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  • Kvist, Jouni; Athanasio, Camila Goncalves; Pfrender, Michael E.; Brown, James B.; Colbourne, John K.; Mirbahai, Leda (2020)
    Background Daphnia species reproduce by cyclic parthenogenesis involving both sexual and asexual reproduction. The sex of the offspring is environmentally determined and mediated via endocrine signalling by the mother. Interestingly, male and female Daphnia can be genetically identical, yet display large differences in behaviour, morphology, lifespan and metabolic activity. Our goal was to integrate multiple omics datasets, including gene expression, splicing, histone modification and DNA methylation data generated from genetically identical female and male Daphnia pulex under controlled laboratory settings with the aim of achieving a better understanding of the underlying epigenetic factors that may contribute to the phenotypic differences observed between the two genders. Results In this study we demonstrate that gene expression level is positively correlated with increased DNA methylation, and histone H3 trimethylation at lysine 4 (H3K4me3) at predicted promoter regions. Conversely, elevated histone H3 trimethylation at lysine 27 (H3K27me3), distributed across the entire transcript length, is negatively correlated with gene expression level. Interestingly, male Daphnia are dominated with epigenetic modifications that globally promote elevated gene expression, while female Daphnia are dominated with epigenetic modifications that reduce gene expression globally. For examples, CpG methylation (positively correlated with gene expression level) is significantly higher in almost all differentially methylated sites in male compared to female Daphnia. Furthermore, H3K4me3 modifications are higher in male compared to female Daphnia in more than 3/4 of the differentially regulated promoters. On the other hand, H3K27me3 is higher in female compared to male Daphnia in more than 5/6 of differentially modified sites. However, both sexes demonstrate roughly equal number of genes that are up-regulated in one gender compared to the other sex. Since, gene expression analyses typically assume that most genes are expressed at equal level among samples and different conditions, and thus cannot detect global changes affecting most genes. Conclusions The epigenetic differences between male and female in Daphnia pulex are vast and dominated by changes that promote elevated gene expression in male Daphnia. Furthermore, the differences observed in both gene expression changes and epigenetic modifications between the genders relate to pathways that are physiologically relevant to the observed phenotypic differences.
  • Ianevski, Aleksandr; Yao, Rouan; Zusinaite, Eva; Lysvand, Hilde; Oksenych, Valentyn; Tenson, Tanel; Bjoras, Magnar; Kainov, Denis (2021)
    Background: Every year, millions of people are hospitalized and thousands die from influenza A virus (FLUAV) infection. Most cases of hospitalizations and death occur among the elderly. Many of these elderly patients are reliant on medical treatment of underlying chronic diseases, such as arthritis, diabetes, and hypertension. We hypothesized that the commonly prescribed medicines for treatment of underlying chronic diseases can affect host responses to FLUAV infection and thus contribute to the morbidity and mortality associated with influenza. Therefore, the aim of this study was to examine whether commonly prescribed medicines could affect host responses to virus infection in vitro. Methods: We first identified 45 active compounds from a list of commonly prescribed medicines. Then, we constructed a drug-target interaction network and identified the potential implication of these interactions for FLUAV-host cell interplay. Finally, we tested the effect of 45 drugs on the viability, transcription, and metabolism of mock- and FLUAV-infected human retinal pigment epithelial (RPE) cells. Results: In silico drug-target interaction analysis revealed that drugs such as atorvastatin, candesartan, and hydroxocobalamin could target and modulate FLUAV-host cell interaction. In vitro experiments showed that at non-cytotoxic concentrations, these compounds affected the transcription and metabolism of FLUAV- and mock-infected cells. Conclusion: Many commonly prescribed drugs were found to modulate FLUAV-host cell interactions in silico and in vitro and could therefore affect their interplay in vivo, thus contributing to the morbidity and mortality of patients with influenza virus infections.
  • Yu, Nancy Y.; Bieder, Andrea; Raman, Amitha; Mileti, Enrichetta; Katayama, Shintaro; Einarsdottir, Elisabet; Fredholm, Bertil B.; Falk, Anna; Tapia-Paez, Isabel; Daub, Carsten O.; Kere, Juha (2017)
    Caffeine is a widely consumed psychoactive substance, but little is known about the effects of caffeine stimulation on global gene expression changes in neurons. Here, we conducted gene expression profiling of human neuroepithelial stem cell-derived neurons, stimulated with normal consumption levels of caffeine (3 mu M and 10 mu M), over a period of 9 h. We found dosage-dependent activation of immediate early genes after 1 h. Neuronal projection development processes were up-regulated and negative regulation of axon extension processes were down-regulated at 3 h. In addition, genes involved in extracellular matrix organization, response for wound healing, and regulation of immune system processes were down-regulated by caffeine at 3 h. This study identified novel genes within the neuronal projection guidance pathways that respond to acute caffeine stimulation and suggests potential mechanisms for the effects of caffeine on neuronal cells.
  • Bokharaie, Honey; Kolch, Walter; Krstic, Aleksandar (2022)
    Alternative splicing (AS) is one of the hallmarks of human cancer. One of the most common mechanisms of vemurafenib resistance in malignant melanoma is AS of BRAF, occurring in 15-30% of patients. The aim of our study was to investigate the transcriptome and AS D04landscape in the isogenic BRAF V600E cell line pair SK-MEL-239, where the vemurafenib-resistant derivative expresses a truncated BRAF transcript that lacks the RAS-binding domain. Transcriptome analysis showed differential expression of spliceosome components between the two cell lines. AS analysis, by four different tools, DEXSeq, rMATS, ASpli, and LeafCutter, has identified genes enriched for cell motility and melanin synthesis in vemurafenib-resistant cells. Overlapping predictions for all four tools have been experimentally validated. Our study expands the understanding of melanoma drug resistance from a new perspective and supports the need to investigate in detail the aberrant AS landscape in patients with malignant melanoma. Alternative mRNA splicing is common in cancers. In BRAF V600E-mutated malignant melanoma, a frequent mechanism of acquired resistance to BRAF inhibitors involves alternative splicing (AS) of BRAF. The resulting shortened BRAF protein constitutively dimerizes and conveys drug resistance. Here, we have analysed AS in SK-MEL-239 melanoma cells and a BRAF inhibitor (vemurafenib)-resistant derivative that expresses an AS, shortened BRAF V600E transcript. Transcriptome analysis showed differential expression of spliceosome components between the two cell lines. As there is no consensus approach to analysing AS events, we used and compared four common AS softwares based on different principles, DEXSeq, rMATS, ASpli, and LeafCutter. Two of them correctly identified the BRAF V600E AS in the vemurafenib-resistant cells. Only 12 AS events were identified by all four softwares. Testing the AS predictions experimentally showed that these overlapping predictions are highly accurate. Interestingly, they identified AS caused alterations in the expression of melanin synthesis and cell migration genes in the vemurafenib-resistant cells. This analysis shows that combining different AS analysis approaches produces reliable results and meaningful, biologically testable hypotheses.
  • Bulanova, Daria; Ianevski, Aleksandr; Bugai, Andrii; Akimov, Yevhen; Kuivanen, Suvi; Paavilainen, Henrik; Kakkola, Laura; Nandania, Jatin; Turunen, Laura; Ohman, Tiina; Ala-Hongisto, Hanna; Pesonen, Hanna M.; Kuisma, Marika S.; Honkimaa, Anni; Walton, Emma L.; Oksenych, Valentyn; Lorey, Martina B.; Guschin, Dmitry; Shim, Jungmin; Kim, Jinhee; Than, Thoa T.; Chang, So Young; Hukkanen, Veijo; Kulesskiy, Evgeny; Marjomaki, Varpu S.; Julkunen, Ilkka; Nyman, Tuula A.; Matikainen, Sampsa; Saarela, Jani S.; Sane, Famara; Hober, Didier; Gabriel, Guelsah; De Brabander, Jef K.; Martikainen, Miika; Windisch, Marc P.; Min, Ji-Young; Bruzzone, Roberto; Aittokallio, Tero; Vaha-Koskela, Markus; Vapalahti, Olli; Pulk, Arto; Velagapudi, Vidya; Kainov, Denis E. (2017)
    Viral diseases remain serious threats to public health because of the shortage of effective means of control. To combat the surge of viral diseases, new treatments are urgently needed. Here we show that small-molecules, which inhibit cellular anti-apoptotic Bcl-2 proteins (Bcl-2i), induced the premature death of cells infected with different RNA or DNA viruses, whereas, at the same concentrations, no toxicity was observed in mock-infected cells. Moreover, these compounds limited viral replication and spread. Surprisingly, Bcl-2i also induced the premature apoptosis of cells transfected with viral RNA or plasmid DNA but not of mock-transfected cells. These results suggest that Bcl-2i sensitizes cells containing foreign RNA or DNA to apoptosis. A comparison of the toxicity, antiviral activity, and side effects of six Bcl-2i allowed us to select A-1155463 as an antiviral lead candidate. Thus, our results pave the way for the further development of Bcl-2i for the prevention and treatment of viral diseases.
  • Toledo, Miriam; Batista-Gonzalez, Ana; Merheb, Emilio; Aoun, Marie Louise; Tarabra, Elena; Feng, Daorong; Sarparanta, Jaakko; Merlo, Paola; Botre, Francesco; Schwartz, Gary J.; Pessin, Jeffrey E.; Singh, Rajat (2018)
    The circadian clock coordinates behavioral and circadian cues with availability and utilization of nutrients. Proteasomal degradation of clock repressors, such as cryptochrome (CRY) 1, maintains periodicity. Whether macroautophagy, a quality control pathway, degrades circadian proteins remains unknown. Here we show that circadian proteins BMAL1, CLOCK, REV-ERB alpha, and CRY1 are lysosomal targets, and that macroautophagy affects the circadian clock by selectively degrading CRY1. Autophagic degradation of CRY1, an inhibitor of gluconeogenesis, occurs in a diurnal window when rodents rely on gluconeogenesis, suggesting that CRY1 degradation is timeimprinted to maintenance of blood glucose. High-fat feeding accelerates autophagic CRY1 degradation and contributes to obesity-associated hyperglycemia. CRY1 contains several light chain 3 (LC3)-interacting region (LIR) motifs, which facilitate the interaction of cargo proteins with the autophagosome marker LC3. Using mutational analyses, we identified two distinct LIRs on CRY1 that exert circadian glycemic control by regulating CRY1 degradation, revealing LIRs as potential targets for controlling hyperglycemia.
  • Hu, Ming; Cebola, Ines; Carrat, Gaelle; Jiang, Shuying; Nawaz, Sameena; Khamis, Amna; Canouil, Mickael; Froguel, Philippe; Schulte, Anke; Solimena, Michele; Ibberson, Mark; Marchetti, Piero; Cardenas-Diaz, Fabian L.; Gadue, Paul J.; Hastoy, Benoit; Almeida-Souza, Leonardo; McMahon, Harvey; Rutter, Guy A. (2021)
    Using chromatin conformation capture, we show that an enhancer cluster in the STARD10 type 2 diabetes (T2D) locus forms a defined 3-dimensional (3D) chromatin domain. A 4.1-kb region within this locus, carrying 5 T2D-associated variants, physically interacts with CTCF-binding regions and with an enhancer possessing strong transcriptional activity. Analysis of human islet 3D chromatin interaction maps identifies the FCHSD2 gene as an additional target of the enhancer cluster. CRISPR-Cas9-mediated deletion of the variant region, or of the associated enhancer, from human pancreas-derived EndoC-bH1 cells impairs glucose- stimulated insulin secretion. Expression of both STARD10 and FCHSD2 is reduced in cells harboring CRISPR deletions, and lower expression of STARD10 and FCHSD2 is associated, the latter nominally, with the possession of risk variant alleles in human islets. Finally, CRISPR-Cas9-mediated loss of STARD10 or FCHSD2, but not ARAP1, impairs regulated insulin secretion. Thus, multiple genes at the STARD10 locus influence b cell function.
  • Kouri, Vesa-Petteri; Olkkonen, Juri; Kaivosoja, Emilia; Ainola, Mari-Mia; Juhila, Juuso; Hovatta, Iiris; Konttinen, Yrjo T.; Mandelin, Jami (2013)
  • Reichhardt, Martin P.; Lundin, Karolina; Lokki, A. Inkeri; Recher, Gaëlle; Vuoristo, Sanna; Katayama, Shintaro; Tapanainen, Juha S.; Kere, Juha; Meri, Seppo; Tuuri, Timo (2019)
    It is essential for early human life that mucosal immunological responses to developing embryos are tightly regulated. An imbalance of the complement system is a common feature of pregnancy complications. We hereby present the first full analysis of the expression and deposition of complement molecules in human pre-implantation embryos. Thus, far, immunological imbalance has been considered in stages of pregnancy following implantation. We here show that complement activation against developing human embryos takes place already at the pre-implantation stage. Using confocal microscopy, we observed deposition of activation products on healthy developing embryos, which highlights the need for strict complement regulation. We show that embryos express complement membrane inhibitors and bind soluble regulators. These findings show that mucosal complement targets human embryos, and indicate potential adverse pregnancy outcomes, if regulation of activation fails. In addition, single-cell RNA sequencing revealed cellular expression of complement activators. This shows that the embryonic cells themselves have the capacity to express and activate C3 and C5. The specific local embryonic expression of complement components, regulators, and deposition of activation products on the surface of embryos suggests that complement has immunoregulatory functions and furthermore may impact cellular homeostasis and differentiation at the earliest stages of life.
  • Latorre, Jèssica; Ortega, Francisco J.; Liñares-Pose, Laura; Moreno-Navarrete, José M.; Lluch, Aina; Comas, Ferran; Oliveras-Cañellas, Núria; Ricart, Wifredo; Höring, Marcus; Zhou, You; Liebisch, Gerhard; Nidhina Haridas, P.A.; Olkkonen, Vesa M.; López, Miguel; Fernández-Real, José M. (2020)
    Background: While the impact of metformin in hepatocytes leads to fatty acid (FA) oxidation and decreased lipogenesis, hepatic microRNAs (miRNAs) have been associated with fat overload and impaired metabolism, contributing to the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Methods: We investigated the expression of hundreds of miRNAs in primary hepatocytes challenged by compounds modulating steatosis, palmitic acid and compound C (as inducers), and metformin (as an inhibitor). Then, additional hepatocyte and rodent models were evaluated, together with transient mimic miRNAs transfection, lipid droplet staining, thin-layer chromatography, quantitative lipidomes, and mitochondrial activity, while human samples outlined the translational significance of this work. Findings: Our results show that treatments triggering fat accumulation and AMPK disruption may compromise the biosynthesis of hepatic miRNAs, while the knockdown of the miRNA-processing enzyme DICER in human hepatocytes exhibited increased lipid deposition. In this context, the ectopic recovery of miR-30b and miR-30c led to significant changes in genes related to FA metabolism, consistent reduction of ceramides, higher mitochondrial activity, and enabled b-oxidation, redirecting FA metabolism fromenergy storage to expenditure. Interpretation: Current findings unravel the biosynthesis of hepatic miR-30b and miR-30c in tackling inadequate FA accumulation, offering a potential avenue for the treatment of NAFLD. Funding: Instituto de Salud Carlos III (ISCIII), Govern de la Generalitat (PERIS2016), Associacio Catalana de Diabetis (ACD), Sociedad Espanola de Diabetes (SED), Fondo Europeo de Desarrollo Regional (FEDER), Xunta de Galicia, Ministerio de Economia y Competitividad (MINECO), "La Caixa" Foundation, and CIBER de la Fisiopatologia de la Obesidad y Nutricion (CIBEROBN). (c) 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4.0/)
  • Munne, Pauliina M.; Martikainen, Lahja; Räty, Iiris; Bertula, Kia; Nonappa; Ruuska, Janika; Ala-Hongisto, Hanna; Peura, Aino; Hollmann, Babette; Euro, Lilya; Yavuz, Kerim; Patrikainen, Linda; Salmela, Maria; Pokki, Juho; Kivento, Mikko; Väänänen, Juho; Suomi, Tomi; Nevalaita, Liina; Mutka, Minna; Kovanen, Panu; Leidenius, Marjut; Meretoja, Tuomo; Hukkinen, Katja; Monni, Outi; Pouwels, Jeroen; Sahu, Biswajyoti; Mattson, Johanna; Joensuu, Heikki; Heikkilä, Päivi; Elo, Laura L.; Metcalfe, Ciara; Junttila, Melissa R.; Ikkala, Olli; Klefström, Juha (2021)
    Breast cancer is now globally the most frequent cancer and leading cause of women's death. Two thirds of breast cancers express the luminal estrogen receptor-positive (ER alpha + ) phenotype that is initially responsive to antihormonal therapies, but drug resistance emerges. A major barrier to the understanding of the ER alpha-pathway biology and therapeutic discoveries is the restricted repertoire of luminal ER alpha + breast cancer models. The ER alpha + phenotype is not stable in cultured cells for reasons not fully understood. We examine 400 patient-derived breast epithelial and breast cancer explant cultures (PDECs) grown in various three-dimensional matrix scaffolds, finding that ER alpha is primarily regulated by the matrix stiffness. Matrix stiffness upregulates the ER alpha signaling via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation. The finding that the matrix stiffness is a central cue to the ER alpha phenotype reveals a mechanobiological component in breast tissue hormonal signaling and enables the development of novel therapeutic interventions. Subject terms: ER-positive (ER + ), breast cancer, ex vivo model, preclinical model, PDEC, stiffness, p38 SAPK. Reliable luminal estrogen receptor (ER alpha+) breast cancer models are limited. Here, the authors use patient derived breast epithelial and breast cancer explant cultures grown in several extracellular matrix scaffolds and show that ER alpha expression is regulated by matrix stiffness via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation.
  • Kuuliala, Krista; Kuuliala, Antti; Koivuniemi, Riitta; Oksanen, Suvi; Hamalainen, Mari; Moilanen, Eeva; Kautiainen, Hannu; Leirisalo-Repo, Marjatta; Repo, Heikki (2015)
    The aim of the present study was to examine constitutive signal transducer and activator of transcription 3 (STAT3) phosphorylation in circulating leukocytes as a candidate biomarker in rheumatoid arthritis (RA). 25 patients with recent-onset, untreated RA provided samples for whole blood flow cytometric determination of intracellular STAT3 phosphorylation, expressed as relative fluorescence units. The occurrence of constitutive STAT3 phosphorylation was evaluated by determining proportion of STAT3-phosphorylated cells among different leukocyte subtypes. Plasma levels of interleukin (IL)-6, IL-17 and IL-21 were measured by immunoassay, radiographs of hands and feet were examined and disease activity score (DAS28) was determined. Biomarkers were restudied and treatment response (according to European League Against Rheumatism) was determined after 12 months of treatment with disease-modifying antirheumatic drugs. At baseline, constitutive phosphorylation of STAT3 occurred in CD4(+) T cells of 14 (56%) patients, CD8(+) T cells of 13 (52%) patients, in CD19+ B cells of 7 (28%) patients, and in CD14(+) monocytes of 12 (48%) patients. STAT3 phosphorylation levels of CD4(+) T cells associated with DAS28, and those of all leukocyte subtypes studied associated with erosive disease. The presence of constitutive STAT3 phosphorylation in CD4(+) T lymphocytes, pSTAT3 fluorescence intensity of CD4(+) and CD8(+) T cells and C-reactive protein (CRP) levels at baseline associated with good treatment response. In conclusion, constitutive STAT3 phosphorylation in circulating CD4(+) T cells is common in recent-onset untreated RA and associates with good treatment response in patients characterized by high disease activity and the presence of systemic inflammation.
  • Percipallea, Piergiorgio; Vartiainen, Maria (2019)
    The emerging role of cytoskeletal proteins in the cell nucleus has become a new frontier in cell biology. Actin and actin-binding proteins regulate chromatin and gene expression, but importantly they are beginning to be essential players in genome organization. These actin-based functions contribute to genome stability and integrity while affecting DNA replication and global transcription patterns. This is likely to occur through interactions of actin with nuclear components including nuclear lamina and subnuclear organelles. An exciting future challenge is to understand how these actin-based genome-wide mechanisms may regulate development and differentiation by interfering with the mechanical properties of the cell nucleus and how regulated actin polymerization plays a role in maintaining nuclear architecture. With a special focus on actin, here we summarize how cytoskeletal proteins operate in the nucleus and how they may be important to consolidate nuclear architecture for sustained gene expression or silencing.
  • Noguchi, Shuhei; Arakawa, Takahiro; Fukuda, Shiro; Furuno, Masaaki; Hasegawa, Akira; Hori, Fumi; Ishikawa-Kato, Sachi; Kaida, Kaoru; Kaiho, Ai; Kanamori-Katayama, Mutsumi; Kawashima, Tsugumi; Kojima, Miki; Kubosaki, Atsutaka; Manabe, Ri-ichiroh; Murata, Mitsuyoshi; Nagao-Sato, Sayaka; Nakazato, Kenichi; Ninomiya, Noriko; Nishiyori-Sueki, Hiromi; Noma, Shohei; Saijyo, Eri; Saka, Akiko; Sakai, Mizuho; Simon, Christophe; Suzuki, Naoko; Tagami, Michihira; Watanabe, Shoko; Yoshida, Shigehiro; Arner, Peter; Axton, Richard A.; Babina, Magda; Baillie, J. Kenneth; Barnett, Timothy C.; Beckhouse, Anthony G.; Blumenthal, Antje; Bodega, Beatrice; Bonetti, Alessandro; Briggs, James; Brombacher, Frank; Carlisle, Ailsa J.; Clevers, Hans C.; Davis, Carrie A.; Detmar, Michael; Dohi, Taeko; Edge, Albert S. B.; Edinger, Matthias; Ehrlund, Anna; Ekwall, Karl; Endoh, Mitsuhiro; Enomoto, Hideki; Eslami, Afsaneh; Fagiolini, Michela; Fairbairn, Lynsey; Farach-Carson, Mary C.; Faulkner, Geoffrey J.; Ferrai, Carmelo; Fisher, Malcolm E.; Forrester, Lesley M.; Fujita, Rie; Furusawa, Jun-ichi; Geijtenbeek, Teunis B.; Gingeras, Thomas; Goldowitz, Daniel; Guhl, Sven; Guler, Reto; Gustincich, Stefano; Ha, Thomas J.; Hamaguchi, Masahide; Hara, Mitsuko; Hasegawa, Yuki; Herlyn, Meenhard; Heutink, Peter; Hitchens, Kelly J.; Hume, David A.; Ikawa, Tomokatsu; Ishizu, Yuri; Kai, Chieko; Kawamoto, Hiroshi; Kawamura, Yuki I.; Kempfle, Judith S.; Kenna, Tony J.; Kere, Juha; Khachigian, Levon M.; Kitamura, Toshio; Klein, Sarah; Klinken, S. Peter; Knox, Alan J.; Kojima, Soichi; Koseki, Haruhiko; Koyasu, Shigeo; Lee, Weonju; Lennartsson, Andreas; Mackay-sim, Alan; Mejhert, Niklas; Mizuno, Yosuke; Morikawa, Hiromasa; Morimoto, Mitsuru; Moro, Kazuyo; Morris, Kelly J.; Motohashi, Hozumi; Mummery, Christine L.; Nakachi, Yutaka; Nakahara, Fumio; Nakamura, Toshiyuki; Nakamura, Yukio; Nozaki, Tadasuke; Ogishima, Soichi; Ohkura, Naganari; Ohno, Hiroshi; Ohshima, Mitsuhiro; Okada-Hatakeyama, Mariko; Okazaki, Yasushi; Orlando, Valerio; Ovchinnikov, Dmitry A.; Passier, Robert; Patrikakis, Margaret; Pombo, Ana; Pradhan-Bhatt, Swati; Qin, Xian-Yang; Rehli, Michael; Rizzu, Patrizia; Roy, Sugata; Sajantila, Antti; Sakaguchi, Shimon; Sato, Hiroki; Satoh, Hironori; Savvi, Suzana; Saxena, Alka; Schmidl, Christian; Schneider, Claudio; Schulze-Tanzil, Gundula G.; Schwegmann, Anita; Sheng, Guojun; Shin, Jay W.; Sugiyama, Daisuke; Sugiyama, Takaaki; Summers, Kim M.; Takahashi, Naoko; Takai, Jun; Tanaka, Hiroshi; Tatsukawa, Hideki; Tomoiu, Andru; Toyoda, Hiroo; van de Wetering, Marc; van den Berg, Linda M.; Verardo, Roberto; Vijayan, Dipti; Wells, Christine A.; Winteringham, Louise N.; Wolvetang, Ernst; Yamaguchi, Yoko; Yamamoto, Masayuki; Yanagi-Mizuochi, Chiyo; Yoneda, Misako; Yonekura, Yohei; Zhang, Peter G.; Zucchelli, Silvia; Abugessaisa, Imad; Arner, Erik; Harshbarger, Jayson; Kondo, Atsushi; Lassmann, Timo; Lizio, Marina; Sahin, Serkan; Sengstag, Thierry; Severin, Jessica; Shimoji, Hisashi; Suzuki, Masanori; Suzuki, Harukazu; Kawai, Jun; Kondo, Naoto; Itoh, Masayoshi; Daub, Carsten O.; Kasukawa, Takeya; Kawaji, Hideya; Carninci, Piero; Forrest, Alistair R. R.; Hayashizaki, Yoshihide (2017)
    In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities.
  • Colis, Laureen; Ernst, Glen; Sanders, Sara; Liu, Hester; Sirajuddin, Paul; Peltonen, Karita; DePasquale, Michael; Barrow, James C.; Laiho, Marikki (2014)
    RNA polymerase I (Pol I) is a dedicated polymerase that transcribes the 45S ribosomal (r) RNA precursor. The 45S rRNA precursor is subsequently processed into the mature 5.8S, 18S, and 28S rRNAs and assembled into ribosomes in the nucleolus. Pol I activity is commonly deregulated in human cancers. On the basis of the discovery of lead molecule BMH-21, a series of pyridoquinazolinecarboxamides have been evaluated as inhibitors of Pol I and activators of the destruction of RPA194, the Poll large catalytic subunit protein. Structure-activity relationships in assays of nucleolar stress and cell viability demonstrate key pharmacophores and their physicochemical properties required for potent activation of Pol I stress and cytotoxidty. This work identifies a set of bioactive compounds that potently cause RPA194 degradation that function in a tightly constrained chemical space. This work has yielded novel derivatives that contribute to the development of Pol I inhibitory cancer therapeutic strategies.
  • Olkkonen, Juri; Kouri, Vesa-Petteri; Hynninen, Joel; Konttinen, Yrjo T.; Mandelin, Jami (2015)
    Objective Patients with rheumatoid arthritis (RA) have altered circadian rhythm of circulating serum cortisol, melatonin and IL-6, as well as disturbance in the expression of clock genes ARNTL2 and NPAS2. In humans, TNF alpha increases the expression ARNTL2 and NPAS2 but paradoxically suppresses clock output genes DPB and PER3. Our objective was to investigate the expression of direct clock suppressors DEC1 and DEC2 (BHLHE 40 and 41 proteins) in response to TNF alpha and investigate their role during inflammation. Methods Cultured primary fibroblasts were stimulated with TNF alpha. Effects on DEC2 were studied using RT-qPCR and immunofluorescence staining. The role of NF-kappa B in DEC2 increase was analyzed using IKK-2 specific inhibitor IMD-0354. Cloned DEC2 was transfected into HEK293 cells to study its effects on gene expression. Transfections into primary human fibroblasts were used to confirm the results. The presence of DEC2 was analyzed in (RA) and osteoarthritis (OA) synovial membranes by immunohistochemistry. Results TNF alpha increased DEC2 mRNA and DEC2 was mainly detected at nuclei after the stimulus. The effects of TNF alpha on DEC2 expression were mediated via NF-kappa B. Overexpression, siRNA and promoter activity studies disclosed that DEC2 directly regulates IL-1 beta, in both HEK293 cells and primary human fibroblasts. DEC2 was increased in synovial membrane in RA compared to OA. Conclusion Not only ARNTL2 and NPAS2 but also DEC2 is regulated by TNF alpha in human fibroblasts. NF-kappa B mediates the effect on DEC2, which upregulates IL-1 beta. Circadian clock has a direct effect on inflammation in human fibroblasts.
  • Skarp, Sini; Xia, Ji-Han; Zhang, Qin; Löija, Marika; Costantini, Alice; Ruddock, Lloyd W.; Mäkitie, Outi; Wei, Gong-Hong; Männikkö, Minna (2020)
    We studied a family with severe primary osteoporosis carrying a heterozygous p.Arg8Phefs*14 deletion in COL1A2, leading to haploinsufficiency. Three affected individuals carried the mutation and presented nearly identical spinal fractures but lacked other typical features of either osteogenesis imperfecta or Ehlers-Danlos syndrome. Although mutations leading to haploinsufficiency in COL1A2 are rare, mutations in COL1A1 that lead to less protein typically result in a milder phenotype. We hypothesized that other genetic factors may contribute to the severe phenotype in this family. We performed whole-exome sequencing in five family members and identified in all three affected individuals a rare nonsense variant (c.1282C > T/p.Arg428*, rs150257846) in ZNF528. We studied the effect of the variant using qPCR and Western blot and its subcellular localization with immunofluorescence. Our results indicate production of a truncated ZNF528 protein that locates in the cell nucleus as per the wild-type protein. ChIP and RNA sequencing analyses on ZNF528 and ZNF528-c.1282C > T indicated that ZNF528 binding sites are linked to pathways and genes regulating bone morphology. Compared with the wild type, ZNF528-c.1282C > T showed a global shift in genomic binding profile and pathway enrichment, possibly contributing to the pathophysiology of primary osteoporosis. We identified five putative target genes for ZNF528 and showed that the expression of these genes is altered in patient cells. In conclusion, the variant leads to expression of truncated ZNF528 and a global change of its genomic occupancy, which in turn may lead to altered expression of target genes. ZNF528 is a novel candidate gene for bone disorders and may function as a transcriptional regulator in pathways affecting bone morphology and contribute to the phenotype of primary osteoporosis in this family together with the COL1A2 deletion. (c) 2020 The Authors.Journal of Bone and Mineral Researchpublished by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
  • Peippo, Minna; Gardberg, Maria; Lamminen, Tarja; Kaipio, Katja; Carpen, Olli; Heuser, Vanina D. (2017)
    The functional properties of actin-regulating formin proteins are diverse and in many cases cell-type specific. FHOD1, a formin expressed predominantly in cells of mesenchymal lineage, bundles actin filaments and participates in maintenance of cell shape, migration and cellular protrusions. FHOD1 participates in cancer associated epithelial to mesenchymal transition (EMT) in oral squamous cell carcinoma and breast cancer. The role of FHOD1 in melanomas has not been characterized. Here, we show that FHOD1 expression is typically strong in cutaneous melanomas and cultured melanoma cells while the expression is low or absent in benign nevi. By using shRNA to knockdown FHOD1 in melanoma cells, we discovered that FHOD1 depleted cells are larger, rounder and have smaller focal adhesions and inferior migratory capacity as compared to control cells. Importantly, we found FHOD1 depleted cells to have reduced colony-forming capacity and attenuated tumor growth in vivo, a finding best explained by the reduced proliferation rate caused by cell cycle arrest. Unexpectedly, FHOD1 depletion did not prevent invasive growth at the tumor margins. These results suggest that FHOD1 participates in key cellular processes that are dysregulated in malignancy, but may not be essential for melanoma cell invasion.
  • Ramilowski, Jordan A.; Yip, Chi Wai; Agrawal, Saumya; Chang, Jen-Chien; Ciani, Yari; Kulakovskiy, Ivan V.; Mendez, Mickael; Ooi, Jasmine Li Ching; Ouyang, John F.; Parkinson, Nick; Petri, Andreas; Roos, Leonie; Severin, Jessica; Yasuzawa, Kayoko; Abugessaisa, Imad; Akalin, Altuna; Antonov, Ivan V.; Arner, Erik; Bonetti, Alessandro; Bono, Hidemasa; Borsari, Beatrice; Brombacher, Frank; Cameron, Chris J. F.; Cannistraci, Carlo Vittorio; Cardenas, Ryan; Cardon, Melissa; Chang, Howard; Dostie, Josee; Ducoli, Luca; Favorov, Alexander; Fort, Alexandre; Garrido, Diego; Gil, Noa; Gimenez, Juliette; Guler, Reto; Handoko, Lusy; Harshbarger, Jayson; Hasegawa, Akira; Hasegawa, Yuki; Hashimoto, Kosuke; Hayatsu, Norihito; Heutink, Peter; Hirose, Tetsuro; Imada, Eddie L.; Itoh, Masayoshi; Kaczkowski, Bogumil; Kanhere, Aditi; Kawabata, Emily; Kawaji, Hideya; Kawashima, Tsugumi; Kelly, S. Thomas; Kojima, Miki; Kondo, Naoto; Koseki, Haruhiko; Kouno, Tsukasa; Kratz, Anton; Kurowska-Stolarska, Mariola; Kwon, Andrew Tae Jun; Leek, Jeffrey; Lennartsson, Andreas; Lizio, Marina; Lopez-Redondo, Fernando; Luginbuhl, Joachim; Maeda, Shiori; Makeev, Vsevolod J.; Marchionni, Luigi; Medvedeva, Yulia A.; Minoda, Aki; Mueller, Ferenc; Munoz-Aguirre, Manuel; Murata, Mitsuyoshi; Nishiyori, Hiromi; Nitta, Kazuhiro R.; Noguchi, Shuhei; Noro, Yukihiko; Nurtdinov, Ramil; Okazaki, Yasushi; Orlando, Valerio; Paquette, Denis; Parr, Callum J. C.; Rackham, Owen J. L.; Rizzu, Patrizia; Martinez, Diego Fernando Sanchez; Sandelin, Albin; Sanjana, Pillay; Semple, Colin A. M.; Shibayama, Youtaro; Sivaraman, Divya M.; Suzuki, Takahiro; Szumowski, Suzannah C.; Tagami, Michihira; Taylor, Martin S.; Terao, Chikashi; Thodberg, Malte; Thongjuea, Supat; Tripathi, Vidisha; Ulitsky, Igor; Verardo, Roberto; Vorontsov, Ilya E.; Yamamoto, Chinatsu; Young, Robert S.; Baillie, J. Kenneth; Forrest, Alistair R. R.; Guigo, Roderic; Hoffman, Michael M.; Hon, Chung Chau; Kasukawa, Takeya; Kauppinen, Sakari; Kere, Juha; Lenhard, Boris; Schneider, Claudio; Suzuki, Harukazu; Yagi, Ken; Hoon, Michiel J. L. de; Shin, Jay W.; Carninci, Piero (2020)
    Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-todate lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.