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Congenital heart disease (CHD) affects ~1% of live births and remains the leading cause of mortality in infants. While large-scale genetic studies have uncovered genes associated with CHD, distinguishing variants that confer risk from the background noise of inconsequential variants remains a challenge. Causative mutations in transcription factors (TF) essential for cardiovascular development, such as NKX2-5, GATA4 and TBX5, have been identified in familial cases of CHD, however they are rare. To expand our understanding of their molecular function and to test whether their interacting proteins may be enriched for variants associated with CHD. We defined the protein-protein interaction (PPI) network of NKX2-5, GATA4 and TBX5 using unbiased mass spectrometry in human iPSC-derived cardiac progenitors (iPS-CPs). This approach yielded a network of 172 proteins. An interdependent gene-regulatory role has been reported at the DNA-binding level for these 3 TF during cardiac development, and we also found interdependent protein interactomes where loss of one TF affected the interactome of the others. Interactomes for each were enriched in proteins involved in similar biological processes, such as chromatin remodeling and gene regulation, or previously unrelated processes such as splicing and mRNA transport. Integration of the iPS-CP-PPI network with the CHD-associated damaging variants found in the Pediatric Cardiac Genomics Consortium whole-exome sequencing cohort revealed statistically significant enrichment in the GATA4 interactome for de novo missense variants. In contrast, neither the TBX5 or NKX2-5 PPIs were enriched for either de novo missense or rare damaging variants. Finally, we developed a framework to rank PPIs with reported damaging variants for functional validation studies. Overall, this work identified novel protein interactors of TFs essential for cardiac development, offering new insights regarding their regulatory roles and the mechanisms through which they may cause CHD.

Summary The Cullin-RING E3 ligase (CRL) family is commonly hijacked by pathogens to redirect the host ubiquitin proteasome machinery to specific targets. During HIV infection, CRL5 is hijacked by HIV Vif to target viral restriction factors of the APOBEC3 family for ubiquitination and degradation. Here, using a quantitative proteomics approach, we identify the E3 ligase ARIH2 as a regulator of CRL5-mediated APOBEC3 degradation. The CUL5Vif/CBFs complex recruits ARIH2 where it acts to transfer ubiquitin directly to the APOBEC3 targets. ARIH2 is essential for CRL5-dependent HIV infectivity in primary CD4+ T cells. Furthermore, we show that ARIH2 cooperates with CRL5 to prime other cellular substrates for polyubiquitination, suggesting this may represent a general mechanism beyond HIV infection and APOBEC3 degradation. Taken together, these data identify ARIH2 as a co-factor in the Vif-hijacked CRL5 complex that contributes to HIV infectivity and demonstrate the operation of the E1-E2-E3/E3-substrate ubiquitination mechanism in a viral infection context.

Background Cardiac glycosides are approved for the treatment of heart failure as Na+/K+ pump inhibitors. Their repurposing in oncology is currently investigated in preclinical and clinical studies. However, the identification of a specific cancer type defined by a molecular signature to design targeted clinical trials with cardiac glycosides remains to be characterized. Here, we demonstrate that cardiac glycoside proscillaridin A specifically targets MYC overexpressing leukemia cells and leukemia stem cells by causing MYC degradation, epigenetic reprogramming and leukemia differentiation through loss of lysine acetylation. Methods Proscillaridin A anticancer activity was investigated against a panel of human leukemia and solid tumor cell lines with different MYC expression levels, overexpression in vitro systems and leukemia stem cells. RNA-sequencing and differentiation studies were used to characterize transcriptional and phenotypic changes. Drug-induced epigenetic changes were studied by chromatin post-translational modification analysis, expression of chromatin regulators, chromatin immunoprecipitation, and mass-spectrometry. Results At a clinically relevant dose, proscillaridin A rapidly altered MYC protein half-life causing MYC degradation and growth inhibition. Transcriptomic profile of leukemic cells after treatment showed a downregulation of genes involved in MYC pathways, cell replication and an upregulation of hematopoietic differentiation genes. Functional studies confirmed cell cycle inhibition and the onset of leukemia differentiation even after drug removal. Proscillaridin A induced a significant loss of lysine acetylation in histone H3 (at lysine 9, 14, 18 and 27) and in non-histone proteins such as MYC itself, MYC target proteins, and a series of histone acetylation regulators. Global loss of acetylation correlated with the rapid downregulation of histone acetyltransferases. Importantly, proscillaridin A demonstrated anticancer activity against lymphoid and myeloid stem cell populations characterized by MYC overexpression. Conclusion Overall, these results strongly support the repurposing of proscillaridin A in MYC overexpressing leukemia. Electronic supplementary material The online version of this article (10.1186/s13046-019-1242-8) contains supplementary material, which is available to authorized users.

Diverse perturbations to endoplasmic reticulum (ER) functions compromise the proper folding and structural maturation of secretory proteins. To study secretory pathway physiology during such “ER stress”, we employed an ER-targeted, redox-responsive, green fluorescent protein—eroGFP—that reports on ambient changes in oxidizing potential. Here we find that diverse ER stress agents cause properly folded, ER-resident eroGFP (and other ER luminal proteins) to “reflux” back to the reducing environment of the cytosol as intact, folded proteins. By utilizing eroGFP in a comprehensive genetic screen inS. cerevisiae, we show that ER protein reflux during ER stress requires specific chaperones and co-chaperones residing in both the ER and the cytosol. Chaperone-mediated ER protein reflux does not require E3 ligase activity, and proceeds even more vigorously when these ER-associated degradation (ERAD) factors are crippled, suggesting that reflux may work in parallel with ERAD. In summary, chaperone-mediated ER-protein reflux may be a conserved protein quality control process that evolved to maintain secretory pathway homeostasis during ER protein-folding stress.SIGNIFICANCEApproximately one third of eukaryotic proteins are synthesized on ribosomes attached to the endoplasmic reticulum (ER) membrane. Many of these polypeptides co- or post-translationally translocate into the ER, wherein they fold and mature. An ER quality-control system proofreads these proteins by facilitating their folding and modification, while eliminating misfolded proteins through ER-associated degradation (ERAD). Yet, the fate of many secretory proteins during ER stress is not completely understood. Here, we uncovered an ER-stress induced “protein reflux” system that delivers intact, folded ER luminal proteins back to the cytosol without degrading them. We found that ER protein reflux works in parallel to ERAD and requires distinct ER-resident and cytosolic chaperones and co-chaperones.

West Nile virus (WNV) is an emerging mosquito-borne flavivirus, related to dengue virus and Zika virus. To gain insight into host pathways involved in WNV infection, we performed a systematic affinity-tag purification mass spectrometry (APMS) study to identify 259 WNV-interacting human proteins. RNA interference screening revealed 26 genes that both interact with WNV proteins and influence WNV infection. We found that WNV, dengue and Zika virus capsids interact with a conserved subset of proteins that impact infection. These include the exon-junction complex (EJC) recycling factor PYM1, which is antiviral against all three viruses. The EJC has roles in nonsense-mediated decay (NMD), and we found that both the EJC and NMD are antiviral and the EJC protein RBM8A directly binds WNV RNA. To counteract this, flavivirus infection inhibits NMD and the capsid-PYM1 interaction interferes with EJC protein function and localization. Depletion of PYM1 attenuates RBM8A binding to viral RNA, suggesting that WNV sequesters PYM1 to protect viral RNA from decay. Together, these data suggest a complex interplay between the virus and host in regulating NMD and the EJC.

Mitochondrial transcription factor A (Tfam)-mediated mtDNA maintenance and transcription, as well as leucine-rich PPR motif-containing protein (Lrpprc)-mediated mtRNA maturation and translation are essential steps of mtDNA-encoded electron transport chain (ETC) protein expression. ETC is essential for mitochondrial thermogenesis, the process of oxygen-dependent heat production inside the mitochondria in brown adipocytes. Here we describe that Tfam or Lrpprc deficiency in brown adipocytes cause non-synchronized ETC mRNA and protein expression (NOSEMPE) and mitochondrial ETC imbalance, ultimately abolish mitochondrial thermogenesis. However, mice with NOSEMPE in brown adipocytes are cold resistant upon an acute 4°C cold challenge, because of augmented nonmitochondrial thermogenesis driven by the “NOSEMPE→ATF4→proteome turnover” pathway. Importantly, mice with either NOSEMPE or ATF4 overexpression in brown adipocytes are protected against high-fat-diet-induced metabolic abnormalities, indicating a positive association between nonmitochondrial thermogenesis in brown adipocytes and metabolic fitness. Thus, although brown adipocytes are defined by their unique ability to produce heat through mitochondrial respiration, our study demonstrates a novel cytosolic nonmitochondrial thermogenesis in brown adipocytes. Targeting this ATF4-dependent nonmitochondrial thermogenesis in brown adipocytes may represent a new therapeutic strategy for combating metabolic disorders.

Influenza A virus (IAV) is a threat to mankind because it generates yearly epidemics and poorly predictable sporadic pandemics with catastrophic potential. IAV has a small RNA genome composed of 8 mini-chromosomes (segments) that constitute a 5’UTR followed by a coding region and a 3’UTR. Transcription of IAV RNA into mRNA depends on host mRNA, as the viral polymerase cleaves 5’m7G-capped nascent transcripts to use as primers to initiate viral mRNA synthesis. We hypothesized that captured host transcripts bearing AUG could drive the expression of upstream ORFs in the viral segments, a phenomenon that would depend on the translatability of the viral 5’UTRs. Here we report the existence of this mechanism, which generates host-virus chimeric proteins. We label these proteins asUpstreamFluORFs (UFO). Depending on the frame, two types of host-virus UFO proteins are made: canonical viral proteins with human-derived N term extensions or novel uncharacterized proteins. Here we show that both types are made during IAV infection. Sequences that enable chimeric protein synthesis are conserved across IAV strains, indicating that selection allowed the expansion of the proteome diversity of IAV in infected cells to include multiple human-virus proteins.

Apolipoprotein (apo) E4, the major genetic risk factor for Alzheimer's disease (AD), alters mitochondrial function and metabolism early in AD pathogenesis. When injured or stressed, neurons increase apoE synthesis. Because of its structural difference from apoE3, apoE4 undergoes neuron-specific proteolysis, generating fragments that enter the cytosol, interact with mitochondria, and cause neurotoxicity. However, apoE4's effect on mitochondrial respiration and metabolism is not understood in detail. Here we used biochemical assays and proteomic profiling to more completely characterize the effects of apoE4 on mitochondrial function and cellular metabolism in Neuro-2a neuronal cells stably expressing apoE4 or apoE3. Under basal conditions, apoE4 impaired respiration and increased glycolysis, but when challenged or stressed, apoE4-expressing neurons had 50% less reserve capacity to generate ATP to meet energy requirements than apoE3-expressing neurons. ApoE4 expression also decreased the NAD+/NADH ratio and increased the levels of reactive oxygen species and mitochondrial calcium. Global proteomic profiling revealed widespread changes in mitochondrial processes in apoE4 cells, including reduced levels of numerous respiratory complex subunits and major disruptions to all detected subunits in complex V (ATP synthase). Also altered in apoE4 cells were levels of proteins related to mitochondrial endoplasmic reticulum-associated membranes, mitochondrial fusion/fission, mitochondrial protein translocation, proteases, and mitochondrial ribosomal proteins. ApoE4-induced bioenergetic deficits led to extensive metabolic rewiring, but despite numerous cellular adaptations, apoE4-expressing neurons remained vulnerable to metabolic stress. Our results provide insights into potential molecular targets of therapies to correct apoE4-associated mitochondrial dysfunction and altered cellular metabolism.

Massive efforts to sequence cancer genomes have compiled an impressive catalogue of cancer mutations, revealing the recurrent exploitation of a handful of 'hallmark cancer pathways'. However, unraveling how sets of mutated proteins in these and other pathways hijack pro-proliferative signaling networks and dictate therapeutic responsiveness remains challenging. Here, we show that cancer driver protein-protein interactions are enriched for additional cancer drivers, highlighting the power of physical interaction maps to explain known, as well as uncover new, disease-promoting pathway interrelationships. We hypothesize that by systematically mapping the protein-protein and genetic interactions in cancer-thereby creating Cancer Cell Maps-we will create resources against which to contextualize a patient's mutations into perturbed pathways/complexes and thereby specify a matching targeted therapeutic cocktail.

Macrophages are highly plastic cells that adopt diverse functional capabilities and play critical roles in immunity, cancer, and tissue homeostasis, but how these different cell fates and activities are triggered in response to their environmental cues is not well understood. We used new proteomic tools to identify protein post-translational modifications (PTMs) that control antibacterial responses of macrophages. Here, we report an unbiased and global analysis of the changes in host protein abundance, phosphorylation, and ubiquitylation, during the first 24-hours of Mycobacterium tuberculosis (Mtb) infection of primary macrophages. We discovered 1379 proteins with changes in their phosphorylation state and 591 proteins with changes in their ubiquitylation in response to Mtb infection. We identified pathways regulated by phosphorylation and ubiquitylation that weren’t reflected by changes in protein abundance, indicating that the activity of these pathways was regulated. These include pathways known to be regulated by ubiquitylation and phosphorylation (e.g. autophagy) as well as pathways that were not known to be regulated during Mtb infection (e.g. nucleocytoplasmic transport and mRNA metabolism). We identified an enrichment in phosphorylation of autophagy receptors (TAX1BP1, p62, optineurin, BNIP3L), several of which were not previously implicated in the host response to Mtb infection. We found that p62 deficiency blocks ubiquitylation and TAX1BP1 deficiency enhances ubiquitylation, suggesting p62 ubiquitylation acts as an amplification loop by promoting downstream adaptor recruitment and serves as a platform for recruitment of ubiquitin. Our results show that TAX1BP1 mediates clearance of ubiquitylated Mtb and targets the bacteria to LC3-positive phagophores. Taken together, our proteomic profiling is likely a valuable resource for initiating mechanistic studies of macrophage biology.

During infection, Legionella pneumophila translocates over 300 effector proteins into the host cytosol, allowing the pathogen to establish an endoplasmic reticulum (ER)-like Legionella-containing vacuole (LCV) that supports bacterial replication. Here, we perform a genome-wide CRISPR-Cas9 screen and secondary targeted screens in U937 human monocyte/macrophage-like cells to systematically identify host factors that regulate killing by L. pneumophila. The screens reveal known host factors hijacked by L. pneumophila, as well as genes spanning diverse trafficking and signaling pathways previously not linked to L. pneumophila pathogenesis. We further characterize C1orf43 and KIAA1109 as regulators of phagocytosis and show that RAB10 and its chaperone RABIF are required for optimal L. pneumophila replication and ER recruitment to the LCV. Finally, we show that Rab10 protein is recruited to the LCV and ubiquitinated by the effectors SidC/SdcA. Collectively, our results provide a wealth of previously undescribed insights into L. pneumophila pathogenesis and mammalian cell function.

Neuroblasts in flies divide asymmetrically by establishing polarity, distributing cell fate determinants asymmetrically, and positioning their spindle for cell division. The apical complex contains aPKC, Bazooka (Par3), and Par6, and its activity depends on L(2)gl. We show that Ankle2 interacts with L(2)gl and affects aPKC. Reducing Ankle2 levels disrupts ER and nuclear envelope morphology, releasing the kinase Ballchen/VRK1 into the cytosol. These defects are associated with reduced phosphorylation of aPKC, disruption of Par complex localization, and spindle alignment defects. Importantly, removal of one copy ofballchen/VRK1orl(2)glsuppresses the loss ofAnkle2and restores viability and brain size. The Zika virus NS4A protein interacts withDrosophilaAnkle2 and VRK1 in dividing neuroblasts. Human mutational studies implicate this neural cell division pathway in microcephaly and motor neuron disease. In summary, NS4A, ANKLE2, VRK1 and LLGL1 define a novel pathway that impinges on asymmetric determinants of neural stem cell division.

Cycles of Cdc53/Cullin1 rubylation (a.k.a NEDDylation) protect ubiquitin-E3 SCF (Skp1-Cullin1-F-box protein) complexes from self-destruction and play an important role in mediating the ubiquitination of key protein substrates involved in cell cycle progression, development, and survival. Cul1 rubylation is balanced by the COP9 signalosome (CSN), a multi-subunit derubylase that shows 1:1 paralogy to the 26S proteasome lid. The turnover of SCF substrates and their relevance to various diseases is well studied, yet, the extent by which environmental perturbations influence Cul1 rubylation/derubylation cycles per se is still unclear. In this study, we show that the level of cellular oxidation serves as a molecular switch, determining Cullin1 rubylation/derubylation ratio. We describe a mutant of the proteasome lid subunit, Rpn11 that exhibits accumulated levels of Cullin1-Rub1 conjugates, a characteristic phenotype of csn mutants. By dissecting between distinct phenotypes of rpn11 mutants, proteasome and mitochondria dysfunction, we were able to recognize the high reactive oxygen species (ROS) production during the transition of cells into mitochondrial respiration, as a checkpoint of Cullin1 rubylation in a reversible manner. Thus, the study adds the rubylation cascade to the list of cellular pathways regulated by redox homeostasis., Graphical abstract fx1, Highlights • Cullin rubylation status inversely correlates with the cellular redox state. • The yeast diauxic shift blocks cullin rubylation in a reversible manner. • Cullin rubylation status is determined by the oxidation state of individual cells. • The activity of rubylation cascade enzymes is perturbed by high ROS. • The loss of cullin rubylation correlates with the appearance of free Rub1 molecules.

As multicellular organisms grow, spatial and temporal patterns of gene expression are strictly regulated to ensure that developmental programs are invoked at appropriate stages. In this work, we describe a putative transcriptional regulator in Arabidopsis, TACO LEAF (TCO), whose overexpression results in the ectopic activation of reproductive genes during vegetative growth. Isolated as an activation-tagged allele, tco-1D displays gene misexpression and phenotypic abnormalities, such as curled leaves and early flowering, characteristic of chromatin regulatory mutants. A role for TCO in this mode of transcriptional regulation is further supported by the subnuclear accumulation patterns of TCO protein and genetic interactions between tco-1D and chromatin modifier mutants. The endogenous expression pattern of TCO and gene misregulation in tco loss-of-function mutants indicate that this factor is involved in seed development. We also demonstrate that specific serine residues of TCO protein are targeted by the ubiquitous kinase CK2. Collectively, these results identify TCO as a novel regulator of gene expression whose activity is likely influenced by phosphorylation, as is the case with many chromatin regulators.

Ebola virus (EBOV) infection often results in fatal illness in humans, yet little is known about how EBOV usurps host pathways during infection. To address this, we used affinity tag-purification mass spectrometry (AP-MS) to generate an EBOV-host protein-protein interaction (PPI) map. We uncovered 194 high-confidence EBOV-human PPIs, including one between the viral transcription regulator VP30 and the host ubiquitin ligase RBBP6. Domain mapping identified a 23 amino acid region within RBBP6 that binds to VP30. A crystal structure of the VP30-RBBP6 peptide complex revealed that RBBP6 mimics the viral nucleoprotein (NP) binding to the same interface of VP30. Knockdown of endogenous RBBP6 stimulated viral transcription and increased EBOV replication, whereas overexpression of either RBBP6 or the peptide strongly inhibited both. These results demonstrate the therapeutic potential of biologics that target this interface and identify additional PPIs that may be leveraged for novel therapeutic strategies.

Although polyubiquitin chains linked through all lysines of ubiquitin exist, specific functions are well-established only for lysine-48 and lysine-63 linkages in Saccharomyces cerevisiae. To uncover pathways regulated by distinct linkages, genetic interactions between a gene deletion library and a panel of lysine-to-arginine ubiquitin mutants were systematically identified. The K11R mutant had strong genetic interactions with threonine biosynthetic genes. Consistently, we found that K11R mutants import threonine poorly. The K11R mutant also exhibited a strong genetic interaction with a subunit of the anaphase-promoting complex (APC), suggesting a role in cell cycle regulation. K11-linkages are important for vertebrate APC function, but this was not previously described in yeast. We show that the yeast APC also modifies substrates with K11-linkages in vitro, and that those chains contribute to normal APC-substrate turnover in vivo. This study reveals comprehensive genetic interactomes of polyubiquitin chains and characterizes the role of K11-chains in two biological pathways.

Summary The multi-subunit bacterial RNA polymerase (RNAP) and its associated regulators carry out transcription and integrate myriad regulatory signals. Numerous studies have interrogated RNAP mechanism, and RNAP mutations drive Escherichia coli adaptation to many health- and industry-relevant environments, yet a paucity of systematic analyses hampers our understanding of the fitness trade-offs from altering RNAP function. Here, we conduct a chemical-genetic analysis of a library of RNAP mutants. We discover phenotypes for non-essential insertions, show that clustering mutant phenotypes increases their predictive power for drawing functional inferences, and demonstrate that some RNA polymerase mutants both decrease average cell length and prevent killing by cell-wall targeting antibiotics. Our findings demonstrate that RNAP chemical-genetic interactions provide a general platform for interrogating structure-function relationships in vivo and for identifying physiological trade-offs of mutations, including those relevant for disease and biotechnology. This strategy should have broad utility for illuminating the role of other important protein complexes.

Components of the proteostasis network malfunction in aging, and reduced protein quality control in neurons has been proposed to promote neurodegeneration. Here, we investigate the role of chaperone-mediated autophagy (CMA), a selective autophagy shown to degrade neurodegeneration-related proteins, in neuronal proteostasis. Using mouse models with systemic and neuronal-specific CMA blockage, we demonstrate that loss of neuronal CMA leads to altered neuronal function, selective changes in the neuronal metastable proteome, and proteotoxicity, all reminiscent of brain aging. Imposing CMA loss on a mouse model of Alzheimer’s disease (AD) has synergistic negative effects on the proteome at risk of aggregation, thus increasing neuronal disease vulnerability and accelerating disease progression. Conversely, chemical enhancement of CMA ameliorates pathology in two different AD experimental mouse models. We conclude that functional CMA is essential for neuronal proteostasis through the maintenance of a subset of the proteome with a higher risk of misfolding than the general proteome.

Structural analysis of host-pathogen protein complexes remains challenging, largely due to their structural heterogeneity. Here, we describe a pipeline for the structural characterization of these complexes using integrative structure modeling based on chemical cross-links and residue-protein contacts inferred from mutagenesis studies. We used this approach on the HIV-1 Vif protein bound to restriction factor APOBEC3G (A3G), the Cullin-5 E3 ring ligase (CRL5), and the cellular transcription factor Core Binding Factor Beta (CBFβ) to determine the structure of the (A3G-Vif-CRL5-CBFβ) complex. Using the MS-cleavable DSSO cross-linker to obtain a set of 132 cross-links within this reconstituted complex along with the atomic structures of the subunits and mutagenesis data, we computed an integrative structure model of the heptameric A3G-Vif-CRL5-CBFβ complex. The structure, which was validated using a series of tests, reveals that A3G is bound to Vif mostly through its N-terminal domain. Moreover, the model ensemble quantifies the dynamic heterogeneity of the A3G C-terminal domain and Cul5 positions. Finally, the model was used to rationalize previous structural, mutagenesis and functional data not used for modeling, including information related to the A3G-bound and unbound structures as well as mapping functional mutations to the A3G-Vif interface. The experimental and computational approach described here is generally applicable to other challenging host-pathogen protein complexes.

Nuclear mRNA export is highly regulated to ensure accurate cellular gene expression. Viral inhibition of cellular mRNA export can enhance viral access to the cellular translation machinery and prevent anti-viral protein production but is generally thought to be nonselective. We report that ORF10 of Kaposi's sarcoma associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. Nuclear export inhibition by ORF10 requires an interaction with an RNA export factor, Rae1. Genome-wide analysis reveals a subset of cellular mRNAs whose nuclear export is blocked by ORF10 with the 3' untranslated regions (3' UTRs) of ORF10-targeted transcripts conferring sensitivity to export inhibition. The Rae1-ORF10 interaction is important for the virus to express viral genes and produce infectious virions. These results suggest that a nuclear DNA virus can selectively interfere with RNA export to restrict host gene expression for optimal replication.

Summary: New genetic tools are needed to understand the functional interactions between HIV and human host factors in primary cells. We recently developed a method to edit the genome of primary CD4+ T cells by electroporation of CRISPR/Cas9 ribonucleoproteins (RNPs). Here, we adapted this methodology to a high-throughput platform for the efficient, arrayed editing of candidate host factors. CXCR4 or CCR5 knockout cells generated with this method are resistant to HIV infection in a tropism-dependent manner, whereas knockout of LEDGF or TNPO3 results in a tropism-independent reduction in infection. CRISPR/Cas9 RNPs can furthermore edit multiple genes simultaneously, enabling studies of interactions among multiple host and viral factors. Finally, in an arrayed screen of 45 genes associated with HIV integrase, we identified several candidate dependency/restriction factors, demonstrating the power of this approach as a discovery platform. This technology should accelerate target validation for pharmaceutical and cell-based therapies to cure HIV infection. : Hultquist et al. report a high-throughput platform for the efficient, multiplex editing of host factors that control HIV infection in primary CD4+ T cells. Arrayed electroporation of CRISPR/Cas9 ribonucleoproteins (RNPs) permits the rapid generation of isogenic human cells with ablated candidate factors and identifies gene modifications that provide viral resistance. Keywords: CRISPR/Cas9, HIV integrase, primary T cells, genome editing, CXCR4, CCR5, LEDGF, TNPO3, host-pathogen interactions, host dependency factors

Type IV pili (TFP) function as mechanosensors to trigger acute virulence programs in Pseudomonas aeruginosa. On surface contact, TFP retraction activates the Chp chemosensory system phosphorelay to upregulate 3', 5'-cyclic monophosphate (cAMP) production and transcription of virulence-associated genes. To dissect the specific interactions mediating the mechanochemical relay, we used affinity purification/mass spectrometry, directed co-immunoprecipitations in P. aeruginosa, single cell analysis of contact-dependent transcriptional reporters, subcellular localization and bacterial two hybrid assays. We demonstrate that FimL, a Chp chemosensory system accessory protein of unknown function, directly links the integral component of the TFP structural complex FimV, a peptidoglycan binding protein, with one of the Chp system output response regulators PilG. FimL and PilG colocalize at cell poles in a FimV-dependent manner. While PilG phosphorylation is required for TFP function and mechanochemical signaling, it is not required for polar localization or binding to FimL. Phylogenetic analysis reveals other bacterial species simultaneously encode TFP, the Chp system, FimL, FimV and adenylate cyclase homologs, suggesting that surface sensing may be widespread among TFP-expressing bacteria. We propose that FimL acts as a scaffold enabling spatial colocalization of TFP and Chp system components to coordinate signaling leading to cAMP-dependent upregulation of virulence genes on surface contact.

Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function, developmental pathways, and disease mechanisms. Here, we develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn), CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types, dissect developmental pathways, and model disease.

Summary Accurately translating genotype to phenotype requires accounting for the functional impact of genetic variation at many biological scales. Here we present a strategy for genotype-phenotype reasoning based on existing knowledge of cellular subsystems. These subsystems and their hierarchical organization are defined by the Gene Ontology or a complementary ontology inferred directly from previously published datasets. Guided by the ontology’s hierarchical structure, we organize genotype data into an “ontotype,” that is, a hierarchy of perturbations representing the effects of genetic variation at multiple cellular scales. The ontotype is then interpreted using logical rules generated by machine learning to predict phenotype. This approach substantially outperforms previous, non-hierarchical methods for translating yeast genotype to cell growth phenotype, and it accurately predicts the growth outcomes of two new screens of 2,503 double gene knockouts impacting DNA repair or nuclear lumen. Ontotypes also generalize to larger knockout combinations, setting the stage for interpreting the complex genetics of disease.

Several systems-level datasets designed to dissect host-pathogen interactions during influenza A infection have been reported. However, apparent discordance among these data has hampered their full utility toward advancing mechanistic and therapeutic knowledge. To collectively reconcile these datasets, we performed a meta-analysis of data from eight published RNAi screens and integrated these data with three protein interaction datasets, including one generated within the context of this study. Further integration of these data with global virus-host interaction analyses revealed a functionally validated biochemical landscape of the influenza-host interface, which can be queried through a simplified and customizable web portal (http://www.metascape.org/IAV). Follow-up studies revealed that the putative ubiquitin ligase UBR4 associates with the viral M2 protein and promotes apical transport of viral proteins. Taken together, the integrative analysis of influenza OMICs datasets illuminates a viral-host network of high-confidence human proteins that are essential for influenza A virus replication.

The Cancer Genome Atlas and similar projects have now analyzed over 10,000 tumor genomes, providing a catalog of the gene mutations, copy number variants and other genetic alterations that are present cancer. In many cases though, it remains unclear which are the key driver mutations or dependencies in a given cancer and how these influence pathogenesis and response to therapy. Although tumors of similar types and clinical outcomes can have patterns of mutations that are strikingly different, it is becoming apparent that these mutations recurrently hijack the same hallmark molecular pathways and networks. For this reason, cancer research and treatment is increasingly dependent on knowledge of biological networks of multiple types, including physical interactions among proteins and both synthetic-lethal and epistatic interactions among genes. Founded in 2015 by labs at UC San Diego and UCSF, the mission of the Cancer Cell Map Initiative (CCMI) is to enable a new era of cancer discovery and treatment based on the complete elucidation of the molecular networks underlying cancer. We believe that this information will be critical for developing computational models of cancer cells that will enable both basic research and clinical decision-making. Our initial research efforts are focused on head and neck cancer and breast cancer but we believe that our approach is widely applicable. Here, I will discuss results from three of the CCMI's main research efforts. One major focus is using affinity purification followed by mass spectrometry (AP/MS) to map protein-protein interactions of frequently mutated genes in multiple, relevant cell culture models. These efforts for approximately 40 genes in both head and neck cancer and breast cancer have largely been completed leading to a range of validation and follow-up studies. To complement these context-specific structural maps, a second main research effort is to generate functional maps by measuring genetic interactions using CRISPR/Cas9 in many of the same cell culture models. Many of these screens have now been completed with data analysis ongoing. Along with these two experimental efforts, a third focus has been to use bioinformatics approaches to assemble a hierarchical map of biological subsystems in cancer, to identify which systems in which cohorts are mutated more often than expected by chance and to explore these potentially driver systems in more detail through a range of experimental studies. To share these and other network maps, the CCMI's bioinformatics core has created the network data exchange (NDEx, ndexbio.org), an open-source framework where scientists and organizations can share, store, manipulate and publish biological network knowledge. Citation Format: Jason F. Kreisberg, Fan Zheng, Samson Fong, Brent M. Kuenzi, Kyle Ford, Danielle Swaney, Minkyu Kim, Jisoo Park, Zhiyong Wang, Dexter Pratt, Natalia Jura, Silvio Gutkind, Prashant Mali, Nevan Krogan, Trey Ideker. Using physical, genetic and integrated cancer cell maps to investigate head and neck cancer and breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 223.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. Advanced HCC has proven particularly difficult to treat because of a scarcity of clear genetic drivers of cancer progression; thus, there are currently no predictive markers that guide HCC therapy. HCC arises in the context of co-morbid hepatitis due to hepatitis B virus (HBV), hepatitis C (HCV) or fatty liver disease. We hypothesize that protein-protein interactions (PPIs) between viral proteins and HCC genes may contribute to tumor initiation and maintenance. In order to characterize these PPIs, we performed affinity purification - mass spectrometry (APMS), defining 145 HBV/host PPIs including known and novel interacting partners. We next used a network propagation algorithm to identify host genes and protein complexes that were preferentially mutated in the absence of HBV infection. HBV is a small DNA virus, with 4 genes of which only one has enzymatic activity, raising a question as to how HBV interaction modifies host behavior. Using AP-MS of host proteins, we found that the HBV X protein (HBx) remodels multiple host protein complexes through direct interaction. These physical effects on complex components result in distinct biochemical behavior from the CRL4 E3 ubiquitin ligase complex as well as the phosphatase PP2A, as determined through global phosphoproteomics and ubiquitin analysis. We show that this remodeling driven by HBx substantially changes cellular protein turnover and downstream signaling dynamics. We followed this up with assessments of cellular viability and proliferation in response to pharmacological inhibition or CRISPRi-based knockdown of HBx effectors. Our data support a model where HBV proteins alter the components and behavior of key regulatory protein complexes in the cell, altering tumor behavior and raising the possibility of precision therapeutics for HCC. Citation Format: John D. Gordan, Adriana Pitea, Manon Eckhardt, Gwendolyn Jang, Rigney E. Turnham, Alex L M. Choi, John Von Dollen, Huat C. Lim, Elizabeth F. Thayer, R. Katie Kelley, Danielle L. Swaney, Wei Zhang, Fabian J. Theis, Trey Ideker, Nevan J. Krogan. Hepatitis B virus remodels host protein interaction networks to generate distinct cellular dependencies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4891.

Deficiency in one or more subsystems of the DNA damage response and repair system hierarchy is a hallmark of cancer and makes DNA repair an attractive target for physical or chemical therapies. An accurate, complete, yet parsimonious model of the systems and pathways comprising the hierarchy of DNA repair mechanisms can be expected to further our understanding of cancer genesis and progression and to facilitate the development of improved cancer therapies. Previous attempts in building a hierarchical model of DNA repair include methods based on literature curation (Gene Ontology [1]) and expert consensus (2). However, as these models rely on manual curation, rapid integration of novel omics-scale experimental data sets is not practicable. Moreover, Gene Ontology is by its definition only concerned with the modeling of pathways in the healthy cell and does not aim to reflect cancer-specific aspects. To bridge this gap, we here describe the generation of an entirely data-driven model of DNA repair, inferred from a large meta-compendium describing the physical interactions between proteins based on an integration of over 110 experimental data sets from different omics levels (genome, transcriptome, proteome). Augmenting a technique that we developed earlier, Active Interaction Mapping (AIM) (3), we define a hierarchical model of DNA repair consisting of 293 genes organized in 40 systems. Briefly, we integrate the interaction evidence using a random forest regressor to generate a weighted, integrated network; we then transform the network into a consensus matrix and feed this into an algorithm based on previous work (4) to identify the hierarchical structure embedded in the network. The hierarchical model takes the form of a directed acyclic graph (DAG), which allows us to model pleiotropic aspects of molecular systems, as some proteins and systems play functional roles in different ancestor systems. Using a network biology approach, we suggest a list of 293 genes playing central roles in DNA repair and find evidence that genome replication and RNA splicing processes are more intimately connected to DNA repair than previously appreciated. We find novel roles for about one third of the genes in our model, identify novel subsystems of already known systems in DNA repair, and can describe novel interactions between already known DNA repair systems. Finally, we perform perturbations of the DNA repair hierarchy in different cell lines, measure the impact on the protein-protein and genetic interaction networks, and use the identified interactions to search for impacted connections within and between systems of the DNA repair model. References: 1. Ashburner M et al., Nature Genetics 2000;25:25. 2. Pearl LH et al., Nature Reviews Cancer 2015;15:166. 3. Kramer MH et al., Molecular Cell 2017;65:761. 4. Kramer M, Dutkowski J, Yu M, Bafna V, Ideker T. Bioinformatics 2014;30:i34. This abstract is also being presented as Poster B43. Citation Format: Anton Kratz, Fan Zheng, Min Kyu, Nevan Krogan, Trey Ideker. A hierarchical model of DNA repair inferred from omics-scale genetic interaction data reveals the dynamics of DNA damage induction [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PHB03.

Interpreting cancer genomes requires a broad understanding of the composition and organization of cellular processes under selective pressure for mutations. Here, we integrate systematic screens for protein interaction with tumor genetic analysis to elucidate a multiscale hierarchical map of 378 protein systems recurrently altered in one or more cancer types. Diverse gene mutations converge on commonly mutated systems, from small protein complexes in specific tumor types to large molecular assemblies and organelles disrupted in most cancers. Unexpected findings include collagen structural alterations with significant prognostic value; mutations impacting the desmosome in 65% melanomas; complexes related to splicing and DNA repair; and an expanded actomyosin cluster involving PIK3CA. The map implicates 556 cancer genes, many well supported by functional assays, including canonical genes implicated in new tissues (e.g., BRCA1/2 mutations in bladder cancer). This work explains mutational heterogeneity as a collection of convergence points across scales of cell biology. This abstract is also being presented as Poster B52. Citation Format: Fan Zheng, Keiichiro Ono, Erica Silva, Danielle Swaney, Minkyu Kim, Julia Shangguan, Dexter Pratt, Xiaolin Nan, Nevan Krogan, Trey Ideker. A multiscale map of recurrently mutated systems in cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR14.

Chromatin remodeling complexes instruct cellular differentiation and lineage specific transcription. The BRG1/BRM-associated factor (BAF) complexes are important for several aspects of differentiation. We show that the catalytic subunit gene Brg1 has a specific role in cardiac precursors (CPs) to initiate cardiac gene expression programs and repress non-cardiac expression. Using immunopurification with mass spectrometry, we have determined the dynamic composition of BAF complexes during mammalian cardiac differentiation, identifying several cell-type specific subunits. We focused on the CP- and cardiomyocyte (CM)-enriched subunits BAF60c (SMARCD3) and BAF170 (SMARCC2). Baf60c and Baf170 co-regulate gene expression with Brg1 in CPs, and in CMs their loss results in broadly deregulated cardiac gene expression. BRG1, BAF60c and BAF170 modulate chromatin accessibility, to promote accessibility at activated genes while closing chromatin at repressed genes. BAF60c and BAF170 are required for proper BAF complex composition, and BAF170 loss leads to retention of BRG1 at CP-specific sites. Thus, dynamic interdependent BAF complex subunit assembly modulates chromatin states and thereby participates in directing temporal gene expression programs in cardiogenesis., Summary: BRG1/BRM-associated factor (BAF) chromatin remodeling complexes include specific subunits during cardiac differentiation that are important for BAF complex composition and for regulating cardiac gene expression.

SUMMARY Multiple posttranslational modifications of the RNA polymerase II C-terminal domain (CTD) coordinate passage through the transcription cycle. The crosstalk between different modifications is poorly understood. Here, we show how acetylation of lysine residues at position 7 of characteristic heptad repeats (K7ac), a modification only found in higher eukaryotes, regulates phosphorylation of serines at position 5 (S5p), a conserved mark of polymerases initiating transcription. Using mass spectrometry, we identified Regulator of Pre-mRNA Domain-containing (RPRD) proteins as reader proteins of K7ac. K7ac enhanced in vitro binding of CTD peptides to the CTD-interacting domain (CID) of RPRD1A and RPRD1B proteins and inversely regulated S5p levels genome-wide. Treatment with deacetylase inhibitors globally enhanced levels of K7ac‐ and decreased levels of S5-phosphorylated polymerases ≥500 base pairs downstream of transcription start sites of expressed genes, consistent with acetylation-dependent S5 dephosphorylation via a previously identified RPRD-associated S5 phosphatase. Consistent with this model, RPRD1B knockdown increased S5p, but also enhanced K7ac levels, indicating that RPRD proteins recruit a K7 deacetylase. Collectively, our data identify RPRD CIDs as K7ac reader domains and reveal auto-regulatory crosstalk between K7ac and S5p via RPRD proteins at the transition from transcription initiation to elongation in higher eukaryotes.

Understanding of repair outcomes after Cas9-induced DNA cleavage is still limited, especially in primary human cells. We sequence repair outcomes at 1,656 on-target genomic sites in primary human T cells and use these data to train a machine learning model, which we have called CRISPR Repair Outcome (SPROUT). SPROUT accurately predicts the length, probability and sequence of nucleotide insertions and deletions, and will facilitate design of SpCas9 guide RNAs in therapeutically important primary human cells.

Targeting MYC oncogene remains a major therapeutic goal in cancer chemotherapy. Here, we demonstrate that proscillaridin, a cardiac glycoside approved for heart failure treatment exhibit anticancer selectivity towards high MYC expressing leukemic cell lines and leukemia stem cells. At a clinically relevant concentration, proscillaridin induced a rapid downregulation of MYC protein level, due to a significant decrease in MYC protein half-life. Proscillaridin treatment induced a downregulation of gene sets involved in MYC pathway, and a concomitant upregulation of genes involved in hematopoietic differentiation. Proscillaridin induced a significant loss of lysine acetylation in histone H3 (K9, K14, K18 and K27) and in non-histone proteins such as MYC, MYC target proteins, and a series of histone acetylation regulators. Loss of lysine acetylation correlated with a rapid downregulation of histone acetyltransferase protein levels, involved in histone and MYC acetylation (CBP, P300, GCN5, TIP60, and MOZ), preferentially in MYC overexpressing leukemia as compared to other cancer cells. These results support the repurposing of proscillaridin in MYC overexpressing leukemia and propose a novel strategy to target MYC in cancer.

SUMMARYPTEN is a tumor suppressor that is often inactivated in cancer and possesses both lipid and protein phosphatase activities. We report the metabolic regulator PDHK1 (pyruvate dehydrogenase kinase1) is a synthetic-essential gene in PTEN-deficient cancer and normal cells. The predominant mechanism of PDHK1 regulation and dependency is the PTEN protein phosphatase dephosphorylates NFκ;B activating protein (NKAP) and limits NFκB activation to suppress expression of PDHK1, a NFκB target gene. Loss of the PTEN protein phosphatase upregulates PDHK1 to drive aerobic glycolysis and induce PDHK1 cellular dependence. PTEN-deficient human tumors harbor increased PDHK1, which is a biomarker of decreased patient survival, establishing clinical relevance. This study uncovers a PTEN-regulated signaling pathway and reveals PDHK1 as a potential target in PTEN-deficient cancers.SIGNIFICANCEThe tumor suppressor PTEN is widely inactivated in cancers and tumor syndromes. PTEN antagonizes PI3K/AKT signaling via its lipid phosphatase activity. The modest success of PI3K/AKT inhibition in PTEN-deficient cancer patients provides rationale for identifying other vulnerabilities in PTEN-deficient cancers to improve clinical outcomes. We show that PTEN-deficient cells are uniquely sensitive to PDHK1 inhibition. PTEN and PDHK1 co-suppression reduced colony formation and induced cell deathin vitroand tumor regressionin vivo. PDHK1 levels were high in PTEN-deficient patient tumors and associated with inferior patient survival, establishing clinical relevance. Our study identifies a PTEN-regulated signaling pathway linking the PTEN protein phosphatase to the metabolic regulator PDHK1 and provides a mechanistic basis for PDHK1 targeting in PTEN-deficient cancers.

Zika virus (ZIKV) belongs to the family Flaviviridae, and is related to other viruses that cause human diseases. Unlike other flaviviruses, ZIKV infection can cause congenital neurological disorders and replicates efficiently in reproductive tissues1-3. Here we show that the envelope protein (E) of ZIKV is polyubiquitinated by the E3 ubiquitin ligase TRIM7 through Lys63 (K63)-linked polyubiquitination. Accordingly, ZIKV replicates less efficiently in the brain and reproductive tissues of Trim7-/- mice. Ubiquitinated E is present on infectious virions of ZIKV when they are released from specific cell types, and enhances virus attachment and entry into cells. Specifically, K63-linked polyubiquitin chains directly interact with the TIM1 (also known as HAVCR1) receptor of host cells, which enhances virus entry in cells as well as in brain tissue in vivo. Recombinant ZIKV mutants that lack ubiquitination are attenuated in human cells and in wild-type mice, but not in live mosquitoes. Monoclonal antibodies against K63-linked polyubiquitin specifically neutralize ZIKV and reduce viraemia in mice. Our results demonstrate that the ubiquitination of ZIKV E is an important determinant of virus entry, tropism and pathogenesis.

Recognition of nucleic acids enables detection of diverse pathogens by a limited number of innate immune receptors but also exposes the host to potential autoimmunity. At least two members of the Toll-like receptor (TLR) family, TLR7 and TLR9, can recognize self RNA or DNA, respectively. Despite the structural and functional similarities between these receptors, their contribution to autoimmune diseases such as SLE can be quite different. However, mechanisms of negative regulation that differentiate between TLR7 and TLR9 have not been described. Here we report a new function for the TLR trafficking chaperone Unc93b1 that specifically limits TLR7 signaling and prevents TLR7-dependent autoimmunity. Unc93b1 is known to traffic TLRs from the endoplasmic reticulum to endosomes, but this new regulatory function does not affect TLR7 localization. Instead, Unc93b1 recruits Syntenin-1, which inhibits TLR7, but not TLR9, signaling. Syntenin-1 binding requires phosphorylation of two serine residues on Unc93b1, providing a mechanism for dynamic regulation of the activation threshold of TLR7. Disruption of the Unc93b1/Syntenin-1 interaction in mice results in TLR7-dependent autoimmunity. Thus, Unc93b1 not only enables proper trafficking of nucleic acid sensing TLRs but also sets the activation threshold of these potentially self-reactive receptors.

The Streptococcus pyogenes Cas9 (SpCas9) nuclease has become a ubiquitous genome editing tool due to its ability to target almost any location in DNA and create a double-stranded break1,2. After DNA cleavage, the break is fixed with endogenous DNA repair machinery, either by non-templated mechanisms (e.g. non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ)), or homology directed repair (HDR) using a complementary template sequence3,4. Previous work has shown that the distribution of repair outcomes within a cell population is non-random and dependent on the targeted sequence, and only recent efforts have begun to investigate this further5–11. However, no systematic work to date has been validated in primary human cells5,7. Here, we report DNA repair outcomes from 1,521 unique genomic locations edited with SpCas9 ribonucleoprotein complexes (RNPs) in primary human CD4+ T cells isolated from multiple healthy blood donors. We used targeted deep sequencing to measure the frequency distribution of repair outcomes for each guide RNA and discovered distinct features that drive individual repair outcomes after SpCas9 cleavage. Predictive features were combined into a new machine learning model, CRISPR Repair OUTcome (SPROUT), that predicts the length and probability of nucleotide insertions and deletions with R2 greater than 0.5. Surprisingly, we also observed large insertions at more than 90% of targeted loci, albeit at a low frequency. The inserted sequences aligned to diverse regions in the genome, and are enriched for sequences that are physically proximal to the break site due to chromatin interactions. This suggests a new mechanism where sequences from three-dimensionally neighboring regions of the genome can be inserted during DNA repair after Cas9-induced DNA breaks. Together, these findings provide powerful new predictive tools for Cas9-dependent genome editing and reveal new outcomes that can result from genome editing in primary T cells.

Multi‐subunit cullin‐RING ligases (CRLs) are the largest family of ubiquitin E3 ligases in humans. CRL activity is tightly regulated to prevent unintended substrate degradation or autocatalytic degradation of CRL subunits. Using a proteomics strategy, we discovered that CRL4 AMBRA 1 (CRL substrate receptor denoted in superscript) targets Elongin C (ELOC), the essential adapter protein of CRL5 complexes, for polyubiquitination and degradation. We showed that the ubiquitin ligase function of CRL4 AMBRA 1 is required to disrupt the assembly and attenuate the ligase activity of human CRL5 SOCS 3 and HIV‐1 CRL5 VIF complexes as AMBRA1 depletion leads to hyperactivation of both CRL5 complexes. Moreover, CRL4 AMBRA 1 modulates interleukin‐6/STAT3 signaling and HIV‐1 infectivity that are regulated by CRL5 SOCS 3 and CRL5 VIF , respectively. Thus, by discovering a substrate of CRL4 AMBRA 1 , ELOC, the shared adapter of CRL5 ubiquitin ligases, we uncovered a novel CRL cross‐regulation pathway.

Infection with Mycobacterium tuberculosis continues to cause substantial human mortality, in part because of the emergence of antimicrobial resistance. Antimicrobial resistance in tuberculosis is solely the result of chromosomal mutations that modify drug activators or targets, yet the mechanisms controlling the mycobacterial DNA-damage response (DDR) remain incompletely defined. Here, we identify RecA serine 207 as a multifunctional signaling hub that controls the DDR in mycobacteria. RecA S207 is phosphorylated after DNA damage, which suppresses the emergence of antibiotic resistance by selectively inhibiting the LexA coprotease function of RecA without affecting its ATPase or strand exchange functions. Additionally, RecA associates with the cytoplasmic membrane during the mycobacterial DDR, where cardiolipin can specifically inhibit the LexA coprotease function of unmodified, but not S207 phosphorylated, RecA. These findings reveal that RecA S207 controls mutagenesis and antibiotic resistance in mycobacteria through phosphorylation and cardiolipin-mediated inhibition of RecA coprotease function.

Various physiological stimuli, such as cold environment, diet, and hormones, trigger brown adipose tissue (BAT) to produce heat through sympathetic nervous system (SNS)- and β-adrenergic receptors (βARs). The βAR stimulation increases intracellular cAMP levels through heterotrimeric G proteins and adenylate cyclases, but the processes by which cAMP modulates brown adipocyte function are not fully understood. Here we described that specific ablation of cAMP production in brown adipocytes led to reduced lipolysis, mitochondrial biogenesis, uncoupling protein 1 (Ucp1) expression, and consequently defective adaptive thermogenesis. Elevated cAMP signaling by sympathetic activation inhibited Salt-inducible kinase 2 (Sik2) through protein kinase A (PKA)-mediated phosphorylation in brown adipose tissue. Inhibition of SIKs enhanced Ucp1 expression in differentiated brown adipocytes and Sik2 knockout mice exhibited enhanced adaptive thermogenesis at thermoneutrality in an Ucp1-dependent manner. Taken together, our data indicate that suppressing Sik2 by PKA-mediated phosphorylation is a requisite for SNS-induced Ucp1 expression and adaptive thermogenesis in BAT, and targeting Sik2 may present a novel therapeutic strategy to ramp up BAT thermogenic activity in humans.

Although gene discovery in neuropsychiatric disorders, including autism spectrum disorder, intellectual disability, epilepsy, schizophrenia, and Tourette disorder, has accelerated, resulting in a large number of molecular clues, it has proven difficult to generate specific hypotheses without the corresponding datasets at the protein complex and functional pathway level. Here, we describe one path forward--an initiative aimed at mapping the physical and genetic interaction networks of these conditions, and then using these maps to connect the genomic data to neurobiology and, ultimately, the clinic. These efforts will include a team of geneticists, structural biologists, neurobiologists, systems biologists, and clinicians, leveraging a wide array of experimental approaches and creating a collaborative infrastructure necessary for long-term investigation. This initiative will ultimately intersect with parallel studies that focus on other diseases, as there is a significant overlap with genes implicated in cancer, infectious disease, and congenital heart defects.

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The HIV-1 Tat protein hijacks P-TEFb kinase to activate paused RNA polymerase II (RNAP II) at the viral promoter. Tat binds additional host factors, but it is unclear how they regulate RNAP II elongation. Here, we identify the cytoplasmic ubiquitin ligase UBE2O as critical for Tat transcriptional activity. Tat hijacks UBE2O to ubiquitinate the P-TEFb kinase inhibitor HEXIM1 of the 7SK snRNP, a fraction of which also resides in the cytoplasm bound to P-TEFb. HEXIM1 ubiquitination sequesters it in the cytoplasm and releases P-TEFb from the inhibitory 7SK complex. Free P-TEFb then becomes enriched in chromatin, a process that is also stimulated by treating cells with a CDK9 inhibitor. Finally, we demonstrate that UBE2O is critical for P-TEFb recruitment to the HIV-1 promoter. Together, the data support a unique model of elongation control where non-degradative ubiquitination of nuclear and cytoplasmic 7SK snRNP pools increases P-TEFb levels for transcriptional activation.

Cell-surface tumor marker EpCAM plays a key role in proliferation, differentiation and adhesion processes in stem and epithelial cells. It is established as a cell-cell adhesion molecule, forming intercellular interactions through homophilic association. However, the mechanism by which such interactions arise has not yet been fully elucidated. Here, we first show that EpCAM monomers do not associate into oligomers that would resemble an inter-cellular homo-oligomer, capable of mediating cell-cell adhesion, by using SAXS, XL-MS and bead aggregation assays. Second, we also show that EpCAM forms stable dimers on the surface of a cell with pre-formed cell-cell contacts using FLIM-FRET; however, no inter-cellular homo-oligomers were detectable. Thus, our study provides clear evidence that EpCAM indeed does not function as a homophilic cell adhesion molecule and therefore calls for a significant revision of its role in both normal and cancerous tissues. In the light of this, we strongly support the previously suggested name Epithelial Cell Activating Molecule instead of the Epithelial Cell Adhesion Molecule.

Mutations in several genes encoding centrosomal proteins dramatically decrease the size of the human brain. We show that Aspm (abnormal spindle-like, microcephaly-associated) and Wdr62 (WD repeat-containing protein 62) interact genetically to control brain size, with mice lacking Wdr62, Aspm, or both showing gene dose-related centriole duplication defects that parallel the severity of the microcephaly and increased ectopic basal progenitors, suggesting premature delamination from the ventricular zone. Wdr62 and Aspm localize to the proximal end of the mother centriole and interact physically, with Wdr62 required for Aspm localization, and both proteins, as well as microcephaly protein Cep63, required to localize CENPJ/CPAP/Sas-4, a final common target. Unexpectedly, Aspm and Wdr62 are required for normal apical complex localization and apical epithelial structure, providing a plausible unifying mechanism for the premature delamination and precocious differentiation of progenitors. Together, our results reveal links among centrioles, apical proteins, and cell fate, and illuminate how alterations in these interactions can dynamically regulate brain size.

The human interferon (IFN)-induced MxA protein is a key antiviral host restriction factor exhibiting broad antiviral activity against many RNA viruses, including highly pathogenic avian influenza A viruses (IAV) of the H5N1 and H7N7 subtype. To date the mechanism for how MxA exerts its antiviral activity is unclear, however, additional cellular factors are believed to be essential for this activity. To identify MxA cofactors we performed a genome-wide siRNA-based screen in human airway epithelial cells (A549) constitutively expressing MxA using an H5N1 reporter virus. These data were complemented with a proteomic screen to identify MxA-interacting proteins. The combined data identified SMARCA2, the ATPase subunit of the BAF chromatin remodeling complex, as a crucial factor required for the antiviral activity of MxA against IAV. Intriguingly, our data demonstrate that although SMARCA2 is essential for expression of some IFN-stimulated genes (ISGs), and the establishment of an antiviral state, it is not required for expression of MxA, suggesting an indirect effect on MxA activity. Transcriptome analysis of SMARCA2-depleted A549-MxA cells identified a small set of SMARCA2-regulated factors required for activity of MxA, in particular IFITM2 and IGFBP3. These findings reveal that several virus-inducible factors work in concert to enable MxA restriction of IAV.

Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.

Epistasis mapping, in which the phenotype that emerges from combining pairs of mutations is measured quantitatively, is a powerful tool for unbiased study of gene function. When performed at a large scale, this approach has been used to assign function to previously uncharacterized genes, define functional modules and pathways, and study their cross talk. These experiments rely heavily on methods for rapid sampling of binary combinations of mutant alleles by systematic generation of a series of double mutants. Epistasis mapping technologies now exist in various model systems. Here we provide an overview of different epistasis mapping technologies, including the pombe epistasis mapper (PEM) system designed for the collection of quantitative genetic interaction data in fission yeast Schizosaccharomyces pombe. Comprising a series of high-throughput selection steps for generation and characterization of double mutants, the PEM system has provided insight into a wide range of biological processes as well as facilitated evolutionary analysis of genetic interactomes across different species.