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1.  Tumor-related gene expression levels in pulmonary pleomorphic carcinoma 
Pulmonary pleomorphic carcinoma (PPC) is a rare type of non-small-cell lung cancer that belongs to the family of sarcomatoid carcinomas and is associated with poor prognosis. We investigated the expressions of tumor-related genes in resected PPC specimens.
Specimens resected from patients with PPC from July 2006 through April 2012 were investigated. Tumor segments were collected from the specimens by micro-dissection to extract mRNA, then RT-PCR was performed according to Dannenberg’s tumor profile method for semi-quantitation of tumor-related gene mRNA. To compare with other types of lung cancer, data from stage-matched adenocarcinoma (AC) and squamous cell carcinoma (SCC) cases in our database were also examined.
The gene expression levels of thymidylate synthase were significantly higher in PPC and SCC as compared to the AC specimens (p < 0.001). The levels of dihydropyrimidine dehydrogenase and thymidine phosphorylase mRNA in PPC showed a similar tendency to those in SCC, in contrast to AC. Furthermore, the expression level of excision repair cross-complementation group 1 mRNA in PPC specimens was similar to that reported in NSCLC, while the level of vascular endothelial growth factor (VEGF) expression was higher as compared to that reported for colorectal cancer.
Although gene expression of tumor cannot be directly correlated to its sensitivity for anti-cancer drugs, it is likely that PPC tumors are not sensitive to anti-metabolic drugs. Anti-VEGF therapy may be a candidate for PPC, while cisplatin also remains a viable option.
PMCID: PMC4465152  PMID: 26031756
Lung cancer; Pleomorphic carcinoma; Gene expression
2.  Three Members of Polyamine Modulon under Oxidative Stress Conditions: Two Transcription Factors (SoxR and EmrR) and a Glutathione Synthetic Enzyme (GshA) 
PLoS ONE  2015;10(4):e0124883.
Members of polyamine modulon whose synthesis is enhanced at the level of translation were looked for under oxidative stress conditions caused by 0.6 μM K2TeO3. When an Escherichia coli polyamine-requiring mutant MA261 was cultured in the presence of K2TeO3, the degree of polyamine stimulation of cell growth was greater than in cells cultured in the absence of K2TeO3. Under these conditions, synthesis of SoxR, a transcriptional factor for expression of the superoxide response regulon, EmrR, a negative transcriptional factor for expression of the genes for drug excretion proteins, EmrA and EmrB, and of GshA, γ-glutamylcysteine synthetase necessary for glutathione (GSH) synthesis, were stimulated by polyamines at the level of translation. Polyamine stimulation of SoxR and EmrR synthesis was dependent on the existence of an unusually located Shine-Dalgarno (SD) sequence in soxR and emrR mRNAs. Polyamine stimulation of GshA synthesis was due to the existence of the inefficient initiation codon UUG instead of AUG. Polyamine stimulation of the synthesis of EmrR was mainly observed at the logarithmic phase of growth, while that of the synthesis of SoxR and GshA was at the stationary phase. These results strongly suggest that polyamines are involved in easing of oxidative stress through stimulation of synthesis of SoxR, EmrR and GshA together with RpoS, previously found as a member of polyamine modulon at the stationary phase.
PMCID: PMC4405209  PMID: 25898225
4.  Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics 
Nature Communications  2015;6:6622.
Our ability to model the dynamics of signal transduction networks will depend on accurate methods to quantify levels of protein phosphorylation on a global scale. Here we describe a motif-targeting quantitation method for phosphorylation stoichiometry typing. Proteome-wide phosphorylation stoichiometry can be obtained by a simple phosphoproteomic workflow integrating dephosphorylation and isotope tagging with enzymatic kinase reaction. Proof-of-concept experiments using CK2-, MAPK- and EGFR-targeting assays in lung cancer cells demonstrate the advantage of kinase-targeted complexity reduction, resulting in deeper phosphoproteome quantification. We measure the phosphorylation stoichiometry of >1,000 phosphorylation sites including 366 low-abundance tyrosine phosphorylation sites, with high reproducibility and using small sample sizes. Comparing drug-resistant and sensitive lung cancer cells, we reveal that post-translational phosphorylation changes are significantly more dramatic than those at the protein and messenger RNA levels, and suggest potential drug targets within the kinase–substrate network associated with acquired drug resistance.
Measuring phosphorylation stoichiometry on a proteomic scale remains a challenge. Tsai et al. develop a technique to measure the basal level of phosphorylation stoichiometry in a single human phosphoproteome and identify molecular changes associated with gefitinib resistance in lung cancer cells.
PMCID: PMC4389224  PMID: 25814448
5.  LATS1 and LATS2 Phosphorylate CDC26 to Modulate Assembly of the Tetratricopeptide Repeat Subcomplex of APC/C 
PLoS ONE  2015;10(2):e0118662.
In budding yeast, the Mitotic Exit Network (MEN) regulates anaphase promoting complex/cyclosome (APC/C) via the Dbf2-Cdc14 signaling cascade. Dbf2 kinase phosphorylates and activates Cdc14 phosphatase, which removes the inhibitory phosphorylation of the APC/C cofactor Cdh1. Although each component of the MEN was highly conserved during evolution, there is presently no evidence supporting direct phosphorylation of CDC14 by large tumor suppressor kinase 1 (LATS1), the human counterpart of Dbf2; hence, it is unclear how LATS1 regulates APC/C. Here, we demonstrate that LATS1 phosphorylates the Thr7 (T7) residue of the APC/C component CDC26 directly. Nocodazole-induced phosphorylation of T7 was reduced by knockdown of LATS1 and LATS2 in HeLa cells, indicating that both of these kinases contribute to the phosphorylation of CDC26 in vivo. The T7 residue of CDC26 is critical for its interaction with APC6, a tetratricopeptide repeat-containing subunit of APC/C, and mutation of this residue to Asp (T7D) reduced the interaction of CDC26 with APC6. Replacement of endogenous CDC26 in HeLa cells with exogenous phosphor-mimic T7D-mutated CDC26 increased the elution size of APC/C subunits in a gel filtration assay, implying a change in the APC/C assembly upon phosphorylation of CDC26. Furthermore, T7D-mutated CDC26 promoted the ubiquitination of polo-like kinase 1, a well-known substrate of APC/C. Overall, these results suggest that LATS1/2 are novel kinases involved in APC/C phosphorylation and indicate a direct regulatory link between LATS1/2 and APC/C.
PMCID: PMC4344199  PMID: 25723520
6.  Intracellular Concentrations of 65 Species of Transcription Factors with Known Regulatory Functions in Escherichia coli 
Journal of Bacteriology  2014;196(15):2718-2727.
The expression pattern of the Escherichia coli genome is controlled in part by regulating the utilization of a limited number of RNA polymerases among a total of its approximately 4,600 genes. The distribution pattern of RNA polymerase changes from modulation of two types of protein-protein interactions: the interaction of core RNA polymerase with seven species of the sigma subunit for differential promoter recognition and the interaction of RNA polymerase holoenzyme with about 300 different species of transcription factors (TFs) with regulatory functions. We have been involved in the systematic search for the target promoters recognized by each sigma factor and each TF using the newly developed Genomic SELEX system. In parallel, we developed the promoter-specific (PS)-TF screening system for identification of the whole set of TFs involved in regulation of each promoter. Understanding the regulation of genome transcription also requires knowing the intracellular concentrations of the sigma subunits and TFs under various growth conditions. This report describes the intracellular levels of 65 species of TF with known function in E. coli K-12 W3110 at various phases of cell growth and at various temperatures. The list of intracellular concentrations of the sigma factors and TFs provides a community resource for understanding the transcription regulation of E. coli under various stressful conditions in nature.
PMCID: PMC4135669  PMID: 24837290
7.  Inhibition of endocytic vesicle fusion by Plk1-mediated phosphorylation of vimentin during mitosis 
Cell Cycle  2013;13(1):126-137.
Endocytic vesicle fusion is inhibited during mitosis, but the molecular pathways that mediate the inhibition remain unclear. Here we uncovered an essential role of Polo-like kinase 1 (Plk1) in this mechanism. Phosphoproteomic analysis revealed that Plk1 phosphorylates the intermediate filament protein vimentin on Ser459, which is dispensable for its filament formation but is necessary for the inhibition of endocytic vesicle fusion in mitosis. Furthermore, this mechanism is required for integrin trafficking toward the cleavage furrow during cytokinesis. Our results thus identify a novel mechanism for fusion inhibition in mitosis and implicate its role in vesicle trafficking after anaphase onset.
PMCID: PMC3925722  PMID: 24196446
endosome; mitosis; Plk1; vesicle fusion; vimentin
8.  Phosphorylation of Mitochondrial Polyubiquitin by PINK1 Promotes Parkin Mitochondrial Tethering 
PLoS Genetics  2014;10(12):e1004861.
The kinase PINK1 and the E3 ubiquitin (Ub) ligase Parkin participate in mitochondrial quality control. The phosphorylation of Ser65 in Parkin's ubiquitin-like (UBl) domain by PINK1 stimulates Parkin activation and translocation to damaged mitochondria, which induces mitophagy generating polyUb chain. However, Parkin Ser65 phosphorylation is insufficient for Parkin mitochondrial translocation. Here we report that Ser65 in polyUb chain is also phosphorylated by PINK1, and that phosphorylated polyUb chain on mitochondria tethers Parkin at mitochondria. The expression of Tom70MTS-4xUb SE, which mimics phospho-Ser65 polyUb chains on the mitochondria, activated Parkin E3 activity and its mitochondrial translocation. An E3-dead form of Parkin translocated to mitochondria with reduced membrane potential in the presence of Tom70MTS-4xUb SE, whereas non-phospho-polyUb mutant Tom70MTS-4xUb SA abrogated Parkin translocation. Parkin binds to the phospho-polyUb chain through its RING1-In-Between-RING (IBR) domains, but its RING0-linker is also required for mitochondrial translocation. Moreover, the expression of Tom70MTS-4xUb SE improved mitochondrial degeneration in PINK1-deficient, but not Parkin-deficient, Drosophila. Our study suggests that the phosphorylation of mitochondrial polyUb by PINK1 is implicated in both Parkin activation and mitochondrial translocation, predicting a chain reaction mechanism of mitochondrial phospho-polyUb production by which rapid translocation of Parkin is achieved.
Author Summary
Parkinson's disease is a neurodegenerative disorder caused by degeneration of the midbrain dopaminergic system in addition to other nervous systems. PINK1 and parkin, which encode mitochondrial protein kinase and cytosolic Ub ligase, respectively, were identified as the genes responsible for the autosomal recessive form of juvenile Parkinson's disease. Activation of PINK1 upon reduction of mitochondrial membrane potential recruits Parkin from the cytosol activating its Ub ligase activity, which ensures removal of damaged mitochondria through mitophagy. However, how PINK1 recruits Parkin to the damaged mitochondria remained unclear. Here, we describe that the phosphorylation of polyUb chain by PINK1 is a key event to recruit Parkin on the mitochondria. Parkin binds to, and is activated by, phospho-polyUb generated by Parkin in collaboration with PINK1. Expression of a phospho-polyUb mimetic protein on mitochondria rescued mitochondrial degeneration caused by loss of PINK1 in Drosophila. Our study suggests the existence of an amplification cascade of Parkin activation and mitochondrial translocation, in which a ‘seed' of phosphorylated polyUb on the mitochondria, generated by PINK1 and Parkin, triggers a chain reaction of Parkin recruitment and activation.
PMCID: PMC4256268  PMID: 25474007
9.  Characterization of the YdeO Regulon in Escherichia coli 
PLoS ONE  2014;9(11):e111962.
Enterobacteria are able to survive under stressful conditions within animals, such as acidic conditions in the stomach, bile salts during transfer to the intestine and anaerobic conditions within the intestine. The glutamate-dependent (GAD) system plays a major role in acid resistance in Escherichia coli, and expression of the GAD system is controlled by the regulatory cascade consisting of EvgAS > YdeO > GadE. To understand the YdeO regulon in vivo, we used ChIP-chip to interrogate the E. coli genome for candidate YdeO binding sites. All of the seven operons identified by ChIP-chip as being potentially regulated by YdeO were confirmed as being under the direct control of YdeO using RT-qPCR, EMSA, DNaseI-footprinting and reporter assays. Within this YdeO regulon, we identified four stress-response transcription factors, DctR, NhaR, GadE, and GadW and enzymes for anaerobic respiration. Both GadE and GadW are involved in regulation of the GAD system and NhaR is an activator for the sodium/proton antiporter gene. In conjunction with co-transcribed Slp, DctR is involved in protection against metabolic endoproducts under acidic conditions. Taken all together, we suggest that YdeO is a key regulator of E. coli survival in both acidic and anaerobic conditions.
PMCID: PMC4222967  PMID: 25375160
10.  A Novel Mouse Model of Soft-Tissue Infection Using Bioluminescence Imaging Allows Noninvasive, Real-Time Monitoring of Bacterial Growth 
PLoS ONE  2014;9(9):e106367.
Musculoskeletal infections, including surgical-site and implant-associated infections, often cause progressive inflammation and destroy areas of the soft tissue. Treating infections, especially those caused by multi-antibiotic resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) remains a challenge. Although there are a few animal models that enable the quantitative evaluation of infection in soft tissues, these models are not always reproducible or sustainable. Here, we successfully established a real-time, in vivo, quantitative mouse model of soft-tissue infection in the superficial gluteus muscle (SGM) using bioluminescence imaging. A bioluminescent strain of MRSA was inoculated into the SGM of BALB/c adult male mice, followed by sequential measurement of bacterial photon intensity and serological and histological analyses of the mice. The mean photon intensity in the mice peaked immediately after inoculation and remained stable until day 28. The serum levels of interleukin-6, interleukin-1 and C-reactive protein at 12 hours after inoculation were significantly higher than those prior to inoculation, and the C-reactive protein remained significantly elevated until day 21. Histological analyses showed marked neutrophil infiltration and abscesses containing necrotic and fibrous tissues in the SGM. With this SGM mouse model, we successfully visualized and quantified stable bacterial growth over an extended period of time with bioluminescence imaging, which allowed us to monitor the process of infection without euthanizing the experimental animals. This model is applicable to in vivo evaluations of the long-term efficacy of novel antibiotics or antibacterial implants.
PMCID: PMC4153648  PMID: 25184249
11.  Repression of Flagellar Genes in Exponential Phase by CsgD and CpxR, Two Crucial Modulators of Escherichia coli Biofilm Formation 
Journal of Bacteriology  2014;196(3):707-715.
Escherichia coli adapts its lifestyle to the variations of environmental growth conditions, swapping between swimming motility or biofilm formation. The stationary-phase sigma factor RpoS is an important regulator of this switch, since it stimulates adhesion and represses flagellar biosynthesis. By measuring the dynamics of gene expression, we show that RpoS inhibits the transcription of the flagellar sigma factor, FliA, in exponential growth phase. RpoS also partially controls the expression of CsgD and CpxR, two transcription factors important for bacterial adhesion. We demonstrate that these two regulators repress the transcription of fliA, flgM, and tar and that this regulation is dependent on the growth medium. CsgD binds to the flgM and fliA promoters around their −10 promoter element, strongly suggesting direct repression. We show that CsgD and CpxR also affect the expression of other known modulators of cell motility. We propose an updated structure of the regulatory network controlling the choice between adhesion and motility.
PMCID: PMC3911157  PMID: 24272779
12.  Unprecedented High-Resolution View of Bacterial Operon Architecture Revealed by RNA Sequencing 
mBio  2014;5(4):e01442-14.
We analyzed the transcriptome of Escherichia coli K-12 by strand-specific RNA sequencing at single-nucleotide resolution during steady-state (logarithmic-phase) growth and upon entry into stationary phase in glucose minimal medium. To generate high-resolution transcriptome maps, we developed an organizational schema which showed that in practice only three features are required to define operon architecture: the promoter, terminator, and deep RNA sequence read coverage. We precisely annotated 2,122 promoters and 1,774 terminators, defining 1,510 operons with an average of 1.98 genes per operon. Our analyses revealed an unprecedented view of E. coli operon architecture. A large proportion (36%) of operons are complex with internal promoters or terminators that generate multiple transcription units. For 43% of operons, we observed differential expression of polycistronic genes, despite being in the same operons, indicating that E. coli operon architecture allows fine-tuning of gene expression. We found that 276 of 370 convergent operons terminate inefficiently, generating complementary 3′ transcript ends which overlap on average by 286 nucleotides, and 136 of 388 divergent operons have promoters arranged such that their 5′ ends overlap on average by 168 nucleotides. We found 89 antisense transcripts of 397-nucleotide average length, 7 unannotated transcripts within intergenic regions, and 18 sense transcripts that completely overlap operons on the opposite strand. Of 519 overlapping transcripts, 75% correspond to sequences that are highly conserved in E. coli (>50 genomes). Our data extend recent studies showing unexpected transcriptome complexity in several bacteria and suggest that antisense RNA regulation is widespread.
We precisely mapped the 5′ and 3′ ends of RNA transcripts across the E. coli K-12 genome by using a single-nucleotide analytical approach. Our resulting high-resolution transcriptome maps show that ca. one-third of E. coli operons are complex, with internal promoters and terminators generating multiple transcription units and allowing differential gene expression within these operons. We discovered extensive antisense transcription that results from more than 500 operons, which fully overlap or extensively overlap adjacent divergent or convergent operons. The genomic regions corresponding to these antisense transcripts are highly conserved in E. coli (including Shigella species), although it remains to be proven whether or not they are functional. Our observations of features unearthed by single-nucleotide transcriptome mapping suggest that deeper layers of transcriptional regulation in bacteria are likely to be revealed in the future.
PMCID: PMC4161252  PMID: 25006232
13.  Redetermination of di­aqua­tris­(4-oxo­pent-2-en-2-olato-κ2 O,O′)lanthanum(III) 
The structure of the title compound, [La(C5H7O2)3(H2O)2], has been redetermined to modern standards with anisotropic displacement parameters for all non-H atoms and the hydrogen-bonding pattern unambiguously established [for the previous study, see Phillips et al. (1968 ▶). Inorg. Chem. 7, 2295–2299]. The La3+ ion is coordinated by three O,O′-bidentate acetyl­acetate (acac−) ligands and two water mol­ecules, resulting in a fairly regular square-anti­prismatic LaO8 coordination geometry, with both aqua ligands part of the same square face. In the crystal, the neutral complex mol­ecules are linked into [110] chains by O—H⋯O hydrogen bonds.
PMCID: PMC4120584  PMID: 25161523
14.  Crisis of Japanese Vascular Flora Shown By Quantifying Extinction Risks for 1618 Taxa 
PLoS ONE  2014;9(6):e98954.
Although many people have expressed alarm that we are witnessing a mass extinction, few projections have been quantified, owing to limited availability of time-series data on threatened organisms, especially plants. To quantify the risk of extinction, we need to monitor changes in population size over time for as many species as possible. Here, we present the world's first quantitative projection of plant species loss at a national level, with stochastic simulations based on the results of population censuses of 1618 threatened plant taxa in 3574 map cells of ca. 100 km2. More than 500 lay botanists helped monitor those taxa in 1994–1995 and in 2003–2004. We projected that between 370 and 561 vascular plant taxa will go extinct in Japan during the next century if past trends of population decline continue. This extinction rate is approximately two to three times the global rate. Using time-series data, we show that existing national protected areas (PAs) covering ca. 7% of Japan will not adequately prevent population declines: even core PAs can protect at best <60% of local populations from decline. Thus, the Aichi Biodiversity Target to expand PAs to 17% of land (and inland water) areas, as committed to by many national governments, is not enough: only 29.2% of currently threatened species will become non-threatened under the assumption that probability of protection success by PAs is 0.5, which our assessment shows is realistic. In countries where volunteers can be organized to monitor threatened taxa, censuses using our method should be able to quantify how fast we are losing species and to assess how effective current conservation measures such as PAs are in preventing species extinction.
PMCID: PMC4055661  PMID: 24922311
15.  Atg38 is required for autophagy-specific phosphatidylinositol 3-kinase complex integrity 
The Journal of Cell Biology  2013;203(2):299-313.
Atg38 provides a physical linkage between the Vps15–Vps34 and Atg14–Vps30 subcomplexes to facilitate PI3-kinase complex I formation.
Autophagy is a conserved eukaryotic process of protein and organelle self-degradation within the vacuole/lysosome. Autophagy is characterized by the formation of an autophagosome, for which Vps34-dervied phosphatidylinositol 3-phosphate (PI3P) is essential. In yeast, Vps34 forms two distinct protein complexes: complex I, which functions in autophagy, and complex II, which is involved in protein sorting to the vacuole. Here we identify and characterize Atg38 as a stably associated subunit of complex I. In atg38Δ cells, autophagic activity was significantly reduced and PI3-kinase complex I dissociated into the Vps15–Vps34 and Atg14–Vps30 subcomplexes. We find that Atg38 physically interacted with Atg14 and Vps34 via its N terminus. Further biochemical analyses revealed that Atg38 homodimerizes through its C terminus and that this homodimer formation is indispensable for the integrity of complex I. These data suggest that the homodimer of Atg38 functions as a physical linkage between the Vps15–Vps34 and Atg14–Vps30 subcomplexes to facilitate complex I formation.
PMCID: PMC3812978  PMID: 24165940
16.  Identification of the Set of Genes, Including Nonannotated morA, under the Direct Control of ModE in Escherichia coli 
Journal of Bacteriology  2013;195(19):4496-4505.
ModE is the molybdate-sensing transcription regulator that controls the expression of genes related to molybdate homeostasis in Escherichia coli. ModE is activated by binding molybdate and acts as both an activator and a repressor. By genomic systematic evolution of ligands by exponential enrichment (SELEX) screening and promoter reporter assays, we have identified a total of nine operons, including the hitherto identified modA, moaA, dmsA, and napF operons, of which six were activated by ModE and three were repressed. In addition, two promoters were newly identified and direct transcription of novel genes, referred to as morA and morB, located on antisense strands of yghW and torY, respectively. The morA gene encodes a short peptide, MorA, with an unusual initiation codon. Surprisingly, overexpression of the morA 5′ untranslated region exhibited an inhibitory influence on colony formation of E. coli K-12.
PMCID: PMC3807462  PMID: 23913318
17.  The Whole Set of Constitutive Promoters Recognized by RNA Polymerase RpoD Holoenzyme of Escherichia coli 
PLoS ONE  2014;9(3):e90447.
The promoter selectivity of Escherichia coli RNA polymerase is determined by the sigma subunit with promoter recognition activity. The model prokaryote Escherichia coli contains seven species of the sigma subunit, each recognizing a specific set of promoters. The major sigma subunit, sigma-70 encoded by rpoD, plays a major role in transcription of growth-related genes. Concomitant with the increase in detection of promoters functioning in vivo under various stressful conditions, the variation is expanding in the consensus sequence of RpoD promoters. In order to identify the canonical sequence of “constitutive promoters” that are recognized by the RNA polymerase holoenzyme containing RpoD sigma in the absence of supporting transcription factors, an in vitro mixed transcription assay was carried out using a whole set of variant promoters, each harboring one base replacement, within the model promoter with the conserved -35 and -10 sequences of RpoD promoters. The consensus sequences, TTGACA(-35) and TATAAT(-10), were identified to be ideal for the maximum level of open complex formation and the highest rate of promoter opening, respectively. For identification of the full range of constitutive promoters on the E. coli genome, a total of 2,701 RpoD holoenzyme-binding sites were identified by Genomic SELEX screening, and using the reconfirmed consensus promoter sequence, a total of maximum 669 constitutive promoters were identified, implying that the majority of hitherto identified promoters represents the TF-dependent “inducible promoters”. One unique feature of the constitutive promoters is the high level of promoter sequence conservation, about 85% carrying five-out-of-six agreements with -35 or -10 consensus sequence. The list of constitutive promoters provides the community resource toward estimation of the inducible promoters that operate under various stressful conditions in nature.
PMCID: PMC3946193  PMID: 24603758
18.  Involvement of Cyclic AMP Receptor Protein in Regulation of the rmf Gene Encoding the Ribosome Modulation Factor in Escherichia coli 
Journal of Bacteriology  2013;195(10):2212-2219.
The decrease in overall translation in stationary-phase Escherichia coli is accompanied with the formation of functionally inactive 100S ribosomes mediated by the ribosome modulation factor (RMF). At present, however, little is known regarding the regulation of stationary-phase-coupled RMF expression. In the course of a systematic screening of regulation targets of DNA-binding transcription factors from E. coli, we realized that CRP (cyclic AMP [cAMP] receptor protein), the global regulator for carbon source utilization, participates in regulation of some ribosomal protein genes, including the rmf gene. In this study, we carried out detailed analysis of the regulation of the RMF gene by cAMP-CRP. The cAMP-dependent binding of CRP to the rmf gene promoter was confirmed by gel shift and DNase I footprinting assays. By using a reporter assay system, the expression level of RMF was found to decrease in the crp knockout mutant, indicating the involvement of CRP as an activator of the rmf promoter. In good agreement with the reduction of rmf promoter activity, we observed decreases in RMF production and 100S ribosome dimerization in the absence of CRP. Taken together, we propose that CRP regulates transcription activation of the rmf gene for formation of 100S ribosome dimers. Physiological roles of CRP involvement in RMF production are discussed.
PMCID: PMC3650541  PMID: 23475967
19.  Transcriptional regulation by Pol II(G) involving Mediator and competitive interactions of Gdown1 and TFIIF with Pol II 
Molecular Cell  2012;45(1):51-63.
Pol II(G) is a distinct form of RNA polymerase II that contains the tightly associated Gdown1 polypeptide (encoded by POLR2M). Unlike Pol II, Pol II(G) is highly dependent upon Mediator for robust activator-dependent transcription in a biochemically defined in vitro system. Here, in vitro studies show that Gdown1 competes with TFIIF for binding to the RPB1 and RPB5 subunits of Pol II, thereby inhibiting an essential function of TFIIF in preinitiation complex assembly, but also that Mediator can actually facilitate Pol II(G) binding to the promoter prior to subsequent Mediator functions. Complementary ChIP and RNAi analyses reveal that Pol II(G) is recruited to promoter regions of subsets of actively transcribed genes, where it appears to restrict transcription. These and other results suggest that Pol II(G) may act to modulate some genes while simultaneously, as a poised (non-initiated) polymerase, setting the stage for Mediator-dependent enhancement of their activity.
PMCID: PMC3259531  PMID: 22244332
20.  PINK1-mediated phosphorylation of the Parkin ubiquitin-like domain primes mitochondrial translocation of Parkin and regulates mitophagy 
Scientific Reports  2012;2:1002.
Parkinson's disease genes PINK1 and parkin encode kinase and ubiquitin ligase, respectively. The gene products PINK1 and Parkin are implicated in mitochondrial autophagy, or mitophagy. Upon the loss of mitochondrial membrane potential (ΔΨm), cytosolic Parkin is recruited to the mitochondria by PINK1 through an uncharacterised mechanism – an initial step triggering sequential events in mitophagy. This study reports that Ser65 in the ubiquitin-like domain (Ubl) of Parkin is phosphorylated in a PINK1-dependent manner upon depolarisation of ΔΨm. The introduction of mutations at Ser65 suggests that phosphorylation of Ser65 is required not only for the efficient translocation of Parkin, but also for the degradation of mitochondrial proteins in mitophagy. Phosphorylation analysis of Parkin pathogenic mutants also suggests Ser65 phosphorylation is not sufficient for Parkin translocation. Our study partly uncovers the molecular mechanism underlying the PINK1-dependent mitochondrial translocation and activation of Parkin as an initial step of mitophagy.
PMCID: PMC3525937  PMID: 23256036
21.  A novel regulator RcdA of the csgD gene encoding the master regulator of biofilm formation in Escherichia coli 
MicrobiologyOpen  2012;1(4):381-394.
The FixJ/LuxR family transcription factor CsgD is a master regulator of biofilm formation in Escherichia coli. Previously, we identified more than 10 transcription factors that participate in regulation of the csgD promoter. After genomic SELEX screening of regulation targets, an uncharacterized TetR-type transcription factor YbjK was found to be involved in regulation of the csgD promoter. In addition, a number of stress-response genes were found to be under the direct control of YbjK. Taken together, we propose to rename it to RcdA (regulator of csgD). One unique feature of RcdA is its mode of DNA binding. Gel shift, DNase-I footprinting, and atomic force microscopic (AFM) analyses indicated that RcdA is a DNA-binding protein with a high level of cooperativity, with which it covers the entire surface of probe DNA through protein–protein interaction and moreover it induces the formation of aggregates of DNA–RcdA complexes.
PMCID: PMC3535384  PMID: 23233451
Biofilm formation; csgD promoter; genomic SELEX; regulation network; transcription factor
22.  LATS1/WARTS phosphorylates MYPT1 to counteract PLK1 and regulate mammalian mitotic progression 
The Journal of Cell Biology  2012;197(5):625-641.
Showing convergence with budding yeast mitotic exit network signaling, the LATS1/WARTS kinase phosphorylates the MYPT1 phosphatase to control PLK1 at the G2 DNA damage checkpoint.
In the mitotic exit network of budding yeast, Dbf2 kinase phosphorylates and regulates Cdc14 phosphatase. In contrast, no phosphatase substrates of LATS1/WARTS kinase, the mammalian equivalent of Dbf2, has been reported. To address this discrepancy, we performed phosphoproteomic screening using LATS1 kinase. Screening identified MYPT1 (myosin phosphatase–targeting subunit 1) as a new substrate for LATS1. LATS1 directly and preferentially phosphorylated serine 445 (S445) of MYPT1. An MYPT1 mutant (S445A) failed to dephosphorylate Thr 210 of PLK1 (pololike kinase 1), thereby activating PLK1. This suggests that LATS1 promotes MYPT1 to antagonize PLK1 activity. Consistent with this, LATS1-depleted HeLa cells or fibroblasts from LATS1 knockout mice showed increased PLK1 activity. We also found deoxyribonucleic acid (DNA) damage–induced LATS1 activation caused PLK1 suppression via the phosphorylation of MYPT1 S445. Furthermore, LATS1 knockdown cells showed reduced G2 checkpoint arrest after DNA damage. These results indicate that LATS1 phosphorylates a phosphatase as does the yeast Dbf2 and demonstrate a novel role of LATS1 in controlling PLK1 at the G2 DNA damage checkpoint.
PMCID: PMC3404884  PMID: 22641346
23.  The nucleoid-associated protein Dan organizes chromosomal DNA through rigid nucleoprotein filament formation in E. coli during anoxia 
Nucleic Acids Research  2012;41(2):746-753.
Dan is a transcription factor that regulates the ttd operon encoding tartrate dehydratase. During anaerobic conditions, its copy number increases by 100-fold, making Dan an abundant nucleoid-associated protein. However, little is known about the mode of Dan–DNA interaction. To understand its cellular functions, we used single-molecule manipulation and imaging techniques to show that Dan binds cooperatively along DNA, resulting in formation of a rigid periodic nucleoprotein filament that strongly restricts accessibility to DNA. Furthermore, in the presence of physiologic levels of magnesium, these filaments interact with each other to cause global DNA condensation. Overall, these results shed light on the architectural role of Dan in the compaction of Escherichia coli chromosomal DNA under anaerobic conditions. Formation of the nucleoprotein filament provides a basis in understanding how Dan may play roles in both chromosomal DNA protection and gene regulation.
PMCID: PMC3553945  PMID: 23180762
24.  Prokaryotic genome regulation: A revolutionary paradigm 
After determination of the whole genome sequence, the research frontier of bacterial molecular genetics has shifted to reveal the genome regulation under stressful conditions in nature. The gene selectivity of RNA polymerase is modulated after interaction with two groups of regulatory proteins, 7 sigma factors and 300 transcription factors. For identification of regulation targets of transcription factors in Escherichia coli, we have developed Genomic SELEX system and subjected to screening the binding sites of these factors on the genome. The number of regulation targets by a single transcription factor was more than those hitherto recognized, ranging up to hundreds of promoters. The number of transcription factors involved in regulation of a single promoter also increased to as many as 30 regulators. The multi-target transcription factors and the multi-factor promoters were assembled into complex networks of transcription regulation. The most complex network was identified in the regulation cascades of transcription of two master regulators for planktonic growth and biofilm formation.
PMCID: PMC3511978  PMID: 23138451
transcription regulation; genome regulation; transcription factor; regulation network; genomic SELEX; Escherichia coli
25.  Metabolomic profiling of lung and prostate tumor tissues by capillary electrophoresis time-of-flight mass spectrometry 
Metabolomics  2012;9(2):444-453.
Metabolic microenvironment of tumor cells is influenced by oncogenic signaling and tissue-specific metabolic demands, blood supply, and enzyme expression. To elucidate tumor-specific metabolism, we compared the metabolomics of normal and tumor tissues surgically resected pairwise from nine lung and seven prostate cancer patients, using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Phosphorylation levels of enzymes involved in central carbon metabolism were also quantified. Metabolomic profiles of lung and prostate tissues comprised 114 and 86 metabolites, respectively, and the profiles not only well distinguished tumor from normal tissues, but also squamous cell carcinoma from the other tumor types in lung cancer and poorly differentiated tumors from moderately differentiated tumors in prostate cancer. Concentrations of most amino acids, especially branched-chain amino acids, were significantly higher in tumor tissues, independent of organ type, but of essential amino acids were particularly higher in poorly differentiated than moderately differentiated prostate cancers. Organ-dependent differences were prominent at the levels of glycolytic and tricarboxylic acid cycle intermediates and associated energy status. Significantly high lactate concentrations and elevated activating phosphorylation levels of phosphofructokinase and pyruvate kinase in lung tumors confirmed hyperactive glycolysis. We highlighted the potential of CE-TOFMS-based metabolomics combined with phosphorylated enzyme analysis for understanding tissue-specific tumor microenvironments, which may lead to the development of more effective and specific anticancer therapeutics.
Electronic supplementary material
The online version of this article (doi:10.1007/s11306-012-0452-2) contains supplementary material, which is available to authorized users.
PMCID: PMC3608864  PMID: 23543897
Metabolomics; CE-MS; Phosphoproteomics; Lung cancer; Prostate cancer; Tumor microenvironment

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