Publications

In Press
Zhang, W, Messing J.  In Press.  PacBio RS for gene family studies. Methods in Molecular Biology. Haplotyping.
Wu, Y, Messing J.  In Press.  Understanding and improving protein traits in maize seeds. Achieving Sustainable Maize Cultivation.
2017
Boucher, HW, Ambrose PG, Chambers HF, Ebright RH, Jezek A, Murray BE, Newland JG, Ostrowsky B, Rex JH.  2017.  White Paper: Developing Antimicrobial Drugs for Resistant Pathogens, Narrow-spectrum Indications, and Unmet Needs.. Journal of Infectious Diseases. Abstract
Despite progress in antimicrobial drug development, a critical need persists for new, feasible pathways to develop antibacterial agents to treat people infected with drug-resistant bacteria. Infections due to resistant Gram-negative bacilli continue to cause unacceptable morbidity and mortality. Antibacterial agents have been traditionally studied in non-inferiority clinical trials that focus on one site of infection (eg, complicated urinary tract infections, intra-abdominal infections), yet these designs may not be optimal, and often are not feasible, for study of infections caused by drug-resistant bacteria. Over the past several years, multiple stakeholders have worked to develop consensus regarding paths forward with a goal of facilitating timely conduct of antimicrobial development. Here we advocate for a novel and pragmatic approach and, towards this end, present feasible trial designs for antibacterial agents that could enable conduct of narrow-spectrum, organism-specific clinical trials and ultimately approval of critically needed new antibacterial agents.
Walker, SS, Degen D, Nickbarg E, Carr D, Soriano A, Mandal M B, Painter RE, Sheth PR, Xiao L, Sher X et al..  2017.  Affinity selection-mass spectrometry identifies a novel antibacterial RNA polymerase inhibitor.. ACS Chemical Biology. 12:1346-1352. Abstract
The growing prevalence of drug-resistant Gram-negative bacteria is a significant global threat to human health. Rifampicin, an RNA polymerase-targeting agent, is an important part of the antibacterial armamentarium; however the emergence of resistance requires that it be used against only certain infections and usually in combination with another antibiotic. While rifampicin has significant clinical limitations, it does show that bacterial RNA polymerase can be an effective target for antibacterial intervention. To find new RNA polymerase inhibitors we initiated a screen of a collection of antibacterial bioactive molecules using affinity selection-mass spectrometry and purified Escherichia coli core RNA polymerase (subunits α, β, β', ω). Affinity selection screening identified a novel small molecule, MRL-436, that binds selectively to and inhibits RNA polymerase in vitro and inhibits RNA synthesis in the cell. Selection for resistance followed by whole genome sequencing identified a missense mutation in rpoC (β' subunit) and, separately, frameshift mutations in rpoZ (ω subunit) suggesting that MRL-436 targets RNA polymerase in the cell. In addition, cells lacking the rpoZ gene or purified RNA polymerase containing either a specific substitution in β' or lacking ω are selectively resistant to MRL-436. Molecular modeling indicates that the location of the substitution mutation in β' is closely juxtaposed with ω in a region of the complex thought to be important for transcription regulation during cellular response to amino acid starvation.
Lin, W, Mandal S, Degen D, Liu Y, Ebright YW, Li S, Feng Y, Zhang Y, Mandal S, Jiang Y et al..  2017.  Structural basis of Mycobacterium tuberculosis transcription and transcription inhibition.. Molecular Cell. 166:169-179. Abstract
Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which kills 1.8 million annually. Mtb RNA polymerase (RNAP) is the target of the first-line antituberculosis drug rifampin (Rif). We report crystal structures of Mtb RNAP, alone and in complex with Rif, at 3.8-4.4 Å resolution. The results identify an Mtb-specific structural module of Mtb RNAP and establish that Rif functions by a steric-occlusion mechanism that prevents extension of RNA. We also report non-Rif-related compounds-Nα-aroyl-N-aryl-phenylalaninamides (AAPs)-that potently and selectively inhibit Mtb RNAP and Mtb growth, and we report crystal structures of Mtb RNAP in complex with AAPs. AAPs bind to a different site on Mtb RNAP than Rif, exhibit no cross-resistance with Rif, function additively when co-administered with Rif, and suppress resistance emergence when co-administered with Rif.
Bird, JG, Nickels BE, Ebright RH.  2017.  RNA Capping by Transcription Initiation with Non-canonical Initiating Nucleotides (NCINs): Determination of Relative Efficiencies of Transcription Initiation with NCINs and NTPs.. Bio-protocol. 7(12) Abstract
It recently has been established that adenine-containing cofactors, including nicotinamide adenine dinucleotide (NAD(+)), reduced nicotinamide adenine dinucleotide (NADH), and 3'-desphospho-coenzyme A (dpCoA), can serve as 'non-canonical initiating nucleotides' (NCINs) for transcription initiation by bacterial and eukaryotic cellular RNA polymerases (RNAPs) and that the efficiency of the reaction is determined by promoter sequence (Bird et al., 2016). Here we describe a protocol to quantify the relative efficiencies of transcription initiation using an NCIN vs. transcription initiation using a nucleoside triphosphate (NTP) for a given promoter sequence.
Maffioli, SI, Zhang Y, Degen D, Carzaniga T, Del Gatto G, Serina S, Monciardini P, Mazzetti C, Guglierame P, Candiani G et al..  2017.  Antibacterial Nucleoside-Analog Inhibitor of Bacterial RNA Polymerase.. Cell. 169(7):1240-1248.e23. Abstract
Drug-resistant bacterial pathogens pose an urgent public-health crisis. Here, we report the discovery, from microbial-extract screening, of a nucleoside-analog inhibitor that inhibits bacterial RNA polymerase (RNAP) and exhibits antibacterial activity against drug-resistant bacterial pathogens: pseudouridimycin (PUM). PUM is a natural product comprising a formamidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 6'-amino-pseudouridine. PUM potently and selectively inhibits bacterial RNAP in vitro, inhibits bacterial growth in culture, and clears infection in a mouse model of Streptococcus pyogenes peritonitis. PUM inhibits RNAP through a binding site on RNAP (the NTP addition site) and mechanism (competition with UTP for occupancy of the NTP addition site) that differ from those of the RNAP inhibitor and current antibacterial drug rifampin (Rif). PUM exhibits additive antibacterial activity when co-administered with Rif, exhibits no cross-resistance with Rif, and exhibits a spontaneous resistance rate an order-of-magnitude lower than that of Rif. PUM is a highly promising lead for antibacterial therapy.
Jiao, X, Doamekpor S, Bird JG, Nickels BE, Tong L, Hart RP, Kiledjian M.  2017.  5′-end Nicotinamide Adenine Dinucleotide cap in human cells promotes RNA decay through DXO-mediated deNADding.. Cell. 168(6):1015-1027.
Chatterjee, M, Liu Q, Menello C, Galli M, Gallavotti A.  2017.  The Combined Action of Duplicated Boron Transporters Is Required for Maize Growth in Boron Deficient Conditions. Genetics. 206:2041-2051. AbstractWebsite
The micronutrient boron is essential in maintaining the structure of plant cell walls and is critical for high yields in crop species. Boron can move into plants by diffusion or by active and facilitated transport mechanisms. We recently showed that mutations in the maize boron efflux transporter ROTTEN EAR (RTE) cause severe developmental defects and sterility. RTE is part of a small gene family containing five additional members (RTE2-RTE6) that show tissue specific expression. The close paralogous gene RTE2 encodes a protein with 95% amino acid identity with RTE and is similarly expressed in shoot and root cells surrounding the vasculature. Despite sharing similar functions with RTE, mutations in the RTE2 gene do not cause growth defects in the shoot, even in boron deficient conditions. However, rte2 mutants strongly enhance the rte phenotype in soils with low boron content, producing shorter plants that fail to form all reproductive structures. The joint action of RTE and RTE2 is also required in root development. These defects can be fully complemented by supplying boric acid, suggesting that diffusion or additional transport mechanisms overcome active boron transport deficiencies in the presence of an excess of boron. Overall, these results suggest that RTE2 and RTE function are essential for maize shoot and root growth in boron deficient conditions.
Garner, AL, Rammohan J, Huynh JP, Onder LM, Chen J, Bae B, Jensen D, Weiss LA, Manzano AR, Darst SA et al..  2017.  Effects of Increasing the Affinity of CarD for RNA Polymerase on Mycobacterium tuberculosis Growth, rRNA Transcription, and Virulence. Journal of Bacteriology. 199:e00698-16..
Bartlett, A, O'Malley R, Huang SC, Galli M, Nery JR, Gallavotti A, Ecker JR.  2017.  Mapping genome-wide transcription factor binding sites using DAP-seq. Nature Protocols. 12(8):1659-1672. AbstractWebsite
To enable low-cost, high-throughput generation of cistrome and epicistrome maps for any organism, we developed DNA affinity purification sequencing (DAP-seq), a transcription factor (TF)-binding site (TFBS) discovery assay that couples affinity-purified TFs with next-generation sequencing of a genomic DNA library. The method is fast, inexpensive, and more easily scaled than chromatin immunoprecipitation sequencing (ChIP-seq). DNA libraries are constructed using native genomic DNA from any source of interest, preserving cell- and tissue-specific chemical modifications that are known to affect TF binding (such as DNA methylation) and providing increased specificity as compared with in silico predictions based on motifs from methods such as protein-binding microarrays (PBMs) and systematic evolution of ligands by exponential enrichment (SELEX). The resulting DNA library is incubated with an affinity-tagged in vitro-expressed TF, and TF–DNA complexes are purified using magnetic separation of the affinity tag. Bound genomic DNA is eluted from the TF and sequenced using next-generation sequencing. Sequence reads are mapped to a reference genome, identifying genome-wide binding locations for each TF assayed, from which sequence motifs can then be derived. A researcher with molecular biology experience should be able to follow this protocol, processing up to 400 samples per week.
Huang, Q, Zhong S, Bleckmann A, Huang J, Guo X, Lin Q, Gu H, Dong J, Dresslhaus T, Qu LJ.  2017.  Sperm cells are passive cargo of the pollen tube in plant fertilization.. Nature Plants. 3:17079
Savage-Dunn, C, Padgett RW.  2017.  The TGF-β Family in Caenorhabditis elegans. The Biology of the TGF-β Family.
2016
Gurdon, C, Svab Z, Feng Y, Kumar D, Maliga P.  2016.  Cell-to-cell movement of mitochondria in plants. Proc Natl Acad Sci U S A. 113:3395-400. AbstractWebsite
We report cell-to-cell movement of mitochondria through a graft junction. Mitochondrial movement was discovered in an experiment designed to select for chloroplast transfer fromNicotiana sylvestrisintoNicotiana tabacumcells. The alloplasmicN. tabacumline we used carriesNicotiana undulatacytoplasmic genomes, and its flowers are male sterile due to the foreign mitochondrial genome. Thus, rare mitochondrial DNA transfer fromN. sylvestristoN. tabacumcould be recognized by restoration of fertile flower anatomy. Analyses of the mitochondrial genomes revealed extensive recombination, tentatively linking male sterility toorf293, a mitochondrial gene causing homeotic conversion of anthers into petals. Demonstrating cell-to-cell movement of mitochondria reconstructs the evolutionary process of horizontal mitochondrial DNA transfer and enables modification of the mitochondrial genome by DNA transmitted from a sexually incompatible species. Conversion of anthers into petals is a visual marker that can be useful for mitochondrial transformation.
Sun, S, Irvine KD.  2016.  Cellular Organization and Cytoskeletal Regulation of the Hippo Signaling Network.. Trends in cell biology. 26(9):694-704. Abstract
The Hippo signaling network integrates diverse upstream signals to control cell fate decisions and regulate organ growth. Recent studies have provided new insights into the cellular organization of Hippo signaling, its relationship to cell-cell junctions, and how the cytoskeleton modulates Hippo signaling. Cell-cell junctions serve as platforms for Hippo signaling by localizing scaffolding proteins that interact with core components of the pathway. Interactions of Hippo pathway components with cell-cell junctions and the cytoskeleton also suggest potential mechanisms for the regulation of the pathway by cell contact and cell polarity. As our understanding of the complexity of Hippo signaling increases, a future challenge will be to understand how the diverse inputs into the pathway are integrated and to define their respective contributions in vivo.
Radford, SJ, McKim KS.  2016.  Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes.. Journal of visualized experiments : JoVE. (116) Abstract
Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of chromosome behavior in oocytes from model organisms holds much promise to uncover the molecular basis of the susceptibility of human oocytes to aneuploidy. Drosophila melanogaster is amenable to genetic manipulation, with over 100 years of research, community, and technique development. Visualizing chromosome behavior and spindle assembly in Drosophila oocytes has particular challenges, however, due primarily to the presence of membranes surrounding the oocyte that are impenetrable to antibodies. We describe here protocols for the collection, preparation, and imaging of meiosis I spindle assembly and chromosome behavior in Drosophila oocytes, which allow the molecular dissection of chromosome segregation in this important model organism.
Pan, Y, Heemskerk I, Ibar C, Shraiman BI, Irvine KD.  2016.  Differential growth triggers mechanical feedback that elevates Hippo signaling.. Proceedings of the National Academy of Sciences of the United States of America. Abstract
Mechanical stress can influence cell proliferation in vitro, but whether it makes a significant contribution to growth control in vivo, and how it is modulated and experienced by cells within developing tissues, has remained unclear. Here we report that differential growth reduces cytoskeletal tension along cell junctions within faster-growing cells. We propose a theoretical model to explain the observed reduction of tension within faster-growing clones, supporting it by computer simulations based on a generalized vertex model. This reduced tension modulates a biomechanical Hippo pathway, decreasing recruitment of Ajuba LIM protein and the Hippo pathway kinase Warts, and decreasing the activity of the growth-promoting transcription factor Yorkie. These observations provide a specific mechanism for a mechanical feedback that contributes to evenly distributed growth, and we show that genetically suppressing mechanical feedback alters patterns of cell proliferation in the developing Drosophila wing. By providing experimental support for the induction of mechanical stress by differential growth, and a molecular mechanism linking this stress to the regulation of growth in developing organs, our results confirm and extend the mechanical feedback hypothesis.
Misra, JR, Irvine KD.  2016.  Vamana Couples Fat Signaling to the Hippo Pathway.. Developmental cell. 39(2):254-266. Abstract
The protocadherins Dachsous and Fat initiate a signaling pathway that controls growth and planar cell polarity by regulating the membrane localization of the atypical myosin Dachs. How Dachs is regulated by Fat signaling has remained unclear. Here we identify the vamana gene as playing a crucial role in regulating membrane localization of Dachs and in linking Fat and Dachsous to Dachs regulation. Vamana, an SH3-domain-containing protein, physically associates with and co-localizes with Dachs and promotes its membrane localization. Vamana also associates with the Dachsous intracellular domain and with a region of the Fat intracellular domain that is essential for controlling Hippo signaling and levels of Dachs. Epistasis experiments, structure-function analysis, and physical interaction experiments argue that Fat negatively regulates Dachs in a Vamana-dependent process. Our findings establish Vamana as a crucial component of the Dachsous-Fat pathway that transmits Fat signaling by regulating Dachs.
Radford, SJ, Nguyen AL, Schindler K, McKim KS.  2016.  The chromosomal basis of meiotic acentrosomal spindle assembly and function in oocytes.. Chromosoma. Abstract
Several aspects of meiosis are impacted by the absence of centrosomes in oocytes. Here, we review four aspects of meiosis I that are significantly affected by the absence of centrosomes in oocyte spindles. One, microtubules tend to assemble around the chromosomes. Two, the organization of these microtubules into a bipolar spindle is directed by the chromosomes. Three, chromosome bi-orientation and attachment to microtubules from the correct pole require modification of the mechanisms used in mitotic cells. Four, chromosome movement to the poles at anaphase cannot rely on polar anchoring of spindle microtubules by centrosomes. Overall, the chromosomes are more active participants during acentrosomal spindle assembly in oocytes, compared to mitotic and male meiotic divisions where centrosomes are present. The chromosomes are endowed with information that can direct the meiotic divisions and dictate their own behavior in oocytes. Processes beyond those known from mitosis appear to be required for their bi-orientation at meiosis I. As mitosis occurs without centrosomes in many systems other than oocytes, including all plants, the concepts discussed here may not be limited to oocytes. The study of meiosis in oocytes has revealed mechanisms that are operating in mitosis and will probably continue to do so.
Vvedenskaya, IO, Vahedian-Movahed H, Zhang Y, Taylor DM, Ebright RH, Nickels BE.  2016.  Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection.. Proceedings of the National Academy of Sciences of the United States of America. 113:E2899-E2905. Abstract
During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein-DNA interactions with the downstream part of the nontemplate strand of the transcription bubble ("core recognition element," CRE). Here, we investigated whether sequence-specific RNAP-CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP-CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP-CRE interactions on TSS selection in vitro and in vivo for a library of 4(7) (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP-CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5' merodiploid native-elongating-transcript sequencing, 5' mNET-seq, we assessed effects of RNAP-CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP-CRE interactions determine TSS selection. Our findings establish RNAP-CRE interactions are a functional determinant of TSS selection. We propose that RNAP-CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).
Mao, Y, Kuta A, Crespo-Enriquez I, Whiting D, Martin T, Mulvaney J, Irvine KD, Francis-West P.  2016.  Dchs1-Fat4 regulation of polarized cell behaviours during skeletal morphogenesis.. Nature communications. 7:11469. Abstract
Skeletal shape varies widely across species as adaptation to specialized modes of feeding and locomotion, but how skeletal shape is established is unknown. An example of extreme diversity in the shape of a skeletal structure can be seen in the sternum, which varies considerably across species. Here we show that the Dchs1-Fat4 planar cell polarity pathway controls cell orientation in the early skeletal condensation to define the shape and relative dimensions of the mouse sternum. These changes fit a model of cell intercalation along differential Dchs1-Fat4 activity that drives a simultaneous narrowing, thickening and elongation of the sternum. Our results identify the regulation of cellular polarity within the early pre-chondrogenic mesenchyme, when skeletal shape is established, and provide the first demonstration that Fat4 and Dchs1 establish polarized cell behaviour intrinsically within the mesenchyme. Our data also reveal the first indication that cell intercalation processes occur during ventral body wall elongation and closure.
Winkelman, JT, Vvedenskaya IO, Zhang Y, Zhang Y, Bird JG, Taylor DM, Gourse RL, Ebright RH, Nickels BE.  2016.  Multiplexed protein-DNA cross-linking: Scrunching in transcription start site selection.. Science (New York, N.Y.). 351(6277):1090-3. Abstract
In bacterial transcription initiation, RNA polymerase (RNAP) selects a transcription start site (TSS) at variable distances downstream of core promoter elements. Using next-generation sequencing and unnatural amino acid-mediated protein-DNA cross-linking, we have determined, for a library of 4(10) promoter sequences, the TSS, the RNAP leading-edge position, and the RNAP trailing-edge position. We find that a promoter element upstream of the TSS, the "discriminator," participates in TSS selection, and that, as the TSS changes, the RNAP leading-edge position changes, but the RNAP trailing-edge position does not change. Changes in the RNAP leading-edge position, but not the RNAP trailing-edge position, are a defining hallmark of the "DNA scrunching" that occurs concurrent with RNA synthesis in initial transcription. We propose that TSS selection involves DNA scrunching prior to RNA synthesis.
Joshi, KK, Matlack TL, Rongo C.  2016.  Dopamine signaling promotes the xenobiotic stress response and protein homeostasis.. The EMBO journal. Abstract
Multicellular organisms encounter environmental conditions that adversely affect protein homeostasis (proteostasis), including extreme temperatures, toxins, and pathogens. It is unclear how they use sensory signaling to detect adverse conditions and then activate stress response pathways so as to offset potential damage. Here, we show that dopaminergic mechanosensory neurons in C. elegans release the neurohormone dopamine to promote proteostasis in epithelia. Signaling through the DA receptor DOP-1 activates the expression of xenobiotic stress response genes involved in pathogenic resistance and toxin removal, and these genes are required for the removal of unstable proteins in epithelia. Exposure to a bacterial pathogen (Pseudomonas aeruginosa) results in elevated removal of unstable proteins in epithelia, and this enhancement requires DA signaling. In the absence of DA signaling, nematodes show increased sensitivity to pathogenic bacteria and heat-shock stress. Our results suggest that dopaminergic sensory neurons, in addition to slowing down locomotion upon sensing a potential bacterial feeding source, also signal to frontline epithelia to activate the xenobiotic stress response so as to maintain proteostasis and prepare for possible infection.
Wang, W, Wu Y, Messing J.  2016.  Genome-wide analysis of pentatricopeptide-repeat proteins of an aquatic plant.. Planta. Abstract
A large proportion of genes in plant genomes are organized as gene families. Whereas most gene families in the aquative plant Spirodela are reduced in their copy number, the PPR gene family is expanded, which match the RNA editing sites in organelles, providing us with new insights in the evolution of flowering plants. Pentatricopeptide-repeat proteins (PPRs) are nuclear-encoded proteins that are targeted to mitochondria and plastids to stabilize and edit mRNA transcribed from organellar genomes. They have been described for many terrestrial plant species from a diverse spectrum of sequenced genomes. To further increase our understanding of the evolution of this gene family across angiosperms, we analyzed the PPR genes in the aquatic species Spirodela polyrhiza in the order of the Alismatales (monocotyledonous plants). Because we had generated next generation sequencing data from transcripts and had sequenced the genome of Spirodela polyrhiza, we were able to identify its PPR genes and determine the level of their expression. In total, we could identify 556 PPR proteins, of which 238 members belong to the P (P motif) subfamily that is mainly involved in RNA stabilization and 318 ones to the PLS (P, Longer P, shorter P motif) subfamily responsible for RNA editing. Compared to other angiosperms, this is a large increase in the copy number of the PLS-PPRs subfamily and the expansion correlates with the increase of the number of RNA editing sites of organellar transcripts. Expression of PPR was generally stable even during growing and dormant stages, indicating that their function was critical throughout development. However, PPRs, especially those of the PLS subfamily, were expressed at relatively low levels, suggesting a delicate fine-tuning of its trans-acting function in the post-transcriptional regulation of gene expression. Thus, understanding PPR evolution and expression will help decipher the PPR code for their binding sites, which could genetically engineer RNA-binding proteins toward desired sequence.
Feng, Y, Zhang Y, Ebright RH.  2016.  Structural basis of transcription activation.. Science (New York, N.Y.). 352(6291):1330-3. AbstractWebsite
Class II transcription activators function by binding to a DNA site overlapping a core promoter and stimulating isomerization of an initial RNA polymerase (RNAP)-promoter closed complex into a catalytically competent RNAP-promoter open complex. Here, we report a 4.4 angstrom crystal structure of an intact bacterial class II transcription activation complex. The structure comprises Thermus thermophilus transcription activator protein TTHB099 (TAP) [homolog of Escherichia coli catabolite activator protein (CAP)], T. thermophilus RNAP σ(A) holoenzyme, a class II TAP-dependent promoter, and a ribotetranucleotide primer. The structure reveals the interactions between RNAP holoenzyme and DNA responsible for transcription initiation and reveals the interactions between TAP and RNAP holoenzyme responsible for transcription activation. The structure indicates that TAP stimulates isomerization through simple, adhesive, stabilizing protein-protein interactions with RNAP holoenzyme.