Signaling pathways involved in regulating T cell proliferation and survival are not well understood. Here we have investigated a possible role of the nuclear factor (NF)-kappaB pathway in regulating mature T cell function by using CD4+ T cells from p50-/- cRel-/- mice, which exhibit virtually no inducible kappaB site binding activity. Studies with these mice indicate an essential role of T cell receptor (TCR)-induced NF-kappaB in regulating interleukin (IL)-2 expression, cell cycle entry, and survival of T cells. Our results further indicate that NF-kappaB regulates TCR-induced expression of antiapoptotic Bcl-2 family members. Strikingly, retroviral transduction of CD4+ T cells with the NF-kappaB-inducing IkappaB kinase beta showed that NF-kappaB activation is not only necessary but also sufficient for T cell survival. In contrast, our results indicate a lack of involvement of NF-kappaB in both IL-2 and Akt-induced survival pathways. In vivo, p50-/- cRel-/- mice showed impaired superantigen-induced T cell responses as well as decreased numbers of effector/memory and regulatory CD4+ T cells. These findings provide the first demonstration of a role for NF-kappaB proteins in regulating T cell function in vivo and establish a critically important function of NF-kappaB in TCR-induced regulation of survival.

Phosphorylation is universally used for controlling protein function, but knowledge of the phosphoproteome in vertebrate embryos has been limited. However, recent technical advances make it possible to define an organism's phosphoproteome at a more comprehensive level. Xenopus laevis offers established advantages for analyzing the regulation of protein function by phosphorylation. Functionally unbiased, comprehensive information about the Xenopus phosphoproteome would provide a powerful guide for future studies of phosphorylation in a developmental context. To this end, we performed a phosphoproteomic analysis of Xenopus oocytes, eggs, and embryos using recently developed mass spectrometry methods. We identified 1,441 phosphorylation sites present on 654 different Xenopus proteins, including hundreds of previously unknown phosphorylation sites. This approach identified several phosphorylation sites described in the literature and/or evolutionarily conserved in other organisms, validating the data's quality. These data will serve as a powerful resource for the exploration of phosphorylation and protein function within a developmental context. Developmental Dynamics 238:1433-1443, 2009. (c) 2009 Wiley-Liss, Inc.

Myelin is formed by specialized myelinating glia: oligodendrocytes and Schwann cells in the central and peripheral nervous systems, respectively. While there are distinct developmental aspects and regulatory pathways in these two cell types, myelination in both systems requires the transcriptional activator Sox10. Sox10 interacts with cell type-specific transcription factors at some loci to induce myelin gene expression, but it is largely unknown how Sox10 transcriptional networks globally compare between oligodendrocytes and Schwann cells. We used in vivo ChIP-Seq analysis of spinal cord and peripheral nerve (sciatic nerve) to identify unique and shared Sox10 binding sites and assess their correlation with active enhancers and transcriptional profiles in oligodendrocytes and Schwann cells. Sox10 binding sites overlap with active enhancers and critical cell type-specific regulators of myelination, such as Olig2 and Myrf in oligodendrocytes, and Egr2/Krox20 in Schwann cells. Sox10 sites also associate with genes critical for myelination in both oligodendrocytes and Schwann cells and are found within super-enhancers previously defined in brain. In Schwann cells, Sox10 sites contain binding motifs of putative partners in the Sp/Klf, Tead, and nuclear receptor protein families. Specifically, siRNA analysis of nuclear receptors Nr2f1 and Nr2f2 revealed downregulation of myelin genes Mbp and Ndrg1 in primary Schwann cells. Our analysis highlights different mechanisms that establish cell type-specific genomic occupancy of Sox10, which reflects the unique characteristics of oligodendrocyte and Schwann cell differentiation. GLIA 2015;63:1897-1914.

We describe the comprehensive analysis of the yeast proteome in just over one hour of optimized analysis. We achieve this expedited proteome characterization with improved sample preparation, chromatographic separations, and by using a new Orbitrap hybrid mass spectrometer equipped with a mass filter, a collision cell, a high-field Orbitrap analyzer, and, finally, a dual cell linear ion trap analyzer (Q-OT-qIT, Orbitrap Fusion). This system offers high MS(2) acquisition speed of 20 Hz and detects up to 19 peptide sequences within a single second of operation. Over a 1.3 h chromatographic method, the Q-OT-qIT hybrid collected an average of 13,447 MS(1) and 80,460 MS(2) scans (per run) to produce 43,400 (x) peptide spectral matches and 34,255 (x) peptides with unique amino acid sequences (1% false discovery rate (FDR)). On average, each one hour analysis achieved detection of 3,977 proteins (1% FDR). We conclude that further improvements in mass spectrometer scan rate could render comprehensive analysis of the human proteome within a few hours.

A mass spectrometry (MS) method is described here that can reproducibly identify hundreds of peptides across multiple experiments. The method uses intelligent data acquisition to precisely target peptides while simultaneously identifying thousands of other, nontargeted peptides in a single nano-LC-MS/MS experiment. We introduce an online peptide elution order alignment algorithm that targets peptides based on their relative elution order, eliminating the need for retention-time-based scheduling. We have applied this method to target 500 mouse peptides across six technical replicate nano-LC-MS/MS experiments and were able to identify 440 of these in all six, compared with only 256 peptides using data-dependent acquisition (DDA). A total of 3757 other peptides were also identified within the same experiment, illustrating that this hybrid method does not eliminate the novel discovery advantages of DDA. The method was also tested on a set of mice in biological quadruplicate and increased the number of identified target peptides in all four mice by over 80% (826 vs 459) compared with the standard DDA method. We envision real-time data analysis as a powerful tool to improve the quality and reproducibility of proteomic data sets.

Intrinsic terminators, which encode GC-rich RNA hairpins followed immediately by a 7-to-9-nucleotide (nt) U-rich "U-tract," play principal roles of punctuating and regulating transcription in most bacteria. However, canonical intrinsic terminators with strong U-tracts are underrepresented in some bacterial lineages, notably mycobacteria, leading to proposals that their RNA polymerases stop at noncanonical intrinsic terminators encoding various RNA structures lacking U-tracts. We generated recombinant forms of mycobacterial RNA polymerase and its major elongation factors NusA and NusG to characterize mycobacterial intrinsic termination. Using in vitro transcription assays devoid of possible mycobacterial contaminants, we established that mycobacterial RNA polymerase terminates more efficiently than Escherichia coli RNA polymerase at canonical terminators with imperfect U-tracts but does not terminate at putative terminators lacking U-tracts even in the presence of mycobacterial NusA and NusG. However, mycobacterial NusG exhibits a novel termination-stimulating activity that may allow intrinsic terminators with suboptimal U-tracts to function efficiently. IMPORTANCE Bacteria rely on transcription termination to define and regulate units of gene expression. In most bacteria, precise termination and much regulation by attenuation are accomplished by intrinsic terminators that encode GC-rich hairpins and U-tracts necessary to disrupt stable transcription elongation complexes. Thus, the apparent dearth of canonical intrinsic terminators with recognizable U-tracts in mycobacteria is of significant interest both because noncanonical intrinsic terminators could reveal novel routes to destabilize transcription complexes and because accurate understanding of termination is crucial for strategies to combat mycobacterial diseases and for computational bioinformatics generally. Our finding that mycobacterial RNA polymerase requires U-tracts for intrinsic termination, which can be aided by NusG, will guide future study of mycobacterial transcription and aid improvement of predictive algorithms to annotate bacterial genome sequences.

Mitochondrial dysfunction is associated with many human diseases, including cancer and neurodegeneration, that are often linked to proteins and pathways that are not well-characterized. To begin defining the functions of such poorly characterized proteins, we used mass spectrometry to map the proteomes, lipidomes, and metabolomes of 174 yeast strains, each lacking a single gene related to mitochondrial biology. 144 of these genes have human homologs, 60 of which are associated with disease and 39 of which are uncharacterized. We present a multi-omic data analysis and visualization tool that we use to find covariance networks that can predict molecular functions, correlations between profiles of related gene deletions, gene-specific perturbations that reflect protein functions, and a global respiration deficiency response. Using this multi-omic approach, we link seven proteins including Hfd1p and its human homolog ALDH3A1 to mitochondrial coenzyme Q (CoQ) biosynthesis, an essential pathway disrupted in many human diseases. This Resource should provide molecular insights into mitochondrial protein functions.

FNR is a well-studied global regulator of anaerobiosis, which is widely conserved across bacteria. Despite the importance of FNR and anaerobiosis in microbial lifestyles, the factors that influence its function on a genome-wide scale are poorly understood. Here, we report a functional genomic analysis of FNR action. We find that FNR occupancy at many target sites is strongly influenced by nucleoid-associated proteins (NAPs) that restrict access to many FNR binding sites. At a genome-wide level, only a subset of predicted FNR binding sites were bound under anaerobic fermentative conditions and many appeared to be masked by the NAPs H-NS, IHF and Fis. Similar assays in cells lacking H-NS and its paralog StpA showed increased FNR occupancy at sites bound by H-NS in WT strains, indicating that large regions of the genome are not readily accessible for FNR binding. Genome accessibility may also explain our finding that genome-wide FNR occupancy did not correlate with the match to consensus at binding sites, suggesting that significant variation in ChIP signal was attributable to cross-linking or immunoprecipitation efficiency rather than differences in binding affinities for FNR sites. Correlation of FNR ChIP-seq peaks with transcriptomic data showed that less than half of the FNR-regulated operons could be attributed to direct FNR binding. Conversely, FNR bound some promoters without regulating expression presumably requiring changes in activity of condition-specific transcription factors. Such combinatorial regulation may allow Escherichia coli to respond rapidly to environmental changes and confer an ecological advantage in the anaerobic but nutrient-fluctuating environment of the mammalian gut.

Age-associated insulin resistance (IR) and obesity-associated IR are two physiologically distinct forms of adult-onset diabetes. While macrophage-driven inflammation is a core driver of obesity-associated IR, the underlying mechanisms of the obesity-independent yet highly prevalent age-associated IR are largely unexplored. Here we show, using comparative adipo-immune profiling in mice, that fat-resident regulatory T cells, termed fTreg cells, accumulate in adipose tissue as a function of age, but not obesity. Supporting the existence of two distinct mechanisms underlying IR, mice deficient in fTreg cells are protected against age-associated IR, yet remain susceptible to obesity-associated IR and metabolic disease. By contrast, selective depletion of fTreg cells via anti-ST2 antibody treatment increases adipose tissue insulin sensitivity. These findings establish that distinct immune cell populations within adipose tissue underlie ageing- and obesity-associated IR, and implicate fTreg cells as adipo-immune drivers and potential therapeutic targets in the treatment of age-associated IR.

Microbial conversion of lignocellulosic feedstocks into biofuels remains an attractive means to produce sustainable energy. It is essential to produce lignocellulosic hydrolysates in a consistent manner in order to study microbial performance in different feedstock hydrolysates. Because of the potential to introduce microbial contamination from the untreated biomass or at various points during the process, it can be difficult to control sterility during hydrolysate production. In this study, we compared hydrolysates produced from AFEX-pretreated corn stover and switchgrass using two different methods to control contamination: either by autoclaving the pretreated feedstocks prior to enzymatic hydrolysis, or by introducing antibiotics during the hydrolysis of non-autoclaved feedstocks. We then performed extensive chemical analysis, chemical genomics, and comparative fermentations to evaluate any differences between these two different methods used for producing corn stover and switchgrass hydrolysates.

Imidazolium ionic liquids (IILs) underpin promising technologies that generate fermentable sugars from lignocellulose for future biorefineries. However, residual IILs are toxic to fermentative microbes such as Saccharomyces cerevisiae, making IIL-tolerance a key property for strain engineering. To enable rational engineering, we used chemical genomic profiling to understand the effects of IILs on S. cerevisiae.

Spores are an essential cell type required for long-term survival across diverse organisms in the tree of life and are a hallmark of fungal reproduction, persistence, and dispersal. Among human fungal pathogens, spores are presumed infectious particles, but relatively little is known about this robust cell type. Here we used the meningitis-causing fungus Cryptococcus neoformans to determine the roles of spore-resident proteins in spore biology. Using highly sensitive nanoscale liquid chromatography/mass spectrometry, we compared the proteomes of spores and vegetative cells (yeast) and identified eighteen proteins specifically enriched in spores. The genes encoding these proteins were deleted, and the resulting strains were evaluated for discernable phenotypes. We hypothesized that spore-enriched proteins would be preferentially involved in spore-specific processes such as dormancy, stress resistance, and germination. Surprisingly, however, the majority of the mutants harbored defects in sexual development, the process by which spores are formed. One mutant in the cohort was defective in the spore-specific process of germination, showing a delay specifically in the initiation of vegetative growth. Thus, by using this in-depth proteomics approach as a screening tool for cell type-specific proteins and combining it with molecular genetics, we successfully identified the first germination factor in C. neoformans. We also identified numerous proteins with previously unknown functions in both sexual development and spore composition. Our findings provide the first insights into the basic protein components of infectious spores and reveal unexpected molecular connections between infectious particle production and spore composition in a pathogenic eukaryote.

Protein kinase signaling along the kinetochore-centromere axis is crucial to assure mitotic fidelity, yet the details of its spatial coordination are obscure. Here, we examined how pools of human Polo-like kinase 1 (Plk1) within this axis control signaling events to elicit mitotic functions. To do this, we restricted active Plk1 to discrete subcompartments within the kinetochore-centromere axis using chemical genetics and decoded functional and phosphoproteomic signatures of each. We observe distinct phosphoproteomic and functional roles, suggesting that Plk1 exists and functions in discrete pools along this axis. Deep within the centromere, Plk1 operates to assure proper chromosome alignment and segregation. Thus, Plk1 at the kinetochore is a conglomerate of an observable bulk pool coupled with additional functional pools below the threshold of microscopic detection or resolution. Although complex, this multiplicity of locales provides an opportunity to decouple functional and phosphoproteomic signatures for a comprehensive understanding of Plk1's kinetochore functions.

Otitis media is a middle ear disease common in children under three years old. Otitis media can occur in normal individuals with no other symptoms or syndromes, but it is often seen in individuals clinically diagnosed with genetic diseases such as CHARGE syndrome, a complex genetic disease caused by mutation in the Chd7 gene and characterized by multiple birth defects. Although otitis media is common in human CHARGE syndrome patients, it has not been reported in mouse models of CHARGE syndrome. In this study, we report a mouse model with a spontaneous deletion mutation in the Chd7 gene and with chronic otitis media of early onset age accompanied by hearing loss. These mice also exhibit morphological alteration in the Eustachian tubes, dysregulation of epithelial proliferation, and decreased density of middle ear cilia. Gene expression profiling revealed up-regulation of Muc5ac, Muc5b and Tgf-β1 transcripts, the products of which are involved in mucin production and TGF pathway regulation. This is the first mouse model of CHARGE syndrome reported to show otitis media with effusion and it will be valuable for studying the etiology of otitis media and other symptoms in CHARGE syndrome.

This study reports on the release of a novel natriuretic peptide, CD-NP, from an in situ polymer precipitation delivery system. Following extensive screening of in-vitro release profiles, an in-vivo evaluation of the efficacy of the delivery system was carried out in Wistar rats. Gel injection was performed subcutaneously on the back of the rats. A secondary messenger, cyclic Guanosine 3'5' Monophosphate (cGMP), was tested for verification of CD-NP bioactivity, in addition to direct measurements of CD-NP levels in plasma and urine using a radio-immuno assay. Plasma evaluation showed an elevated level of CD-NP over 3 weeks' duration. Unexpectedly, plasma cGMP level followed a decreasing trend over the same duration despite high CD-NP level. Loss of drug bioactivity was ruled out as a high level of CD-NP and cGMP excretion was observed in the treatment group as compared to baseline readings. This unexpected low-plasma cGMP levels and high-urinary cGMP excretion suggest that there might be other compensatory responses to regulation of the CDNP bioactivity as a result of the high drug dosing. The results stress the importance of assessing the overall bioactivity of released drug (in-vivo) concurrently in addition to measuring its concentrations, to determine the correct release profile.

Myelin is essential for the rapidity of saltatory nerve conduction, and also provides trophic support for axons to prevent axonal degeneration. Two critical determinants of myelination are SOX10 and EGR2/KROX20. SOX10 is required for specification of Schwann cells from neural crest, and is required at every stage of Schwann cell development. Egr2/Krox20 expression is activated by axonal signals in myelinating Schwann cells, and is required for cell cycle arrest and myelin formation. To elucidate the integrated function of these two transcription factors during peripheral nerve myelination, we performed in vivo ChIP-Seq analysis of myelinating peripheral nerve. Integration of these binding data with loss-of-function array data identified a range of genes regulated by these factors. In addition, although SOX10 itself regulates Egr2/Krox20 expression, leading to coordinate activation of several major myelin genes by the two factors, there is a large subset of genes that are activated independent of EGR2. Finally, the results identify a set of SOX10-dependent genes that are expressed in early Schwann cell development, but become subsequently repressed by EGR2/KROX20.

Coenzyme Q (CoQ) lipids are ancient electron carriers that, in eukaryotes, function in the mitochondrial respiratory chain. In mitochondria, CoQ lipids are built by an inner membrane-associated, multicomponent, biosynthetic pathway via successive steps of isoprenyl tail polymerization, 4-hydroxybenzoate head-to-tail attachment, and head modification, resulting in the production of CoQ. In yeast, we discovered that head-modifying CoQ pathway components selectively colocalize to multiple resolvable domains in vivo, representing supramolecular assemblies. In cells engineered with conditional ON or OFF CoQ pathways, domains were strictly correlated with CoQ production and substrate flux, respectively, indicating that CoQ lipid intermediates are required for domain formation. Mitochondrial CoQ domains were also observed in human cells, underscoring their conserved functional importance. CoQ domains within cells were highly enriched adjacent to ER-mitochondria contact sites. Together, our data suggest that CoQ domains function to facilitate substrate accessibility for processive and efficient CoQ production and distribution in cells.

In bacteria, disassembly of elongating transcription complexes (ECs) can occur at intrinsic terminators in a 2- to 3-nucleotide window after transcription of multiple kilobase pairs of DNA. Intrinsic terminators trigger pausing on weak RNA-DNA hybrids followed by formation of a strong, GC-rich stem-loop in the RNA exit channel of RNA polymerase (RNAP), inactivating nucleotide addition and inducing dissociation of RNA and RNAP from DNA. Although the movements of RNA and DNA during intrinsic termination have been studied extensively leading to multiple models, the effects of RNAP conformational changes remain less well defined. RNAP contains a clamp domain that closes around the nucleic acid scaffold during transcription initiation and can be displaced by either swiveling or opening motions. Clamp opening is proposed to promote termination by releasing RNAP-nucleic acid contacts. We developed a cysteine crosslinking assay to constrain clamp movements and study effects on intrinsic termination. We found that biasing the clamp into different conformations perturbed termination efficiency, but that perturbations were due primarily to changes in elongation rate, not the competing rate at which ECs commit to termination. After commitment, however, inhibiting clamp movements slowed release of DNA but not of RNA from the EC. We also found that restricting trigger-loop movements with the RNAP inhibitor microcin J25 prior to commitment inhibits termination, in agreement with a recently proposed multistate-multipath model of intrinsic termination. Together our results support views that termination commitment and DNA release are separate steps and that RNAP may remain associated with DNA after termination.

Current Hi-C analysis approaches are unable to account for reads that align to multiple locations, and hence underestimate biological signal from repetitive regions of genomes. We developed and validated mHi-C, a multi-read mapping strategy to probabilistically allocate Hi-C multi-reads. mHi-C exhibited superior performance over utilizing only uni-reads and heuristic approaches aimed at rescuing multi-reads on benchmarks. Specifically, mHi-C increased the sequencing depth by an average of 20% resulting in higher reproducibility of contact matrices and detected interactions across biological replicates. The impact of the multi-reads on the detection of significant interactions is influenced marginally by the relative contribution of multi-reads to the sequencing depth compared to uni-reads, cis-to-trans ratio of contacts, and the broad data quality as reflected by the proportion of mappable reads of datasets. Computational experiments highlighted that in Hi-C studies with short read lengths, mHi-C rescued multi-reads can emulate the effect of longer reads. mHi-C also revealed biologically supported bona fide promoter-enhancer interactions and topologically associating domains involving repetitive genomic regions, thereby unlocking a previously masked portion of the genome for conformation capture studies.

The genetics of individual lipid species and their relevance in disease is largely unresolved. We profiled a subset of storage, signaling, membrane, and mitochondrial liver lipids across 385 mice from 47 strains of the BXD mouse population fed chow or high-fat diet and integrated these data with complementary multi-omics datasets. We identified several lipid species and lipid clusters with specific phenotypic and molecular signatures and, in particular, cardiolipin species with signatures of healthy and fatty liver. Genetic analyses revealed quantitative trait loci for 68% of the lipids (lQTL). By multi-layered omics analyses, we show the reliability of lQTLs to uncover candidate genes that can regulate the levels of lipid species. Additionally, we identified lQTLs that mapped to genes associated with abnormal lipid metabolism in human GWASs. This work provides a foundation and resource for understanding the genetic regulation and physiological significance of lipid species.