Systematic exploration of ubiquitin sequence, E1 activation efficiency, and experimental fitness in yeast
The complexity of biological interaction networks poses a challenge to understanding the function of individual connections in the overall network. To address this challenge, we developed a high-throughput reverse engineering strategy to analyze how thousands of specific perturbations (encompassing all point mutations in a central gene) impact both a specific edge (interaction to a directly connected node) and an overall network function. We analyzed the effects of ubiquitin mutations on activation by the E1 enzyme and compared these to effects on yeast growth rate. Using this approach, we delineated ubiquitin mutations that selectively impacted the ubiquitin-E1 edge. We find that the elasticity function relating the efficiency of ubiquitin-E1 interaction to growth rate is non-linear and that a greater than 50-fold decrease in E1 activation efficiency is required to reduce growth rate by 2-fold. Despite the robustness of fitness to decreases in E1 activation efficiency, the effects of most ubiquitin mutations on E1 activation paralleled the effects on growth rate. Our observations indicate that most ubiquitin mutations that disrupt E1 activation also disrupt other functions. The structurally characterized ubiquitin-E1 interface encompasses the interfaces of ubiquitin with most other known binding partners, and we propose that this enables E1 in wild-type cells to selectively activate ubiquitin protein molecules capable of binding to other partners from the cytoplasmic pool of ubiquitin protein that will include molecules with chemical damage and/or errors from transcription and translation.
Susceptibility to HLA-DM protein is determined by a dynamic conformation of major histocompatibility complex class II molecule bound with peptide
HLA-DM mediates the exchange of peptides loaded onto MHCII molecules during antigen presentation by a mechanism that remains unclear and controversial. Here, we investigated the sequence and structural determinants of HLA-DM interaction. Peptides interacting nonoptimally in the P1 pocket exhibited low MHCII binding affinity and kinetic instability and were highly susceptible to HLA-DM-mediated peptide exchange. These changes were accompanied by conformational alterations detected by surface plasmon resonance, SDS resistance assay, antibody binding assay, gel filtration, dynamic light scattering, small angle x-ray scattering, and NMR spectroscopy. Surprisingly, all of those changes could be reversed by substitution of the P9 pocket anchor residue. Moreover, MHCII mutations outside the P1 pocket and the HLA-DM interaction site increased HLA-DM susceptibility. These results indicate that a dynamic MHCII conformational determinant rather than P1 pocket occupancy is the key factor determining susceptibility to HLA-DM-mediated peptide exchange and provide a molecular mechanism for HLA-DM to efficiently target unstable MHCII-peptide complexes for editing and exchange those for more stable ones.
Chitosan, the deacetylated derivative of chitin, can be found in the cell wall of some fungi and is used in translational applications. We have shown that highly purified preparations of chitosan, but not chitin, activate the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in primed mouse bone marrow-derived macrophages (BMMPhi), inducing a robust IL-1beta response. In this article, we further define specific cell types that are activated and delineate mechanisms of activation. BMMPhi differentiated to promote a classically activated (M1) phenotype released more IL-1beta in response to chitosan than intermediate or alternatively activated macrophages (M2). Chitosan, but not chitin, induced a robust IL-1beta response in mouse dendritic cells, peritoneal macrophages, and human PBMCs. Three mechanisms for NLRP3 inflammasome activation may contribute: K(+) efflux, reactive oxygen species, and lysosomal destabilization. The contributions of these mechanisms were tested using a K(+) efflux inhibitor, high extracellular potassium, a mitochondrial reactive oxygen species inhibitor, lysosomal acidification inhibitors, and a cathepsin B inhibitor. These studies revealed that each of these pathways participated in optimal NLRP3 inflammasome activation by chitosan. Finally, neither chitosan nor chitin stimulated significant release from unprimed BMMPhi of any of 22 cytokines and chemokines assayed. This study has the following conclusions: 1) chitosan, but not chitin, stimulates IL-1beta release from multiple murine and human cell types; 2) multiple nonredundant mechanisms appear to participate in inflammasome activation by chitosan; and 3) chitin and chitosan are relatively weak stimulators of inflammatory mediators from unprimed BMMPhi. These data have implications for understanding the nature of the immune response to microbes and biomaterials that contain chitin and chitosan.
Regulatory T (Treg) cells are important in the maintenance of self-tolerance, and the depletion of Treg cells correlates with autoimmune development. It has been shown that type I interferon (IFN) responses induced early in the infection of mice can drive memory (CD44hi) CD8 and CD4 T cells into apoptosis, and we questioned here whether the apoptosis of CD44-expressing Treg cells might be involved in the infection-associated autoimmune development. Instead, we found that Treg cells were much more resistant to apoptosis than CD44hi CD8 and CD4 T cells at days 2 to 3 after lymphocytic choriomeningitis virus infection, when type I IFN levels are high. The infection caused a downregulation of the interleukin-7 (IL-7) receptor, needed for survival of conventional T cells, while increasing on Treg cells the expression of the high-affinity IL-2 receptor, needed for STAT5-dependent survival of Treg cells. The stably maintained Treg cells early during infection may explain the relatively low incidence of autoimmune manifestations among infected patients.
IMPORTANCE: Autoimmune diseases are controlled in part by regulatory T cells (Treg) and are thought to sometimes be initiated by viral infections. We tested the hypothesis that Treg may die off at early stages of infection, when virus-induced factors kill other lymphocyte types. Instead, we found that Treg resisted this cell death, perhaps reducing the tendency of viral infections to cause immune dysfunction and induce autoimmunity.
Virus-specific CD8+ T cells in the lymphoid organs contract at the resolution of virus infections by apoptosis or by dissemination into peripheral tissues, and those residing in nonlymphoid organs, including the peritoneal cavity and fat pads, are more resistant to apoptosis than those in the spleen and lymph nodes. This stability of memory T cells in the nonlymphoid tissues may enhance protection to secondary challenges. Here, we show that lymphocytic choriomeningitis virus (LCMV)-specific CD8+ T cells in nonlymphoid tissues were enriched for memory precursors (expressing high levels of interleukin-7 receptor and low levels of killer cell lectin-like receptor G1 [IL-7Rhi KLRG1lo]) and had higher expression of CD27, CXCR3, and T cell factor-1 (TCF-1), each a marker that is individually correlated with decreased apoptosis. CD8+ T cells in the peritoneal cavity of TCF-1-deficient mice had decreased survival, suggesting a role for TCF-1 in promoting survival in the nonlymphoid tissues. CXCR3+ CD8+ T cells resisted apoptosis and accumulated in the lymph nodes of mice treated with FTY720, which blocks the export of lymph node cells into peripheral tissue. The peritoneal exudate cells (PEC) expressed increased amounts of CXCR3 ligands, CXCL9 and CXCL10, which may normally recruit these nonapoptotic cells from the lymph nodes. In addition, adoptive transfer of splenic CD8+ T cells into PEC or spleen environments showed that the peritoneal environment promoted survival of CD8+ T cells. Thus, intrinsic stability of T cells which are present in the nonlymphoid tissues along with preferential migration of apoptosis-resistant CD8+ T cells into peripheral sites and the availability of tissue-specific factors that enhance memory cell survival may collectively account for the tissue-dependent apoptotic differences.
IMPORTANCE: Most infections are initiated at nonlymphoid tissue sites, and the presence of memory T cells in nonlymphoid tissues is critical for protective immunity in various viral infection models. Virus-specific CD8+ T cells in the nonlymphoid tissues are more resistant to apoptosis than those in lymphoid organs during the resolution and memory phase of the immune response to acute LCMV infection. Here, we investigated the mechanisms promoting stability of T cells in the nonlymphoid tissues. This increased resistance to apoptosis of virus-specific CD8+ T cells in nonlymphoid tissues was due to several factors. Nonlymphoid tissues were enriched in memory phenotype CD8+ T cells, which were intrinsically resistant to apoptosis irrespective of the tissue environment. Furthermore, apoptosis-resistant CD8+ T cells preferentially migrated into the nonlymphoid tissues, where the availability of tissue-specific factors may enhance memory cell survival. Our findings are relevant for the generation of long-lasting vaccines providing protection at peripheral infection sites.
Substantial evidence has suggested that the brain structures of the medial prefrontal cortex (mPFC) and amygdala (AMYG) are implicated in the pathophysiology of stress-related disorders. However, little is known with respect to the system-level adaptation of their neural circuitries to the perturbations of traumatic stressors. By utilizing behavioral tests and an awake animal imaging approach, in the present study we non-invasively investigated the impact of single-episode predator odor exposure in an inescapable environment on behaviors and neural circuits in rodents. We found that predator odor exposure significantly increased the freezing behavior. In addition, animals exhibited heightened anxiety levels seven days after the exposure. Intriguingly, we also found that the intrinsic functional connectivity within the AMYG-mPFC circuit was considerably compromised seven days after the traumatic event. Our data provide neuroimaging evidence suggesting that prolonged neuroadaptation induced by a single episode of traumatic stress can be non-invasively detected in rodents. These results also support the face validity and construction validity of using the paradigm of single trauma exposure in an inescapable environment as an animal model for post-traumatic stress disorder. Taken together, the present study has opened a new avenue to investigating animal models of stress-related mental disorders by going beyond static neuroanatomy, and ultimately bridging the gap between basic biomedical and human imaging research.
Folding of globular proteins can be envisioned as the contraction of a random coil unfolded state toward the native state on an energy surface rough with local minima trapping frustrated species. These substructures impede productive folding and can serve as nucleation sites for aggregation reactions. However, little is known about the relationship between frustration and its underlying sequence determinants. Chemotaxis response regulator Y (CheY), a 129-amino acid bacterial protein, has been shown previously to populate an off-pathway kinetic trap in the microsecond time range. The frustration has been ascribed to premature docking of the N- and C-terminal subdomains or, alternatively, to the formation of an unproductive local-in-sequence cluster of branched aliphatic side chains, isoleucine, leucine, and valine (ILV). The roles of the subdomains and ILV clusters in frustration were tested by altering the sequence connectivity using circular permutations. Surprisingly, the stability and buried surface area of the intermediate could be increased or decreased depending on the location of the termini. Comparison with the results of small-angle X-ray-scattering experiments and simulations points to the accelerated formation of a more compact, on-pathway species for the more stable intermediate. The effect of chain connectivity in modulating the structures and stabilities of the early kinetic traps in CheY is better understood in terms of the ILV cluster model. However, the subdomain model captures the requirement for an intact N-terminal domain to access the native conformation. Chain entropy and aliphatic-rich sequences play crucial roles in biasing the early events leading to frustration in the folding of CheY.
It has been some 40 years since repeating subunits in eukaryotic chromatin, initially termed "nu bodies," were described. Four decades of study have characterized the structural organization of the nucleosome, from multiple crystal structures of individual nucleosomes to genome-wide maps of nucleosome positions in scores of organisms. Nucleosome positioning can impact essentially all DNA-templated processes, making an appreciation of the forces shaping the nucleosomal landscape in eukaryotes key to fully understanding genomic regulation. Here, we review the factors impacting nucleosome positioning and the ways that nucleosomes can control the output of the genome.
Peptide binding to major histocompatibility complex class II (MHCII) molecules is a key process in antigen presentation and CD4+ T cell epitope selection. This unit describes a fairly simple but powerful fluorescence polarization-based binding competition assay to measure peptide binding to soluble recombinant MHCII molecules. The binding of a peptide of interest to MHCII molecules is assessed based on its ability to inhibit the binding of a fluorescence-labeled probe peptide, with the strength of binding characterized as IC50 (concentration required for 50% inhibition of probe peptide binding). Data analysis related to this method is discussed. In addition, this unit includes a support protocol for fluorescence labeling peptide using an amine-reactive probe. The advantage of this protocol is that it allows simple, fast, and high-throughput measurements of binding for a large set of peptides to MHCII molecules.
Kinome-wide functional analysis highlights the role of cytoskeletal remodeling in somatic cell reprogramming
The creation of induced pluripotent stem cells (iPSCs) from somatic cells by ectopic expression of transcription factors has galvanized the fields of regenerative medicine and developmental biology. Here, we report a kinome-wide RNAi-based analysis to identify kinases that regulate somatic cell reprogramming to iPSCs. We prepared 3,686 small hairpin RNA (shRNA) lentiviruses targeting 734 kinase genes covering the entire mouse kinome and individually examined their effects on iPSC generation. We identified 59 kinases as barriers to iPSC generation and characterized seven of them further. We found that shRNA-mediated knockdown of the serine/threonine kinases TESK1 or LIMK2 promoted mesenchymal-to-epithelial transition, decreased COFILIN phosphorylation, and disrupted Actin filament structures during reprogramming of mouse embryonic fibroblasts. Similarly, knockdown of TESK1 in human fibroblasts also promoted reprogramming to iPSCs. Our study reveals the breadth of kinase networks regulating pluripotency and identifies a role for cytoskeletal remodeling in modulating the somatic cell reprogramming process.
Insulin receptor substrate 2-mediated phosphatidylinositol 3-kinase signaling selectively inhibits glycogen synthase kinase 3beta to regulate aerobic glycolysis
Insulin receptor substrate 1 (IRS-1) and IRS-2 are cytoplasmic adaptor proteins that mediate the activation of signaling pathways in response to ligand stimulation of upstream cell surface receptors. Despite sharing a high level of homology and the ability to activate PI3K, only Irs-2 positively regulates aerobic glycolysis in mammary tumor cells. To determine the contribution of Irs-2-dependent PI3K signaling to this selective regulation, we generated an Irs-2 mutant deficient in the recruitment of PI3K. We identified four tyrosine residues (Tyr-649, Tyr-671, Tyr-734, and Tyr-814) that are essential for the association of PI3K with Irs-2 and demonstrate that combined mutation of these tyrosines inhibits glucose uptake and lactate production, two measures of aerobic glycolysis. Irs-2-dependent activation of PI3K regulates the phosphorylation of specific Akt substrates, most notably glycogen synthase kinase 3beta (Gsk-3beta). Inhibition of Gsk-3beta by Irs-2-dependent PI3K signaling promotes glucose uptake and aerobic glycolysis. The regulation of unique subsets of Akt substrates by Irs-1 and Irs-2 may explain their non-redundant roles in mammary tumor biology. Taken together, our study reveals a novel mechanism by which Irs-2 signaling preferentially regulates tumor cell metabolism and adds to our understanding of how this adaptor protein contributes to breast cancer progression.
Allosteric regulation is an essential function of many proteins that control a variety of different processes such as catalysis, signal transduction, and gene regulation. Structural rearrangements have historically been considered the main means of communication between different parts of a protein. Recent studies have highlighted the importance, however, of changes in protein flexibility as an effective way to mediate allosteric communication across a protein. Scapharca dimeric hemoglobin (HbI) is the simplest possible allosteric system, with cooperative ligand binding between two identical subunits. Thermodynamic equilibrium studies of the binding of oxygen to HbI have shown that cooperativity is an entropically driven effect. The change in entropy of the system observed upon ligand binding may arise from changes in the protein, the ligand, or the water of the system. The goal of this study is to determine the contribution of the change in entropy of the protein backbone to HbI cooperative binding. Molecular dynamics simulations and nuclear magnetic resonance relaxation techniques have revealed that the fast internal motions of HbI contribute to the cooperative binding to carbon monoxide in two ways: (1) by contributing favorably to the free energy of the system and (2) by participating in the cooperative mechanism at the HbI subunit interface. The internal dynamics of the weakly cooperative HbI mutant, F97Y, were also investigated with the same methods. The changes in backbone NH dynamics observed for F97Y HbI upon ligand binding are not as large as for the wild type, in agreement with the reduced cooperativity observed for this mutant. The results of this study indicate that interface flexibility and backbone conformational entropy of HbI participate in and are important for the cooperative mechanism of carbon monoxide binding.
T cell development in the thymus produces multiple lineages of cells, including innate T cells. Studies in mice harboring alterations in TCR signaling proteins or transcriptional regulators have revealed an expanded population of CD4(+) innate T cells in the thymus that produce IL-4 and express the transcription factor promyelocytic leukemia zinc finger (PLZF). In these mice, IL-4 produced by the CD4(+)PLZF(+) T cell population leads to the conversion of conventional CD8(+) thymocytes into innate CD8(+) T cells resembling memory T cells expressing eomesodermin. The expression of PLZF, the signature invariant NKT cell transcription factor, in these innate CD4(+) T cells suggests that they might be a subset of alphabeta or gammadelta TCR(+) NKT cells or mucosal-associated invariant T (MAIT) cells. To address these possibilities, we characterized the CD4(+)PLZF(+) innate T cells in itk(-/-) mice. We show that itk(-/-) innate PLZF(+)CD4(+) T cells are not CD1d-dependent NKT cells, MR1-dependent MAIT cells, or gammadelta T cells. Furthermore, although the itk(-/-) innate PLZF(+)CD4(+) T cells express alphabeta TCRs, neither beta2-microglobulin-dependent MHC class I nor any MHC class II molecules are required for their development. In contrast to invariant NKT cells and MAIT cells, this population has a highly diverse TCRalpha-chain repertoire. Analysis of peripheral tissues indicates that itk(-/-) innate PLZF(+)CD4(+) T cells preferentially home to spleen and mesenteric lymph nodes owing to increased expression of gut-homing receptors, and that their expansion is regulated by commensal gut flora. These data support the conclusion that itk(-/-) innate PLZF(+)CD4(+) T cells are a novel subset of innate T cells.
mu-Opioid inhibition of Ca2+ currents and secretion in isolated terminals of the neurohypophysis occurs via ryanodine-sensitive Ca2+ stores
mu-Opioid agonists have no effect on calcium currents (I(Ca)) in neurohypophysial terminals when recorded using the classic whole-cell patch-clamp configuration. However, mu-opioid receptor (MOR)-mediated inhibition of I(Ca) is reliably demonstrated using the perforated-patch configuration. This suggests that the MOR-signaling pathway is sensitive to intraterminal dialysis and is therefore mediated by a readily diffusible second messenger. Using the perforated patch-clamp technique and ratio-calcium-imaging methods, we describe a diffusible second messenger pathway stimulated by the MOR that inhibits voltage-gated calcium channels in isolated terminals from the rat neurohypophysis (NH). Our results show a rise in basal intracellular calcium ([Ca(2+)]i) in response to application of [D-Ala(2)-N-Me-Phe(4),Gly5-ol]-Enkephalin (DAMGO), a MOR agonist, that is blocked by D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), a MOR antagonist. Buffering DAMGO-induced changes in [Ca(2+)]i with BAPTA-AM completely blocked the inhibition of both I(Ca) and high-K(+)-induced rises in [Ca(2+)]i due to MOR activation, but had no effect on kappa-opioid receptor (KOR)-mediated inhibition. Given the presence of ryanodine-sensitive stores in isolated terminals, we tested 8-bromo-cyclic adenosine diphosphate ribose (8Br-cADPr), a competitive inhibitor of cyclic ADP-ribose (cADPr) signaling that partially relieves DAMGO inhibition of I(Ca) and completely relieves MOR-mediated inhibition of high-K(+)-induced and DAMGO-induced rises in [Ca(2+)]i. Furthermore, antagonist concentrations of ryanodine completely blocked MOR-induced increases in [Ca(2+)]i and inhibition of I(Ca) and high-K(+)-induced rises in [Ca(2+)]i while not affecting KOR-mediated inhibition. Antagonist concentrations of ryanodine also blocked MOR-mediated inhibition of electrically-evoked increases in capacitance. These results strongly suggest that a key diffusible second messenger mediating the MOR-signaling pathway in NH terminals is [Ca(2+)]i released by cADPr from ryanodine-sensitive stores.
HTT-lowering reverses Huntington's disease immune dysfunction caused by NFkappaB pathway dysregulation
Huntington's disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The peripheral innate immune system contributes to Huntington's disease pathogenesis and has been targeted successfully to modulate disease progression, but mechanistic understanding relating this to mutant huntingtin expression in immune cells has been lacking. Here we demonstrate that human Huntington's disease myeloid cells produce excessive inflammatory cytokines as a result of the cell-intrinsic effects of mutant huntingtin expression. A direct effect of mutant huntingtin on the NFkappaB pathway, whereby it interacts with IKKgamma, leads to increased degradation of IkappaB and subsequent nuclear translocation of RelA. Transcriptional alterations in intracellular immune signalling pathways are also observed. Using a novel method of small interfering RNA delivery to lower huntingtin expression, we show reversal of disease-associated alterations in cellular function-the first time this has been demonstrated in primary human cells. Glucan-encapsulated small interfering RNA particles were used to lower huntingtin levels in human Huntington's disease monocytes/macrophages, resulting in a reversal of huntingtin-induced elevated cytokine production and transcriptional changes. These findings improve our understanding of the role of innate immunity in neurodegeneration, introduce glucan-encapsulated small interfering RNA particles as tool for studying cellular pathogenesis ex vivo in human cells and raise the prospect of immune cell-directed HTT-lowering as a therapeutic in Huntington's disease.
Glial wingless/Wnt regulates glutamate receptor clustering and synaptic physiology at the Drosophila neuromuscular junction
Glial cells are emerging as important regulators of synapse formation, maturation, and plasticity through the release of secreted signaling molecules. Here we use chromatin immunoprecipitation along with Drosophila genomic tiling arrays to define potential targets of the glial transcription factor Reversed polarity (Repo). Unexpectedly, we identified wingless (wg), a secreted morphogen that regulates synaptic growth at the Drosophila larval neuromuscular junction (NMJ), as a potential Repo target gene. We demonstrate that Repo regulates wg expression in vivo and that local glial cells secrete Wg at the NMJ to regulate glutamate receptor clustering and synaptic function. This work identifies Wg as a novel in vivo glial-secreted factor that specifically modulates assembly of the postsynaptic signaling machinery at the Drosophila NMJ.
Genome-wide mutant fitness profiling identifies nutritional requirements for optimal growth of Yersinia pestis in deep tissue
Rapid growth in deep tissue is essential to the high virulence of Yersinia pestis, causative agent of plague. To better understand the mechanisms underlying this unusual ability, we used transposon mutagenesis and high-throughput sequencing (Tn-seq) to systematically probe the Y. pestis genome for elements contributing to fitness during infection. More than a million independent insertion mutants representing nearly 200,000 unique genotypes were generated in fully virulent Y. pestis. Each mutant in the library was assayed for its ability to proliferate in vitro on rich medium and in mice following intravenous injection. Virtually all genes previously established to contribute to virulence following intravenous infection showed significant fitness defects, with the exception of genes for yersiniabactin biosynthesis, which were masked by strong intercellular complementation effects. We also identified more than 30 genes with roles in nutrient acquisition and metabolism as experiencing strong selection during infection. Many of these genes had not previously been implicated in Y. pestis virulence. We further examined the fitness defects of strains carrying mutations in two such genes-encoding a branched-chain amino acid importer (brnQ) and a glucose importer (ptsG)-both in vivo and in a novel defined synthetic growth medium with nutrient concentrations matching those in serum. Our findings suggest that diverse nutrient limitations in deep tissue play a more important role in controlling bacterial infection than has heretofore been appreciated. Because much is known about Y. pestis pathogenesis, this study also serves as a test case that assesses the ability of Tn-seq to detect virulence genes.
IMPORTANCE: Our understanding of the functions required by bacteria to grow in deep tissues is limited, in part because most growth studies of pathogenic bacteria are conducted on laboratory media that do not reflect conditions prevailing in infected animal tissues. Improving our knowledge of this aspect of bacterial biology is important as a potential pathway to the development of novel therapeutics. Yersinia pestis, the plague bacterium, is highly virulent due to its rapid dissemination and growth in deep tissues, making it a good model for discovering bacterial adaptations that promote rapid growth during infection. Using Tn-seq, a genome-wide fitness profiling technique, we identified several functions required for fitness of Y. pestis in vivo that were not previously known to be important. Most of these functions are needed to acquire or synthesize nutrients. Interference with these critical nutrient acquisition pathways may be an effective strategy for designing novel antibiotics and vaccines.
Genome-wide functional analysis reveals factors needed at the transition steps of induced reprogramming
Although transcriptome analysis can uncover the molecular changes that occur during induced reprogramming, the functional requirements for a given factor during stepwise cell-fate transitions are left unclear. Here, we used a genome-wide RNAi screen and performed integrated transcriptome analysis to identify key genes and cellular events required at the transition steps in reprogramming. Genes associated with cell signaling pathways (e.g., Itpr1, Itpr2, and Pdia3) constitute the major regulatory networks before cells acquire pluripotency. Activation of a specific gene set (e.g., Utf1 or Tdgf1) is important for mature induced pluripotent stem cell formation. Strikingly, a major proportion of RNAi targets ( approximately 53% to 70%) includes genes whose expression levels are unchanged during reprogramming. Among these non-differentially expressed genes, Dmbx1, Hnf4g, Nobox, and Asb4 are important, whereas Nfe2, Cdkn2aip, Msx3, Dbx1, Lzts1, Gtf2i, and Ankrd22 are roadblocks to reprogramming. Together, our results provide a wealth of information about gene functions required at transition steps during reprogramming.
RCAS viruses are replication-competent in avian cells, but are replication-deficient in mammalian cells. Therefore, high-titer RCAS virus stocks can be generated only in avian cells. The chicken fibroblast cell line DF1 is well suited for this purpose. Successful infection of target mammalian cells, particularly in vivo, is dependent on the production of high titer viruses by DF1 cells. Moreover, consistency in viral titer helps to ensure uniformity in results produced following the use of independent lots of virus producer cells. Therefore, it is critical to determine the viral titer before initiating these experiments. Because several factors, including insert size and the effect of the inserted gene product on the viability of DF1 cells, influence viral titer, the production of high virus titers cannot be assumed. For RCASBP-A-based viruses, a titer of > 1 x 10(7) IU/mL is considered appropriate. Importantly, the virus reverse transcriptase is error prone; errors will accumulate in the virus produced over time. Therefore, virus producer cells should not be cultured for > 4-6 wk before being replaced with fresh producer cells. Low passage virus producer cells may be frozen and stored at -80 degrees C; thawed cells will not display a reduction in virus titer. Virus can be collected regularly, concentrated, and stored at -80 degrees C for long-term use; thawed viral stocks typically show a 10-fold decrease in titer.
Nucleotide excision repair (NER) is critical for the repair of DNA lesions induced by UV radiation, but its contribution in replicating cells is less clear. Here, we show that dual incision by NER endonucleases, including XPF and XPG, promotes the S-phase accumulation of the BRCA1 and Fanconi anemia-associated DNA helicase FANCJ to sites of UV-induced damage. FANCJ promotes replication protein A phosphorylation and the arrest of DNA synthesis following UV irradiation. Interaction defective mutants of FANCJ reveal that BRCA1 binding is not required for FANCJ localization, whereas interaction with the mismatch repair (MMR) protein MLH1 is essential. Correspondingly, we find that FANCJ, its direct interaction with MLH1, and the MMR protein MSH2 function in a common pathway in response to UV irradiation. FANCJ-deficient cells are not sensitive to killing by UV irradiation, yet we find that DNA mutations are significantly enhanced. Thus, we considered that FANCJ deficiency could be associated with skin cancer. Along these lines, in melanoma we found several somatic mutations in FANCJ, some of which were previously identified in hereditary breast cancer and Fanconi anemia. Given that, mutations in XPF can also lead to Fanconi anemia, we propose collaborations between Fanconi anemia, NER, and MMR are necessary to initiate checkpoint activation in replicating human cells to limit genomic instability.