Publications

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Montano, SP, Pierce M, Cot'e ML, Vershon AK, Georgiadis MM.  2002.  Crystallographic Studies of a Novel DNA-binding Domain from the Yeast Transcriptional Activator Ndt80. Acta Crystallogr D Biol Crystallogr. 58:2127-2130. Abstract
The Ndt80 protein is a transcriptional activator that plays a key role in the progression of the meiotic divisions in the yeast Saccharomyces cerevisiae. Ndt80 is strongly induced during the middle stages of the sporulation pathway and binds specifically to a promoter element called the MSE to activate transcription of genes required for the meiotic divisions. Here, the preliminary structural and functional studies to characterize the DNA-binding activity of this protein are reported. Through deletion analysis and limited proteolysis studies of Ndt80, a novel 32 kDa DNA-binding domain that is sufficient for DNA-binding in vitro has been defined. Crystals of the DNA-binding domain of Ndt80 in two distinct lattices have been obtained, for which diffraction data extend to 2.3 A resolution.
McKim, KS, Joyce EF, Jang JK.  2009.  Cytological analysis of meiosis in fixed Drosophila ovaries. Methods Mol Biol. 558:197-216. AbstractWebsite
Methods are described to analyze two different parts of the Drosophila ovary, which correspond to early stages (pachytene) and late stages (metaphase I and beyond) of meiosis. In addition to taking into account morphology, the techniques differ by fixation conditions and the method to isolate the tissue. Most of these methods are whole mounts, which preserve the three-dimensional structure.
Manheim, EA, Jang JK, Dominic D, McKim KS.  2002.  Cytoplasmic localization and evolutionary conservation of MEI-218, a protein required for meiotic crossing over in Drosophila. Mol. Biol. Cell. 13:84-95.
Rauskolb, C, Sun S, Sun G, Pan Y, Irvine KD.  2014.  Cytoskeletal Tension Inhibits Hippo Signaling through an Ajuba-Warts Complex.. Cell. 158:143-156. AbstractWebsite
Mechanical forces have been proposed to modulate organ growth, but a molecular mechanism that links them to growth regulation in vivo has been lacking. We report that increasing tension within the cytoskeleton increases Drosophila wing growth, whereas decreasing cytoskeletal tension decreases wing growth. These changes in growth can be accounted for by changes in the activity of Yorkie, a transcription factor regulated by the Hippo pathway. The influence of myosin activity on Yorkie depends genetically on the Ajuba LIM protein Jub, a negative regulator of Warts within the Hippo pathway. We further show that Jub associates with α-catenin and that its localization to adherens junctions and association with α-catenin are promoted by cytoskeletal tension. Jub recruits Warts to junctions in a tension-dependent manner. Our observations delineate a mechanism that links cytoskeletal tension to regulation of Hippo pathway activity, providing a molecular understanding of how mechanical forces can modulate organ growth.
Chang, HC, Rongo C.  2005.  Cytosolic tail sequences and subunit interactions are critical for synaptic localization of glutamate receptors. J Cell Sci. 118:1945-56. AbstractWebsite
AMPA-type glutamate receptors mediate excitatory synaptic transmission in the nervous system. The receptor subunit composition and subcellular localization play an important role in regulating synaptic strength. GLR-1 and GLR-2 are the Caenorhabditis elegans subunits most closely related to the mammalian AMPA-type receptors. These subunits are expressed in overlapping sets of interneurons, and contain type-I PDZ binding motifs in their carboxy-terminal cytosolic tail sequences. We report that GLR-1 and GLR-2 may form a heteromeric complex, the localization of which depends on either GLR-1 or GLR-2 tail sequences. Subunit interactions alone can mediate synaptic localization as endogenous GLR-1, or GLR-2 subunits can rescue the localization defects of subunits lacking tail sequences. Moreover, GLR-2 cytosolic tail sequences are sufficient to confer synaptic localization on a heterologous reporter containing a single-transmembrane domain. The localization of this GLR-2 reporter requires both a PDZ-binding motif in the GLR-2 tail sequence, and sequences outside of this motif. The PDZ protein LIN-10 regulates the localization of the reporter through the sequences outside of the PDZ-binding motif. Our results suggest that multiple synaptic localization signals reside in the cytosolic tail sequence of the receptor subunits, and that channel assembly can rescue the synaptic localization defects of individual mutant subunits as long as there are also wild-type subunits in the receptor complex.
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Mao, Y, Rauskolb C, Cho E, Hu W-L, Hayter H, Minihan G, Katz FN, Irvine KD.  2006.  Dachs: an unconventional myosin that functions downstream of Fat to regulate growth, affinity and gene expression in Drosophila. Development (Cambridge, England). 133:2539-51. AbstractWebsite
The dachs gene was first identified almost a century ago based on its requirements for appendage growth, but has been relatively little studied. Here, we describe the phenotypes of strong dachs mutations, report the cloning of the dachs gene, characterize the localization of Dachs protein, and investigate the relationship between Dachs and the Fat pathway. Mutation of dachs reduces, but does not abolish, the growth of legs and wings. dachs encodes an unconventional myosin that preferentially localizes to the membrane of imaginal disc cells. dachs mutations suppress the effects of fat mutations on gene expression, cell affinity and growth in imaginal discs. Dachs protein localization is influenced by Fat, Four-jointed and Dachsous, consistent with its genetic placement downstream of fat. However, dachs mutations have only mild tissue polarity phenotypes, and only partially suppress the tissue polarity defects of fat mutants. Our results implicate Dachs as a crucial downstream component of a Fat signaling pathway that influences growth, affinity and gene expression during development.
Barr, MM, Androwski RJ, Rashid A, Lee H, Lee J, Barr MM.  2013.  Dauer-specific dendrite arborization in C. elegans is regulated by KPC-1/Furin.. Current biology : CB. 23(16):1527-35. Abstract
Dendrites often display remarkably complex and diverse morphologies that are influenced by developmental and environmental cues. Neuroplasticity in response to adverse environmental conditions entails both hypertrophy and resorption of dendrites. How dendrites rapidly alter morphology in response to unfavorable environmental conditions is unclear. The nematode Caenorhabditis elegans enters into a stress-resistant dauer larval stage in response to an adverse environment.
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.
Luo, C, Dong J, Zhang Y, Lam E.  2014.  Decoding the role of chromatin architecture in development: coming closer to the end of the tunnel.. Frontiers in Plant Science. 5 AbstractWebsite
Form and function in biology are intimately related aspects that are often difficult to untangle. While the structural aspects of chromatin organization were apparent from early cytological observations long before the molecular details of chromatin functions were deciphered, the extent to which genome architecture may impact its output remains unclear. A major roadblock to resolve this issue is the divergent scales, both temporal and spatial, of the experimental approaches for examining these facets of chromatin biology. Recent advances in high-throughput sequencing and informatics to model and monitor genome-wide chromatin contact sites provide the much-needed platform to close this gap. This mini-review will focus on discussing recent efforts applying new technologies to elucidate the roles of genome architecture in coordinating global gene expression output. Our discussion will emphasize the potential roles of differential genome 3-D structure as a driver for cell fate specification of multicellular organisms. An integrated approach that combines multiple new methodologies may finally have the necessary temporal and spatial resolution to provide clarity on the roles of chromatin architecture during development.
Lang, Z, Wills DM, Lemmon ZH, Shannon LM, Bukowski R, Wu Y, Messing J, Doebley JF.  2014.  Defining the Role of prolamin-box binding factor1 Gene During Maize Domestication. J Hered. AbstractWebsite
The prolamin-box binding factor1 (pbf1) gene encodes a transcription factor that controls the expression of seed storage protein (zein) genes in maize. Prior studies show that pbf1 underwent selection during maize domestication although how it affected trait change during domestication is unknown. To assay how pbf1 affects phenotypic differences between maize and teosinte, we compared nearly isogenic lines (NILs) that differ for a maize versus teosinte allele of pbf1. Kernel weight for the teosinte NIL (162mg) is slightly but significantly greater than that for the maize NIL (156mg). RNAseq data for developing kernels show that the teosinte allele of pbf1 is expressed at about twice the level of the maize allele. However, RNA and protein assays showed no difference in zein profile between the two NILs. The lower expression for the maize pbf1 allele suggests that selection may have favored this change; however, how reduced pbf1 expression alters phenotype remains unknown. One possibility is that pbf1 regulates genes other than zeins and thereby is a domestication trait. The observed drop in seed weight associated with the maize allele of pbf1 is counterintuitive but could represent a negative pleiotropic effect of selection on some other aspect of kernel composition.
Cho, E, Feng Y, Rauskolb C, Maitra S, Fehon RG, Irvine KD.  2006.  Delineation of a Fat tumor suppressor pathway. Nature Genetics. 38:1142-50. AbstractWebsite
Recent studies in Drosophila melanogaster of the protocadherins Dachsous and Fat suggest that they act as ligand and receptor, respectively, for an intercellular signaling pathway that influences tissue polarity, growth and gene expression, but the basis for signaling downstream of Fat has remained unclear. Here, we characterize functional relationships among D. melanogaster tumor suppressors and identify the kinases Discs overgrown and Warts as components of a Fat signaling pathway. fat, discs overgrown and warts regulate a common set of downstream genes in multiple tissues. Genetic experiments position the action of discs overgrown upstream of the Fat pathway component dachs, whereas warts acts downstream of dachs. Warts protein coprecipitates with Dachs, and Warts protein levels are influenced by fat, dachs and discs overgrown in vivo, consistent with its placement as a downstream component of the pathway. The tumor suppressors Merlin, expanded, hippo, salvador and mob as tumor suppressor also share multiple Fat pathway phenotypes but regulate Warts activity independently. Our results functionally link what had been four disparate groups of D. melanogaster tumor suppressors, establish a basic framework for Fat signaling from receptor to transcription factor and implicate Warts as an integrator of multiple growth control signals.
Hanlon, SE, Norris DN, Vershon AK.  2003.  Depletion of H2A-H2b Dimers in Saccharomyces Cerevisiae Triggers Meiotic Arrest by Reducing IME1 Expression and Activating the BUB2-dependent Branch of the Spindle Checkpoint. Genetics. 164:1333-1344. Abstract
In the yeast Saccharomyces cerevisiae, diploid strains carrying homozygous hta1-htb1Delta mutations express histone H2A-H2B dimers at a lower level than do wild-type cells. Although this mutation has only minor effects on mitotic growth, it causes an arrest in sporulation prior to the first meiotic division. In this report, we show that the hta1-htb1Delta mutant exhibits reduced expression of early and middle-sporulation-specific genes and that the meiotic arrest of the hta1-htb1Delta mutant can be partially bypassed by overexpression of IME1. Additionally, deletions of BUB2 or BFA1, components of one branch of the spindle checkpoint pathway, bypass the meiotic arrest. Mutations in the other branch of the pathway or in the pachytene checkpoint are unable to suppress the meiotic block. These observations indicate that depletion of the H2A-H2B dimer blocks sporulation by at least two mechanisms: disruption of the expression of meiotic regulatory genes and activation of the spindle checkpoint. Our results show that the failure to progress through the meiotic pathway is not the result of global chromosomal alterations but that specific aspects of meiosis are sensitive to depletion of the H2A-H2B dimer.
Zhang, XP, Gunasekera A, Ebright YW, Ebright RH.  1991.  Derivatives of CAP having no solvent-accessible cysteine residues, or having a unique solvent-accessible cysteine residue at amino acid 2 of the helix-turn-helix motif.. Journal of biomolecular structure & dynamics. 9(3):463-73. Abstract
The Escherichia coli catabolite gene activator protein (CAP) is a helix-turn-helix motif sequence-specific DNA binding protein. CAP contains a unique solvent-accessible cysteine residue at amino acid 10 of the helix-turn-helix motif. In published work, we have constructed a prototype semi-synthetic site-specific DNA cleavage agent from CAP by use of cysteine-specific chemical modification to incorporate a nucleolytic chelator-metal complex at amino acid 10 of the helix-turn-helix motif [Ebright, R., Ebright, Y., Pendergrast, P.S. and Gunasekera, A., Proc. Natl. Acad. Sci. USA 87, 2882-2886 (1990)]. Construction of second-generation semi-synthetic site-specific DNA cleavage agents from CAP requires the construction of derivatives of CAP having unique solvent-accessible cysteine residues at sites within CAP other than amino acid 10 of the helix-turn-helix motif. In the present work, we have constructed and characterized two derivatives of CAP having no solvent-accessible cysteine residues: [Ser178]CAP and [Leu178]CAP. In addition, in the present work, we have constructed and characterized one derivative of CAP having a unique solvent-accessible cysteine residue at amino acid 2 of the helix-turn-helix motif: [Cys170;Ser178]CAP.
Heyduk, T, Heyduk E, Severinov K, Tang H, Ebright RH.  1996.  Determinants of RNA polymerase alpha subunit for interaction with beta, beta', and sigma subunits: hydroxyl-radical protein footprinting.. Proceedings of the National Academy of Sciences of the United States of America. 93(19):10162-6. Abstract
Escherichia coli RNA polymerase (RNAP) alpha subunit serves as the initiator for RNAP assembly, which proceeds according to the pathway 2 alpha-->alpha 2-->alpha 2 beta-->alpha 2 beta beta'-->alpha 2 beta beta' sigma. In this work, we have used hydroxyl-radical protein footprinting to define determinants of alpha for interaction with beta, beta', and sigma. Our results indicate that amino acids 30-75 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta (i.e., in alpha 2 beta, alpha 2 beta beta', and alpha 2 beta beta' sigma), and amino acids 175-210 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta' (i.e., in alpha 2 beta beta' and alpha 2 beta beta' sigma). The protected regions are conserved in the alpha homologs of prokaryotic, eukaryotic, archaeal, and chloroplast RNAPs and contain sites of substitutions that affect RNAP assembly. We conclude that the protected regions define determinants of alpha for direct functional interaction with beta and beta'. The observed maximal magnitude of protection upon interaction with beta and the observed maximal magnitude of protection upon interaction with beta' both correspond to the expected value for complete protection of one of the two alpha protomers of RNAP (i.e., 50% protection). We propose that only one of the two alpha protomers of RNAP interacts with beta and that only one of the two alpha protomers of RNAP interacts with beta'.
Savery, NJ, Lloyd GS, Busby SJW, Thomas MS, Ebright RH, Gourse RL.  2002.  Determinants of the C-terminal domain of the Escherichia coli RNA polymerase alpha subunit important for transcription at class I cyclic AMP receptor protein-dependent promoters.. Journal of bacteriology. 184(8):2273-80. Abstract
Alanine scanning of the Escherichia coli RNA polymerase alpha subunit C-terminal domain (alphaCTD) was used to identify amino acid side chains important for class I cyclic AMP receptor protein (CRP)-dependent transcription. Key residues were investigated further in vivo and in vitro. Substitutions in three regions of alphaCTD affected class I CRP-dependent transcription from the CC(-61.5) promoter and/or the lacP1 promoter. These regions are (i) the 287 determinant, previously shown to contact CRP during class II CRP-dependent transcription; (ii) the 265 determinant, previously shown to be important for alphaCTD-DNA interactions, including those required for class II CRP-dependent transcription; and (iii) the 261 determinant. We conclude that CRP contacts the same target in alphaCTD, the 287 determinant, at class I and class II CRP-dependent promoters. We also conclude that the relative contributions of individual residues within the 265 determinant depend on promoter sequence, and we discuss explanations for effects of substitutions in the 261 determinant.
Pendergrast, PS, Chen Y, Ebright YW, Ebright RH.  1992.  Determination of the orientation of a DNA binding motif in a protein-DNA complex by photocrosslinking.. Proceedings of the National Academy of Sciences of the United States of America. 89(21):10287-91. Abstract
We have developed a straightforward biochemical method to determine the orientation of the DNA binding motif of a sequence-specific DNA binding protein relative to the DNA site in the protein-DNA complex. The method involves incorporation of a photoactivatable crosslinking agent at a single site within the DNA binding motif of the sequence-specific DNA binding protein, formation of the derivatized protein-DNA complex, UV-irradiation of the derivatized protein-DNA complex, and determination of the nucleotide(s) at which crosslinking occurs. We have applied the method to catabolite gene activator protein (CAP). We have constructed and analyzed two derivatives of CAP: one having a phenyl azide photoactivatable crosslinking agent at amino acid 2 of the helix-turn-helix motif of CAP, and one having a phenyl azide photoactivatable crosslinking agent at amino acid 10 of the helix-turn-helix motif of CAP. The results indicate that amino acid 2 of the helix-turn-helix motif is close to the top-strand nucleotides of base pairs 3 and 4 of the DNA half site in the CAP-DNA complex, and that amino acid 10 of the helix-turn-helix motif is close to the bottom-strand nucleotide of base pair 10 of the DNA half site in the CAP-DNA complex. The results define unambiguously the orientation of the helix-turn-helix motif relative to the DNA half site in the CAP-DNA complex. Comparison of the results to the crystallographic structure of the CAP-DNA complex [Schultz, S., Shields, S. & Steitz, T. (1991) Science 253, 1001-1007] indicates that the method provides accurate, high-resolution proximity and orientation information.
Dismukes, CG, Brimblecombe R, Felton GAN, Pryadun RS, Sheats JE, Spiccia L, Swiegers GF.  2009.  Development of Bioinspired Mn4O4−Cubane Water Oxidation Catalysts: Lessons from Photosynthesis. Accounts of Chemical Research. 42:1935-1943. AbstractWebsite
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Anderson, KV, Irvine KD.  2009.  Developmental biology moves forward in the 21st century. Current opinion in genetics & development. 19:299-301.Website
Kirihara, JA, Hunsperger JP, Mahoney WC, Messing JW.  1988.  Differential expression of a gene for a methionine-rich storage protein in maize. Molecular & general genetics : MGG. 211:477-84. AbstractWebsite
A methionine-rich 10 kDa zein storage protein from maize was isolated and the sequence of the N-terminal 30 amino acids was determined. Based on the amino acid sequence, two mixed oligonucleotides were synthesized and used to probe a maize endosperm cDNA library. A full-length cDNA clone encoding the 10 kDa zein was isolated by this procedure. The nucleotide sequence of the cDNA clone predicts a polypeptide of 129 amino acids, preceded by a signal peptide of 21 amino acids. The predicted polypeptide is unique in its extremely high content of methionine (22.5%). The maize inbred line BSSS-53, which has increased seed methionine due to overproduction of this protein, was compared to W23, a standard inbred line. Northern blot analysis showed that the relative RNA levels for the 10 kDa zein were enhanced in developing seeds of BSSS-53, providing a molecular basis for the overproduction of the protein. Southern blot analysis indicated that there are one or two 10 kDa zein genes in the maize genome.
Miclaus, M, Xu JH, Messing J.  2011.  Differential gene expression and epiregulation of alpha zein gene copies in maize haplotypes. PLoS Genet. 7:e1002131. AbstractWebsite
Multigenic traits are very common in plants and cause diversity. Nutritional quality is such a trait, and one of its factors is the composition and relative expression of storage protein genes. In maize, they represent a medium-size gene family distributed over several chromosomes and unlinked locations. Two inbreds, B73 and BSSS53, both from the Iowa Stiff Stock Synthetic collection, have been selected to analyze allelic and non-allelic variability in these regions that span between 80-500 kb of chromosomal DNA. Genes were copied to unlinked sites before and after allotetraploidization of maize, but before transposition enlarged intergenic regions in a haplotype-specific manner. Once genes are copied, expression of donor genes is reduced relative to new copies. Epigenetic regulation seems to contribute to silencing older copies, because some of them can be reactivated when endosperm is maintained as cultured cells, indicating that copy number variation might contribute to a reserve of gene copies. Bisulfite sequencing of the promoter region also shows different methylation patterns among gene clusters as well as differences between tissues, suggesting a possible position effect on regulatory mechanisms as a result of inserting copies at unlinked locations. The observations offer a potential paradigm for how different gene families evolve and the impact this has on their expression and regulation of their members.
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.
Goldman, SR, Ebright RH, Nickels BE.  2009.  Direct detection of abortive RNA transcripts in vivo. Science. 324:927-8. AbstractWebsite
During transcription initiation in vitro, prokaryotic and eukaryotic RNA polymerase (RNAP) can engage in abortive initiation-the synthesis and release of short (2 to 15 nucleotides) RNA transcripts-before productive initiation. It has not been known whether abortive initiation occurs in vivo. Using hybridization with locked nucleic acid probes, we directly detected abortive transcripts in bacteria. In addition, we show that in vivo abortive initiation shows characteristics of in vitro abortive initiation: Abortive initiation increases upon stabilizing interactions between RNAP and either promoter DNA or sigma factor, and also upon deleting elongation factor GreA. Abortive transcripts may have functional roles in regulating gene expression in vivo.
Goldman, SR, Ebright RH, Nickels BE.  2009.  Direct detection of abortive RNA transcripts in vivo.. Science (New York, N.Y.). 324(5929):927-8. Abstract
During transcription initiation in vitro, prokaryotic and eukaryotic RNA polymerase (RNAP) can engage in abortive initiation-the synthesis and release of short (2 to 15 nucleotides) RNA transcripts-before productive initiation. It has not been known whether abortive initiation occurs in vivo. Using hybridization with locked nucleic acid probes, we directly detected abortive transcripts in bacteria. In addition, we show that in vivo abortive initiation shows characteristics of in vitro abortive initiation: Abortive initiation increases upon stabilizing interactions between RNAP and either promoter DNA or sigma factor, and also upon deleting elongation factor GreA. Abortive transcripts may have functional roles in regulating gene expression in vivo.
Pushkar, Y, Long X, Glatzel P, Brudvig G W, Dismukes  CG, Collins T J, Yachandra V K, Yano J, Bergmann U.  2010.  Direct Detection of Oxygen Ligation to the Mn4Ca Cluster of Photosystem II by X-ray Emission Spectroscopy. Angewandte Chemie International Edition. 49:800-803.Website