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Chattopadhyay, Gopinath, Jayantika Bhowmick, Kavyashree Manjunath, Shahbaz Ahmed, Parveen Goyal, and Raghavan Varadarajan. "Mechanistic insights into global suppressors of protein folding defects." PLOS Genetics 18, no. 8 (August 29, 2022): e1010334. http://dx.doi.org/10.1371/journal.pgen.1010334.
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Most amino acid substitutions in a protein either lead to partial loss of function or are near neutral. Several studies have shown the existence of second-site mutations that can rescue defects caused by diverse loss of function mutations. Such global suppressor mutations are key drivers of protein evolution. However, the mechanisms responsible for such suppression remain poorly understood. To address this, we characterized multiple suppressor mutations both in isolation and in combination with inactive mutants. We examined six global suppressors of the bacterial toxin CcdB, the known M182T global suppressor of TEM-1 β-lactamase, the N239Y global suppressor of p53-DBD and three suppressors of the SARS-CoV-2 spike Receptor Binding Domain. When coupled to inactive mutants, they promote increased in-vivo solubilities as well as regain-of-function phenotypes. In the case of CcdB, where novel suppressors were isolated, we determined the crystal structures of three such suppressors to obtain insight into the specific molecular interactions responsible for the observed effects. While most individual suppressors result in small stability enhancements relative to wildtype, which can be combined to yield significant stability increments, thermodynamic stabilisation is neither necessary nor sufficient for suppressor action. Instead, in diverse systems, we observe that individual global suppressors greatly enhance the foldability of buried site mutants, primarily through increase in refolding rate parameters measured in vitro. In the crowded intracellular environment, mutations that slow down folding likely facilitate off-pathway aggregation. We suggest that suppressor mutations that accelerate refolding can counteract this, enhancing the yield of properly folded, functional protein in vivo.
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Shortle, David, and Beth Lin. "GENETIC ANALYSIS OF STAPHYLOCOCCAL NUCLEASE: IDENTIFICATION OF THREE INTRAGENIC "GLOBAL" SUPPRESSORS OF NUCLEASE-MINUS MUTATIONS." Genetics 110, no. 4 (August 1, 1985): 539–55. http://dx.doi.org/10.1093/genetics/110.4.539.
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ABSTRACT A collection of 77 unique missense mutations distributed across the gene encoding staphylococcal nuclease (nuc) has been assembled. These mutations were induced by random gap misrepair mutagenesis of the cloned gene and were identified in E. coli transformants expressing reduced levels of nuclease activity. Four nuc - mutations which alter amino acid residues at positions outside of the active site region of the enzyme were submitted to a second round of mutagenesis, and characterization of several independent NUC+ isolates lead to the identification of three second-site suppressor mutations within the protein-coding sequence of the nuc gene. On separation from the mutation originally suppressed and recombination with a number of other nuc - mutations, all three suppressors displayed the property of "global" suppression, i.e., phenotypic suppression of the nuclease-minus character of multiple different alleles. A simple and generally applicable strategy was used to obtain efficient homologous recombination between plasmids for purposes of mapping nuc - mutations, mapping second-site suppressors and constructing double mutant combinations from pairs of single mutations.
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Herring, Christopher D., and Frederick R. Blattner. "Global Transcriptional Effects of a Suppressor tRNA and the Inactivation of the Regulator frmR." Journal of Bacteriology 186, no. 20 (October 15, 2004): 6714–20. http://dx.doi.org/10.1128/jb.186.20.6714-6720.2004.
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ABSTRACT Expression of an amber suppressor tRNA should result in read-through of the 326 open reading frames (ORFs) that terminate with amber stop codons in the Escherichia coli genome, including six pseudogenes. Abnormal extension of an ORF might alter the activities of the protein and have effects on cellular physiology, while suppression of a pseudogene could lead to a gain of function. We used oligonucleotide microarrays to determine if any effects were apparent at the level of transcription in glucose minimal medium. Surprisingly, only eight genes had significantly different expression in the presence of the suppressor. Among these were the genes yaiN, adhC, and yaiM, forming a single putative operon whose likely function is the degradation of formaldehyde. Expression of wild-type yaiN was shown to result in repression of the operon, while a suppression-mimicking allele lacking the amber stop codon and extended 7 amino acids did not. The operon was shown to be induced by formaldehyde, and the genes have been renamed frmR, frmA, and frmB, respectively.
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Baroni, T. E., T. Wang, H. Qian, L. R. Dearth, L. N. Truong, J. Zeng, A. E. Denes, S. W. Chen, and R. K. Brachmann. "A global suppressor motif for p53 cancer mutants." Proceedings of the National Academy of Sciences 101, no. 14 (March 22, 2004): 4930–35. http://dx.doi.org/10.1073/pnas.0401162101.
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Csink, A. K., R. Linsk, and J. A. Birchler. "Mosaic suppressor, a gene in Drosophila that modifies retrotransposon expression and interacts with zeste." Genetics 136, no. 2 (February 1, 1994): 573–83. http://dx.doi.org/10.1093/genetics/136.2.573.
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Abstract A newly identified locus in Drosophila melanogaster, Mosaic suppressor (Msu), is described. This gene modifies the expression of white-apricot (wa), which is a copia retrotransposon-induced allele of the white gene. In addition to suppressing wa in a mosaic fashion, this mutation suppresses or enhances the expression of several other retrotransposon induced white alleles. Mutations in Msu alter copia transcript abundance and may regulate the expression of several other retrotransposons. While each of the two Msu isolates is homozygous lethal, heteroallelic escapers occur at a low frequency. These escapers act not only as strong suppressors of wa, but also as a recessive enhancer of synaptic-dependent gene expression at white. The mutation described here suggests a connection between the regulation of specific transcriptional units such as retrotransposons and more global synapsis dependent regulatory effects.
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Cheng, Chin Leng, Michael K. Wong, and Mark Hochstrasser. "Yeast Nst1 is a novel component of P-bodies and is a specific suppressor of proteasome base assembly defects." Molecular Biology of the Cell 32, no. 20 (October 1, 2021): ar6. http://dx.doi.org/10.1091/mbc.e21-04-0178.
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We identified negative salt tolerance 1 (Nst1) as a dosage suppressor of the temperature sensitivity and proteasome assembly defects in multiple base mutants. Both endogenous and overexpressed Nst1 could localize to processing bodies. Nst1 overexpression inhibited global protein translation that in turn suppressed aggregation and proteasome assembly defects in base assembly mutants.
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Huang, W., and T. Palzkill. "A natural polymorphism in -lactamase is a global suppressor." Proceedings of the National Academy of Sciences 94, no. 16 (August 5, 1997): 8801–6. http://dx.doi.org/10.1073/pnas.94.16.8801.
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Irie, K., K. Yamaguchi, K. Kawase, and K. Matsumoto. "The yeast MOT2 gene encodes a putative zinc finger protein that serves as a global negative regulator affecting expression of several categories of genes, including mating-pheromone-responsive genes." Molecular and Cellular Biology 14, no. 5 (May 1994): 3150–57. http://dx.doi.org/10.1128/mcb.14.5.3150-3157.1994.
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The STE4 gene encodes the beta subunit of a heterotrimeric G protein that is an essential component of the pheromone signal transduction pathway. To identify downstream component(s) of Ste4, we sought pseudo-revertants that restored mating competence to ste4 mutants. The suppressor mot2 was isolated as a recessive mutation that restored conjugational competence to a temperature-sensitive ste4 mutant and simultaneously conferred a temperature-sensitive growth phenotype. The MOT2 gene encodes a putative zinc finger protein, the deletion of which resulted in temperature-sensitive growth, increased expression of FUS1 in the absence of pheromones, and suppression of a deletion of the alpha-factor receptor. On the other hand, sterility resulting from deletion of STE4 was not suppressed by the mot2 deletion. These phenotypes are similar to those associated with temperature-sensitive mutations in CDC36 and CDC39, which are proposed to encode general negative regulators of transcription rather than factors involved in the pheromone response pathway. Deletion of MOT2 also caused increased transcription of unrelated genes such as GAL7 and PHO84. Overexpression of MOT2 suppresses the growth defect of temperature-sensitive mutations in CDC36 and CDC39. These observations suggest that Mot2 functions as a general negative regulator of transcription in the same processes as Cdc36 and Cdc39.
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Irie, K., K. Yamaguchi, K. Kawase, and K. Matsumoto. "The yeast MOT2 gene encodes a putative zinc finger protein that serves as a global negative regulator affecting expression of several categories of genes, including mating-pheromone-responsive genes." Molecular and Cellular Biology 14, no. 5 (May 1994): 3150–57. http://dx.doi.org/10.1128/mcb.14.5.3150.
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The STE4 gene encodes the beta subunit of a heterotrimeric G protein that is an essential component of the pheromone signal transduction pathway. To identify downstream component(s) of Ste4, we sought pseudo-revertants that restored mating competence to ste4 mutants. The suppressor mot2 was isolated as a recessive mutation that restored conjugational competence to a temperature-sensitive ste4 mutant and simultaneously conferred a temperature-sensitive growth phenotype. The MOT2 gene encodes a putative zinc finger protein, the deletion of which resulted in temperature-sensitive growth, increased expression of FUS1 in the absence of pheromones, and suppression of a deletion of the alpha-factor receptor. On the other hand, sterility resulting from deletion of STE4 was not suppressed by the mot2 deletion. These phenotypes are similar to those associated with temperature-sensitive mutations in CDC36 and CDC39, which are proposed to encode general negative regulators of transcription rather than factors involved in the pheromone response pathway. Deletion of MOT2 also caused increased transcription of unrelated genes such as GAL7 and PHO84. Overexpression of MOT2 suppresses the growth defect of temperature-sensitive mutations in CDC36 and CDC39. These observations suggest that Mot2 functions as a general negative regulator of transcription in the same processes as Cdc36 and Cdc39.
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Nahar, Rahul, Parham Ramezani-Rad, Sinisa Dovat, Maike Buchner, Thomas G. Graeber, and Markus Muschen. "Mechanisms of Ikaros-Mediated Tumor Suppression." Blood 118, no. 21 (November 18, 2011): 408. http://dx.doi.org/10.1182/blood.v118.21.408.408.
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Abstract Abstract 408 Background: The Ikaros (IKZF1) tumor suppressor is deleted in >80% of the cases of Ph+ ALL. While Ikaros cooperates with pre-B cell receptor signaling to induce cell cycle exit in Ph+ ALL (Trageser et al., J Exp Med, 2009), the mechanism of Ikaros-mediated tumor suppression is poorly understood. Here we report on a series of genetic experiments that show that Ikaros (i) interferes with key survival pathways downstream of the BCR-ABL1 kinase, (ii) inhibits leukemia cell proliferation through interaction with the pre-B cell receptor signaling pathway and (iii) activates the tumor suppressors p53, p21 and p27. Results: To elucidate the mechanism of Ikaros-dependent tumor suppression in BCR-ABL1-driven B cell lineage leukemia, we studied regulation of critical phosphorylation events downstream of the BCR-ABL1 kinase as a central mediators of survival and proliferation. Reconstitution of Ikaros expression in BCR-ABL1-transformed pre-B ALL cells resulted in rapid and global dephosphorylation comparable to the effect of Imatinib. A detailed analysis showed that Ikaros-induced dephosphorylation events affect activation of Stat5 (Y694), AKT (S473), ERK1/2 (T202 and Y204) and SRC (Y416). Interestingly, both Imatinib-treatment and reconstitution of pre-B cell receptor signaling using retroviral vectors for expression of the m heavy chain or the BLNK adapter molecule have the same effects as reconstitution of Ikaros. In fact, a comprehensive gene expression analysis demonstrated that Ikaros reconstitution resulted in similar gene expression changes as reconstitution of pre-B cell receptor signaling (m heavy chain or BLNK), reconstitution of PAX5, Cre-mediated deletion of Stat5 or Myc, or treatment with Imatinib. The signature of common gene expression changes shared between reconstitution of Ikaros, Pax5, m heavy chain, BLNK and inducible deletion of Stat5 or Myc and Imatinib-treatment involves known tumor suppressors including SPIB, BTG1, and BTG2. These findings suggest that reconstitution of tumor suppressive transcription factor (Ikaros, Pax5) converges with pre-B cell receptor-mediated tumor suppression. To better understand how pre-B cell receptor signaling and Ikaros intersect, we combined reconstitution of Ikaros with genetic deletion of either the (more proximal) SYK kinase or the (more distal) BLNK adapter molecule. While inducible Cre-mediated deletion of Syk had no effect on Ikaros-mediated tumor suppression, deletion of the BLNK adapter compromised the ability of Ikaros to function as tumor suppressor. These findings were confirmed in an in vivo transplantation experiment. While mice transplanted with Ikaros+ BLNK+ leukemia cells survived indefinitely, mice transplanted with Ikaros- BLNK+, Ikaros+ BLNK- or Ikaros- BLNK- leukemia cells died after 24 to 31 days post transplantation. While these findings provide genetic evidence for collaboration between the Ikaros and pre-B cell receptor tumor suppressor pathways, Ikaros and pre-B cell receptor signaling differ with respect to activation of classical tumor suppressor pathways. While reconstitution of pre-B cell receptor signaling failed to activate Arf, p53 or p27, protein levels of all these molecules were strongly upregulated by Ikaros. In agreement with these findings, reconstitution of pre-B cell receptor signaling had the same tumor suppressive effect in wildtype leukemia cells as in Arf−/−, p53−/− as well as p27−/− leukemia cells. Conversely, deletion of Arf and p53 significantly diminished the ability of Ikaros to function as tumor suppressor. Conclusion: Ikaros deletion represents a near-obligatory lesion in the pathogenesis of Ph+ ALL. Here we provide genetic evidence for three novel pathways of Ikaros-mediated tumor suppression. Like PAX5, Ikaros reconstitution results in multiple dephosphorylation events (Stat5, AKT, ERK1/2 and SRC are affected). In collaboration with the pre-B cell receptor and its downstream adapter molecule BLNK, Ikaros suppressed MYC and inhibits cell cycle progression. Induction of the Arf/p53 pathway represents a distinct function of Ikaros, which is not shared with the pre-B cell receptor signaling pathway. Disclosures: No relevant conflicts of interest to declare.
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Ansseau, M., P. Papart, W. Pitchot, M. Timsit-Berthier, JJ Legros, and R. von Frenckell. "Dexamethasone suppression test and prediction of treatment response to selective antidepressants." European Psychiatry 7, no. 4 (1992): 191–94. http://dx.doi.org/10.1017/s0924933800005307.
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SummaryThe usefulness of the dexamethasone suppression test (DST) in the prediction of treatment response to selective antidepressants was tested in 56 major depressive inpatients. Following DST, patients were randomly assigned to treatment by either nomifensine, a catecholaminergic antidepressant, or zimeldine, a serotonergic antidepressant during a 3-week period and assessed by means of the second part of the Clinical Global Impressions (CGI-2). No significant difference was present between the 27 DST suppressor and the 29 DST non-suppressor patients in their overall clinical outcome. Moreover, no preferential response to nomifensine or zimeldine was noted in any of the two groups defined according to DST status. Therefore, these results do not support the usefulness of the DST in the prediction of the treatment response to antidepressants in general and to selective antidepressants in particular.
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Wells, Derek H., and Sharon R. Long. "Mutations in rpoBC Suppress the Defects of a Sinorhizobium meliloti relA Mutant." Journal of Bacteriology 185, no. 18 (September 15, 2003): 5602–10. http://dx.doi.org/10.1128/jb.185.18.5602-5610.2003.
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ABSTRACT The nitrogen-fixing symbiosis between Sinorhizobium meliloti and Medicago sativa requires complex physiological adaptation by both partners. One method by which bacteria coordinately control physiological adaptation is the stringent response, which is triggered by the presence of the nucleotide guanosine tetraphosphate (ppGpp). ppGpp, produced by the RelA enzyme, is thought to bind to and alter the ability of RNA polymerase (RNAP) to initiate and elongate transcription and affect the affinity of the core enzyme for various sigma factors. An S. meliloti relA mutant which cannot produce ppGpp was previously shown to be defective in the ability to form nodules. This mutant also overproduces a symbiotically necessary exopolysaccharide called succinoglycan (38). The work presented here encompasses the analysis of suppressor mutants, isolated from host plants, that suppress the symbiotic defects of the relA mutant. All suppressor mutations are extragenic and map to either rpoB or rpoC, which encode the β and β′ subunits of RNAP. Phenotypic, structural, and gene expression analyses reveal that suppressor mutants can be divided into two classes; one is specific in its effect on stringent response-regulated genes and shares striking similarity with suppressor mutants of Escherichia coli strains that lack ppGpp, and another reduces transcription of all genes tested in comparison to that in the relA parent strain. Our findings indicate that the ability to successfully establish symbiosis is tightly coupled with the bacteria's ability to undergo global physiological adjustment via the stringent response.
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Kinscherf, Thomas G., and David K. Willis. "Global Regulation by gidA in Pseudomonas syringae." Journal of Bacteriology 184, no. 8 (April 15, 2002): 2281–86. http://dx.doi.org/10.1128/jb.184.8.2281-2286.2002.
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ABSTRACT Analysis of two virulence mutants of Pseudomonas syringae B728a revealed that the Tn 5 sites of insertion were within the gidA open reading frame (ORF). These mutations were pleiotropic, affecting diverse phenotypic traits, such as lipodepsipeptide (syringomycin and syringopeptin) antibiotic production, swarming, presence of fluorescent pigment, and virulence. Site-specific recombination of a disrupted gidA gene into the chromosome resulted in the same phenotypic pattern as transposon insertion. Mutant phenotypes were restored by the gidA ORF on a plasmid. The salA gene, a copy number suppressor of the syringomycin-deficient phenotype in gacS and gacA mutants, was also found to suppress the antibiotic-negative phenotypes of gidA mutants, suggesting that gidA might play some role in salA regulation. Reporter studies with chromosomal salA-lacZ translational fusions confirmed that salA reporter expression decreased approximately fivefold in a gidA mutant background, with a concurrent decrease in the expression of the syringomycin biosynthetic reporter fusion syrB-lacZ. Wild-type levels of reporter expression were restored by supplying an intact gidA gene on a plasmid. Often described as being involved in cell division, more recent evidence suggests a role for gidA in moderating translational fidelity, suggesting a mechanism by which global regulation might occur. The gidA gene is essentially universal in the domains Bacteria and Eucarya but has no counterparts in Archaea, probably reflecting specific differences in the translational machinery between the former and latter domains.
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Kaiser, Martin F., David C. Johnson, Ping Wu, Brian A. Walker, Annamaria Brioli, Fabio Mirabella, Christopher P. Wardell, Lorenzo Melchor, Faith E. Davies, and Gareth J. Morgan. "Global methylation analysis identifies prognostically important epigenetically inactivated tumor suppressor genes in multiple myeloma." Blood 122, no. 2 (July 11, 2013): 219–26. http://dx.doi.org/10.1182/blood-2013-03-487884.
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Key Points Epigenetic inactivation of tumor suppressor genes is associated with an unfavorable prognosis in multiple myeloma. Drug response and microenvironment interaction pathways are affected by epigenetic inactivation, linking tumor biology to prognosis.
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Augustus, Anne Marie, Teresa Celaya, Fasahath Husain, Matthew Humbard, and Rajeev Misra. "Antibiotic-Sensitive TolC Mutants and Their Suppressors." Journal of Bacteriology 186, no. 6 (March 15, 2004): 1851–60. http://dx.doi.org/10.1128/jb.186.6.1851-1860.2004.
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ABSTRACT The TolC protein of Escherichia coli, through its interaction with AcrA and AcrB, is thought to form a continuous protein channel that expels inhibitors from the cell. Consequently, tolC null mutations display a hypersensitive phenotype. Here we report the isolation and characterization of tolC missense mutations that direct the synthesis of mutant TolC proteins partially disabled in their efflux role. All alterations, consisting of single amino acid substitutions, were localized within the periplasmic α-helical domain. In two mutants carrying an I106N or S350F substitution, the hypersensitivity phenotype may be in part due to aberrant TolC assembly. However, two other alterations, R367H and R390C, disrupted efflux function by affecting interactions among the helices surrounding TolC's periplasmic tunnel. Curiously, these two TolC mutants were sensitive to a large antibiotic, vancomycin, and exhibited a Dex+ phenotype. These novel phenotypes of TolCR367H and TolCR390C were likely the result of a general influx of molecules through a constitutively open tunnel aperture, which normally widens only when TolC interacts with other proteins during substrate translocation. An intragenic suppressor alteration (T140A) was isolated from antibiotic-resistant revertants of the hypersensitive TolCR367H mutant. T140A also reversed, either fully (R390C) or partially (I106N and S350F), the hypersensitivity phenotype of other TolC mutants. Our data suggest that this global suppressor phenotype of T140A is the result of impeded antibiotic influx caused by tapering of the tunnel passage rather than by correcting individual mutational defects. Two extragenic suppressors of TolCR367H, mapping in the regulatory region of acrAB, uncoupled the AcrR-mediated repression of the acrAB genes. The resulting overexpression of AcrAB reduced the hypersensitivity phenotype of all the TolC mutants. Similar results were obtained when the chromosomal acrR gene was deleted or the acrAB genes were expressed from a plasmid. Unlike the case for the intragenic suppressor T140A, the overexpression of AcrAB diminished hypersensitivity towards only erythromycin and novobiocin, which are substrates of the TolC-AcrAB efflux pump, but not towards vancomycin, which is not a substrate of this pump. This showed that the two types of suppressors produced their effects by fundamentally different means, as the intragenic suppressor decreased the general influx while extragenic suppressors increased the efflux of TolC-AcrAB pump-specific antibiotics.
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Krüger, Larissa, Christina Herzberg, Hermann Rath, Tiago Pedreira, Till Ischebeck, Anja Poehlein, Jan Gundlach, et al. "Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis." PLOS Genetics 17, no. 1 (January 22, 2021): e1009092. http://dx.doi.org/10.1371/journal.pgen.1009092.
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In order to adjust to changing environmental conditions, bacteria use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Cyclic di-adenosine monophosphate (c-di-AMP) is the only known essential nucleotide second messenger. In addition to the well-established role of this second messenger in the control of potassium homeostasis, we observed that glutamate is as toxic as potassium for a c-di-AMP-free strain of the Gram-positive model bacterium Bacillus subtilis. In this work, we isolated suppressor mutants that allow growth of a c-di-AMP-free strain under these toxic conditions. Characterization of glutamate resistant suppressors revealed that they contain pairs of mutations, in most cases affecting glutamate and potassium homeostasis. Among these mutations, several independent mutations affected a novel glutamate transporter, AimA (Amino acid importer A, formerly YbeC). This protein is the major transporter for glutamate and serine in B. subtilis. Unexpectedly, some of the isolated suppressor mutants could suppress glutamate toxicity by a combination of mutations that affect phospholipid biosynthesis and a specific gain-of-function mutation of a mechanosensitive channel of small conductance (YfkC) resulting in the acquisition of a device for glutamate export. Cultivation of the c-di-AMP-free strain on complex medium was an even greater challenge because the amounts of potassium, glutamate, and other osmolytes are substantially higher than in minimal medium. Suppressor mutants viable on complex medium could only be isolated under anaerobic conditions if one of the two c-di-AMP receptor proteins, DarA or DarB, was absent. Also on complex medium, potassium and osmolyte toxicity are the major bottlenecks for the growth of B. subtilis in the absence of c-di-AMP. Our results indicate that the essentiality of c-di-AMP in B. subtilis is caused by the global impact of the second messenger nucleotide on different aspects of cellular physiology.
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Wang, Jen-Yeu, Altaf Hossain Sarker, Priscilla K. Cooper, and Michael R. Volkert. "The Single-Strand DNA Binding Activity of Human PC4 Prevents Mutagenesis and Killing by Oxidative DNA Damage." Molecular and Cellular Biology 24, no. 13 (July 1, 2004): 6084–93. http://dx.doi.org/10.1128/mcb.24.13.6084-6093.2004.
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ABSTRACT Human positive cofactor 4 (PC4) is a transcriptional coactivator with a highly conserved single-strand DNA (ssDNA) binding domain of unknown function. We identified PC4 as a suppressor of the oxidative mutator phenotype of the Escherichia coli fpg mutY mutant and demonstrate that this suppression requires its ssDNA binding activity. Saccharomyces cerevisiae mutants lacking their PC4 ortholog Sub1 are sensitive to hydrogen peroxide and exhibit spontaneous and peroxide-induced hypermutability. PC4 expression suppresses the peroxide sensitivity of the yeast sub1Δ mutant, suggesting that the human protein has a similar function. A role for yeast and human proteins in DNA repair is suggested by the demonstration that Sub1 acts in a peroxide resistance pathway involving Rad2 and by the physical interaction of PC4 with the human Rad2 homolog XPG. We show that XPG recruits PC4 to a bubble-containing DNA substrate with a resulting displacement of XPG and formation of a PC4-DNA complex. We discuss the possible requirement for PC4 in either global or transcription-coupled repair of oxidative DNA damage to mediate the release of XPG bound to its substrate.
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Trizzino, Marco, Elisa Barbieri, Ana Petracovici, Shuai Wu, Sarah A. Welsh, Tori A. Owens, Silvia Licciulli, Rugang Zhang, and Alessandro Gardini. "The Tumor Suppressor ARID1A Controls Global Transcription via Pausing of RNA Polymerase II." Cell Reports 23, no. 13 (June 2018): 3933–45. http://dx.doi.org/10.1016/j.celrep.2018.05.097.
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Knights, Alexander J., Lu Yang, Manan Shah, Laura J. Norton, Gamran S. Green, Elizabeth S. Stout, Emily J. Vohralik, Merlin Crossley, and Kate G. R. Quinlan. "Krüppel-like factor 3 (KLF3) suppresses NF-κB–driven inflammation in mice." Journal of Biological Chemistry 295, no. 18 (March 25, 2020): 6080–91. http://dx.doi.org/10.1074/jbc.ra120.013114.
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Bacterial products such as lipopolysaccharides (or endotoxin) cause systemic inflammation, resulting in a substantial global health burden. The onset, progression, and resolution of the inflammatory response to endotoxin are usually tightly controlled to avoid chronic inflammation. Members of the NF-κB family of transcription factors are key drivers of inflammation that activate sets of genes in response to inflammatory signals. Such responses are typically short-lived and can be suppressed by proteins that act post-translationally, such as the SOCS (suppressor of cytokine signaling) family. Less is known about direct transcriptional regulation of these responses, however. Here, using a combination of in vitro approaches and in vivo animal models, we show that endotoxin treatment induced expression of the well-characterized transcriptional repressor Krüppel-like factor 3 (KLF3), which, in turn, directly repressed the expression of the NF-κB family member RELA/p65. We also observed that KLF3-deficient mice were hypersensitive to endotoxin and exhibited elevated levels of circulating Ly6C+ monocytes and macrophage-derived inflammatory cytokines. These findings reveal that KLF3 is a fundamental suppressor that operates as a feedback inhibitor of RELA/p65 and may be important in facilitating the resolution of inflammation.
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Fan, Sijia, Jing Wang, Guangqing Yu, Fangjing Rong, Dawei Zhang, Chenxi Xu, Juan Du, Zhi Li, Gang Ouyang, and Wuhan Xiao. "TET is targeted for proteasomal degradation by the PHD-pVHL pathway to reduce DNA hydroxymethylation." Journal of Biological Chemistry 295, no. 48 (September 22, 2020): 16299–313. http://dx.doi.org/10.1074/jbc.ra120.014538.
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Hypoxia-inducible factors are heterodimeric transcription factors that play a crucial role in a cell's ability to adapt to low oxygen. The von Hippel-Lindau tumor suppressor (pVHL) acts as a master regulator of HIF activity, and its targeting of prolyl hydroxylated HIF-α for proteasomal degradation under normoxia is thought to be a major mechanism for pVHL tumor suppression and cellular response to oxygen. Whether pVHL regulates other targets through a similar mechanism is largely unknown. Here, we identify TET2/3 as novel targets of pVHL. pVHL induces proteasomal degradation of TET2/3, resulting in reduced global 5-hydroxymethylcytosine levels. Conserved proline residues within the LAP/LAP-like motifs of these two proteins are hydroxylated by the prolyl hydroxylase enzymes (PHD2/EGLN1 and PHD3/EGLN3), which is prerequisite for pVHL-mediated degradation. Using zebrafish as a model, we determined that global 5-hydroxymethylcytosine levels are enhanced in vhl-null, egln1a/b-double-null, and egln3-null embryos. Therefore, we reveal a novel function for the PHD-pVHL pathway in regulating TET protein stability and activity. These data extend our understanding of how TET proteins are regulated and provide new insight into the mechanisms of pVHL in tumor suppression.
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Hajek, Michael, Asel Biktasova, Andrew Sewell, Cyril Gary, Paul Cantalupo, Karen S. Anderson, Wendell G. Yarbrough, and Natalia Issaeva. "Global Genome Demethylation Causes Transcription-Associated DNA Double Strand Breaks in HPV-Associated Head and Neck Cancer Cells." Cancers 13, no. 1 (December 23, 2020): 21. http://dx.doi.org/10.3390/cancers13010021.
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High levels of DNA methylation at CpG loci are associated with transcriptional repression of tumor suppressor genes and dysregulation of DNA repair genes. Human papilloma virus (HPV)-associated head and neck squamous cell carcinomas (HNSCC) have high levels of DNA methylation and methylation has been associated with dampening of an innate immune response in virally infected cells. We have been exploring demethylation as a potential treatment in HPV+ HNSCC and recently reported results of a window clinical trial showing that HNSCCs are particularly sensitive to demethylating agent 5-azacytidine (5-aza). Mechanistically, sensitivity is partially due to downregulation of HPV genes expression and restoration of tumor suppressors p53 and Rb. Here, for the first time, we show that 5-azaC treatment of HPV+ HNSCC induces replication and transcription-associated DNA double strand breaks (DSBs) that occur preferentially at demethylated genomic DNA. Blocking replication or transcription prevented formation of DNA DSBs and reduced sensitivity of HPV-positive head and neck cancer cells to 5-azaC, demonstrating that both replication and active transcription are required for formation of DSBs associated with 5-azaC.
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Nagaraja, Ankur K., Chad J. Creighton, Zhifeng Yu, Huifeng Zhu, Preethi H. Gunaratne, Jeffrey G. Reid, Emuejevoke Olokpa, et al. "A Link between mir-100 and FRAP1/mTOR in Clear Cell Ovarian Cancer." Molecular Endocrinology 24, no. 2 (February 1, 2010): 447–63. http://dx.doi.org/10.1210/me.2009-0295.
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Abstract MicroRNAs (miRNAs) are small noncoding RNAs that direct gene regulation through translational repression and degradation of complementary mRNA. Although miRNAs have been implicated as oncogenes and tumor suppressors in a variety of human cancers, functional roles for individual miRNAs have not been described in clear cell ovarian carcinoma, an aggressive and chemoresistant subtype of ovarian cancer. We performed deep sequencing to comprehensively profile miRNA expression in 10 human clear cell ovarian cancer cell lines compared with normal ovarian surface epithelial cultures and discovered 54 miRNAs that were aberrantly expressed. Because of the critical roles of the phosphatidylinositol 3-kinase/v-akt murine thymoma viral oncogene homolog 1/mammalian target of rapamycin (mTOR) pathway in clear cell ovarian cancer, we focused on mir-100, a putative tumor suppressor that was the most down-regulated miRNA in our cancer cell lines, and its up-regulated target, FRAP1/mTOR. Overexpression of mir-100 inhibited mTOR signaling and enhanced sensitivity to the rapamycin analog RAD001 (everolimus), confirming the key relationship between mir-100 and the mTOR pathway. Furthermore, overexpression of the putative tumor suppressor mir-22 repressed the EVI1 oncogene, which is known to suppress apoptosis by stimulating phosphatidylinositol 3-kinase/v-akt murine thymoma viral oncogene homolog 1 signaling. In addition to these specific effects, reversing the expression of mir-22 and the putative oncogene mir-182 had widespread effects on target and nontarget gene populations that ultimately caused a global shift in the cancer gene signature toward a more normal state. Our experiments have revealed strong candidate miRNAs and their target genes that may contribute to the pathogenesis of clear cell ovarian cancer, thereby highlighting alternative therapeutic strategies for the treatment of this deadly cancer.
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Kucuk, Can, Xiaozhou Hu, Bei Jiang, Philippe Gaulard, David Klinkebiel, Huimin Geng, Alyssa Bouska, Javeed Iqbal, Timothy McKeithan, and Wing C. Chan. "Global Methylation Analysis Reveals Novel Candidate Tumor Suppressor Genes In Natural Killer Cell Lymphomas." Blood 122, no. 21 (November 15, 2013): 1262. http://dx.doi.org/10.1182/blood.v122.21.1262.1262.
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Abstract Background Natural killer cell lymphomas (NKCLs) are aggressive neoplasms with poor prognosis. A few candidate tumor suppressor genes were identified with locus specific methylation analysis. However, no genome-wide study has been performed on NKCL cases to identify the spectrum of epigenetically silenced candidate genes contributing to the neoplastic transformation of NK cells. Methods Here, we analyzed genome-wide methylation profile in NKCL cases (n=12) and NK cell lines (n=2) by performing methyl-sensitive cut counting (MSCC). The methylation profile of malignant NK samples (n=14) were compared with IL2 activated normal NK cells (n=3) to identify genes with promoter hypermethylation. HG U133+ II DNA Microarray was performed on 6 NKCL cases, 2 NK cell lines and resting or activated normal NK cells (n=4).q-MSP was performed on NK cell lines (n=7) or NKCL cases (n=8) for selected genes to validate the promoter methylation identified by MSSC. q-RT-PCR was performed on NK cell lines to validate epigenetic silencing. Decitabine treatment and q-RT-PCR was performed on NK cell lines to assess reactivation of genes with promoter methylation. Apoptosis assay was performed by Annexin V-PE staining. Cell survival assay was performed on Asparaginase treated NK cell lines to show the correlation of Asparagine synthetase (ASNS) mRNA expression and sensitivity to the asparaginase treatment. Results We observed significant global promoter hypermethylation in malignant NK samples compared with activated NK cells. Using stringent criteria which included significant correlation between promoter methylation and gene expression, we identified 152 genes showing recurrent promoter hypermethylation and transcriptional repression in NKCL cases compared with normal NK cells. 34.4% (51 of 152) of the silenced genes have been previously shown to be tumor suppressor gene in at least one cancer type. Gene Ontology and pathway analysis of the methylated/downregulated genes showed enrichment of the cell cycle inhibitors, pro-apoptotic genes such as BIM and DAPK1, DNA methylation modifiers such as TET2 and DNMT3A, metabolic pathway genes, transcription factors, negative regulators of JAK/STAT signaling such as SOCS6 and IL12Rb2, WNT pathway and NK-cell activation pathways. Decitabine treatment of NK cell lines reactivated expression of all 10 selected genes. Ectopic expression of BIM in two BIM-null NK cell lines led to increased apoptosis and eventual elimination of BIM-transduced cells. Reintroduction of BIM also sensitized NK cell lines to chemotherapy-induced apoptosis. NK cell lines with ASNS methylation and repression showed increased sensitivity to treatment with L-asparaginase, an agent highly effective in the treatment of NKCLs. Conclusions We identified novel candidate tumor suppressor genes epigenetically silenced in NKCLs which may contribute to the pathogenesis of NKCLs. Reactivation of these silenced genes may be therapeutically effective in NKCL patients. In addition, we showed that epigenetic inactivation of ASNS has an association with response to Asparaginase treatment. Promoter methylation status of ASNS in NKCL patients may be used in the decision of which patients are more likely to respond to asparaginase containing chemotherapeutic regimens. Disclosures: No relevant conflicts of interest to declare.
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Moggs, J. G., T. C. Murphy, F. L. Lim, D. J. Moore, R. Stuckey, K. Antrobus, I. Kimber, and G. Orphanides. "Anti-proliferative effect of estrogen in breast cancer cells that re-express ERα is mediated by aberrant regulation of cell cycle genes." Journal of Molecular Endocrinology 34, no. 2 (April 2005): 535–51. http://dx.doi.org/10.1677/jme.1.01677.
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Estrogen receptor (ER)-negative breast carcinomas do not respond to hormone therapy, making their effective treatment very difficult. The re-expression of ERα in ER-negative MDA-MB-231 breast cancer cells has been used as a model system, in which hormone-dependent responses can be restored. Paradoxically, in contrast to the mitogenic activity of 17β-estradiol (E2) in ER-positive breast cancer cells, E2 suppresses proliferation in ER-negative breast cancer cells in which ERα has been re-expressed. We have used global gene expression profiling to investigate the mechanism by which E2 suppresses proliferation in MDA-MB-231 cells that express ERα through adenoviral infection. We show that a number of genes known to promote cell proliferation and survival are repressed by E2 in these cells. These include genes encoding the anti-apoptosis factor SURVIVIN, positive cell cycle regulators (CDC2, CYCLIN B1, CYCLIN B2, CYCLIN G1, CHK1, BUB3, STK6, SKB1, CSE1 L) and chromosome replication proteins (MCM2, MCM3, FEN1, RRM2, TOP2A, RFC1). In parallel, E2-induced the expression of the negative cell cycle regulators KIP2 and QUIESCIN Q6, and the tumour-suppressor genes E-CADHERIN and NBL1. Strikingly, the expression of several of these genes is regulated in the opposite direction by E2 compared with their regulation in ER-positive MCF-7 cells. Together, these data suggest a mechanism for the E2-dependent suppression of proliferation in ER-negative breast cancer cells into which ERα has been reintroduced.
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Luense, Svenja, Philip Denner, Amaury Fernández-Montalván, Ingo Hartung, Manfred Husemann, Carlo Stresemann, and Stefan Prechtl. "Quantification of Histone H3 Lys27 Trimethylation (H3K27me3) by High-Throughput Microscopy Enables Cellular Large-Scale Screening for Small-Molecule EZH2 Inhibitors." Journal of Biomolecular Screening 20, no. 2 (November 19, 2014): 190–201. http://dx.doi.org/10.1177/1087057114559668.
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EZH2 inhibition can decrease global histone H3 lysine 27 trimethylation (H3K27me3) and thereby reactivates silenced tumor suppressor genes. Inhibition of EZH2 is regarded as an option for therapeutic cancer intervention. To identify novel small-molecule (SMOL) inhibitors of EZH2 in drug discovery, trustworthy cellular assays amenable for phenotypic high-throughput screening (HTS) are crucial. We describe a reliable approach that quantifies changes in global levels of histone modification marks using high-content analysis (HCA). The approach was validated in different cell lines by using small interfering RNA and SMOL inhibitors. By automation and miniaturization from a 384-well to 1536-well plate, we demonstrated its utility in conducting phenotypic HTS campaigns and assessing structure-activity relationships (SAR). This assay enables screening of SMOL EZH2 inhibitors and can advance the mechanistic understanding of H3K27me3 suppression, which is crucial with regard to epigenetic therapy. We observed that a decrease in global H3K27me3, induced by EZH2 inhibition, comprises two distinct mechanisms: (1) inhibition of de novo DNA methylation and (II) inhibition of dynamic, replication-independent H3K27me3 turnover. This report describes an HCA assay for primary HTS to identify, profile, and optimize cellular active SMOL inhibitors targeting histone methyltransferases, which could benefit epigenetic drug discovery.
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Kobayashi, Hideki. "Inducible Suppression of Global Translation by Overuse of Rare Codons." Applied and Environmental Microbiology 81, no. 7 (January 30, 2015): 2544–53. http://dx.doi.org/10.1128/aem.03708-14.
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ABSTRACTRecently, artificial gene networks have been developed in synthetic biology to control gene expression and make organisms as controllable as robots. Here, I present an artificial posttranslational gene-silencing system based on the codon usage bias and low tRNA content corresponding to minor codons. I engineered the green fluorescent protein (GFP) gene to inhibit translation indirectly with the lowest-usage codons to monopolize various minor tRNAs (lgfp). The expression oflgfpinterfered nonspecifically with the growth ofEscherichia coli,Saccharomyces cerevisiae, human HeLa cervical cancer cells, MCF7 breast cancer cells, and HEK293 kidney cells, as well as phage and adenovirus expansion. Furthermore, insertion oflgfpdownstream of a phage response promoter conferred phage resistance onE. coli. Such engineered gene silencers could act as components of biological networks capable of functioning with suitable promoters inE. coli,S. cerevisiae, and human cells to control gene expression. The results presented here show general suppressor artificial genes for live cells and viruses. This robust system provides a gene expression or cell growth control device for artificially synthesized gene networks.
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Küçük, Can, Xiaozhou Hu, Bei Jiang, David Klinkebiel, Huimin Geng, Qiang Gong, Alyssa Bouska, et al. "Global Promoter Methylation Analysis Reveals Novel Candidate Tumor Suppressor Genes in Natural Killer Cell Lymphoma." Clinical Cancer Research 21, no. 7 (January 22, 2015): 1699–711. http://dx.doi.org/10.1158/1078-0432.ccr-14-1216.
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Marciano, David C., Jeanine M. Pennington, Xiaohu Wang, Jian Wang, Yu Chen, Veena L. Thomas, Brian K. Shoichet, and Timothy Palzkill. "Genetic and Structural Characterization of an L201P Global Suppressor Substitution in TEM-1 β-Lactamase." Journal of Molecular Biology 384, no. 1 (December 2008): 151–64. http://dx.doi.org/10.1016/j.jmb.2008.09.009.
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Kanduri, Meena, Nicola Cahill, Hanna Göransson, Camilla Enström, Fergus Ryan, Anders Isaksson, and Richard Rosenquist. "Differential genome-wide array–based methylation profiles in prognostic subsets of chronic lymphocytic leukemia." Blood 115, no. 2 (January 14, 2010): 296–305. http://dx.doi.org/10.1182/blood-2009-07-232868.
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Abstract Global hypomethylation and regional hypermethylation are well-known epigenetic features of cancer; however, in chronic lymphocytic leukemia (CLL), studies on genome-wide epigenetic modifications are limited. Here, we analyzed the global methylation profiles in CLL, by applying high-resolution methylation microarrays (27 578 CpG sites) to 23 CLL samples, belonging to the immunoglobulin heavy-chain variable (IGHV) mutated (favorable) and IGHV unmutated/IGHV3-21 (poor-prognostic) subsets. Overall, results demonstrated significant differences in methylation patterns between these subgroups. Specifically, in IGHV unmutated CLL, we identified methylation of 7 known or candidate tumor suppressor genes (eg, VHL, ABI3, and IGSF4) as well as 8 unmethylated genes involved in cell proliferation and tumor progression (eg, ADORA3 and PRF1 enhancing the nuclear factor-κB and mitogen-activated protein kinase pathways, respectively). In contrast, these latter genes were silenced by methylation in IGHV mutated patients. The array data were validated for selected genes using methylation-specific polymerase chain reaction, quantitative reverse transcriptase–polymerase chain reaction, and bisulfite sequencing. Finally, the significance of DNA methylation in regulating gene promoters was shown by reinducing 4 methylated tumor suppressor genes (eg, VHL and ABI3) in IGHV unmutated samples using the methyl-inhibitor 5-aza-2′-deoxycytidine. Taken together, our data for the first time reveal differences in global methylation profiles between prognostic subsets of CLL, which may unfold epigenetic silencing mechanisms involved in CLL pathogenesis.
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Pramodh, Sreepoorna, Ritu Raina, Arif Hussain, Sali Abubaker Bagabir, Shafiul Haque, Syed Tasleem Raza, Mohammad Rehan Ajmal, Shalini Behl, and Deepika Bhagavatula. "Luteolin Causes 5′CpG Demethylation of the Promoters of TSGs and Modulates the Aberrant Histone Modifications, Restoring the Expression of TSGs in Human Cancer Cells." International Journal of Molecular Sciences 23, no. 7 (April 6, 2022): 4067. http://dx.doi.org/10.3390/ijms23074067.
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Cancer progression is linked to abnormal epigenetic alterations such as DNA methylation and histone modifications. Since epigenetic alterations, unlike genetic changes, are heritable and reversible, they have been considered as interesting targets for cancer prevention and therapy by dietary compounds such as luteolin. In this study, epigenetic modulatory behaviour of luteolin was analysed on HeLa cells. Various assays including colony forming and migration assays, followed by biochemical assays of epigenetic enzymes including DNA methyltransferase, histone methyl transferase, histone acetyl transferase, and histone deacetylases assays were performed. Furthermore, global DNA methylation and methylation-specific PCR for examining the methylation status of CpG promoters of various tumour suppressor genes (TSGs) and the expression of these TSGs at transcript and protein level were performed. It was observed that luteolin inhibited migration and colony formation in HeLa cells. It also modulated DNA methylation at promoters of TSGs and the enzymatic activity of DNMT, HDAC, HMT, and HAT and reduced the global DNA methylation. Decrease in methylation resulted in the reactivation of silenced tumour suppressor genes including FHIT, DAPK1, PTEN, CDH1, SOCS1, TIMPS, VHL, TP53, TP73, etc. Hence, luteolin-targeted epigenetic alterations provide a promising approach for cancer prevention and intervention.
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Iizuka, Masayoshi, Olga F. Sarmento, Takao Sekiya, Heidi Scrable, C. David Allis, and M. Mitchell Smith. "Hbo1 Links p53-Dependent Stress Signaling to DNA Replication Licensing." Molecular and Cellular Biology 28, no. 1 (October 22, 2007): 140–53. http://dx.doi.org/10.1128/mcb.00662-07.
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ABSTRACT Hbo1 is a histone acetyltransferase (HAT) that is required for global histone H4 acetylation, steroid-dependent transcription, and chromatin loading of MCM2-7 during DNA replication licensing. It is the catalytic subunit of protein complexes that include ING and JADE proteins, growth regulatory factors and candidate tumor suppressors. These complexes are thought to act via tumor suppressor p53, but the molecular mechanisms and links between stress signaling and chromatin, are currently unknown. Here, we show that p53 physically interacts with Hbo1 and negatively regulates its HAT activity in vitro and in cells. Two physiological stresses that stabilize p53, hyperosmotic shock and DNA replication fork arrest, also inhibit Hbo1 HAT activity in a p53-dependent manner. Hyperosmotic stress during G1 phase specifically inhibits the loading of the MCM2-7 complex, providing an example of the chromatin output of this pathway. These results reveal a direct regulatory connection between p53-responsive stress signaling and Hbo1-dependent chromatin pathways.
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Chen, Y., and B. K. Tye. "The yeast Mcm1 protein is regulated posttranscriptionally by the flux of glycolysis." Molecular and Cellular Biology 15, no. 8 (August 1995): 4631–39. http://dx.doi.org/10.1128/mcb.15.8.4631.
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Mcm1 is a multifunctional protein which plays a role both in the initiation of DNA replication and in the transcriptional regulation of diverse genes in Saccharomyces cerevisiae. The mcm1-1 mutation results in instability of minichromosomes and alpha-specific sterility. Second-site suppressors that restore minichromosome stability but not fertility to the mcm1-1 mutant were isolated. Two of the suppressors, pgm1-1 and pgm1-2, are mutant alleles of PGM1 which encodes a glycolytic enzyme, phosphoglycerate mutase. We show that the pgm1-1 mutation suppresses the minichromosome maintenance (Mcm) defect by increasing the protein activity or level of Mcm1-1 posttranscriptionally. This increase in the intracellular Mcm1-1 activity is sufficient to suppress the Mcm defect but only minimally suppresses the mating defect. Mutations in genes encoding other glycolytic enzymes, such as eno2::URA3, can also suppress the Mcm phenotype of mcm1-1. Suppression by these glycolytic enzyme mutations correlates with a reduced rate of glycolysis rather than a reduced rate of cell growth. This study suggests that in response to changes in their nutritional states yeast cells may attain homeostasis by modulating the activity of global regulators like Mcm1, which plays a central role in the regulation of energy-expensive anabolic processes.
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Ding, Yali, Bing He, Jonathon Payne, Dhimant Desai, Arati Sharma, Daniel Bogush, Elanora Dovat, et al. "Regulation of Heterochromatin Landscape in T-Cell Acute Lymphoblastic Leukemia." Blood 138, Supplement 1 (November 5, 2021): 2217. http://dx.doi.org/10.1182/blood-2021-154388.
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Abstract Ikzf1 encodes a zinc finger, DNA-binding protein that functions as a tumor suppressor in acute lymphoblastic leukemia (ALL). Deletion and/or loss of Ikaros function results in the development of high-risk leukemia. In the nucleus, Ikaros forms complexes with histone deacetylase complex, NuRD, and it participates in the formation of heterochromatin. The role of Ikaros-mediated formation of heterochromatin in tumor suppression in leukemia is unknown. We determined global genomic occupancy of Ikaros, global heterochromatin distribution, chromatin accessibility, DNA methylation landscape, and gene expression in primary human T-cell ALL (T-ALL), as well as in mouse T-ALL to analyze how Ikaros regulates heterochromatin landscape and gene expression in T-ALL. Results showed that Ikaros DNA occupancy is essential for the recruitment of histone deacetylase 1 (HDAC1), Polycomb repressive complex 2 (PRC2) and formation of facultative heterochromatin, as well as the formation of constitutive heterochromatin (characterized by H3K9me3 occupancy). T-ALL cells with deletion of both Ikzf1 alleles have severely impaired HDAC1 DNA occupancy and reduced H3K27me3. Re-introduction of Ikzf1 via retroviral transduction resulted in the restoration of H3K27me3 facultative heterochromatin, along with HDAC1 DNA occupancy. The H3K27me3 genomic distribution following Ikzf1 re-introduction showed high homology to the H3K27me3 genomic distribution in normal thymocytes. Analysis of H3K9me3 genomic distribution showed that Ikzf1 deletion results in dramatic redistribution of H3K9me3 global occupancy, with reduced H3K9me3 occupancy at pericentromeric loci. Reintroduction of Ikzf1 enhances H3K9me3 enrichment in pericentromeric loci, as well as at the promoters of genes that are involves in cellular proliferation. Analysis of DNA methylation distribution showed that Ikzf1 expression regulates global DNA methylation landscape. The presence of facultative heterochromatin, with enrichment of H3K27me3, inversely correlated with DNA methylation. Global analysis of chromatin accessibility revealed that Ikaros binding resulted in the loss of chromatin accessibility at over 3400 previously-accessible chromatin sites. Dynamic analyses demonstrate the long-lasting effects of Ikaros's DNA binding on heterochromatin distribution and chromatin accessibility. Analysis of gene expression in T-ALL with both Ikzf1 alleles and in Ikzf1-defficient cells (from Ikzf1-defficient T-ALL, and from Ikzf1-wild-type T-ALL following Ikzf1 deletion by CRISPR) showed that Ikaros-induced redistribution of facultative and constitutive heterochromatin results in the repression of several genes that are critical for cell cycle progression, PI3K-AKT-mTOR, and WNT signaling pathway. In conclusion, results suggest that Ikaros' tumor suppressor function in T-ALL occurs via global regulation of the heterochromatin, DNA methylation landscape, and chromatin accessibility, as well as via epigenetic regulation of transcription of the genes that play essential roles in signaling pathways that promote cellular proliferation. Disclosures No relevant conflicts of interest to declare.
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Osorio-Pérez, Sofía Madeline, Carolina Estrada-Meza, Luis M. Ruiz-Manriquez, María Goretti Arvizu-Espinosa, Aashish Srivastava, Ashutosh Sharma, and Sujay Paul. "Thymoquinone Potentially Modulates the Expression of Key Onco- and Tumor Suppressor miRNAs in Prostate and Colon Cancer Cell Lines: Insights from PC3 and HCT-15 Cells." Genes 14, no. 9 (August 30, 2023): 1730. http://dx.doi.org/10.3390/genes14091730.
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Prostate cancer (PC) and colon cancer significantly contribute to global cancer-related morbidity and mortality. Thymoquinone (TQ), a naturally occurring phytochemical found in black cumin, has shown potential as an anticancer compound. This study aimed to investigate the effects of TQ on the expression profile of key tumor suppressor and onco-suppressor miRNAs in PC3 prostate cancer cells and HCT-15 colon cancer cells. Cell viability assays revealed that TQ inhibited the growth of both cell lines in a dose-dependent manner, with IC50 values of approximately 82.59 μM for HCT-15 and 55.83 μM for PC3 cells. Following TQ treatment at the IC50 concentrations, miRNA expression analysis demonstrated that TQ significantly downregulated miR-21-5p expression in HCT-15 cells and upregulated miR-34a-5p, miR-221-5p, miR-17-5p, and miR-21-5p expression in PC3 cells. However, no significant changes were observed in the expression levels of miR-34a-5p and miR-200a-5p in HCT-15 cells. The current findings suggest that TQ might exert its antiproliferative effects by modulating specific tumor suppressor and onco-suppressor miRNAs in prostate and colon cancer cells. Further investigations are warranted to elucidate the precise underlying mechanisms and to explore the therapeutic potential of TQ in cancer treatment. To the best of our knowledge, this is the first report regarding the effect of TQ on the miRNA expression profile in colon and prostate cancer cell lines.
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Kawamata, Norihiko, Takayuki Saitoh, Sakura Sakajiri, and Phillip H. Koeffler. "Identification of Candidate Tumor Suppressor Genes Silenced Epigenetically in Mantle Cell Lymphoma." Blood 106, no. 11 (November 16, 2005): 3001. http://dx.doi.org/10.1182/blood.v106.11.3001.3001.
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Abstract Many tumor suppressor genes are silenced by epigenetic mechanisms in human cancers, including mantle cell lymphoma (MCL). In this study, we have used a variety of research tools to screen for genes that are epigenetically silenced in MCL. Changes in the global gene expression profile of the MCL cell line, Jeko1, were analyzed after treatment with the combination of the demethylating agent, 5-aza-2′-deoxycytidine, and the histone deacetylase inhibitor, suberoyl anilide bishydroxamide, by DNA microarray technique. By screening over 22,000 genes, we identified 26 candidate tumor suppressor genes, expression of which were enhanced by the treatment, in the MCL line. Basal expression of these 26 genes were low in Jeko1 cells. The treatment enhanced the expression more than 2 folds and the enhancement was also confirmed by real-time PCR. Methylation status of these 26 genes were examined by bisulfite sequencing and/or combined bisulfite and restriction enzyme digestion assay in Jeko1 cells. We found hypermethylation of a CpG island in the middle of the INPP5F gene. We also found the hypermethylation of that region of INPP5F in normal peripheral blood. We also examined expression levels of these 26 genes in normal mantle cells by real-time PCR and found only 11 genes showed high levels of transcription in laser-dissected normal mantle cells. We examined expression of these 11 genes in eight MCL clinical samples by real-time PCR and found that only three genes, INPP5F, DUSP10 and FGD2 showed very low expression levels. We conclude that expression of INPP5F, DUSP10 and FGD2 genes were suppressed in MCL cells although the expression of these genes are high in normal mantle cells. INPP5F is a inositol phosphatase and could be involved in PI3K pathway. DUSP10 is a dual specific phosphatase and could be involved in JNK pathway. FGD2 is a RAS-GAP gene and could be involved in RAS pathway. These three genes may be candidate tumor suppressor genes in MCL and further functional analysis is ongoing.
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Rodrigue, Amélie, Guillaume Margaillan, Thiago Torres Gomes, Yan Coulombe, Gemma Montalban, Simone da Costa e Silva Carvalho, Larissa Milano, et al. "A global functional analysis of missense mutations reveals two major hotspots in the PALB2 tumor suppressor." Nucleic Acids Research 47, no. 20 (October 5, 2019): 10662–77. http://dx.doi.org/10.1093/nar/gkz780.
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Abstract While biallelic mutations in the PALB2 tumor suppressor cause Fanconi anemia subtype FA-N, monoallelic mutations predispose to breast and familial pancreatic cancer. Although hundreds of missense variants in PALB2 have been identified in patients to date, only a few have clear functional and clinical relevance. Herein, we investigate the effects of 44 PALB2 variants of uncertain significance found in breast cancer patients and provide detailed analysis by systematic functional assays. Our comprehensive functional analysis reveals two hotspots for potentially deleterious variations within PALB2, one at each terminus. PALB2 N-terminus variants p.P8L [c.23C>T], p.Y28C [c.83A>G], and p.R37H [c.110G>A] compromised PALB2-mediated homologous recombination. At the C-terminus, PALB2 variants p.L947F [c.2841G>T], p.L947S [c.2840T>C], and most strikingly p.T1030I [c.3089C>T] and p.W1140G [c.3418T>C], stood out with pronounced PARP inhibitor sensitivity and cytoplasmic accumulation in addition to marked defects in recruitment to DNA damage sites, interaction with BRCA2 and homologous recombination. Altogether, our findings show that a combination of functional assays is necessary to assess the impact of germline missense variants on PALB2 function, in order to guide proper classification of their deleteriousness.
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Seong, Rak-Kyun, Jae Kyung Lee, and Ok Sarah Shin. "Zika Virus-Induction of the Suppressor of Cytokine Signaling 1/3 Contributes to the Modulation of Viral Replication." Pathogens 9, no. 3 (February 27, 2020): 163. http://dx.doi.org/10.3390/pathogens9030163.
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Zika virus (ZIKV) is a mosquito-borne flavivirus that has emerged and caused global outbreaks since 2007. Although ZIKV proteins have been shown to suppress early anti-viral innate immune responses, little is known about the exact mechanisms. This study demonstrates that infection with either the African or Asian lineage of ZIKV leads to a modulated expression of suppressor of cytokine signaling (SOCS) genes encoding SOCS1 and SOCS3 in the following cell models: A549 human lung adenocarcinoma cells; JAr human choriocarcinoma cells; human neural progenitor cells. Studies of viral gene expression in response to SOCS1 or SOCS3 demonstrated that the knockdown of these SOCS proteins inhibited viral NS5 or ZIKV RNA expression, whereas overexpression resulted in an increased expression. Moreover, the overexpression of SOCS1 or SOCS3 inhibited the retinoic acid-inducible gene-I-like receptor-mediated activation of both type I and III interferon pathways. These results imply that SOCS upregulation following ZIKV infection modulates viral replication, possibly via the regulation of anti-viral innate immune responses.
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Lycan, D., G. Mikesell, M. Bunger, and L. Breeden. "Differential effects of Cdc68 on cell cycle-regulated promoters in Saccharomyces cerevisiae." Molecular and Cellular Biology 14, no. 11 (November 1994): 7455–65. http://dx.doi.org/10.1128/mcb.14.11.7455-7465.1994.
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Swi4 and Swi6 form a complex which is required for Start-dependent activation of HO and for high-level expression of G1 cyclin genes CLN1 and CLN2. To identify other regulators of this pathway, we screened for dominant, recessive, conditional, and allele-specific suppressors of swi4 mutants. We isolated 16 recessive suppressors that define three genes, SSF1, SSF5, and SSF9 (suppressor of swi four). Mutations in all three genes bypass the requirement for both Swi4 and Swi6 for HO transcription and activate transcription from reporter genes lacking upstream activating sequences (UASs). SSF5 is allelic with SIN4 (TSF3), a gene implicated in global repression of transcription and chromatin structure, and SSF9 is likely to be a new global repressor of transcription. SSF1 is allelic with CDC68 (SPT16). cdc68 mutations have been shown to increase expression from defective promoters, while preventing transcription from other intact promoters, including CLN1 and CLN2. We find that CDC68 is a required activator of both SWI4 and SWI6, suggesting that CDC68's role at the CLN promoters may be indirect. The target of CDC68 within the SWI4 promoter is complex in that known activating elements (MluI cell cycle boxes) in the SWI4 promoter are required for CDC68 dependence but only within the context of the full-length promoter. This result suggests that there may be both a chromatin structure and a UAS-specific component to Cdc68 function at SWI4. We suggest that Cdc68 functions both in the assembly of repressive complexes that form on many intact promoters in vivo and in the relief of this repression during gene activation.
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Lycan, D., G. Mikesell, M. Bunger, and L. Breeden. "Differential effects of Cdc68 on cell cycle-regulated promoters in Saccharomyces cerevisiae." Molecular and Cellular Biology 14, no. 11 (November 1994): 7455–65. http://dx.doi.org/10.1128/mcb.14.11.7455.
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Swi4 and Swi6 form a complex which is required for Start-dependent activation of HO and for high-level expression of G1 cyclin genes CLN1 and CLN2. To identify other regulators of this pathway, we screened for dominant, recessive, conditional, and allele-specific suppressors of swi4 mutants. We isolated 16 recessive suppressors that define three genes, SSF1, SSF5, and SSF9 (suppressor of swi four). Mutations in all three genes bypass the requirement for both Swi4 and Swi6 for HO transcription and activate transcription from reporter genes lacking upstream activating sequences (UASs). SSF5 is allelic with SIN4 (TSF3), a gene implicated in global repression of transcription and chromatin structure, and SSF9 is likely to be a new global repressor of transcription. SSF1 is allelic with CDC68 (SPT16). cdc68 mutations have been shown to increase expression from defective promoters, while preventing transcription from other intact promoters, including CLN1 and CLN2. We find that CDC68 is a required activator of both SWI4 and SWI6, suggesting that CDC68's role at the CLN promoters may be indirect. The target of CDC68 within the SWI4 promoter is complex in that known activating elements (MluI cell cycle boxes) in the SWI4 promoter are required for CDC68 dependence but only within the context of the full-length promoter. This result suggests that there may be both a chromatin structure and a UAS-specific component to Cdc68 function at SWI4. We suggest that Cdc68 functions both in the assembly of repressive complexes that form on many intact promoters in vivo and in the relief of this repression during gene activation.
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Scholl, Amanda, Alexander Muselman, and Dong-Er Zhang. "An Intronic Suppressor Element Regulates RUNX1 Alternative Polyadenylation." Blood 126, no. 23 (December 3, 2015): 3578. http://dx.doi.org/10.1182/blood.v126.23.3578.3578.
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Abstract Polyadenylation is a post-transcriptional modification where the 3' end of an mRNA is cleaved and 250-300 adenines are added. It is predicted that 70-75% of human genes have more than one polyadenylation sequence (PAS) and are subject to alternative polyadenylation (APA). APA events affect the coding sequence of a gene when a proximal PAS is located within an intron, constitutive exon, or alternative exon. Gene expression is also affected if there are multiple PAS within the distal 3' untranslated region (UTR); proximal PAS usage shortens the 3'UTR, which can remove cis-regulatory regions such as miRNA and RNA-binding protein (RBP) sites. Furthermore, global changes in APA are linked to cellular state-proximal PAS usage is associated with immature developmental phases, cell proliferation, and cancerous phenotypes. Consequently, APA is a pertinent post-transcriptional modification that regulates gene expression and isoform generation across developmental stages and tissue types. Despite its significance, there are few APA studies in the hematology field, and those that exist have focused on global shifts in PAS usage. In this study, we uniquely focus on the APA mechanism of a single gene, RUNX1, and how this event can alter hematopoietic stem cell (HSC) homeostasis and hematopoiesis. There are three main isoforms of RUNX1 that differ in promoter and/or PAS usage. RUNX1b/c use different promoters, but have identical C-terminal regions. RUNX1a utilizes the same promoter as RUNX1b, but differs from both RUNX1b/c due to usage of a proximal PAS located in alternative exon 7a. RUNX1b/c are robustly expressed in most progenitor populations and differentiated blood cell lineages, whereas RUNX1a is restricted to human CD34+ HSCs. Functionally, RUNX1b/c promote HSC differentiation and lineage commitment, whereas RUNX1a expands HSCs and their engraftment potential, a property with therapeutic advantages but leukemic potential. Due to the difference in expression pattern and distinct functionality of RUNX1a compared to RUNX1b/c, it is relevant to study the APA event that dictates isoform generation. Elucidating this mechanism could provide valuable insight into the transient control of the HSC population for therapeutic benefit and illuminate new leukemogenic pathways. To study RUNX1 APA, we cloned alternative terminal exon 7a (RUNX1a) and constitutive exon 7b (RUNX1b/c) in between the two exons of a split GFP minigene reporter, along with 500 bp of their upstream and downstream flanking introns. We hypothesized that exon 7a would be skipped during processing of the minigene construct because the proximal PAS is rarely used in vivo. Conversely, exon 7b, the penultimate exon in RUNX1b/c, would be spliced in between the GFP exons, disrupting the GFP protein. These constructs were tested in KG-1a and U937 cells. Flow cytometry for GFP fluorescence supported our hypothesis as the exon 7a minigene produced a robust GFP signal and the exon 7b minigene produced no GFP signal. We confirmed that the GFP changes were due to the hypothesized mRNA processing events by performing RT-PCR using primers specific to the two GFP exons. These data show that important cis-regulatory elements that determine RUNX1 APA are located within exon 7a, 7b, and the cloned intronic regions. Next, we altered these minigenes by strategically making chimeric constructs that consist of either exon 7a or 7b with all combinations of upstream/downstream flanking introns. We discovered that replacing the intron upstream of exon 7a confers 2-5 fold greater splicing and polyadenylation of exon 7a, indicative of RUNX1a isoform generation. Therefore, a suppressor cis-element is located in this upstream intronic region. However, placing this intron upstream of exon 7b is not sufficient to reduce its inclusion between the GFP exons. Instead, both the upstream and downstream intronic regions flanking exon 7a are required. This suggests an RNA-looping mechanism that prevents splicing and usage of the exon 7a proximal PAS. Cleavage factor (CFIm) and Polypyrimidine-tract binding protein 1 (PTBP1) are RBPs involved in splicing and polyadenylation that alter mRNA processing by RNA-looping. We aim to narrow down the suppressor region upstream of exon 7a to identify a consensus sequence and the respective RBP that diminishes RUNX1 proximal PAS usage. This knowledge can be leveraged to enhance RUNX1a production and expand HSCs for therapeutic benefit. Disclosures No relevant conflicts of interest to declare.
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Kampa, Kerstin M., Sandra Mueller, Michael Bonin, Marcus M. Schittenhelm, and Charles D. Lopez. "Insights Into the Role of ASPP2 (Apoptosis Stimulating Protein of p53-2) in Lymphomagenesis." Blood 114, no. 22 (November 20, 2009): 1967. http://dx.doi.org/10.1182/blood.v114.22.1967.1967.
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Abstract Abstract 1967 Poster Board I-990 ASPP2 is a member of a family of p53 binding proteins that enhance apoptosis, in part through selective stimulation of p53 transactivation of pro-apoptotic target genes. Low ASPP2 expression is found in many human cancers and has been associated with poor clinical outcome in patients with aggressive lymphoma. Using an ASPP2+/- mouse model, we have previously demonstrated that ASPP2 is a haploinsufficient tumor suppressor and that reduced ASPP2 expression results in attenuated damage-response thresholds (Kampa et al., PNAS 2009). While ASPP2-/- mice are not viable, ASPP2+/- mice have an increased incidence of -irradiation-induced tumors compared to ASPP2+/+ mice.γspontaneous and ASPP2+/- mice develop high-grade thymic T-cell lymphomas after -irradiation. Moreover, primary ASPP2+/- thymocytes have an attenuatedγ -irradiation compared to ASPP2+/+ thymocytes.γapoptotic response after To explore the mechanisms of how attenuated ASPP2 expression could increase thymic lymphomagenesis and attenuate apoptosis, we performed global gene expression profiling on unirradiated, and 5 Gy irradiated ASPP2+/+ and ASPP2+/- thymocytes using an Affymetrix Mouse GeneChip® Array. We found significant differences in gene expression between ASPP2+/+ and ASPP2+/- thymocytes, in both unirradiated and irradiated sets. Using Ingenuity Pathway Analysis software, we found that amongst the highest scoring pathways displaying differences were those associated with cell growth, tumor formation, hematologic malignancies, immune response, cell death and cell cycle regulation. We additionally studied global phosphorylation patterns using 2-dimensional gel electrophoresis, fluorescent phosphoprotein dye Pro-Q Diamond staining, and liquid chromatography tandem mass spectrometry to determine the posttranscriptional mechanisms mediated by attenuated ASPP2 expression. Analysis of the phosphoproteome of ASPP2+/+ and ASPP2+/- mouse embryonic fibroblasts (with and without irradiation) revealed differences in the phosphorylation status of 108 peptides/proteins including those involved in regulating cell cycle checkpoints, T-cell receptor signaling, cell stress response, DNA repair mechanisms, cell growth, translation and transcription. Differential expression of the identified genes and proteins was verified by PCR and Western Blot. Thus, reduced ASPP2 expression affects global transcriptional as well as post-transcriptional networks intimately involved in the development of hematologic disorders–suggesting that ASPP2 function is by far more complex than solely enhancing the expression of pro-apoptotic p53 target genes. Given that ASPP2 is a bona fide tumor suppressor, reduced ASPP2 levels result in global dysregulation of pathways engaged in tumor suppression networks and the cellular damage response, which may ultimately promote genomic instability and tumor formation. Our findings provide insights into the role of ASPP2 in lymphomagenesis and reveal possible new targets for cancer therapy. Disclosures: No relevant conflicts of interest to declare.
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Fernandez-Zapico, M. E., S. Tsuji, and R. Urrutia. "GLOBAL FUNCTIONAL ANALYSIS OF SP/KLF PROTEINS IDENTIFY KLF11 AS NOVEL TUMOR SUPPRESSOR CANDIDATE FOR PANCREATIC CANCER." Pancreas 31, no. 4 (November 2005): 441. http://dx.doi.org/10.1097/01.mpa.0000193667.32396.85.
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Capuozzo, Maurizio, Mariachiara Santorsola, Marco Bocchetti, Francesco Perri, Marco Cascella, Vincenza Granata, Venere Celotto, et al. "p53: From Fundamental Biology to Clinical Applications in Cancer." Biology 11, no. 9 (September 6, 2022): 1325. http://dx.doi.org/10.3390/biology11091325.
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p53 tumour suppressor gene is our major barrier against neoplastic transformation. It is involved in many cellular functions, including cell cycle arrest, senescence, DNA repair, apoptosis, autophagy, cell metabolism, ferroptosis, immune system regulation, generation of reactive oxygen species, mitochondrial function, global regulation of gene expression, miRNAs, etc. Its crucial importance is denounced by the high percentage of amino acid sequence identity between very different species (Homo sapiens, Drosophila melanogaster, Rattus norvegicus, Danio rerio, Canis lupus familiaris, Gekko japonicus). Many of its activities allowed life on Earth (e.g., repair from radiation-induced DNA damage) and directly contribute to its tumour suppressor function. In this review, we provide paramount information on p53, from its discovery, which is an interesting paradigm of science evolution, to potential clinical applications in anti-cancer treatment. The description of the fundamental biology of p53 is enriched by specific information on the structure and function of the protein as well by tumour/host evolutionistic perspectives of its role.
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Menghi, Francesca, Koichiro Inaki, XingYi Woo, Pooja A. Kumar, Krzysztof R. Grzeda, Ankit Malhotra, Vinod Yadav, et al. "The tandem duplicator phenotype as a distinct genomic configuration in cancer." Proceedings of the National Academy of Sciences 113, no. 17 (April 7, 2016): E2373—E2382. http://dx.doi.org/10.1073/pnas.1520010113.
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Next-generation sequencing studies have revealed genome-wide structural variation patterns in cancer, such as chromothripsis and chromoplexy, that do not engage a single discernable driver mutation, and whose clinical relevance is unclear. We devised a robust genomic metric able to identify cancers with a chromotype called tandem duplicator phenotype (TDP) characterized by frequent and distributed tandem duplications (TDs). Enriched only in triple-negative breast cancer (TNBC) and in ovarian, endometrial, and liver cancers, TDP tumors conjointly exhibit tumor protein p53 (TP53) mutations, disruption of breast cancer 1 (BRCA1), and increased expression of DNA replication genes pointing at rereplication in a defective checkpoint environment as a plausible causal mechanism. The resultant TDs in TDP augment global oncogene expression and disrupt tumor suppressor genes. Importantly, the TDP strongly correlates with cisplatin sensitivity in both TNBC cell lines and primary patient-derived xenografts. We conclude that the TDP is a common cancer chromotype that coordinately alters oncogene/tumor suppressor expression with potential as a marker for chemotherapeutic response.
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McCoy, James M., Rebecca J. Stewart, Alessandro D. Uboldi, Dongdi Li, Jan Schröder, Nicollas E. Scott, Anthony T. Papenfuss, Adele M. Lehane, Leonard J. Foster, and Christopher J. Tonkin. "A forward genetic screen identifies a negative regulator of rapid Ca2+-dependent cell egress (MS1) in the intracellular parasite Toxoplasma gondii." Journal of Biological Chemistry 292, no. 18 (March 3, 2017): 7662–74. http://dx.doi.org/10.1074/jbc.m117.775114.
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Toxoplasma gondii, like all apicomplexan parasites, uses Ca2+ signaling pathways to activate gliding motility to power tissue dissemination and host cell invasion and egress. A group of “plant-like” Ca2+-dependent protein kinases (CDPKs) transduces cytosolic Ca2+ flux into enzymatic activity, but how they function is poorly understood. To investigate how Ca2+ signaling activates egress through CDPKs, we performed a forward genetic screen to isolate gain-of-function mutants from an egress-deficient cdpk3 knockout strain. We recovered mutants that regained the ability to egress from host cells that harbored mutations in the gene Suppressor of Ca2+-dependent Egress 1 (SCE1). Global phosphoproteomic analysis showed that SCE1 deletion restored many Δcdpk3-dependent phosphorylation events to near wild-type levels. We also show that CDPK3-dependent SCE1 phosphorylation is required to relieve its suppressive activity to potentiate egress. In summary, our work has uncovered a novel component and suppressor of Ca2+-dependent cell egress during Toxoplasma lytic growth.
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Hirsch, Heather A., Gauri W. Jawdekar, Kang-Ae Lee, Liping Gu, and R. William Henry. "Distinct Mechanisms for Repression of RNA Polymerase III Transcription by the Retinoblastoma Tumor Suppressor Protein." Molecular and Cellular Biology 24, no. 13 (July 1, 2004): 5989–99. http://dx.doi.org/10.1128/mcb.24.13.5989-5999.2004.
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ABSTRACT The retinoblastoma (RB) protein represses global RNA polymerase III transcription of genes that encode nontranslated RNAs, potentially to control cell growth. However, RNA polymerase III-transcribed genes exhibit diverse promoter structures and factor requirements for transcription, and a universal mechanism explaining global repression is uncertain. We show that RB represses different classes of RNA polymerase III-transcribed genes via distinct mechanisms. Repression of human U6 snRNA (class 3) gene transcription occurs through stable promoter occupancy by RB, whereas repression of adenovirus VAI (class 2) gene transcription occurs in the absence of detectable RB-promoter association. Endogenous RB binds to a human U6 snRNA gene in both normal and cancer cells that maintain functional RB but not in HeLa cells whose RB function is disrupted by the papillomavirus E7 protein. Both U6 promoter association and transcriptional repression require the A/B pocket domain and C region of RB. These regions of RB contribute to U6 promoter targeting through numerous interactions with components of the U6 general transcription machinery, including SNAPC and TFIIIB. Importantly, RB also concurrently occupies a U6 promoter with RNA polymerase III during repression. These observations suggest a novel mechanism for RB function wherein RB can repress U6 transcription at critical steps subsequent to RNA polymerase III recruitment.
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Wanga, Xin-zhi, Jia-li Gu, Ming Gao, Yong Bian, Jiang-yu Liang, Hong-mei Wen, and Hao Wu. "Peperomin E Induces Promoter Hypomethylation of Metastatic-Suppressor Genes and Attenuates Metastasis in Poorly Differentiated Gastric Cancer." Cellular Physiology and Biochemistry 50, no. 6 (2018): 2341–64. http://dx.doi.org/10.1159/000495096.
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Background/Aims: Peperomin E (PepE), a natural secolignan isolated from the whole plant of Peperomia dindygulensis, has been reported by ourselves and others to display potent anti-cancer effects in many types cancer cells, especially gastric cancer. However, the effects of PepE on the metastasis of poorly-differentiated gastric cancer cells and the underlying molecular mechanisms have not been well elucidated. Methods: We evaluated PepE effects on gastric cancer cell invasion and migration in vitro via wound healing and transwell assays and those on growth and metastasis in vivo using an orthotopic xenograft NOD-SCID mouse model. DNA methyltransferase (DNMT) activity was determined using a colorimetric DNMT activity/inhibition assay kit. PepE binding kinetics to DNMTs were determined using the bio-layer interferometry binding assay. Gene and protein levels of DNMTs, AMPKα-Sp1 signaling molecules, and metastatic-suppressor genes in PepE-treated gastric cancer cells were determined using quantitative reverse transcription-PCR arrays and western blotting. The effect of PepE on Sp1 binding to the DNMT promoter was determined by electrophoretic mobility-shift assay. Global DNA methylation levels were determined using liquid chromatography coupled with electrospray ionization tandem mass spectrometry. The methylation status of silenced metastatic-suppressor genes (MSGs) in gastric cancer cells was investigated by methylation-specific PCR. Results: PepE can dose-dependently suppress invasion and migration of poorly-differentiated gastric cancer cells in vitro and in vivo with low toxicity against normal cells. Mechanistically, PepE not only covalently binds to the catalytic domain of DNMT1 and inhibits its activity (IC50 value 3.61 μM) but also down-regulates DNMT1, 3a, and 3b mRNA and protein expression in in gastric cancer cells, by disruption of the physical interaction of Sp1 with the DNMT1, 3a, and 3b promoter and mediation of the AMPKα-Sp1 signaling pathway. The dual inhibition activity of PepE toward DNMTs renders a relative global DNA hypomethylation, which induces MSG promoter hypomethylation (e.g., E-cadherin and TIMP3) and enhances their expression in gastric cancer cells. Conclusion: Collectively, our data indicated that PepE may represent a promising therapeutic lead compound for intervention in gastric cancer metastasis and may also exhibit potential as a DNA methylation inhibitor for use in epigenetic cancer therapy.
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Crang, Nick, Khushboo Borah, Euan K. James, Beatriz Jorrín, Patrick Green, Andrzej Tkacz, Alison K. East, and Philip S. Poole. "Role and Regulation of Poly-3-Hydroxybutyrate in Nitrogen Fixation in Azorhizobium caulinodans." Molecular Plant-Microbe Interactions® 34, no. 12 (December 2021): 1390–98. http://dx.doi.org/10.1094/mpmi-06-21-0138-r.
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An Azorhizobium caulinodans phaC mutant (OPS0865) unable to make poly-3-hydroxybutyrate (PHB), grows poorly on many carbon sources and cannot fix nitrogen in laboratory culture. However, when inoculated onto its host plant, Sesbania rostrata, the phaC mutant consistently fixed nitrogen. Upon reisolation from S. rostrata root nodules, a suppressor strain (OPS0921) was isolated that has significantly improved growth on a variety of carbon sources and also fixes nitrogen in laboratory culture. The suppressor retains the original mutation and is unable to synthesize PHB. Genome sequencing revealed a suppressor transition mutation, G to A (position 357,354), 13 bases upstream of the ATG start codon of phaR in its putative ribosome binding site (RBS). PhaR is the global regulator of PHB synthesis but also has other roles in regulation within the cell. In comparison with the wild type, translation from the phaR native RBS is increased approximately sixfold in the phaC mutant background, suggesting that the level of PhaR is controlled by PHB. Translation from the phaR mutated RBS (RBS*) of the suppressor mutant strain (OPS0921) is locked at a low basal rate and unaffected by the phaC mutation, suggesting that RBS* renders the level of PhaR insensitive to regulation by PHB. In the original phaC mutant (OPS0865), the lack of nitrogen fixation and poor growth on many carbon sources is likely to be due to increased levels of PhaR causing dysregulation of its complex regulon, because PHB formation, per se, is not required for effective nitrogen fixation in A. caulinodans. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .
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Li, Wei, and Chung-Dar Lu. "Regulation of Carbon and Nitrogen Utilization by CbrAB and NtrBC Two-Component Systems in Pseudomonas aeruginosa." Journal of Bacteriology 189, no. 15 (June 1, 2007): 5413–20. http://dx.doi.org/10.1128/jb.00432-07.
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ABSTRACT The global effect of the CbrAB and NtrBC two-component systems on the control of carbon and nitrogen utilization in Pseudomonas aeruginosa was characterized by phenotype microarray analyses with single and double mutants and the isogenic parent strain. The tested compounds were clustered based on the growth phenotypes of these strains, and the results clearly demonstrated the pivotal roles of CbrAB and NtrBC in carbon and nitrogen utilization, respectively. Growth of the cbrAB deletion mutant on arginine, histidine, and polyamines used as the sole carbon source was abolished, while growth on the tricarboxylic acid (TCA) cycle intermediates was sustained. In this study, suppressors of the cbr mutant were selected from minimal medium containing l-arginine as the sole carbon and nitrogen source. These mutants fell into two groups according to the ability to utilize histidine. The genomic library of a histidine-positive suppressor mutant was constructed, and the corresponding suppressor gene was identified by complementation as an ntrB allele. Similar results were obtained from four additional suppressor mutants, and point mutations of these ntrB alleles resulting in the following changes in residues were identified, with implications for reduced phosphatase activities: L126W, D227A, P228L, and S229I. The Ntr systems of these ntrB mutants became constitutively active, as revealed by the activity profiles of glutamate dehydrogenase, glutamate synthase, and glutamine synthetase. As a result, these mutants not only regain the substrate-specific induction on catabolic arginine and histidine operons but are also expressed to higher levels than the wild type. While the ΔcbrAB ntrB(Con) mutant restored growth on many N-containing compounds used as the carbon sources, its capability to grow on TCA cycle intermediates and glucose was compromised when ammonium served as the sole nitrogen source, mostly due to an extreme imbalance of carbon and nitrogen regulatory systems. In summary, this study supports the notion that CbrAB and NtrBC form a network to control the C/N balance in P. aeruginosa. Possible molecular mechanisms of these two regulatory elements in the control of arginine and histidine operons used as the model systems are discussed.
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Burgon, Trever B., Jomaquai A. Jenkins, Stephen B. Deitz, Jeannie F. Spagnolo, and Karla Kirkegaard. "Bypass Suppression of Small-Plaque Phenotypes by a Mutation in Poliovirus 2A That Enhances Apoptosis." Journal of Virology 83, no. 19 (July 22, 2009): 10129–39. http://dx.doi.org/10.1128/jvi.00642-09.
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ABSTRACT The rate of protein secretion in host cells is inhibited during infection with several different picornaviruses, with consequences likely to have significant effects on viral growth, spread, and pathogenesis. This Sin + (secretion inhibition) phenotype has been documented for poliovirus, foot-and-mouth disease virus, and coxsackievirus B3 and can lead to reduced cell surface expression of major histocompatibility complex class I and tumor necrosis factor receptor as well as reduced extracellular secretion of induced cytokines such as interleukin-6 (IL-6), IL-8, and beta interferon. The inhibition of protein secretion is global, affecting the movement of all tested cargo proteins through the cellular secretion apparatus. To test the physiological significance of the Sin − and Sin + phenotypes in animal models, Sin − mutant viruses are needed that fail to inhibit host protein secretion and also exhibit robust growth properties. To identify such Sin − mutant polioviruses, we devised a fluorescence-activated cell sorter-based screen to select virus-infected cells that nevertheless expressed newly synthesized surface proteins. After multiple rounds of selection, candidate Sin − mutant viruses were screened for genetic stability, increased secretion of cargo molecules and wild-type translation and growth properties. A newly identified Sin − mutant poliovirus that contained coding changes in nonstructural proteins 2A (N32D) and 2C (E253G) was characterized. In this virus, the 2C mutation is responsible for the Sin − phenotype and the 2A mutation suppresses a resulting growth defect by increasing the rate of cell death and therefore the rate of viral spread. The 2A-N32D suppressor mutation was not allele specific and, by increasing the rate of cellular apoptosis, affected a completely different pathway than the 2C-E253G Sin − mutation. Therefore, the 2A mutation suppresses the 2C-E253G mutant phenotype by a bypass suppression mechanism.