of 12 proteins, but are typified by mutually exclusive RNase III endonucleases with distinct cleavage specificities and unique partner proteins. Previous studies identified a network of protein-protein interactions among a subset of common editosome proteins, but interactions among the endonucleases and their partner proteins, and their interactions with common subunits were not identified. Here, chemical cross-linking and mass spectrometry, comparative structural modeling, and genetic and biochemical analyses were used to define the molecular architecture and subunit organization of purified editosomes. We identified intra- and interprotein cross-links for all editosome subunits that are fully consistent with editosome protein structures and previously identified interactions, which we validated by genetic and biochemical studies. The results were used to create a highly detailed map of editosome protein domain proximities, leading to identification of molecular interactions between subunits, insights into the functions of noncatalytic editosome proteins, and a global understanding of editosome architecture.

Regulation of the Rhp26ERCC6/CSB chromatin remodeler by a novel conserved leucine latch motif.

CSB/ERCC6 (Cockayne syndrome B protein/excision repair cross-complementation group 6), a member of a subfamily of SWI2/SNF2 (SWItch/sucrose nonfermentable)-related chromatin remodelers, plays crucial roles in gene expression and the maintenance of genome integrity. Here, we report the mechanism of the autoregulation of Rhp26, which is the homolog of CSB/ERCC6 in Schizosaccharomyces pombe. We identified a novel conserved protein motif, termed the "leucine latch," at the N terminus of Rhp26. The leucine latch motif mediates the autoinhibition of the ATPase and chromatin-remodeling activities of Rhp26 via its interaction with the core ATPase domain. Moreover, we found that the C terminus of the protein counteracts this autoinhibition and that both the N- and C-terminal regions of Rhp26 are needed for its proper function in DNA repair in vivo. The presence of the leucine latch motif in organisms ranging from yeast to humans suggests a conserved mechanism for the autoregulation of CSB/ERCC6 despite the otherwise highly divergent nature of the N- and C-terminal regions.

Structural insights into the human NuA4/TIP60 acetyltransferase and chromatin remodeling complex.

The human nucleosome acetyltransferase of histone H4 (NuA4)/Tat-interactive protein, 60 kilodalton (TIP60) coactivator complex, a fusion of the yeast switch/sucrose nonfermentable related 1 (SWR1) and NuA4 complexes, both incorporates the histone variant H2A.Z into nucleosomes and acetylates histones H4, H2A, and H2A.Z to regulate gene expression and maintain genome stability. Our cryo-electron microscopy studies show that, within the NuA4/TIP60 complex, the E1A binding protein P400 (EP400) subunit serves as a scaffold holding the different functional modules in specific positions, creating a distinct arrangement of the actin-related protein (ARP) module. EP400 interacts with the transformation/transcription domain-associated protein (TRRAP) subunit by using a footprint that overlaps with that of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, preventing the formation of a hybrid complex. Loss of the TRRAP subunit leads to mislocalization of NuA4/TIP60, resulting in the redistribution of H2A.Z and its acetylation across the genome, emphasizing the dual functionality of NuA4/TIP60 as a single macromolecular assembly.

The AT-hook is an evolutionarily conserved auto-regulatory domain of SWI/SNF required for cell lineage priming.

The SWI/SNF ATP-dependent chromatin remodeler is a master regulator of the epigenome, controlling pluripotency and differentiation. Towards the C-terminus of the catalytic subunit of SWI/SNF is a motif called the AT-hook that is evolutionary conserved. The AT-hook is present in many chromatin modifiers and generally thought to help anchor them to DNA. We observe however that the AT-hook regulates the intrinsic DNA-stimulated ATPase activity aside from promoting SWI/SNF recruitment to DNA or nucleosomes by increasing the reaction velocity a factor of 13 with no accompanying change in substrate affinity (KM). The changes in ATP hydrolysis causes an equivalent change in nucleosome movement, confirming they are tightly coupled. The catalytic subunit's AT-hook is required in vivo for SWI/SNF remodeling activity in yeast and mouse embryonic stem cells. The AT-hook in SWI/SNF is required for transcription regulation and activation of stage-specific enhancers critical in cell lineage priming. Similarly, growth assays suggest the AT-hook is required in yeast SWI/SNF for activation of genes involved in amino acid biosynthesis and metabolizing ethanol. Our findings highlight the importance of studying SWI/SNF attenuation versus eliminating the catalytic subunit or completely shutting down its enzymatic activity.

An embryonic stem cell chromatin remodeling complex, esBAF, is essential for embryonic stem cell self-renewal and pluripotency.

Mammalian SWI/SNF [also called BAF (Brg/Brahma-associated factors)] ATP-dependent chromatin remodeling complexes are essential for formation of the totipotent and pluripotent cells of the early embryo. In addition, subunits of this complex have been recovered in screens for genes required for nuclear reprogramming in Xenopus and mouse embryonic stem cell (ES) morphology. However, the mechanism underlying the roles of these complexes is unclear. Here, we show that BAF complexes are required for the self-renewal and pluripotency of mouse ES cells but not for the proliferation of fibroblasts or other cells. Proteomic studies reveal that ES cells express distinctive complexes (esBAF) defined by the presence of Brg (Brahma-related gene), BAF155, and BAF60A, and the absence of Brm (Brahma), BAF170, and BAF60C. We show that this specialized subunit composition is required for ES cell maintenance and pluripotency. Our proteomic analysis also reveals that esBAF complexes interact directly with key regulators of pluripotency, suggesting that esBAF complexes are specialized to interact with ES cell-specific regulators, providing a potential explanation for the requirement of BAF complexes in pluripotency.

PARK7/DJ-1 promotes pyruvate dehydrogenase activity and maintains Treg homeostasis during ageing.

Pyruvate dehydrogenase (PDH) is the gatekeeper enzyme of the tricarboxylic acid (TCA) cycle. Here we show that the deglycase DJ-1 (encoded by PARK7, a key familial Parkinson's disease gene) is a pacemaker regulating PDH activity in CD4+ regulatory T cells (Treg cells). DJ-1 binds to PDHE1-ß (PDHB), inhibiting phosphorylation of PDHE1-α (PDHA), thus promoting PDH activity and oxidative phosphorylation (OXPHOS). Park7 (Dj-1) deletion impairs Treg survival starting in young mice and reduces Treg homeostatic proliferation and cellularity only in aged mice. This leads to increased severity in aged mice during the remission of experimental autoimmune encephalomyelitis (EAE). Dj-1 deletion also compromises differentiation of inducible Treg cells especially in aged mice, and the impairment occurs via regulation of PDHB. These findings provide unforeseen insight into the complicated regulatory machinery of the PDH complex. As Treg homeostasis is dysregulated in many complex diseases, the DJ-1-PDHB axis represents a potential target to maintain or re-establish Treg homeostasis.

Assembly of SNAPc, Bdp1, and TBP on the U6 snRNA Gene Promoter in Drosophila melanogaster.

U6 snRNA is transcribed by RNA polymerase III (Pol III) and has an external upstream promoter that consists of a TATA sequence recognized by the TBP subunit of the Pol III basal transcription factor IIIB and a proximal sequence element (PSE) recognized by the small nuclear RNA activating protein complex (SNAPc). Previously, we found that Drosophila melanogaster SNAPc (DmSNAPc) bound to the U6 PSE can recruit the Pol III general transcription factor Bdp1 to form a stable complex with the DNA. Here, we show that DmSNAPc-Bdp1 can recruit TBP to the U6 promoter, and we identify a region of Bdp1 that is sufficient for TBP recruitment. Moreover, we find that this same region of Bdp1 cross-links to nucleotides within the U6 PSE at positions that also cross-link to DmSNAPc. Finally, cross-linking mass spectrometry reveals likely interactions of specific DmSNAPc subunits with Bdp1 and TBP. These data, together with previous findings, have allowed us to build a more comprehensive model of the DmSNAPc-Bdp1-TBP complex on the U6 promoter that includes nearly all of DmSNAPc, a portion of Bdp1, and the conserved region of TBP.

Identification of Cross-linked Peptides Using Isotopomeric Cross-linkers.

Chemical cross-linking combined with mass spectrometry (CL-MS) is a powerful method for characterizing the architecture of protein assemblies and for mapping protein-protein interactions. Despite its proven utility, confident identification of cross-linked peptides remains a formidable challenge, especially when the peptides are derived from complex mixtures. MS cleavable cross-linkers are gaining importance for CL-MS as they permit reliable identification of cross-linked peptides by whole proteome database searching using MS/MS information. Here we introduce a novel class of MS cleavable cross-linkers called isotopomeric cross-linkers (ICLs), which allow for confident and efficient identification of cross-linked peptides by whole proteome database searching. ICLs are simple, symmetrical molecules that asymmetrically incorporate heavy and light stable isotopes into the two arms of the cross-linker. As a result of this property, ICLs automatically generate pairs of isotopomeric cross-linked peptides, which differ only by the positions of the heavy and light isotopes. Upon fragmentation during MS analysis, these isotopomeric cross-linked peptides generate unique isotopic doublet ions that correspond to the individual peptides in the cross-link. The doublet ion information is used to determine the masses of the two cross-linked peptides from the same MS2 spectrum that is also used for peptide spectrum matching (PSM) by sequence database searching. Here we present the rationale for and mechanism of cross-linked peptide identification by ICL-MS. We describe the synthesis of the ICL-1 reagent, the ICL-MS workflow, and the performance characteristics of ICL-MS for identifying cross-linked peptides derived from increasingly complex mixtures by whole proteome database searching.

Architecture of the chromatin remodeler RSC and insights into its nucleosome engagement.

Eukaryotic DNA is packaged into nucleosome arrays, which are repositioned by chromatin remodeling complexes to control DNA accessibility. The Saccharomyces cerevisiae RSC (Remodeling the Structure of Chromatin) complex, a member of the SWI/SNF chromatin remodeler family, plays critical roles in genome maintenance, transcription, and DNA repair. Here, we report cryo-electron microscopy (cryo-EM) and crosslinking mass spectrometry (CLMS) studies of yeast RSC complex and show that RSC is composed of a rigid tripartite core and two flexible lobes. The core structure is scaffolded by an asymmetric Rsc8 dimer and built with the evolutionarily conserved subunits Sfh1, Rsc6, Rsc9 and Sth1. The flexible ATPase lobe, composed of helicase subunit Sth1, Arp7, Arp9 and Rtt102, is anchored to this core by the N-terminus of Sth1. Our cryo-EM analysis of RSC bound to a nucleosome core particle shows that in addition to the expected nucleosome-Sth1 interactions, RSC engages histones and nucleosomal DNA through one arm of the core structure, composed of the Rsc8 SWIRM domains, Sfh1 and Npl6. Our findings provide structural insights into the conserved assembly process for all members of the SWI/SNF family of remodelers, and illustrate how RSC selects, engages, and remodels nucleosomes.

Histone Octamer Structure Is Altered Early in ISW2 ATP-Dependent Nucleosome Remodeling.

Nucleosomes are the fundamental building blocks of chromatin that regulate DNA access and are composed of histone octamers. ATP-dependent chromatin remodelers like ISW2 regulate chromatin access by translationally moving nucleosomes to different DNA regions. We find that histone octamers are more pliable than previously assumed and distorted by ISW2 early in remodeling before DNA enters nucleosomes and the ATPase motor moves processively on nucleosomal DNA. Uncoupling the ATPase activity of ISW2 from nucleosome movement with deletion of the SANT domain from the C terminus of the Isw2 catalytic subunit traps remodeling intermediates in which the histone octamer structure is changed. We find restricting histone movement by chemical crosslinking also traps remodeling intermediates resembling those seen early in ISW2 remodeling with loss of the SANT domain. Other evidence shows histone octamers are intrinsically prone to changing their conformation and can be distorted merely by H3-H4 tetramer disulfide crosslinking.

Molecular structure of promoter-bound yeast TFIID.

Transcription preinitiation complex assembly on the promoters of protein encoding genes is nucleated in vivo by TFIID composed of the TATA-box Binding Protein (TBP) and 13 TBP-associate factors (Tafs) providing regulatory and chromatin binding functions. Here we present the cryo-electron microscopy structure of promoter-bound yeast TFIID at a resolution better than 5 Å, except for a flexible domain. We position the crystal structures of several subunits and, in combination with cross-linking studies, describe the quaternary organization of TFIID. The compact tri lobed architecture is stabilized by a topologically closed Taf5-Taf6 tetramer. We confirm the unique subunit stoichiometry prevailing in TFIID and uncover a hexameric arrangement of Tafs containing a histone fold domain in the Twin lobe.

Transcription Activation Domains of the Yeast Factors Met4 and Ino2: Tandem Activation Domains with Properties Similar to the Yeast Gcn4 Activator.

Eukaryotic transcription activation domains (ADs) are intrinsically disordered polypeptides that typically interact with coactivator complexes, leading to stimulation of transcription initiation, elongation, and chromatin modifications. Here we examined the properties of two strong and conserved yeast ADs: Met4 and Ino2. Both factors have tandem ADs that were identified by conserved sequence and functional studies. While the AD function of both factors depended on hydrophobic residues, Ino2 further required key conserved acidic and polar residues for optimal function. Binding studies showed that the ADs bound multiple Med15 activator-binding domains (ABDs) with similar orders of micromolar affinity and similar but distinct thermodynamic properties. Protein cross-linking data show that no unique complex was formed upon Met4-Med15 binding. Rather, we observed heterogeneous AD-ABD contacts with nearly every possible AD-ABD combination. Many of these properties are similar to those observed with yeast activator Gcn4, which forms a large heterogeneous, dynamic, and fuzzy complex with Med15. We suggest that this molecular behavior is common among eukaryotic activators.

Gcn4-Mediator Specificity Is Mediated by a Large and Dynamic Fuzzy Protein-Protein Complex.

Transcription activation domains (ADs) are inherently disordered proteins that often target multiple coactivator complexes, but the specificity of these interactions is not understood. Efficient transcription activation by yeast Gcn4 requires its tandem ADs and four activator-binding domains (ABDs) on its target, the Mediator subunit Med15. Multiple ABDs are a common feature of coactivator complexes. We find that the large Gcn4-Med15 complex is heterogeneous and contains nearly all possible AD-ABD interactions. Gcn4-Med15 forms via a dynamic fuzzy protein-protein interface, where ADs bind the ABDs in multiple orientations via hydrophobic regions that gain helicity. This combinatorial mechanism allows individual low-affinity and specificity interactions to generate a biologically functional, specific, and higher affinity complex despite lacking a defined protein-protein interface. This binding strategy is likely representative of many activators that target multiple coactivators, as it allows great flexibility in combinations of activators that can cooperate to regulate genes with variable coactivator requirements.

Structure of human TFIID and mechanism of TBP loading onto promoter DNA.

The general transcription factor IID (TFIID) is a critical component of the eukaryotic transcription preinitiation complex (PIC) and is responsible for recognizing the core promoter DNA and initiating PIC assembly. We used cryo-electron microscopy, chemical cross-linking mass spectrometry, and biochemical reconstitution to determine the complete molecular architecture of TFIID and define the conformational landscape of TFIID in the process of TATA box-binding protein (TBP) loading onto promoter DNA. Our structural analysis revealed five structural states of TFIID in the presence of TFIIA and promoter DNA, showing that the initial binding of TFIID to the downstream promoter positions the upstream DNA and facilitates scanning of TBP for a TATA box and the subsequent engagement of the promoter. Our findings provide a mechanistic model for the specific loading of TBP by TFIID onto the promoter.

Loss of Snf5 Induces Formation of an Aberrant SWI/SNF Complex.

The SWI/SNF chromatin remodeling complex is highly conserved from yeast to human, and aberrant SWI/SNF complexes contribute to human disease. The Snf5/SMARCB1/INI1 subunit of SWI/SNF is a tumor suppressor frequently lost in pediatric rhabdoid cancers. We examined the effects of Snf5 loss on the composition, nucleosome binding, recruitment, and remodeling activities of yeast SWI/SNF. The Snf5 subunit is shown by crosslinking-mass spectrometry (CX-MS) and subunit deletion analysis to interact with the ATPase domain of Snf2 and to form a submodule consisting of Snf5, Swp82, and Taf14. Snf5 promotes binding of the Snf2 ATPase domain to nucleosomal DNA and enhances the catalytic and nucleosome remodeling activities of SWI/SNF. Snf5 is also required for SWI/SNF recruitment by acidic transcription factors. RNA-seq analysis suggests that both the recruitment and remodeling functions of Snf5 are required in vivo for SWI/SNF regulation of gene expression. Thus, loss of SNF5 alters the structure and function of SWI/SNF.

Proteomic and bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in human malignancy.

Subunits of mammalian SWI/SNF (mSWI/SNF or BAF) complexes have recently been implicated as tumor suppressors in human malignancies. To understand the full extent of their involvement, we conducted a proteomic analysis of endogenous mSWI/SNF complexes, which identified several new dedicated, stable subunits not found in yeast SWI/SNF complexes, including BCL7A, BCL7B and BCL7C, BCL11A and BCL11B, BRD9 and SS18. Incorporating these new members, we determined mSWI/SNF subunit mutation frequency in exome and whole-genome sequencing studies of primary human tumors. Notably, mSWI/SNF subunits are mutated in 19.6% of all human tumors reported in 44 studies. Our analysis suggests that specific subunits protect against cancer in specific tissues. In addition, mutations affecting more than one subunit, defined here as compound heterozygosity, are prevalent in certain cancers. Our studies demonstrate that mSWI/SNF is the most frequently mutated chromatin-regulatory complex (CRC) in human cancer, exhibiting a broad mutation pattern, similar to that of TP53. Thus, proper functioning of polymorphic BAF complexes may constitute a major mechanism of tumor suppression.