Two-way mixed-effects methods for joint association analysis using both host and pathogen genomes
Miaoyan Wang, Fabrice Roux, Claudia Bartoli, Carine Huard-Chauveau, Christopher Meyer, Hana Lee, Dominique Roby, Mary Sara McPeek, and Joy Bergelson
Genome-wide association (GWA) mapping is a powerful tool for identification of genetic variants underlying complex traits. However, existing methods typically performGWA mapping within a single species; methods allowing the description of the genomic landscape of interspecies interactions are only beginning to be developed. Here, we present a method to simultaneously perform GWA mapping on two interacting species.We applied our approach to the Arabidopsis thaliana–Xanthomonas arboricola pathosystem and identified candidate genes conferring host–pathogen specificity. By integrating the whole-genome sequence data available for pairs of interacting species, we can decipher the genetic architecture of complex traits in finer detail than has previously been possible. (See pp. E5440–E5449.)
Mapping an ancient city with a century of remotely sensed data
David Stott, Søren Munch Kristiansen, Achim Lichtenberger, and Rubina Raja
Understanding how people in the past adapted to environmental and economic challenges can help us anticipate and meet these challenges in the present. However, these very processes threaten the physical remains embodying this information worldwide: Urban expansion and resource exploitation mean that the quantity and quality of archaeological information are diminishing daily. In this work, we demonstrate how multitemporal aerial photography and modern airborne laser scanning are invaluable tools for mapping the remaining archaeological features extant in the present and for adding context to them from what has been lost. This knowledge enables cultural heritage administrators and archaeologists to actively monitor, understand, and manage the existing remains to make sure important information is not lost to posterity. (See pp. E5450–E5458.)
Gene cluster conservation provides insight into cercosporin biosynthesis and extends production to the genus Colletotrichum
Ronnie de Jonge, Malaika K. Ebert, Callie R. Huitt-Roehl, Paramita Pal, Jeffrey C. Suttle, Rebecca E. Spanner, Jonathan D. Neubauer, Wayne M. Jurick II, Karina A. Stott, Gary A. Secor, Bart P. H. J. Thomma, Yves Van de Peer, Craig A. Townsend, and Melvin D. Bolton
Species in the fungal genus Cercospora cause diseases in many important crops worldwide. Their success as pathogens is largely due to the secretion of cercosporin during infection. We report that the cercosporin toxin biosynthesis (CTB) gene cluster is ancient and was horizontally transferred to diverse fungal plant pathogens. Because our analyses revealed genes adjacent to the established CTB cluster with similar evolutionary trajectories, we evaluated their role in Cercospora beticola to show that four are necessary for cercosporin biosynthesis. Lastly, we confirmed that the apple pathogen Colletotrichum fioriniae produces cercosporin, the first case outside the family Mycosphaerellaceae. Other Colletotrichum plant pathogens also harbor the CTB cluster, which points to a wider role that this toxin may play in virulence. (See pp. E5459–E5466.)
Potent and selective antitumor activity of a T cell-engaging bispecific antibody targeting a membrane-proximal epitope of ROR1
Junpeng Qi, Xiuling Li, Haiyong Peng, Erika M. Cook, Eman L. Dadashian, Adrian Wiestner, HaJeung Park, and Christoph Rader
Harnessing and enhancing the innate and adaptive immune system to fight cancer represents one of the most promising strategies in contemporary cancer therapy. Although bispecific antibodies (biAbs) that combine a T cell-engaging arm with a tumor cell-binding arm are particularly potent cancer immunotherapeutic agents, they rely on the identification of tumor antigens with highly restricted expression. The receptor tyrosine kinase ROR1 is expressed by numerous cancers and is largely absent from postnatal healthy cells and tissues. Here we show that T cell-engaging biAbs that target ROR1 are highly potent in in vitro, in vivo, and ex vivo models of cancer, in particular when targeting a conserved site on ROR1 close to the tumor cell membrane we precisely mapped by X-ray crystallography. (See pp. E5467–E5476.)
In vitro reconstitution of sortase-catalyzed pilus polymerization reveals structural elements involved in pilin cross-linking
Chungyu Chang, Brendan R. Amer, Jerzy Osipiuk, Scott A. McConnell, I-Hsiu Huang, Van Hsieh, Janine Fu, Hong H. Nguyen, John Muroski, Erika Flores, Rachel R. Ogorzalek Loo, Joseph A. Loo, John A. Putkey, Andrzej Joachimiak, Asis Das, Robert T. Clubb, and Hung Ton-That
Gram-positive sortase enzymes represent two broad functional categories—those that cross-link proteins to the cell wall and those that can catalyze this reaction and polymerize proteins to build adhesive pilus fibers. Here we report an in vitro reproduction of a robust pilus polymerization reaction using a variant of a corynebacterial pilus-specific sortase in which the catalytic center is unmasked. By molecular modeling, we uncovered a conserved structural element of pilus-specific sortases critical for protein ligation in vitro and further demonstrated that the activated sortase ligates the isolated domains of the pilin harboring the donor and acceptor motifs for ligation. Besides enabling future molecular studies and antibiotic development, our system provides a powerful platform for bioconjugation and protein engineering. (See pp. E5477–E5486.)
Sequential activation of human signal recognition particle by the ribosome and signal sequence drives efficient protein targeting
Jae Ho Lee, Sowmya Chandrasekar, SangYoon Chung, Yu-Hsien Hwang Fu, Demi Liu, Shimon Weiss, and Shu-ou Shan
The universally conserved signal recognition particle (SRP) delivers ∼30% of the proteome to the appropriate cellular membrane. How SRP achieves efficient and selective protein targeting in eukaryotes remains elusive. Here, we show that a functional signal sequence on the nascent polypeptide confers a significant kinetic privilege to ribosome-bound SRP during the recruitment of SRP receptor, thereby enabling rapid and selective membrane targeting of SRP-dependent substrates. In addition, single-molecule spectroscopy revealed ribosome- and signal sequence-induced global conformational rearrangements in human SRP, which, together with biochemical analyses, provides a molecular model for substrate-induced activation of mammalian SRP. Analogous mechanisms have been described for replication, transcription, and translation, and may be envisioned for other pathways in which biological fidelity exceeds substrate binding specificity. (See pp. E5487–E5496.)
Protein kinase Cα gain-of-function variant in Alzheimer’s disease displays enhanced catalysis by a mechanism that evades down-regulation
Julia A. Callender, Yimin Yang, Gema Lordén, Natalie L. Stephenson, Alexander C. Jones, John Brognard, and Alexandra C. Newton
This work unveils how an Alzheimer’s disease-associated mutation (M489V) in protein kinase Cα (PKCα) enhances catalytic activity without sensitizing the protein to the cell’s homeostatic degradation of aberrantly active PKCα. The active conformation of wild-type PKC is sensitive to degradation, and therefore constitutively activated PKC paradoxically manifests as loss of function. We show that PKCα-M489V enhances the intrinsic catalytic rate of the kinase without altering the equilibrium between the autoinhibited (protected) conformation and the activated (degradation-sensitive) conformation. Thus, the on/off dynamics are unchanged, but reactions are catalyzed at a faster rate when the enzyme is on. These findings are significant because they provide a mechanism through which a disease mutation in PKCα causes aberrant activation without resulting in paradoxical loss of function via degradation. (See pp. E5497–E5505.)
Structural basis for MTR4–ZCCHC8 interactions that stimulate the MTR4 helicase in the nuclear exosome-targeting complex
M. Rhyan Puno and Christopher D. Lima
Aberrant or unwanted transcripts can be degraded by the RNA exosome with the help of the nuclear exosome-targeting (NEXT) complex. NEXT, composed of RNA-binding protein RBM7, scaffold ZCCHC8, and helicase MTR4, is implicated in stress response, neurodegeneration, and viral ribogenesis. Here, we characterize the activities of NEXT that support its role in exosome-mediated decay. NEXT catalyzes 3′→5′ helicase activity and disrupts RNA:RNA and DNA:RNA duplexes more efficiently than MTR4. Optimal activity is observed when substrates include a uridine-rich motif, for interactions with RBM7, and a 3′ poly(A) tail. The ZCCHC8 C-terminal domain binds the helicase core and can stimulate MTR4 helicase/ATPase activities. Our results highlight the interplay among NEXT subunits to ensure effective targeting of substrates. (See pp. E5506–E5515.)
Dynamic action potential clamp predicts functional separation in mild familial and severe de novo forms of SCN2A epilepsy
Géza Berecki, Katherine B. Howell, Yadeesha H. Deerasooriya, Maria Roberta Cilio, Megan K. Oliva, David Kaplan, Ingrid E. Scheffer, Samuel F. Berkovic, and Steven Petrou
SCN2A, encoding the voltage-gated sodium channel Nav1.2, has emerged as a major gene implicated in neonatal-, infantile-, and even childhood-onset epilepsies. Many of these epilepsies are also associated with cognitive and behavioral impairments that range in type and severity. The biophysical, neurophysiological, and clinical impacts of SCN2A mutations are poorly understood. Here, we use clinical evaluation and biophysical analyses to explore the mechanisms underpinning distinctive phenotypes produced by SCN2A variants associated with mild familial or severe de novo forms of epilepsy. We show that dynamic clamp analysis provides clear benefits over conventional voltage clamp for a rapid and definitive prediction of neuron-scale phenotypic consequences, and is well positioned to impact diagnosis and drug discovery in genetic epilepsy. (See pp. E5516–E5525.)
Transcription factor profiling reveals molecular choreography and key regulators of human retrotransposon expression
Xiaoji Sun, Xuya Wang, Zuojian Tang, Mark Grivainis, David Kahler, Chi Yun, Paolo Mita, David Fenyö, and Jef D. Boeke
Retrotransposons replicate through RNA intermediates that are reverse transcribed and inserted at new genomic locations. LINE-1 (L1) elements constitute ∼17% of the human genome, making them the most successful retrotransposons in the human genome by mass. The activity of L1s was shown first in the germline or during early embryogenesis. More recent studies demonstrate a wider prevalence of L1 expression in somatic cells including neurons, aging cells, and different types of cancer. In this study, we developed the MapRRCon pipeline and performed a comprehensive computational analysis of L1 transcriptional regulators using ENCODE ChIP-seq datasets. We revealed the binding of various transcription factors, including Myc and CTCF, to the 5′ UTR promoter of the youngest human L1 family (L1HS) and their potential functional impact on L1HS expression. (See pp. E5526–E5535.)
Activated integrins identify functional antigen-specific CD8+ T cells within minutes after antigen stimulation
Stoyan Dimitrov, Cécile Gouttefangeas, Luciana Besedovsky, Anja T. R. Jensen, P. Anoop Chandran, Elisa Rusch, Ramona Businger, Michael Schindler, Tanja Lange, Jan Born, and Hans-Georg Rammensee
Assessing antigen-specific T cells is crucial for our understanding of immune reactions against pathogens and tumors, and for evaluating immunotherapies in patients. Existing techniques to evaluate the functionality of T lymphocytes all rely on de novo expression of proteins, typically intracellular cytokines, and therefore require elaborated protocols and reagents. We have established a simple flow cytometry-based method to assess the functionality of CD8+ T cells by identifying immediate changes in the conformation and valency of cell surface integrins that occur within minutes following antigenic stimulation. Because of its robustness, sensitivity, and broad applicability, the assay can be rapidly implemented for the measurement and isolation of functional T cells for basic research and in the clinical setting. (See pp. E5536–E5545.)
Mesenchymal MAPKAPK2/HSP27 drives intestinal carcinogenesis
Ana Henriques, Vasiliki Koliaraki, and George Kollias
Although MK2 inhibition has been proposed as a therapy in cancer, its exact role, as well as the cellular and molecular mechanisms underlying it, in the intestine is not known. Here, we show that complete MK2 deletion leads to decreased epithelial cell proliferation, associated with reduced tumor growth and invasive potential in the Apcmin/+ and colitis-associated cancer model. Notably, this function of MK2 is not mediated by its well-described immunomodulatory roles in inflammatory cells. Instead, MK2 modulates tumor progression mainly via modulating mesenchymal-specific Hsp27-mediated activation of protumorigenic mediators. Our results advance our understanding of mesenchymal MAPK signaling in intestinal cancer progression and demonstrate the value of MK2 inhibition in the treatment of cancer. (See pp. E5546–E5555.)
Role of CD40 and ADAMTS13 in von Willebrand factor-mediated endothelial cell–platelet–monocyte interaction
Miruna Popa, Sibgha Tahir, Julia Elrod, Su Hwan Kim, Florian Leuschner, Thorsten Kessler, Peter Bugert, Ulrich Pohl, Andreas H. Wagner, and Markus Hecker
In this study we have identified a mechanism that links pathophysiological ADAMTS13 deficiency to innate immunity and hemostasis by facilitating monocyte extravasation as a prerequisite for their subsequent on-site differentiation into proinflammatory macrophages under conditions of both physiological laminar flow and disturbed reduced flow, e.g., as occurs at atherosclerosis predilection sites. Our bioassay approach using patients’ plasma may become a useful diagnostic tool to assess ADAMTS13 activity ex vivo under conditions of near-physiological to atherosclerosis-prone arterial blood flow, which seems to be more relevant than the conventional static FRET-based assay method. (See pp. E5556–E5565.)
KDM4B protects against obesity and metabolic dysfunction
Yingduan Cheng, Quan Yuan, Laurent Vergnes, Xin Rong, Ji Youn Youn, Jiong Li, Yongxin Yu, Wei Liu, Hua Cai, Jiandie D. Lin, Peter Tontonoz, Christine Hong, Karen Reue, and Cun-Yu Wang
Obesity has become a major epidemic around the globe, with fatal comorbidities such as type 2 diabetes, heart disease, and cancer that have led to alarming health concerns in modern medicine. Although excess adipose tissue is associated with negative metabolic outcomes, the physiological functions of epigenetic regulators in adipose tissue and metabolism are unclear. Here we show that KDM4B in adipose tissues epigenetically controls energy expenditure, oxidation, lipolysis, and thermogenesis. Loss of Kdm4b impairs energy expenditure, adaptive thermogenesis, and adipose tissue lipolysis, resulting in obesity and associated metabolic dysfunction. Our results provide insights into control of obesity and suggest that modulation of KDM4B levels or activity may be a potential therapeutic target for human obesity. (See pp. E5566–E5575.)
Metaproteomics method to determine carbon sources and assimilation pathways of species in microbial communities
Manuel Kleiner, Xiaoli Dong, Tjorven Hinzke, Juliane Wippler, Erin Thorson, Bernhard Mayer, and Marc Strous
To understand the roles that microorganisms play in diverse environments such as the open ocean or the human intestinal tract, we need an understanding of their metabolism and physiology. A variety of methods such as metagenomics and metaproteomics exist to assess the metabolism of environmental microorganisms based on gene content and gene expression. These methods often only provide indirect evidence for which substrates are used by a microorganism in a community. The direct protein stable isotope fingerprint (SIF) method that we developed allows linking microbial species in communities to the environmental carbon sources they consume by determining their stable carbon isotope signature. Direct protein-SIF also allows assessing which carbon fixation pathway is used by autotrophic microorganisms that directly assimilate CO2. (See pp. E5576–E5584.)
Maturation of polycistronic mRNAs by the endoribonuclease RNase Y and its associated Y-complex in Bacillus subtilis
Aaron DeLoughery, Jean-Benoît Lalanne, Richard Losick, and Gene-Wei Li
Bacterial operons must solve a fundamental problem: how to produce discordant amounts of proteins from cotranscribed genes. Here, we used a genome-wide approach to map operon mRNA isoforms with nucleotide resolution in Bacillus subtilis and to quantify their abundances in different genetic backgrounds. These results revealed RNA endonucleolytic cleavages located in between cotranscribed genes that lead to differential transcript stability and abundance. The RNA endonuclease’s activity toward operon mRNA maturation requires a three-protein Y-complex. Interestingly, the Y-complex has few effects on other targets of the RNA endonuclease. The dichotomy of targets suggests that the Y-complex acts as a specificity factor for the general endonuclease, a role that could be broadly conserved in other Firmicutes, including the human pathogen Staphylococcus aureus. (See pp. E5585–E5594.)
NT3-chitosan enables de novo regeneration and functional recovery in monkeys after spinal cord injury
Jia-Sheng Rao, Can Zhao, Aifeng Zhang, Hongmei Duan, Peng Hao, Rui-Han Wei, Junkui Shang, Wen Zhao, Zuxiang Liu, Juehua Yu, Kevin S. Fan, Zhaolong Tian, Qihua He, Wei Song, Zhaoyang Yang, Yi Eve Sun, and Xiaoguang Li
Spinal cord injury (SCI) is a severe medical condition often leading to permanent loss of sensory, motor, and autonomic functions, currently with no cure. This study provides evidence that in monkeys with severe SCI, a bioactive and biodegradable material, NT3-chitosan, elicited robust de novo neural regeneration including, for example, long-distance axonal growth of cortical motor neurons in the cortical spinal tract, as well as sensory and motor functional recovery. In this study, we utilized a battery of minimally invasive outcome measures including fMRI, magnetic resonance diffusion tensor imaging, and kinematics walking analyses, which are all clinical trial-compatible. Essentially, success in this approach in nonhuman primate SCI models provides a solid foundation for its potential therapeutic application. (See pp. E5595–E5604.)
Doc2-mediated superpriming supports synaptic augmentation
Renhao Xue, David A. Ruhl, Joseph S. Briguglio, Alexander G. Figueroa, Robert A. Pearce, and Edwin R. Chapman
Plastic changes in synaptic connections constitute the basis of learning and memory. Different forms of synaptic plasticity are generally distinguished experimentally by their timescales, but it is unclear whether each form of plasticity corresponds to a distinct biological process with a dedicated molecular mechanism. In the present study, we show that the Ca2+-binding protein, Doc2, “superprimes” a subset of already primed synaptic vesicles to make them more likely to release, and this process selectively contributes to augmentation (on the scale of seconds). The underlying molecular mechanism does not mediate other forms of short-term enhancement (that occur on the timescale of milliseconds or minutes). This work establishes a function of Doc2 in maintaining synaptic plasticity within a narrow time window. (See pp. E5605–E5613.)
A theta rhythm in macaque visual cortex and its attentional modulation
Georgios Spyropoulos, Conrado Arturo Bosman, and Pascal Fries
Theta rhythms, ≈3–8 Hz, have been found in many different parts of the brain. They are predominant in the rodent hippocampus, yet have also been described in the neocortex, primarily in frontal and parietal areas in relation to executive functions. Here, we show a ≈4-Hz theta rhythm in awake macaque monkey area V4 and primary visual cortex. This theta rhythm was spatially coextensive with visually induced gamma-band activity, and gamma power was modulated by theta phase. The strength of theta and of theta-rhythmic gamma modulation was markedly reduced by selective attention. Theta rhythmicity has been observed in microsaccade sequences, and microsaccades influence early visual activity. Yet, removing (the effects of) microsaccades did not influence the results. (See pp. E5614–E5623.)
Mediator subunit MED31 is required for radial patterning of Arabidopsis roots
Xiaoyue Zhang, Wenkun Zhou, Qian Chen, Mingming Fang, Shuangshuang Zheng, Ben Scheres, and Chuanyou Li
SHORTROOT (SHR) and SCARECROW (SCR) orchestrate a transcriptional program that is essential for ground tissue patterning. The regulation of the transcriptional output of SHR/SCR remains unclear. We show that MED31, a subunit of the plant Mediator coactivator complex, bridges the communication between SHR/SCR and the RNA polymerase II general transcriptional machinery. MED31 and SHR bind to the same protein domain of SCR, and the relative abundance of MED31 and SHR determines the dynamic formation of a MED31/SCR/SHR ternary complex. We studied the role of the MED31/SCR/SHR ternary complex in controlling the spatiotemporal expression of CYCLIND6;1 during ground tissue patterning. This study provides insight into the mechanisms by which master transcriptional regulators control organ patterning. (See pp. E5624–E5633.)