A Structural Model of the Endogenous Human BAF Complex Informs Disease Mechanisms.

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.

Modular Organization and Assembly of SWI/SNF Family Chromatin Remodeling Complexes.

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.

Absolute Quantification of Transcription Factors Reveals Principles of Gene Regulation in Erythropoiesis.

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.

Systematic mutagenesis of TFIIH subunit p52/Tfb2 identifies residues required for XPB/Ssl2 subunit function and genetic interactions with TFB6.

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.

Loss of the neural-specific BAF subunit ACTL6B relieves repression of early response genes and causes recessive autism.

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.

Proteomic/transcriptomic analysis of erythropoiesis.

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.

An LXR-NCOA5 gene regulatory complex directs inflammatory crosstalk-dependent repression of macrophage cholesterol efflux.

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.

Architecture of the Saccharomyces cerevisiae SAGA transcription coactivator complex.

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.

Mechanism for microbial population collapse in a fluctuating resource environment.

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.

Quantitative proteomic analysis of purified yeast kinetochores identifies a PP1 regulatory subunit.

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.

Tension directly stabilizes reconstituted kinetochore-microtubule attachments.

Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes. Accurate segregation depends on selective stabilization of correct 'bi-oriented' kinetochore-microtubule attachments, which come under tension as the result of opposing forces exerted by microtubules. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for >30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules. Moreover, tension increases the lifetimes of the reconstituted attachments directly, through a catch bond-like mechanism that does not require Aurora B. On the basis of these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.

The Mub1/Ubr2 ubiquitin ligase complex regulates the conserved Dsn1 kinetochore protein.

The kinetochore is the macromolecular complex that assembles onto centromeric DNA and orchestrates the segregation of duplicated chromosomes. More than 60 components make up the budding yeast kinetochore, including inner kinetochore proteins that bind to centromeric chromatin and outer proteins that directly interact with microtubules. However, little is known about how these components assemble into a functional kinetochore and whether there are quality control mechanisms that monitor kinetochore integrity. We previously developed a method to isolate kinetochore particles via purification of the conserved Dsn1 kinetochore protein. We find that the Mub1/Ubr2 ubiquitin ligase complex associates with kinetochore particles through the CENP-C(Mif2) protein. Although Mub1/Ubr2 are not stable kinetochore components in vivo, they regulate the levels of the conserved outer kinetochore protein Dsn1 via ubiquitylation. Strikingly, a deletion of Mub1/Ubr2 restores the levels and viability of a mutant Dsn1 protein, reminiscent of quality control systems that target aberrant proteins for degradation. Consistent with this, Mub1/Ubr2 help to maintain viability when kinetochores are defective. Together, our data identify a previously unknown regulatory mechanism for the conserved Dsn1 kinetochore protein. We propose that Mub1/Ubr2 are part of a quality control system that monitors kinetochore integrity, thus ensuring genomic stability.

Systematic measurement of transcription factor-DNA interactions by targeted mass spectrometry identifies candidate gene regulatory proteins.

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.

An integrated chemical cross-linking and mass spectrometry approach to study protein complex architecture and function.

Knowledge of protein structures and protein-protein interactions is essential for understanding biological processes. Chemical cross-linking combined with mass spectrometry is an attractive approach for studying protein-protein interactions and protein structure, but to date its use has been limited largely by low yields of informative cross-links (because of inefficient cross-linking reactions) and by the difficulty of confidently identifying the sequences of cross-linked peptide pairs from their fragmentation spectra. Here we present an approach based on a new MS labile cross-linking reagent, BDRG (biotin-aspartate-Rink-glycine), which addresses these issues. BDRG incorporates a biotin handle (for enrichment of cross-linked peptides prior to MS analysis), two pentafluorophenyl ester groups that react with peptide amines, and a labile Rink-based bond between the pentafluorophenyl groups that allows cross-linked peptides to be separated during MS and confidently identified by database searching of their fragmentation spectra. We developed a protocol for the identification of BDRG cross-linked peptides derived from purified or partially purified protein complexes, including software to aid in the identification of different classes of cross-linker-modified peptides. Importantly, our approach permits the use of high accuracy precursor mass measurements to verify the database search results. We demonstrate the utility of the approach by applying it to purified yeast TFIIE, a heterodimeric transcription factor complex, and to a single-step affinity-purified preparation of the 12-subunit RNA polymerase II complex. The results show that the method is effective at identifying cross-linked peptides derived from purified and partially purified protein complexes and provides complementary information to that from other structural approaches. As such, it is an attractive approach to study the topology of protein complexes.

Glycocapture-assisted global quantitative proteomics (gagQP) reveals multiorgan responses in serum toxicoproteome.

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.

Index-ion triggered MS2 ion quantification: a novel proteomics approach for reproducible detection and quantification of targeted proteins in complex mixtures.

Biomedical research requires protein detection technology that is not only sensitive and quantitative, but that can reproducibly measure any set of proteins in a biological system in a high throughput manner. Here we report the development and application of a targeted proteomics platform termed index-ion triggered MS2 ion quantification (iMSTIQ) that allows reproducible and accurate peptide quantification in complex mixtures. The key feature of iMSTIQ is an approach called index-ion triggered analysis (ITA) that permits the reproducible acquisition of full MS2 spectra of targeted peptides independent of their ion intensities. Accurate quantification is achieved by comparing the relative intensities of multiple pairs of fragment ions derived from isobaric targeted peptides during MS2 analysis. Importantly, the method takes advantage of the favorable performance characteristics of the LTQ-Orbitrap, which include high mass accuracy, resolution, and throughput. As such it provides an attractive targeted proteomics tool to meet the demands of systems biology research and biomarker studies.

The study of macromolecular complexes by quantitative proteomics.

We describe a generic strategy for determining the specific composition, changes in the composition, and changes in the abundance of protein complexes. It is based on the use of isotope-coded affinity tag (ICAT) reagents and mass spectrometry to compare the relative abundances of tryptic peptides derived from suitable pairs of purified or partially purified protein complexes. In a first application, the genuine protein components of a large RNA polymerase II (Pol II) preinitiation complex (PIC) were distinguished from a background of co-purifying proteins by comparing the relative abundances of peptides derived from a control sample and the specific complex that was purified from nuclear extracts by a single-step promoter DNA affinity procedure. In a second application, peptides derived from immunopurified STE12 protein complexes isolated from yeast cells in different states were used to detect quantitative changes in the abundance of the complexes, and to detect dynamic changes in the composition of the samples. The use of quantitative mass spectrometry to guide identification of specific complex components in partially purified samples, and to detect quantitative changes in the abundance and composition of protein complexes, provides the researcher with powerful new tools for the comprehensive analysis of macromolecular complexes.

Identification of TFB5, a new component of general transcription and DNA repair factor IIH.

We previously described the use of quantitative proteomics to study macromolecular complexes. Applying the method to analyze a yeast RNA polymerase II preinitiation complex, we identified a new 8-kDa protein, encoded by the uncharacterized open reading frame YDR079c-a, as a potential new component of the preinitiation complex. Here we show that YDR079c-a is a bona fide component of polymerase II preinitiation complexes and investigate its role in transcription. YDR079c-a is recruited to promoters both in vivo and in vitro and is required for efficient transcription in vitro and for normal induction of GAL genes. In addition, YDR079c-a is a core component of general transcription and DNA repair factor IIH and is required for efficient recruitment of TFIIH to a promoter. Yeast lacking YDR079c-a grow slowly, and, like strains carrying mutations in core TFIIH subunits, are sensitive to ultraviolet radiation. YDR079c-a is conserved throughout evolution, and mutations in the human ortholog account for a DNA repair-deficient form of the tricothiodystrophy disorder called TTD-A(2). The identification of a new, evolutionarily conserved, core TFIIH subunit is essential for our understanding of TFIIH function in transcription, DNA repair and human disease.

A new, tenth subunit of TFIIH is responsible for the DNA repair syndrome trichothiodystrophy group A.

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.