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Kurrle, Nina, Frank Schnütgen, Juliana Heidler, Ina Poser, Frank Wempe, Diego Yepes, Ilka Wittig, et al. "Exploring the Function of Sestrin/Gator As Novel Regulators of Hematopoiesis." Blood 128, no. 22 (December 2, 2016): 1484. http://dx.doi.org/10.1182/blood.v128.22.1484.1484.
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Abstract The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that responds to multiple environmental cues such as reactive oxygen species (ROS) and thereby regulates many fundamental biological processes including cell growth and autophagy. mTOR is found in two distinct multiprotein complexes, mTORC1 and mTORC2, of which mTORC1 has been established to play an important role in the regulation of hematopoiesis. For example, mTORC1 inhibition, combined with activation of canonical Wnt-signaling, was shown to increase long term repopulating (LT)-HSC self-renewal, whereas its activation depletes LT-HSCs. The activity of mTORC1 is tightly controlled by multiple layers of upstream regulators, including the recently discovered GTPase activating protein (GAP) activity towards Rags (GATOR) complex, which is an AMP-Kinase/Tuberous sclerosis complex (TSC)-independent mTORC1 inhibitor induced by amino acid deprivation. GATOR consists of two subcomplexes, GATOR1 and GATOR2, whereby GATOR2 inhibits GATOR1. Inactivation of GATOR2 prevents mTORC1 activation by amino acids, whereas inactivation of GATOR1 constitutively activates mTORC1. Sestrins (Sesn1, Sesn2 and Sesn3) are a family stress-inducible, redox-sensitive proteins that are involved in cellular- or organism-level adaptation to diverse metabolic challenges. They have been identified as direct interactors of GATOR2 and shown to inhibit mTORC1 by preventing GATOR2 from inhibiting GATOR1 in presence of amino acids. In quantitative affinity purification-mass spectrometry (AP-MS) and coimmunoprecipitation experiments with HeLa- and mouse embryonic stem cells harboring in situ GFP-tagged Sesn2, WDR59 and NPRL3 alleles, we could confirm the Sesn2/GATOR interaction under nearly physiological conditions. To analyze the function of Sestrin/GATOR during hematopoiesis in more detail, we isolated Lin- Sca+ hematopoietic cells from the bone marrow of Sesn2-/- mice and performed serial replating experiments and competitive hematopoietic repopulation experiments in lethally irradiated mice and observed that Sesn2-/- progenitor cells proliferate significantly faster than their wild type counterparts albeit only during the initial engraftment phase. At later stages, the wild type cells took over, exceeding the Sesn2-/- cells by 3-4 fold in peripheral blood, bone marrow and spleen three months after transplantation. This suggests that the increased proliferative potential of Sesn2-/- progenitor cells leads to a depletion of the LT-HSC pool, strikingly resembling the phenotype of activated mTORC1. Disclosures No relevant conflicts of interest to declare.
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Solanki, Sumeet, Jun-Hee Lee, and Yatrik Shah. "AMINO ACID SENSING PATHWAYS IN INFLAMMATORY BOWEL DISEASE." Inflammatory Bowel Diseases 28, Supplement_1 (January 22, 2022): S23—S24. http://dx.doi.org/10.1093/ibd/izac015.036.
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Abstract IBD is a chronic inflammatory disease of the gastrointestinal tract affecting millions of people worldwide. The last few decades have seen rapid increase of IBD cases in the US contributing to exorbitant health-care costs and morbidity rates. IBD comprises of two major subtypes: ulcerative colitis and Crohn’s disease. Although the etiology of IBD is not completely understood, a complex interaction of environmental, genetic, immune, and gut microbial factors contributes to its pathogenesis. These factors ultimately converge to disrupt intestinal epithelial cell homeostasis, compromising mucosal barrier function, leading to unresolved relapsing inflammatory injury. Poor dietary habits such as Western diet are thought to play a pivotal role in the development of colitis. A healthy and balanced diet is important in management of the disease. Epidemiological and experimental studies demonstrate that high-protein diet triggers inflammatory flares and colitis patients are often advised to reduce animal dietary protein. However, colitis patients are also at high risk of a protein malnutrition. Thus, the precise mechanisms by which low dietary protein increases incidence of colitis and/or aggravate already established disease remains unknown. Our preliminary findings suggest that low dietary protein intake aggravated dextran sulfate sodium (DSS)-induced colitis with reduced body weight, colon length and increased histological injury. Dietary habits impact intestinal epithelial cell homeostasis and regeneration process in the event of an injury. The mechanistic target of rapamycin complex 1 (mTORC1), a ‘master’ regulator of cell growth participates in intestinal tissue regeneration. Remarkably, studies inhibiting mTORC1 activity have shown to disrupt the regenerative capacity of intestinal epithelium and increased susceptibility to colitis while activating mTORC1 had a positive effect. As amino acids potently activate mTORC1, we assessed and found that low dietary protein significantly reduce colonic mTORC1 activation. The newly discovered GAP activity towards Rags (GATOR1 & GATOR2) complexes act as amino acid sensing pathways by which mTORC1 activity is modulated. GATOR1 is a negative regulator, whereas GATOR2 is a positive regulator of mTORC1. CRISPR/CAS9 knockout of Wdr24 (GATOR2) led to inactivation of mTORC1 under amino acid culture conditions. Next, we investigated the role of amino acid sensing pathway in colitis models and found that disruption of intestinal epithelial specific GATOR2 complex (Wdr24ΔIE) attenuated mTORC1 activity and increased susceptibility to colitis. These findings suggest that low protein diet impacts colitis by modulating the intestinal epithelial amino acid sensing pathway.
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Padi, Sathish K. R., Neha Singh, Jeremiah J. Bearss, Virginie Olive, Jin H. Song, Marina Cardó-Vila, Andrew S. Kraft, and Koichi Okumura. "Phosphorylation of DEPDC5, a component of the GATOR1 complex, releases inhibition of mTORC1 and promotes tumor growth." Proceedings of the National Academy of Sciences 116, no. 41 (September 23, 2019): 20505–10. http://dx.doi.org/10.1073/pnas.1904774116.
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The Pim and AKT serine/threonine protein kinases are implicated as drivers of cancer. Their regulation of tumor growth is closely tied to the ability of these enzymes to mainly stimulate protein synthesis by activating mTORC1 (mammalian target of rapamycin complex 1) signaling, although the exact mechanism is not completely understood. mTORC1 activity is normally suppressed by amino acid starvation through a cascade of multiple regulatory protein complexes, e.g., GATOR1, GATOR2, and KICSTOR, that reduce the activity of Rag GTPases. Bioinformatic analysis revealed that DEPDC5 (DEP domain containing protein 5), a component of GATOR1 complex, contains Pim and AKT protein kinase phosphorylation consensus sequences. DEPDC5 phosphorylation by Pim and AKT kinases was confirmed in cancer cells through the use of phospho-specific antibodies and transfection of phospho-inactive DEPDC5 mutants. Consistent with these findings, during amino acid starvation the elevated expression of Pim1 overcame the amino acid inhibitory protein cascade and activated mTORC1. In contrast, the knockout of DEPDC5 partially blocked the ability of small molecule inhibitors against Pim and AKT kinases both singly and in combination to suppress tumor growth and mTORC1 activity in vitro and in vivo. In animal experiments knocking in a glutamic acid (S1530E) in DEPDC5, a phospho mimic, in tumor cells induced a significant level of resistance to Pim and the combination of Pim and AKT inhibitors. Our results indicate a phosphorylation-dependent regulatory mechanism targeting DEPDC5 through which Pim1 and AKT act as upstream effectors of mTORC1 to facilitate proliferation and survival of cancer cells.
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Wei, Youheng, Brad Reveal, Weili Cai, and Mary A. Lilly. "The GATOR1 Complex Regulates Metabolic Homeostasis and the Response to Nutrient Stress in Drosophila melanogaster." G3 Genes|Genomes|Genetics 6, no. 12 (December 1, 2016): 3859–67. http://dx.doi.org/10.1534/g3.116.035337.
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Abstract TORC1 regulates metabolism and growth in response to a large array of upstream inputs. The evolutionarily conserved trimeric GATOR1 complex inhibits TORC1 activity in response to amino acid limitation. In humans, the GATOR1 complex has been implicated in a wide array of pathologies including cancer and hereditary forms of epilepsy. However, the precise role of GATOR1 in animal physiology remains largely undefined. Here, we characterize null mutants of the GATOR1 components nprl2, nprl3, and iml1 in Drosophila melanogaster. We demonstrate that all three mutants have inappropriately high baseline levels of TORC1 activity and decreased adult viability. Consistent with increased TORC1 activity, GATOR1 mutants exhibit a cell autonomous increase in cell growth. Notably, escaper nprl2 and nprl3 mutant adults have a profound locomotion defect. In line with a nonautonomous role in the regulation of systemic metabolism, expressing the Nprl3 protein in the fat body, a nutrient storage organ, and hemocytes but not muscles and neurons rescues the motility of nprl3 mutants. Finally, we show that nprl2 and nprl3 mutants fail to activate autophagy in response to amino acid limitation and are extremely sensitive to both amino acid and complete starvation. Thus, in Drosophila, in addition to maintaining baseline levels of TORC1 activity, the GATOR1 complex has retained a critical role in the response to nutrient stress. In summary, the TORC1 inhibitor GATOR1 contributes to multiple aspects of the development and physiology of Drosophila.
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Shen, Kuang, Rick K. Huang, Edward J. Brignole, Kendall J. Condon, Max L. Valenstein, Lynne Chantranupong, Aimaiti Bomaliyamu, et al. "Architecture of the human GATOR1 and GATOR1–Rag GTPases complexes." Nature 556, no. 7699 (March 28, 2018): 64–69. http://dx.doi.org/10.1038/nature26158.
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Muller, Maéline, Jasmine Bélanger, Imane Hadj-Aissa, Conghao Zhang, Chantelle F. Sephton, and Paul A. Dutchak. "GATOR1 Mutations Impair PI3 Kinase-Dependent Growth Factor Signaling Regulation of mTORC1." International Journal of Molecular Sciences 25, no. 4 (February 8, 2024): 2068. http://dx.doi.org/10.3390/ijms25042068.
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GATOR1 (GAP Activity TOward Rag 1) is an evolutionarily conserved GTPase-activating protein complex that controls the activity of mTORC1 (mammalian Target Of Rapamycin Complex 1) in response to amino acid availability in cells. Genetic mutations in the GATOR1 subunits, NPRL2 (nitrogen permease regulator-like 2), NPRL3 (nitrogen permease regulator-like 3), and DEPDC5 (DEP domain containing 5), have been associated with epilepsy in humans; however, the specific effects of these mutations on GATOR1 function and mTORC1 regulation are not well understood. Herein, we report that epilepsy-linked mutations in the NPRL2 subunit of GATOR1, NPRL2-L105P, -T110S, and -D214H, increase basal mTORC1 signal transduction in cells. Notably, we show that NPRL2-L105P is a loss-of-function mutation that disrupts protein interactions with NPRL3 and DEPDC5, impairing GATOR1 complex assembly and resulting in high mTORC1 activity even under conditions of amino acid deprivation. Furthermore, our studies reveal that the GATOR1 complex is necessary for the rapid and robust inhibition of mTORC1 in response to growth factor withdrawal or pharmacological inhibition of phosphatidylinositol-3 kinase (PI3K). In the absence of the GATOR1 complex, cells are refractory to PI3K-dependent inhibition of mTORC1, permitting sustained translation and restricting the nuclear localization of TFEB, a transcription factor regulated by mTORC1. Collectively, our results show that epilepsy-linked mutations in NPRL2 can block GATOR1 complex assembly and restrict the appropriate regulation of mTORC1 by canonical PI3K-dependent growth factor signaling in the presence or absence of amino acids.
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Gu, Xin, Jose M. Orozco, Robert A. Saxton, Kendall J. Condon, Grace Y. Liu, Patrycja A. Krawczyk, Sonia M. Scaria, J. Wade Harper, Steven P. Gygi, and David M. Sabatini. "SAMTOR is an S-adenosylmethionine sensor for the mTORC1 pathway." Science 358, no. 6364 (November 9, 2017): 813–18. http://dx.doi.org/10.1126/science.aao3265.
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mTOR complex 1 (mTORC1) regulates cell growth and metabolism in response to multiple environmental cues. Nutrients signal via the Rag guanosine triphosphatases (GTPases) to promote the localization of mTORC1 to the lysosomal surface, its site of activation. We identified SAMTOR, a previously uncharacterized protein, which inhibits mTORC1 signaling by interacting with GATOR1, the GTPase activating protein (GAP) for RagA/B. We found that the methyl donor S-adenosylmethionine (SAM) disrupts the SAMTOR-GATOR1 complex by binding directly to SAMTOR with a dissociation constant of approximately 7 μM. In cells, methionine starvation reduces SAM levels below this dissociation constant and promotes the association of SAMTOR with GATOR1, thereby inhibiting mTORC1 signaling in a SAMTOR-dependent fashion. Methionine-induced activation of mTORC1 requires the SAM binding capacity of SAMTOR. Thus, SAMTOR is a SAM sensor that links methionine and one-carbon metabolism to mTORC1 signaling.
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Jang, Ki Beom, Agus Suryawan, Marta L. Fiorotto, and Teresa A. Davis. "PSII-18 Prematurity alters nutrient signaling and protein synthesis in skeletal muscle of neonatal piglets." Journal of Animal Science 102, Supplement_3 (September 1, 2024): 694–95. http://dx.doi.org/10.1093/jas/skae234.783.
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Abstract Extrauterine growth restriction is common in infants born preterm, and negatively affects lean mass accretion and health. Prematurity has been shown to inhibit the feeding-induced stimulation of protein synthesis in skeletal muscle. We hypothesized that nutrient signaling, activation of the mechanistic target of rapamycin complex 1 (mTORC1), and protein synthesis in the muscle are limited in preterm infants which negatively impacts muscle growth. The objective of this study was to examine how prematurity influences the mechanisms involved in insulin- and amino acid-induced signaling and protein synthesis in skeletal muscle of piglets delivered either 9 d preterm (104 d, n = 25) or at term (112 d, n = 26) by cesarean section. After resuscitation, they were surgically implanted with jugular vein and carotid artery catheters and placed in individual incubators. They were randomly allotted to one of three treatments within each preterm and term birth groups: euaminoacidemic-euglycemic (FAST), hyperinsulinemic-euaminoacidemic-euglycemic (INS), or euinsulinemic-hyperaminoacidemic-euglycemic (AA) clamps on 4 d after birth. After the clamp procedure, the piglets were euthanized to collect longissimus dorsi muscle for estimating in vivo fractional protein synthesis rates and the abundance and activation of components related to insulin and amino acid signaling. Data were analyzed using the MIXED procedure of SAS. The leucine sensor, Sestrin1 bound to GATOR2 (P < 0.05) was reduced in response to AA whereas the abundance of the mTOR-RagA and mTOR-RagC complexes increased (P < 0.05). The phosphorylation of Akt was increased (P < 0.05) in response to INS and the phosphorylation of mTORC1 and protein synthesis increased (P < 0.05) in response to both AA and INS. Prematurity did not affect the protein abundances of the AA transporters for glutamine (SLC38A2), leucine (SLC7A5), and arginine (SLC38A9). However, prematurity reduced (P < 0.05) the abundances of the leucine sensors SAR1B, the Sestrin1-GATOR complex, the AA sensor, RAB1A, and the threonine sensor, TARS2 (P < 0.05). The abundances of the glutamine sensor, ARF1, the arginine sensor, CASTOR, and the methionine-SAMTOR GATOR complex were similar. Prematurity reduced (P < 0.05) the abundance of the mTOR-RagA and mTOR-RagC complexes, the phosphorylation of mTORC1 and muscle protein synthesis (P < 0.05). In conclusion, prematurity negatively affects protein synthesis in skeletal muscle of neonatal preterm piglets by blunting anabolic pathways responsible for nutrient-sensing and mTORC1 activation. The reduced anabolic response likely contributes to reduced lean growth and extrauterine growth restriction following preterm birth.
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Smieszek, S. P. "0018 Whole Genome Sequencing Study Identifies Novel Variants Associated with Intrinsic Circadian Period in Humans." Sleep 43, Supplement_1 (April 2020): A7—A8. http://dx.doi.org/10.1093/sleep/zsaa056.017.
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Abstract Introduction Non-24 is a circadian rhythm disorder in which the master body clock runs either slightly earlier or, more commonly in the disorder, longer than 24 hours. Methods We conducted the first whole genome sequencing study of a non-24 population of 174 individuals that we identified as being totally blind with Non-24 Disorder. We have directly tested the association between SNPs and circadian period length (tau) (n=69). Linear regression corrected for PCs and covariates identified a strong signal in HCN1, Brain Cyclic Nucleotide-Gated Channel 1, HCN1. Results HCN1 channel is responsible for the feedback on the rods regulating the dynamic range of light reactivity under dim or intermediate light conditions. Minor allele rs72762058 associated with longer tau, a difference of 12 minutes, and mean tau of 24.71. In Drosophila there is only one HCN channel encoding gene, DmIh. Interestingly, DmIh mutant flies display alterations in the rest:activity pattern, and altered circadian rhythms, specifically, arrhythmic behavior or a shorter period in constant darkness. We report a variant that associated with longer tau. In addition, we identify others variants that strongly associate with tau, such as a missense variant (rs16989535), (minor allele associated with longer tau), within DEPDC5, GATOR Complex Protein). Subjects carrying the rare allele have a period > 25.2. DEPDC5 is part of GATOR1 complex, together with NPRL2 and NPRL3acts to inhibit the mTORC1 pathway. The GATOR1 seizure phenotype consists mostly of focal seizures, often sleep-related and drug-resistant and is associated with focal cortical dysplasia (20%). mTOR signaling is part of the photic entrainment pathway in the SCN, it regulates autonomous clock properties in a variety of circadian oscillators. Light-induced mTORC1 activation appears to be important for photic entrainment of the SCN clock, as rapamycin modulates light-induced phase shifts of wheel-running and body temperature rhythms in mice. Conclusion We identify variants in HCN1 and DEPDC5 implicated in significantly longer tau. Knowledge of the circadian clock and period length is not only essential for understanding of the basic clockwork mechanisms but also could provide insights into mechanistic links between circadian dysfunctions and human diseases such as epilepsy. Support Vanda Pharmaceuticals
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Xu, Dandan, Kevin L. Shimkus, Holly A. Lacko, Lydia Kutzler, Leonard S. Jefferson, and Scot R. Kimball. "Evidence for a role for Sestrin1 in mediating leucine-induced activation of mTORC1 in skeletal muscle." American Journal of Physiology-Endocrinology and Metabolism 316, no. 5 (May 1, 2019): E817—E828. http://dx.doi.org/10.1152/ajpendo.00522.2018.
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Previous studies established that leucine stimulates protein synthesis in skeletal muscle to the same extent as a complete mixture of amino acids, and the effect occurs through activation of the mechanistic target of rapamycin in complex 1 (mTORC1). Recent studies using cells in culture showed that the Sestrins bind leucine and are required for leucine-dependent activation of mTORC1. However, the role they play in mediating leucine-dependent activation of the kinase in vivo has been questioned because the dissociation constant of Sestrin2 for leucine is well below circulating and intramuscular levels of the amino acid. The goal of the present study was to compare expression of the Sestrins in skeletal muscle to other tissues and to assess their relative role in mediating activation of mTORC1 by leucine. The results show that the relative expression of the Sestrin proteins varies widely among tissues and that in skeletal muscle Sestrin1 expression is higher than Sestrin3, whereas Sestrin2 expression is markedly lower. Analysis of the dissociation constants of the Sestrins for leucine as assessed by leucine-induced dissociation of the Sestrin·GAP activity toward Rags 2 (GATOR2) complex revealed that Sestrin1 has the highest affinity for leucine and that Sestrin3 has the lowest affinity. In agreement with the dissociation constants calculated using cells in culture, oral leucine administration promotes disassembly of the Sestrin1·GATOR2 complex but not the Sestrin2 or Sestrin3·GATOR2 complex. Overall, the results presented herein are consistent with a model in which leucine-induced activation of mTORC1 in skeletal muscle in vivo occurs primarily through release of Sestrin1 from GATOR2.
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Loissell-Baltazar, Yahir A., and Svetlana Dokudovskaya. "SEA and GATOR 10 Years Later." Cells 10, no. 10 (October 8, 2021): 2689. http://dx.doi.org/10.3390/cells10102689.
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The SEA complex was described for the first time in yeast Saccharomyces cerevisiae ten years ago, and its human homologue GATOR complex two years later. During the past decade, many advances on the SEA/GATOR biology in different organisms have been made that allowed its role as an essential upstream regulator of the mTORC1 pathway to be defined. In this review, we describe these advances in relation to the identification of multiple functions of the SEA/GATOR complex in nutrient response and beyond and highlight the consequence of GATOR mutations in cancer and neurodegenerative diseases.
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Van ’t Hof, Femke, and Eva Brilstra. "Focale epilepsie en de GATOR1 complex genen." Epilepsie, periodiek voor professionals 19, no. 2 (June 1, 2021): 11–13. http://dx.doi.org/10.54160/epilepsie.11027.
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Een monogene oorzaak bij focale epilepsie is minder zeldzaam dan vroeger werd gedacht. De meest voorkomende groep erfelijke, focale epilepsieën worden veroorzaakt door varianten in de GATOR1 complex genen (DEPDC5, NPRL2 en NPRL3), ook wel de ‘GATORopathieën’ genoemd. Er is steeds meer bekend over de verschillende ziekte-uitingen van deze aandoeningen, en zelfs over de consequenties voor behandeling. Dit maakt genetische diagnostiek belangrijk.
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Laufenberg, Lacee J., Kristen T. Crowell, and Charles H. Lang. "Alcohol Acutely Antagonizes Refeeding-Induced Alterations in the Rag GTPase-Ragulator Complex in Skeletal Muscle." Nutrients 13, no. 4 (April 9, 2021): 1236. http://dx.doi.org/10.3390/nu13041236.
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The Ragulator protein complex is critical for directing the Rag GTPase proteins and mTORC1 to the lysosome membrane mediating amino acid-stimulated protein synthesis. As there is a lack of evidence on alcohol’s effect on the Rag-Ragulator complex as a possible mechanism for the development of alcoholic skeletal muscle wasting, the aim of our study was to examine alterations in various protein–protein complexes in the Rag-Ragulator pathway produced acutely by feeding and how these are altered by alcohol under in vivo conditions. Mice (C57Bl/6; adult males) were fasted, and then provided rodent chow for 30 min (“refed”) or remained food-deprived (“fasted”). Mice subsequently received ethanol (3 g/kg ethanol) or saline intraperitoneally, and hindlimb muscles were collected 1 h thereafter for analysis. Refeeding-induced increases in myofibrillar and sarcoplasmic protein synthesis, and mTOR and S6K1 phosphorylation, were prevented by alcohol. This inhibition was not associated with a differential rise in the intracellular leucine concentration or plasma leucine or insulin levels. Alcohol increased the amount of the Sestrin1•GATOR2 complex in the fasted state and prevented the refeeding-induced decrease in Sestrin1•GATOR2 seen in control mice. Alcohol antagonized the increase in the RagA/C•Raptor complex formation seen in the refed state. Alcohol antagonized the increase in Raptor with immunoprecipitated LAMPTOR1 (part of the Ragulator complex) after refeeding and decreased the association of RagC with LAMPTOR1. Finally, alcohol increased the association of the V1 domain of v-ATPase with LAMPTOR1 and prevented the refeeding-induced decrease in v-ATPase V1 with LAMPTOR1. Overall, these data demonstrate that acute alcohol intake disrupts multiple protein–protein complexes within the Rag-Ragulator complex, which are associated with and consistent with the concomitant decline in nutrient-stimulated muscle protein synthesis under in vivo conditions.
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Kowalsky, Allison Ho, Sim Namkoong, Eric Mettetal, Hwan-Woo Park, Dubek Kazyken, Diane C. Fingar, and Jun Hee Lee. "The GATOR2–mTORC2 axis mediates Sestrin2-induced AKT Ser/Thr kinase activation." Journal of Biological Chemistry 295, no. 7 (January 8, 2020): 1769–80. http://dx.doi.org/10.1074/jbc.ra119.010857.
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Sestrins represent a family of stress-inducible proteins that prevent the progression of many age- and obesity-associated disorders. Endogenous Sestrins maintain insulin-dependent AKT Ser/Thr kinase (AKT) activation during high-fat diet–induced obesity, and overexpressed Sestrins activate AKT in various cell types, including liver and skeletal muscle cells. Although Sestrin-mediated AKT activation improves metabolic parameters, the mechanistic details underlying such improvement remain elusive. Here, we investigated how Sestrin2, the Sestrin homolog highly expressed in liver, induces strong AKT activation. We found that two known targets of Sestrin2, mTOR complex (mTORC) 1 and AMP-activated protein kinase, are not required for Sestrin2-induced AKT activation. Rather, phosphoinositol 3-kinase and mTORC2, kinases upstream of AKT, were essential for Sestrin2-induced AKT activation. Among these kinases, mTORC2 catalytic activity was strongly up-regulated upon Sestrin2 overexpression in an in vitro kinase assay, indicating that mTORC2 may represent the major link between Sestrin2 and AKT. As reported previously, Sestrin2 interacted with mTORC2; however, we found here that this interaction occurs indirectly through GATOR2, a pentameric protein complex that directly interacts with Sestrin2. Deleting or silencing WDR24 (WD repeat domain 24), the GATOR2 component essential for the Sestrin2–GATOR2 interaction, or WDR59, the GATOR2 component essential for the GATOR2–mTORC2 interaction, completely ablated Sestrin2-induced AKT activation. We also noted that Sestrin2 also directly binds to the pleckstrin homology domain of AKT and induces AKT translocation to the plasma membrane. These results uncover a signaling mechanism whereby Sestrin2 activates AKT through GATOR2 and mTORC2.
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Nada, Shigeyuki, and Masato Okada. "Genetic dissection of Ragulator structure and function in amino acid-dependent regulation of mTORC1." Journal of Biochemistry 168, no. 6 (July 11, 2020): 621–32. http://dx.doi.org/10.1093/jb/mvaa076.
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Abstract Ragulator is a heteropentameric protein complex consisting of two roadblock heterodimers wrapped by the membrane anchor p18/Lamtor1. The Ragulator complex functions as a lysosomal membrane scaffold for Rag GTPases to recruit and activate mechanistic target of rapamycin complex 1 (mTORC1). However, the roles of Ragulator structure in the regulation of mTORC1 function remain elusive. In this study, we disrupted Ragulator structure by directly anchoring RagC to lysosomes and monitored the effect on amino acid-dependent mTORC1 activation. Expression of lysosome-anchored RagC in p18-deficient cells resulted in constitutive lysosomal localization and amino acid-independent activation of mTORC1. Co-expression of Ragulator in this system restored the amino acid dependency of mTORC1 activation. Furthermore, ablation of Gator1, a suppressor of Rag GTPases, induced amino acid-independent activation of mTORC1 even in the presence of Ragulator. These results demonstrate that Ragulator structure is essential for amino acid-dependent regulation of Rag GTPases via Gator1. In addition, our genetic analyses revealed new roles of amino acids in the regulation of mTORC1 as follows: amino acids could activate a fraction of mTORC1 in a Rheb-independent manner, and could also drive negative-feedback regulation of mTORC1 signalling via protein phosphatases. These intriguing findings contribute to our overall understanding of the regulatory mechanisms of mTORC1 signalling.
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Drissen, Roy, Boris Guyot, Lin Zhang, Ann Atzberger, Jackie Sloane-Stanley, Bill Wood, Catherine Porcher, and Paresh Vyas. "Lineage-specific combinatorial action of enhancers regulates mouse erythroid Gata1 expression." Blood 115, no. 17 (April 29, 2010): 3463–71. http://dx.doi.org/10.1182/blood-2009-07-232876.
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Abstract Precise spatiotemporal control of Gata1 expression is required in both early hematopoietic progenitors to determine erythroid/megakaryocyte versus granulocyte/monocyte lineage output and in the subsequent differentiation of erythroid cells and megakaryocytes. An enhancer element upstream of the mouse Gata1 IE (1st exon erythroid) promoter, mHS−3.5, can direct both erythroid and megakaryocytic expression. However, loss of this element ablates only megakaryocytes, implying that an additional element has erythroid specificity. Here, we identify a double DNaseI hypersensitive site, mHS−25/6, as having erythroid but not megakaryocytic activity in primary cells. It binds an activating transcription factor complex in erythroid cells where it also makes physical contact with the Gata1 promoter. Deletion of mHS−25/6 or mHS−3.5 in embryonic stem cells has only a modest effect on in vitro erythroid differentiation, whereas loss of both elements ablates both primitive and definitive erythropoiesis with an almost complete loss of Gata1 expression. Surprisingly, Gata2 expression was also concomitantly low, suggesting a more complex interaction between these 2 factors than currently envisaged. Thus, whereas mHS−3.5 alone is sufficient for megakaryocytic development, mHS−3.5 and mHS−25/6 collectively regulate erythroid Gata1 expression, demonstrating lineage-specific differences in Gata1 cis-element use important for development of these 2 cell types.
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Vagapova, E. R., P. V. Spirin, T. D. Lebedev, and V. S. Prassolov. "The Role of TAL1 in Hematopoiesis and Leukemogenesis." Acta Naturae 10, no. 1 (March 15, 2018): 15–23. http://dx.doi.org/10.32607/20758251-2018-10-1-15-23.
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TAL1 (SCL/TAL1, T-cell acute leukemia protein 1) is a transcription factor that is involved in the process of hematopoiesis and leukemogenesis. It participates in blood cell formation, forms mesoderm in early embryogenesis, and regulates hematopoiesis in adult organisms. TAL1 is essential in maintaining the multipotency of hematopoietic stem cells (HSC) and keeping them in quiescence (stage G0). TAL1 forms complexes with various transcription factors, regulating hematopoiesis (E2A/HEB, GATA1-3, LMO1-2, Ldb1, ETO2, RUNX1, ERG, FLI1). In these complexes, TAL1 regulates normal myeloid differentiation, controls the proliferation of erythroid progenitors, and determines the choice of the direction of HSC differentiation. The transcription factors TAL1, E2A, GATA1 (or GATA2), LMO2, and Ldb1 are the major components of the SCL complex. In addition to normal hematopoiesis, this complex may also be involved in the process of blood cell malignant transformation. Upregulation of C-KIT expression is one of the main roles played by the SCL complex. Today, TAL1 and its partners are considered promising therapeutic targets in the treatment of T-cell acute lymphoblastic leukemia.
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Papadopoulos, Petros, Laura Gutiérrez, Jeroen Demmers, Elisabeth Scheer, Farzin Pourfarzad, Dimitris N. Papageorgiou, Elena Karkoulia, et al. "TAF10 Interacts with the GATA1 Transcription Factor and Controls Mouse Erythropoiesis." Molecular and Cellular Biology 35, no. 12 (April 13, 2015): 2103–18. http://dx.doi.org/10.1128/mcb.01370-14.
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The ordered assembly of a functional preinitiation complex (PIC), composed of general transcription factors (GTFs), is a prerequisite for the transcription of protein-coding genes by RNA polymerase II. TFIID, comprised of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs), is the GTF that is thought to recognize the promoter sequences allowing site-specific PIC assembly. Transcriptional cofactors, such as SAGA, are also necessary for tightly regulated transcription initiation. The contribution of the two TAF10-containing complexes (TFIID, SAGA) to erythropoiesis remains elusive. By ablating TAF10 specifically in erythroid cellsin vivo, we observed a differentiation block accompanied by deregulated GATA1 target genes, includingGata1itself, suggesting functional cross talk between GATA1 and TAF10. Additionally, we analyzed by mass spectrometry the composition of TFIID and SAGA complexes in mouse and human cells and found that their global integrity is maintained, with minor changes, during erythroid cell differentiation and development. In agreement with our functional data, we show that TAF10 interacts directly with GATA1 and that TAF10 is enriched on theGATA1locus in human fetal erythroid cells. Thus, our findings demonstrate a cross talk between canonical TFIID and SAGA complexes and cell-specific transcription activators during development and differentiation.
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Hesketh, Geoffrey G., Fotini Papazotos, Judy Pawling, Dushyandi Rajendran, James D. R. Knight, Sebastien Martinez, Mikko Taipale, Daniel Schramek, James W. Dennis, and Anne-Claude Gingras. "The GATOR–Rag GTPase pathway inhibits mTORC1 activation by lysosome-derived amino acids." Science 370, no. 6514 (October 15, 2020): 351–56. http://dx.doi.org/10.1126/science.aaz0863.
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The mechanistic target of rapamycin complex 1 (mTORC1) couples nutrient sufficiency to cell growth. mTORC1 is activated by exogenously acquired amino acids sensed through the GATOR–Rag guanosine triphosphatase (GTPase) pathway, or by amino acids derived through lysosomal degradation of protein by a poorly defined mechanism. Here, we revealed that amino acids derived from the degradation of protein (acquired through oncogenic Ras-driven macropinocytosis) activate mTORC1 by a Rag GTPase–independent mechanism. mTORC1 stimulation through this pathway required the HOPS complex and was negatively regulated by activation of the GATOR-Rag GTPase pathway. Therefore, distinct but functionally coordinated pathways control mTORC1 activity on late endocytic organelles in response to distinct sources of amino acids.
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Meng, Jin, and Shawn M. Ferguson. "GATOR1-dependent recruitment of FLCN–FNIP to lysosomes coordinates Rag GTPase heterodimer nucleotide status in response to amino acids." Journal of Cell Biology 217, no. 8 (May 30, 2018): 2765–76. http://dx.doi.org/10.1083/jcb.201712177.
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Folliculin (FLCN) is a tumor suppressor that coordinates cellular responses to changes in amino acid availability via regulation of the Rag guanosine triphosphatases. FLCN is recruited to lysosomes during amino acid starvation, where it interacts with RagA/B as a heterodimeric complex with FLCN-interacting proteins (FNIPs). The FLCN–FNIP heterodimer also has GTPase-activating protein (GAP) activity toward RagC/D. These properties raised two important questions. First, how is amino acid availability sensed to regulate lysosomal abundance of FLCN? Second, what is the relationship between FLCN lysosome localization, RagA/B interactions, and RagC/D GAP activity? In this study, we show that RagA/B nucleotide status determines the FLCN–FNIP1 recruitment to lysosomes. Starvation-induced FLCN–FNIP lysosome localization requires GAP activity toward Rags 1 (GATOR1), the GAP that converts RagA/B to the guanosine diphosphate (GDP)-bound state. This places FLCN–FNIP recruitment to lysosomes under the control of amino acid sensors that act upstream of GATOR1. By binding to RagA/BGDP and acting on RagC/D, FLCN–FNIP can coordinate nucleotide status between Rag heterodimer subunits in response to changes in amino acid availability.
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Freson, Kathleen, Koen Devriendt, Gert Matthijs, Achiel Van Hoof, Rita De Vos, Chantal Thys, Kristien Minner, Marc F. Hoylaerts, Jos Vermylen, and Chris Van Geet. "Platelet characteristics in patients with X-linked macrothrombocytopenia because of a novel GATA1mutation." Blood 98, no. 1 (July 1, 2001): 85–92. http://dx.doi.org/10.1182/blood.v98.1.85.
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Abstract A new mutation is described in the X-linked geneGATA1, resulting in macrothrombocytopenia and mild dyserythropoietic features but no marked anemia in a 4-generation family. The molecular basis for the observed phenotype is a substitution of glycine for aspartate in the strictly conserved codon 218 (D218G) of the amino-terminal zinc finger loop of the transcription factor GATA1. Zinc finger interaction studies demonstrated that this mutation results in a weak loss of affinity of GATA1 for its essential cofactor FOG1, whereas direct D218G-GATA1 binding to DNA was normal. The phenotypic effects of this mutation in the patients' platelets have been studied. Semiquantitative RNA analysis, normalized for β-actin messenger RNA, showed extremely low transcription of the GATA1 target genes GPIbβ and GPIXbut also a significantly lower expression of the nondirectly GATA1-regulated Gsα gene, suggestive of incomplete megakaryocyte maturation. In contrast, GPIIIa expression was close to normal in agreement with its early appearance during megakaryocyte differentiation. Flow cytometric analysis of patient platelets confirmed the existence of a platelet population with abnormal size distribution and reduced GPIb complex levels but with normal GPIIIa expression. It also showed the presence of very immature platelets lacking almost all membrane glycoproteins studied (GPIbα, GPIbβ, GPIIIa, GPIX, and GPV). Patients' platelets showed weak ristocetin-induced agglutination, compatible with the disturbed GPIb complex. Accordingly, electron microscopy of the patients' platelets revealed giant platelets with cytoplasmic clusters consisting of smooth endoplasmic reticulum and abnormal membrane complexes. In conclusion,GATA1 mutations can lead to isolated X-linked macrothrombocytopenia without anemia.
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Krenn, Martin, Matias Wagner, Christoph Hotzy, Elisabeth Graf, Sandrina Weber, Theresa Brunet, Bettina Lorenz-Depiereux, et al. "Diagnostic exome sequencing in non-acquired focal epilepsies highlights a major role of GATOR1 complex genes." Journal of Medical Genetics 57, no. 9 (February 21, 2020): 624–33. http://dx.doi.org/10.1136/jmedgenet-2019-106658.
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BackgroundThe genetic architecture of non-acquired focal epilepsies (NAFEs) becomes increasingly unravelled using genome-wide sequencing datasets. However, it remains to be determined how this emerging knowledge can be translated into a diagnostic setting. To bridge this gap, we assessed the diagnostic outcomes of exome sequencing (ES) in NAFE.Methods112 deeply phenotyped patients with NAFE were included in the study. Diagnostic ES was performed, followed by a screen to detect variants of uncertain significance (VUSs) in 15 well-established focal epilepsy genes. Explorative gene prioritisation was used to identify possible novel candidate aetiologies with so far limited evidence for NAFE.ResultsES identified pathogenic or likely pathogenic (ie, diagnostic) variants in 13/112 patients (12%) in the genes DEPDC5, NPRL3, GABRG2, SCN1A, PCDH19 and STX1B. Two pathogenic variants were microdeletions involving NPRL3 and PCDH19. Nine of the 13 diagnostic variants (69%) were found in genes of the GATOR1 complex, a potentially druggable target involved in the mammalian target of rapamycin (mTOR) signalling pathway. In addition, 17 VUSs in focal epilepsy genes and 6 rare variants in candidate genes (MTOR, KCNA2, RBFOX1 and SCN3A) were detected. Five patients with reported variants had double hits in different genes, suggesting a possible (oligogenic) role of multiple rare variants.ConclusionThis study underscores the molecular heterogeneity of NAFE with GATOR1 complex genes representing the by far most relevant genetic aetiology known to date. Although the diagnostic yield is lower compared with severe early-onset epilepsies, the high rate of VUSs and candidate variants suggests a further increase in future years.
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Hamlett, Isla, Julia Draper, John Strouboulis, Francisco Iborra, Catherine Porcher, and Paresh Vyas. "Characterization of megakaryocyte GATA1-interacting proteins: the corepressor ETO2 and GATA1 interact to regulate terminal megakaryocyte maturation." Blood 112, no. 7 (October 1, 2008): 2738–49. http://dx.doi.org/10.1182/blood-2008-03-146605.
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Abstract The transcription factor GATA1 coordinates timely activation and repression of megakaryocyte gene expression. Loss of GATA1 function results in excessive megakaryocyte proliferation and disordered terminal platelet maturation, leading to thrombocytopenia and leukemia in patients. The mechanisms by which GATA1 does this are unclear. We have used in vivo biotinylated GATA1 to isolate megakaryocyte GATA1-partner proteins. Here, several independent approaches show that GATA1 interacts with several proteins in the megakaryocyte cell line L8057 and in primary megakaryocytes. They include FOG1, the NURD complex, the pentameric complex containing SCL/TAL-1, the zinc-finger regulators GFI1B and ZFP143, and the corepressor ETO2. Knockdown of ETO2 expression promotes megakaryocyte differentiation and enhances expression of select genes expressed in terminal megakaryocyte maturation, eg, platelet factor 4 (Pf4). ETO2-dependent direct repression of the Pf4 proximal promoter is mediated by GATA-binding sites and an E-Box motif. Consistent with this, endogenous ETO2, GATA1, and the SCL pentameric complex all specifically bind the promoter in vivo. Finally, as ETO2 expression is restricted to immature megakaryocytes, these data suggest that ETO2 directly represses inappropriate early expression of a subset of terminally expressed megakaryocyte genes by binding to GATA1 and SCL.
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Figlia, Gianluca, Sandra Müller, Anna M. Hagenston, Susanne Kleber, Mykola Roiuk, Jan-Philipp Quast, Nora ten Bosch, et al. "Brain-enriched RagB isoforms regulate the dynamics of mTORC1 activity through GATOR1 inhibition." Nature Cell Biology 24, no. 9 (September 2022): 1407–21. http://dx.doi.org/10.1038/s41556-022-00977-x.
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AbstractMechanistic target of rapamycin complex 1 (mTORC1) senses nutrient availability to appropriately regulate cellular anabolism and catabolism. During nutrient restriction, different organs in an animal do not respond equally, with vital organs being relatively spared. This raises the possibility that mTORC1 is differentially regulated in different cell types, yet little is known about this mechanistically. The Rag GTPases, RagA or RagB bound to RagC or RagD, tether mTORC1 in a nutrient-dependent manner to lysosomes where mTORC1 becomes activated. Although the RagA and B paralogues were assumed to be functionally equivalent, we find here that the RagB isoforms, which are highly expressed in neurons, impart mTORC1 with resistance to nutrient starvation by inhibiting the RagA/B GTPase-activating protein GATOR1. We further show that high expression of RagB isoforms is observed in some tumours, revealing an alternative strategy by which cancer cells can retain elevated mTORC1 upon low nutrient availability.
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Snow, Jonathan W., and Stuart H. Orkin. "Translational Isoforms of FOG1 Regulate GATA1-interacting Complexes." Journal of Biological Chemistry 284, no. 43 (August 4, 2009): 29310–19. http://dx.doi.org/10.1074/jbc.m109.043497.
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Mishima, Yuta, Satoru Miyagi, Atsunori Saraya, Masamitsu Negishi, Mitsuhiro Endoh, Takaho A. Endo, Tetsuro Toyoda, et al. "The Hbo1-Brd1/Brpf2 complex is responsible for global acetylation of H3K14 and required for fetal liver erythropoiesis." Blood 118, no. 9 (September 1, 2011): 2443–53. http://dx.doi.org/10.1182/blood-2011-01-331892.
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Abstract The histone acetyltransferases (HATs) of the MYST family include TIP60, HBO1, MOZ/MORF, and MOF and function in multisubunit protein complexes. Bromodomain-containing protein 1 (BRD1), also known as BRPF2, has been considered a subunit of the MOZ/MORF H3 HAT complex based on analogy with BRPF1 and BRPF3. However, its physiologic function remains obscure. Here we show that BRD1 forms a novel HAT complex with HBO1 and regulates erythropoiesis. Brd1-deficient embryos showed severe anemia because of impaired fetal liver erythropoiesis. Biochemical analyses revealed that BRD1 bridges HBO1 and its activator protein, ING4. Genome-wide mapping in erythroblasts demonstrated that BRD1 and HBO1 largely colocalize in the genome and target key developmental regulator genes. Of note, levels of global acetylation of histone H3 at lysine 14 (H3K14) were profoundly decreased in Brd1-deficient erythroblasts and depletion of Hbo1 similarly affected H3K14 acetylation. Impaired erythropoiesis in the absence of Brd1 accompanied reduced expression of key erythroid regulator genes, including Gata1, and was partially restored by forced expression of Gata1. Our findings suggest that the Hbo1-Brd1 complex is the major H3K14 HAT required for transcriptional activation of erythroid developmental regulator genes.
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Varricchio, Lilian, Carmela Dell'Aversana, Angela Nebbioso, Giovanni Migliaccio, Lucia Altucci, James J. Bieker, and Anna Rita F. Migliaccio. "Identification of a New Functional HDAC Complex Composed by HDAC5, GATA1 and EKLF in Human Erythroid Cells." Blood 120, no. 21 (November 16, 2012): 979. http://dx.doi.org/10.1182/blood.v120.21.979.979.
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Abstract Abstract 979 Histone deacetylation, the reaction that maintains chromatin in a condensed configuration preventing gene expression, is catalyzed by the histone deacetylase (HDAC) superfamily. The human HDAC family includes 18 different isoforms classified on the basis of their sequence homology to HDACs from Saccharomyces Cerevisiae into class I (HDAC1, −2, −3, and −8), IIa (HDAC4, −5, −7, and −9), IIb (HDAC6 and −10) and IV (HDAC11). Class I HDACs bind the DNA directly while class IIa HDACs shuffles other proteins between nucleus and cytoplasm. While the role of individual class I HDACs in erythropoiesis is starting to emerge, that of class IIa and b HDACs is still largely unknown. To clarify the role played by class IIa HDACs in the control of human erythropoiesis, an extensive analysis of expression, activity, and function of different classes of HDACs during the maturation of erythroblasts derived in vitro from adult blood or cord blood was performed. HDACs expression/activity. Erythroid maturation was associated with increased expression of class I HDACs (both mRNA and protein) which, in the case of HDAC1, was also associated with increased enzymatic activity and association with its NuRD partner GATA1. By contrast, reductions either in expression (HDAC4) or activity (HDAC5) of class IIa HDACs were observed with maturation. In addition, GATA1 and EKLF were consistently found associated in human erythroblasts but EKLF was not found associated with HDAC1. The extent of nuclear-cytoplasmic trafficking of class I (HDAC1 and 2) and IIa (HDAC4 and 5) and of the transcription factors EKLF and GATA1 in response to EPO was determined. HDAC2/EKLF/GATA1 and HDAC4 were found constitutively present in the nucleus and in the cytoplasm, respectively. By contrast, the nuclear concentration of HDAC1 increased while that of HDAC5 and of GATA1fl decreased upon stimulation with EPO. The last two observations suggested that HDAC5, GATA1 and EKLF might be associated in a complex. Identification of the HDAC5/EKLF/GATA1 complex. A series of IPs followed by WB experiments showed that HDAC5 was consistently associated with EKLF and GATA1 and conversely, both GATA1 (preferentially GATA1fl over GATA1s) and EKLF were consistently associated with HDAC5 (Fig 1A and not shown). Interestingly also pERK was detected in IPs with HDAC5, EKLF and GATA1 antibodies. These results indicate that in erythroid cells HDAC5 forms a complex with GATA1, EKLF and pERK. Identification of the biological activity of the HDAC5/GATA1/EKLF/pERK complex. The association between GATA1/EKLF was greater in cells generated with cord blood (which express high HbF levels) than in those derived from adult blood and their association decreased with maturation, suggesting that the complex may regulate HbF expression. To confirm this hypothesis, HDAC5/GATA1 association and γ/(γ+ β) mRNA ratios were determined in erythroid cells induced to mature in the presence of a pan-class II-specific (APHA9, ID50=20 μM for HDAC4) HDAC inhibitor (HDACi) (Fig 1B)1. Cells exposed in parallel to the class I/IIa-specific (UBHA24, ID50 =0.2 and 0.6 μM for HDAC1 and HDAC4, respectively) HDACi, were used as control. Exposure to APHA9 reduced the association between GATA1 and HDAC5 and increased γ/(γ + β) mRNA expression ratio, while this association was not affected by exposure to the class I/II HDACi which, as expected, also increased γ/(γ+ β) mRNA ratio. Conclusions. These data identify a new HDAC complex formed by HDAC5, EKLF and GATA1 that regulates γ/(γ + β) ratio. We hypothesize that the biological role of this new complex is to shuffle GATA1 and EKLF from the cytoplasm to the nucleus, making them able to engage into the NuRD and Sin3A complex respectively, and that inhibition of the activity of this complex affects γ-globin expression indirectly by limiting the amount of GATA1and EKLF available to associate with NuRD and Sin3A. Disclosures: No relevant conflicts of interest to declare.
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Suryawan, Agus, Marko Rudar, Marta L. Fiorotto, and Teresa A. Davis. "Differential regulation of mTORC1 activation by leucine and β-hydroxy-β-methylbutyrate in skeletal muscle of neonatal pigs." Journal of Applied Physiology 128, no. 2 (February 1, 2020): 286–95. http://dx.doi.org/10.1152/japplphysiol.00332.2019.
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Leucine (Leu) and its metabolite β-hydroxy-β-methylbutyrate (HMB) stimulate mechanistic target of rapamycin (mTOR) complex 1 (mTORC1)-dependent protein synthesis in the skeletal muscle of neonatal pigs. This study aimed to determine whether HMB and Leu utilize common nutrient-sensing mechanisms to activate mTORC1. In study 1, neonatal pigs were fed one of five diets for 24 h: low protein (LP), high protein (HP), or LP supplemented with 4 (LP+HMB4), 40 (LP+HMB40), or 80 (LP+HMB80) μmol HMB·kg body wt−1·day−1. In study 2, neonatal pigs were fed for 24 h: LP, LP supplemented with Leu (LP+Leu), or HP diets delivering 9, 18, and 18 mmol Leu·kg body wt−1·day−1, respectively. The upstream signaling molecules that regulate mTORC1 activity were analyzed. mTOR phosphorylation on Ser2448 and Ser2481 was greater in LP+HMB40, LP+HMB80, and LP+Leu than in LP and greater in HP than in HMB-supplemented groups ( P < 0.05), whereas HP and LP+Leu were similar. Rheb-mTOR complex formation was lower in LP than in HP ( P < 0.05), with no enhancement by HMB or Leu supplementation. The Sestrin2-GATOR2 complex was more abundant in LP than in HP and was reduced by Leu ( P < 0.05) but not HMB supplementation. RagA-mTOR and RagC-mTOR complexes were higher in LP+Leu and HP than in LP and HMB groups ( P < 0.05). There were no treatment differences in RagB-SH3BP4, Vps34-LRS, and RagD-LRS complex abundances. Phosphorylation of Erk1/2 and TSC2, but not AMPK, was lower in LP than HP ( P < 0.05) and unaffected by HMB or Leu supplementation. Our results demonstrate that HMB stimulates mTORC1 activation in neonatal muscle independent of the leucine-sensing pathway mediated by Sestrin2 and the Rag proteins. NEW & NOTEWORTHY Dietary supplementation with either leucine or its metabolite β-hydroxy-β-methylbutyrate (HMB) stimulates protein synthesis in skeletal muscle of the neonatal pig. Our results demonstrate that both leucine and HMB stimulate mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) phosphorylation in neonatal muscle. This leucine-stimulated process involves dissociation of the Sestrin2-GATOR2 complex and increased binding of Rag A/C to mTOR. However, HMB’s activation of mTORC1 is independent of this leucine-sensing pathway.
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Tauchmann, Samantha, Frederik Otzen Bagger, Thomas Bock, Roos Krimpenfort, Francesca Aglialoro, Peter Valent, Alexandre Fagnan, Marieke von Lindern, Thomas Mercher, and Juerg Schwaller. "Dissecting GATA1 Protein Interactions in Normal and Malignant Human Erythroblasts." Blood 138, Supplement 1 (November 5, 2021): 3293. http://dx.doi.org/10.1182/blood-2021-148351.
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Abstract Acute erythroid leukemia (AEL) is characterized by uncontrolled accumulation of transformed erythroblasts. Previous analysis of murine and human AEL revealed aberrant regulation of the master regulator GATA1, which controls terminal erythroid differentiation in multi-protein complexes acting as activators or repressors of gene expression. Although most malignant erythroblasts constitutively express abundant GATA1 protein, terminal erythroid differentiation is impaired. Notably, overexpression of GATA1 significantly induced partial or complete terminal erythroid differentiation of the human AEL cell line K562 or immortalised HUDEP2 human erythroblasts, respectively. These observations led us to hypothesize that blocked terminal erythroid differentiation in AEL might be the consequence of titratable dose-dependent aberrant GATA1 protein interactions. We comparatively analysed nuclear extracts from three human AEL cell lines (F36P, K562, KMOE2) and primary cells from an AEL patient. In addition, we analysed HUDEP2 and primary human erythroblasts (hEBST) from healthy donors that retain the potential for complete in vitro terminal erythroid differentiation. We quantified protein expression using a tandem mass tag (TMT) based approach (n=3/cell type) and we compared putative GATA1 interactions by immunoprecipitation (IP) followed by liquid chromatography mass spectrometry (MS) (n=3/cell type). Quantitative proteomics identified 6774 commonly expressed proteins in AEL and "normal" erythroblasts with a high reproducibility (mean coefficients of variation <10%) for all six different cell types. Unsupervised hierarchical clustering displayed a clear separation of the AEL cells from the "normal erythroblasts" (hEBST, HUDEP2). 386 proteins were higher expressed in the AEL group (logFC>=2; q<0.05), whereas 623 were more abundant in normal erythroblasts (logFC>=2; q<0.05). IP-MS analysis of nuclear lysates from the AEL cell lines, the AEL primary sample, HUDEP2 and hEBST resulted in a matrix containing 1616 proteins from which 126 proteins seem to significantly differentially interact with GATA1. 54 proteins were more enriched in the AEL group, whereas 72 proteins were more enriched in "normal" erythroblasts (q<0.5). Principal component analysis (PCA) showed for all cell lines a similar clustering pattern, accounting for 24% and 32% of the variance. Pulled-down proteins in hEBST and HUDEP2 clustered together and were closer to F36P and KMOE, than LAM49 and K562. Notably, we found significant enrichment (validated by immunoblotting) of the SKI protooncogene in AEL cells (logFC=1.82; q=0.013), a finding which not only confirmed previous findings in murine AEL models (MEL cells, erythroblasts from Nsd1 -/- mice) but also speaks for the functionality of our approach. Similarly, the LRPPRC leucine-rich PPR-motif-containing protein overexpressed in several cancers, as well the lactate dehydrogenases A and B (LDHA, LDHB) were significantly enriched in malignant erythroblasts (logFC>2; q<0.05). Furthermore, the ZEB2 zinc finger E-box-binding homeobox 2 protein, was significantly enriched in AEL cells (logFC=2.02; q=0.005). In contrast, the hematopoietic master transcription factor Runt-related transcription factor 1 (RUNX1) (logFC=2.48; q=0.0018) as well as DNA binding protein Ikaros (IKZF1) (logFC=1.71; q=0.13) were significantly enriched (validated by immunoblotting) in HUDEP2 and hEBST. Moreover, the MCM6 DNA binding mini-chromosome maintenance complex component 6 critical for proper DNA replication was enriched in normal erythroblasts (logFC=1.99; q=0.0001). Interestingly, one of the most strongly enriched (and validated by immunoblotting) proteins in normal erythroblasts was the nuclear pore complex protein NUP155 (logFC=6.1; q=0.0000001). Integration of the quantitative proteomics and the IP-MS analysis identified 118 proteins differentially expressed and differentially pulled-down by GATA1-IP, of which 49 were enriched in malignant and 69 proteins in normal erythroblasts (q<0.5). This shows that we reproducibly identified proteins that are differentially associated with GATA1 which are also differentially expressed in AEL cells versus normal erythroblasts. A targeted CRISPR/Cas9 screen is under way to identify GATA1-interacting proteins responsible for impaired erythroid differentiation of AEL cells. Disclosures Valent: Novartis: Honoraria; Pfizer: Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; OAP Orphan Pharmaceuticals: Honoraria.
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Migliaccio, Giovanni, Carmela Dell’Aversana, Angela Nebbioso, Elena Alfani, Lilian arricchio, Antonello Mai, Pratima Chaurasia, et al. "Ontogenic-Specific Increasesin HDAC1 Activity and Transcription Factor Association During the Maturation of Human Adult Erythroblasts in Vitro." Blood 114, no. 22 (November 1, 2009): 1978. http://dx.doi.org/10.1182/blood.v114.22.1978.1978.
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Abstract Abstract 1978 Poster Board I-1000 Histone deacetylation is one of the major pathways that maintains chromatin in a condensed configuration preventing gene expression in eukaryotic cells. The deacetylation reaction is catalyzed by the histone deacetylase (HDAC) superfamily, which includes eighteen distinct enzymes. HDACs perform their biological function as multiprotein complexes (Sin3A, NuRD and CoREST) that include at least two HDAC isoforms, DNA docking factors (transcription factors and methyl-binding proteins) and protein kinases (PKC). Data from murine cell lines suggest that association of HDAC1 with EKLF and/or Gata1, which occurs as part of the Sin3A or NuRD complex, may provide specificity to the regulation exerted by this enzyme during erythroid maturation. The role of HDAC complexes in primary human erythroid cells has remained poorly defined. The objective of this study was to characterize HDAC expression in human erythroblasts (EB) and monitor changes in expression and activity during maturation in response to erythropoietin (EPO). Human immature EB (iEB) were generated by culturing adult blood (AB) and cord blood (CB) mononuclear cells for 10-12 days with SCF, IL-3, EPO, dexamethasone and estradiol and then for 24-72 hrs in cultures containing EPO alone (mature EB, mEB) (Migliaccio et al, BCMC 28:168, 2002). The levels of HDAC isoform mRNAs and proteins expressed by iEB and mEB, as well as levels of HDAC1 and HDAC5 activity and association of HDAC1 with either GATA1 or EKLF, were then determined. By quantitative RT-PCR, iEB expressed detectable levels of mRNA for all HDAC isoforms, including SIRT 1 and 2. Induction of maturation had modest effects on the level of HDAC mRNA expressed by the EB with the exception of the mRNA for SIRT2 (increased by 10-fold), HDAC2 and HDAC6 (both increased by 2-3-fold). The increase in HDAC6 mRNA observed with maturation correlated with that of GATA1 (HDAC6 is immediately downstream to GATA1). By western-blot analyses, iEB expressed high levels only of HDAC1 to 5 and SIRT1 and 2. Induction of maturation did not affect the HDAC2 and HDAC3 but decreased HDAC1, HDAC4 and HDAC5 and increased SIRT2 protein levels. Therefore, the levels of mRNAs for these genes remained constant but their protein levels decreased with maturation. To evaluate the effect of decrements in protein level on enzymatic activity, the activity of complexes immunoprecipitated with antibodies specific for HDAC1 and HDAC5, the enzymes whose content decreased the most with maturation, from similar numbers of iEB and mEB was compared. iEB expressed HDAC1 and HDAC5 activity levels 2-fold greater than the standard (HeLa extracts). In agreement with the protein levels, HDAC5 activity decreased (by 1-log) with maturation. However, the activity of HDAC1 increased by 2-fold upon EPO exposure. To further characterize the interactions between transcription factors with HDAC1 within the complex, western-blot analyses of proteins co-immunoprecipitated with GATA1 (or HDAC1) from iEB and mEB obtained from CB and AB were compared (see Figure). A greater fraction of GATA 1 was associated with HDAC1 and EKLF in iEB obtained from CB than in those obtained from AB and in both cases the association increased with maturation. In conclusion, these results extend those previously observed with cell lines (Chen and Bieker, Mol Cell Biol 24:10416, 2004) and suggest that erythroid maturation of primary cells is associated with the dynamic regulation of the HDAC1-complex that includes increased enzymatic activity and ontogenetic-specific re-organization of transcription factors recruited to the complex. Disclosures: No relevant conflicts of interest to declare.Disclosures: No relevant conflicts of interest to declare.
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Nishikawa, Keizo, Makoto Kobayashi, Atsuko Masumi, Susan E. Lyons, Brant M. Weinstein, P. Paul Liu, and Masayuki Yamamoto. "Self-Association of Gata1 Enhances Transcriptional Activity In Vivo in Zebra Fish Embryos." Molecular and Cellular Biology 23, no. 22 (November 15, 2003): 8295–305. http://dx.doi.org/10.1128/mcb.23.22.8295-8305.2003.
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ABSTRACT Gata1 is a prototype transcription factor that regulates hematopoiesis, yet the molecular mechanisms by which Gata1 transactivates its target genes in vivo remain unclear. We previously showed, in transgenic zebra fish, that Gata1 autoregulates its own expression. In this study, we characterized the molecular mechanisms for this autoregulation by using mutations in the Gata1 protein which impair autoregulation. Of the tested mutations, replacement of six lysine residues with alanine (Gata1KA6), which inhibited self-association activity of Gata1, reduced the Gata1-dependent induction of reporter gene expression driven by the zebra fish gata1 hematopoietic regulatory domain (gata1 HRD). Furthermore, overexpression of wild-type Gata1 but not Gata1KA6 rescued the expression of Gata1 downstream genes in vlad tepes, a germ line gata1 mutant fish. Interestingly, both GATA sites in the double GATA motif in gata1 HRD were critical for the promoter activity and for binding of the self-associated Gata1 complex, whereas only the 3′-GATA site was required for Gata1 monomer binding. These results thus provide the first in vivo evidence that the ability of Gata1 to self-associate critically contributes to the autoregulation of the gata1 gene.
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Hasegawa, Atsushi, Hiroshi Kaneko, Daishi Ishihara, Masahiro Nakamura, Akira Watanabe, Masayuki Yamamoto, Cecelia D. Trainor, and Ritsuko Shimizu. "GATA1 Binding Kinetics on Conformation-Specific Binding Sites Elicit Differential Transcriptional Regulation." Molecular and Cellular Biology 36, no. 16 (May 23, 2016): 2151–67. http://dx.doi.org/10.1128/mcb.00017-16.
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GATA1 organizes erythroid and megakaryocytic differentiation by orchestrating the expression of multiple genes that show diversified expression profiles. Here, we demonstrate that GATA1 monovalently binds to a single GATA motif (Single-GATA) while a monomeric GATA1 and a homodimeric GATA1 bivalently bind to two GATA motifs in palindromic (Pal-GATA) and direct-repeat (Tandem-GATA) arrangements, respectively, and form higher stoichiometric complexes on respective elements. The amino-terminal zinc (N) finger of GATA1 critically contributes to high occupancy of GATA1 on Pal-GATA. GATA1 lacking the N finger-DNA association fails to trigger a rate of target gene expression comparable to that seen with the wild-type GATA1, especially when expressed at low level. This study revealed that Pal-GATA and Tandem-GATA generate transcriptional responses from GATA1 target genes distinct from the response of Single-GATA. Our results support the notion that the distinct alignments in binding motifs are part of a critical regulatory strategy that diversifies and modulates transcriptional regulation by GATA1.
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Suryawan, Agus, and Teresa A. Davis. "Amino Acid- and Insulin-Induced Activation of mTORC1 in Neonatal Piglet Skeletal Muscle Involves Sestrin2-GATOR2, Rag A/C-mTOR, and RHEB-mTOR Complex Formation." Journal of Nutrition 148, no. 6 (May 23, 2018): 825–33. http://dx.doi.org/10.1093/jn/nxy044.
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Abstract Background Feeding stimulates protein synthesis in skeletal muscle of neonates and this response is regulated through activation of mechanistic target of rapamycin complex 1 (mTORC1). The identity of signaling components that regulate mTORC1 activation in neonatal muscle has not been fully elucidated. Objective We investigated the independent effects of the rise in amino acids (AAs) and insulin after a meal on the abundance and activation of potential regulators of mTORC1 in muscle and whether the responses are modified by development. Methods Overnight-fasted 6- and 26-d-old pigs were infused for 2 h with saline (control group) or with a balanced AA mixture (AA group) or insulin (INS group) to achieve fed levels while insulin or AAs, respectively, and glucose were maintained at fasting levels. Muscles were analyzed for potential mTORC1 regulatory mechanisms and results were analyzed by 2-factor ANOVA followed by Tukey's post hoc test. Results The abundances of DEP domain-containing mTOR-interacting protein (DEPTOR), growth factor receptor bound protein 10 (GRB10), and regulated in development and DNA damage response 2 (REDD2) were lower (65%, 73%, and 53%, respectively; P < 0.05) and late endosomal/lysosomal adaptor, MAPK and mTOR activator 1/2 (LAMTOR1/2), vacuolar H+-ATPase (V-ATPase), and Sestrin2 were higher (94%, 141%, 145%, and 127%, respectively; P < 0.05) in 6- than in 26-d-old pigs. Both AA and INS groups increased phosphorylation of GRB10 (P < 0.05) compared with control in 26- but not in 6-d-old pigs. Formation of Ras-related GTP-binding protein A (RagA)-mTOR, RagC-mTOR, and Ras homolog enriched in brain (RHEB)-mTOR complexes was increased (P < 0.05) and Sestrin2-GTPase activating protein activity towards Rags 2 (GATOR2) complex was decreased (P < 0.05) by both AA and INS groups and these responses were greater (P < 0.05) in 6- than in 26-d-old pigs. Conclusion The results suggest that formation of RagA-mTOR, RagC-mTOR, RHEB-mTOR, and Sestrin2-GATOR2 complexes may be involved in the AA- and INS-induced activation of mTORC1 in skeletal muscle of neonates after a meal and that enhanced activation of the mTORC1 signaling pathway in neonatal muscle is in part due to regulation by DEPTOR, GRB10, REDD2, LAMTOR1/2, V-ATPase, and Sestrin2.
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Takayama, Mariko, Rie Fujita, Mikiko Suzuki, Ryuhei Okuyama, Setsuya Aiba, Hozumi Motohashi, and Masayuki Yamamoto. "Genetic Analysis of Hierarchical Regulation for Gata1 and NF-E2 p45 Gene Expression in Megakaryopoiesis." Molecular and Cellular Biology 30, no. 11 (March 29, 2010): 2668–80. http://dx.doi.org/10.1128/mcb.01304-09.
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ABSTRACT GATA1 and NF-E2 p45 are two important regulators of megakaryopoiesis. Whereas GATA1 is known to regulate the p45 gene, details of the GATA1 contribution to the spatiotemporal expression of the p45 gene remain to be elucidated. To clarify the relationship between GATA1 and p45, we performed genetic complementation rescue analysis of p45 function in megakaryocytes utilizing the hematopoietic regulatory domain of the Gata1 gene (G1HRD). We established transgenic mouse lines expressing p45 under G1HRD regulation and crossed the mice with p45-null mice. Compound mutant mice displayed normal platelet counts and no sign of hemorrhage, indicating that G1HRD has the ability to express p45 in a spatiotemporally correct manner. However, deletion of 38 amino acids from the N-terminal region of p45 abrogated the p45 rescue function, suggesting the presence of an essential transactivation activity in the region. We then crossed the G1HRD-p45 transgenic mice with megakaryocyte-specific Gata1 gene knockdown (Gata1 Δ neo Δ HS) mice. The G1HRD-p45 transgene was insufficient for complete rescue of the Gata1 Δ neo Δ HS megakaryocytes, suggesting that GATA1 or other factors regulated by GATA1 are required to cooperate with p45 for normal megakaryopoiesis. This study thus provides a unique in vivo validation of the hierarchical relationship between GATA1 and p45 in megakaryocytes.
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Li, LiQi, Johannes Freudenberg, Kairong Cui, Ryan Dale, Sang-Hyun Song, Ann Dean, Keji Zhao, Raja Jothi, and Paul E. Love. "Ldb1-nucleated transcription complexes function as primary mediators of global erythroid gene activation." Blood 121, no. 22 (May 30, 2013): 4575–85. http://dx.doi.org/10.1182/blood-2013-01-479451.
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Key Points Ldb1 complexes bind to and positively regulate the expression of a large number of erythroid genes including most known Gata1-regulated genes. Ldb1 complexes and Klf1 frequently bind together and coregulate erythroid gene expression.
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Campbell, Amy E., and Gerd A. Blobel. "Linking Transcription Factor Pathways to Disease-Causing GATA1 Mutations." Blood 118, no. 21 (November 18, 2011): 2371. http://dx.doi.org/10.1182/blood.v118.21.2371.2371.
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Abstract Abstract 2371 Missense mutations in the gene encoding hematopoietic transcription factor GATA1 cause congenital anemias and/or thrombocytopenias. Seven such mutations are reported. All of these give rise to amino acid substitutions within the amino terminal zinc finger (NF) of GATA1, producing a range of clinical phenotypes. Thus, V205M, G208R, and D218Y cause severe anemia and thrombocytopenia; G208S, R216Q, and D218G cause thrombocytopenia with minimal anemia; R216W gives rise to thrombocytopenia and congenital erythropoietic porphyria. One of these mutations, R216Q, occurs at the DNA binding interface and alters the ability of GATA1 to recognize a subset of cis motifs in vitro. Other mutations, including V205M, G208S, D218G, and D218Y, occur outside the DNA binding domain of the NF and inhibit interactions with the GATA1 cofactor FOG1 as determined by in vitro binding assays. However, these two mechanisms do not easily explain the broad spectrum of phenotypes associated with the mutations. For example, how do two substitutions of the same residue bring about disparate phenotypes? We examined the effects of each mutation on erythroid maturation, lineage-specific gene expression, in vivo target gene occupancy, and cofactor recruitment by introducing altered forms of GATA1 into murine GATA1-null proerythroblasts. The V205M, G208R, and D218Y mutations severely impaired erythroid maturation, recapitulating patient phenotypes. The G208S mutation also severely impaired erythroid maturation, causing a more pronounced defect than that expected from the clinical presentation. In contrast, R216Q and D218G produced mild effects in erythroid cells consistent with patient phenotypes. The porphyria-associated mutation R216W also produced relatively subtle effects in erythroid cells. We note that among the mutants, failure to activate gene expression strongly correlated with failure to repress gene expression. ChIP assays revealed that the V205M, G208R, and D218Y mutations impaired GATA1 target site occupancy. This indicates that despite normal DNA binding in vitro, the association with cofactor complexes is required for stable binding to chromatinized target sites in vivo. In contrast, the G208S mutant exhibited relatively normal chromatin occupancy, but reduced recruitment of FOG1 and SCL/Tal1 to GATA1-bound sites at erythroid genes. D218G also perturbed cofactor recruitment without greatly affecting GATA1 binding to its target genes. Notably, this mutation diminished SCL/Tal1 recruitment without significantly altering FOG1 occupancy. This implicates the SCL/Tal1 transcription complex in the pathogenesis of disorders caused by certain GATA1 mutations. Moreover, by uncoupling GATA1 chromatin occupancy and cofactor recruitment, G208S and D218G offer potentially useful tools for unraveling site-specific mechanisms of GATA1-regulated gene expression. Finally, both the R216Q and R216W mutants displayed relatively normal GATA1 chromatin occupancy and FOG1 and SCL/Tal1 recruitment at most sites. R216W presents as porphyria, and selective defects in the regulation of heme biosynthetic genes have yet be uncovered. Given that R216Q presents as thrombocytopenia, defects caused by this mutation may be revealed only in the context of megakaryocytes. Studies using similar rescue assays of a GATA1-null megakaryocyte-erythroid progenitor line are underway and will be discussed. In concert, our results reveal that in vivo analysis of GATA1 in its native environment provides mechanistic insights not obtainable from in vitro studies. Moreover, they demonstrate the usefulness of gene complementation assays for the dissection of transcription pathways surrounding normal and altered GATA1 to improve our understanding of disease. Disclosures: No relevant conflicts of interest to declare.
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Suzuki, Mikiko, Takashi Moriguchi, Kinuko Ohneda, and Masayuki Yamamoto. "Differential Contribution of the Gata1 Gene Hematopoietic Enhancer to Erythroid Differentiation." Molecular and Cellular Biology 29, no. 5 (December 22, 2008): 1163–75. http://dx.doi.org/10.1128/mcb.01572-08.
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ABSTRACT GATA1 is a key regulator of erythroid cell differentiation. To examine how Gata1 gene expression is regulated in a stage-specific manner, transgenic mouse lines expressing green fluorescent protein (GFP) reporter from the Gata1 locus in a bacterial artificial chromosome (G1BAC-GFP) were prepared. We found that the GFP reporter expression faithfully recapitulated Gata1 gene expression. Using GFP fluorescence in combination with hematopoietic surface markers, we established a purification protocol for two erythroid progenitor fractions, referred to as burst-forming units-erythroid cell-related erythroid progenitor (BREP) and CFU-erythroid cell-related erythroid progenitor (CREP) fractions. We examined the functions of the Gata1 gene hematopoietic enhancer (G1HE) and the highly conserved GATA box in the enhancer core. Both deletion of the G1HE and substitution mutation of the GATA box caused almost complete loss of GFP expression in the BREP fraction, but the CREP stage expression was suppressed only partially, indicating the critical contribution of the GATA box to the BREP stage expression of Gata1. Consistently, targeted deletion of G1HE from the chromosomal Gata1 locus provoked suppressed expression of the Gata1 gene in the BREP fraction, which led to aberrant accumulation of BREP stage hematopoietic progenitor cells. These results demonstrate the physiological significance of the dynamic regulation of Gata1 gene expression in a differentiation stage-specific manner.
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Ueki, Nobuhide, Leiqing Zhang, and Michael J. Hayman. "Ski Negatively Regulates Erythroid Differentiation through Its Interaction with GATA1." Molecular and Cellular Biology 24, no. 23 (December 1, 2004): 10118–25. http://dx.doi.org/10.1128/mcb.24.23.10118-10125.2004.
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ABSTRACT The Ski oncoprotein dramatically affects cell growth, differentiation, and/or survival. Recently, Ski was shown to act in distinct signaling pathways including those involving nuclear receptors, transforming growth factor β, and tumor suppressors. These divergent roles of Ski are probably dependent on Ski's capacity to bind multiple partners with disparate functions. In particular, Ski alters the growth and differentiation program of erythroid progenitor cells, leading to malignant leukemia. However, the mechanism underlying this important effect has remained elusive. Here we show that Ski interacts with GATA1, a transcription factor essential in erythropoiesis. Using a Ski mutant deficient in GATA1 binding, we show that this Ski-GATA1 interaction is critical for Ski's ability to repress GATA1-mediated transcription and block erythroid differentiation. Furthermore, the repression of GATA1-mediated transcription involves Ski's ability to block DNA binding of GATA1. This finding is in marked contrast to those in previous reports on the mechanism of repression by Ski, which have described a model involving the recruitment of corepressors into DNA-bound transcription complexes. We propose that Ski cooperates in the process of transformation in erythroid cells by interfering with GATA1 function, thereby contributing to erythroleukemia.
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Reinhardt, Katarina, C. Michel Zwaan, Michael Dworzak, Jasmijn D. E. de Rooij, Gertjan Kaspers, Thomas Lehrnbecher, Henrik Hasle, et al. "High Frequency of GATA1 Mutations in Childhood Non-Down Syndrome Acute Megakaryoblastic Leukemia." Blood 120, no. 21 (November 16, 2012): 888. http://dx.doi.org/10.1182/blood.v120.21.888.888.
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Abstract Abstract 888 Introduction: Pediatric acute megakaryoblastic leukemia (AMKL) occurred in 6.6% (84/1271) of the children enrolled to the AML-BFM98 and 2004 studies. Despite a similar phenotype in morphology and immunophenotype, AMKL shows a heterogenous cytogenetic distribution (normal karyotype 23%, complex karyotype 21%, t(1;22) 9%; MLL-rearrangement 8%; monosomy 7 5%, trisomy 8 5%; other aberrations 29%). Mutations of the hematopoietic transcription factor GATA1 have been identified in almost all children suffering myeloid leukemia of Down syndrome (ML-DS). In addition, GATA1 mutations (GATA1mut) could be identified in children with trisomy 21 mosaic. Here, AMKL without evidence of Down syndrome or Down syndrome mosaic were analyzed for mutations in exon 1, 2 or 3 of the transcription factor GATA1. Patients: Seventy-one children from the AML-BFM Study group (n=51; 2000–2011), the Netherlands (n=10), France (n=3) and Scandinavia (n=7) were included. Within the AML-BFM Group the 51 analyzed patients showed similar characteristics compared to the total cohort of 84 children with AMKL of the AML-BFM 98 and 2004 studies. AMKL was confirmed according to the WHO classification by genetics (t(1;22)); morphology and immunophenotyping. Table 1a) summarizes the patientxs characteristics and b) the cytogenetic results. Methods: For GATA1 mutation screening genomic DNA was amplified by PCR reaction for exon 1, 2, and 3. PCR amplicons were analyzed by direct sequencing or following denaturing high-performance liquid chromatography (WAVE). Results: Seven different GATA1 mutations were detected in 8 children (11.1%; table 2). In all GATA1mut leukemia, a trisomy 21 within the leukemic blasts could be detected. Seven out of these 8 children and all other 64 AMKL patients have been treated with intensive chemotherapy regimens according the study group protocols. The results are given in table 2. All achieved continuous complete remission (CCR; 0.4 to 4.2 years). In one newborn with typical morphology and immunophenotype a GATA1mut associated transient leukemia was supposed. The child achieved CCR (follow-up 6 years). In total, allogeneic stem cell transplantation in 1st CR was performed in 6 children with AMKL (GATA1mut leukemia n=1). Conclusions: GATA1 mutations occurred in 11% of children with AMKL without any symptoms or evidence of trisomy 21 or trisomy 21 mosaic. GATA1 mutations are associated with a trisomy 21 within the leukemic blasts. Although non-response occurred, prognosis was significant better compared to other AMKL. Therefore, analysis of GATA1 mutation in infant AMKL is strongly recommended. Whether treatment reduction similar to ML-DS Down syndrome is feasible needs to be confirmed. Disclosures: No relevant conflicts of interest to declare.
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Barbosa, R. C. C., C. B. Gitti, M. C. N. Castro, and F. Mendes-de-Almeida. "Aspectos clínicos e laboratoriais do complexo gengivite-estomatite em gatos domésticos." Arquivo Brasileiro de Medicina Veterinária e Zootecnia 70, no. 6 (December 2018): 1784–92. http://dx.doi.org/10.1590/1678-4162-10037.
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RESUMO Foram incluídos 76 gatos domésticos com diferentes graus (I a IV) de lesões em cavidade oral, os quais foram avaliados clinicamente e tiveram coletadas amostras de sangue e suabes da cavidade oral. A maioria dos gatos portadores de CGE eram machos, castrados, adultos, sem raça definida e com estilo de vida confinado. Os sinais clínicos observados e associados à gravidade da inflamação na cavidade oral foram halitose (98,7%); ptialismo (22,4%); hemorragia bucal (9,2%) e úlcera na parte superior dos lábios (2,6%); desconforto à manipulação da cavidade oral (44,7%) e perda dentária (55,3%). A maioria dos gatos avaliados foi classificada no grau II (43,4%). Não se observou diferença significativa nos resultados do eritrograma dos gatos portadores de CGE, independentemente da gravidade das lesões e da sintomatologia clínica. Entretanto, observou-se neutrofilia (21,1%) e aumento de proteínas plasmáticas totais (47,3%), na maioria dos animais de grau II, sugerindo que esses parâmetros laboratoriais, quando aumentados, possam estar associados a graus menos graves de CGE. A análise das lâminas de citologia da cavidade oral dos gatos demonstrou que a presença de Simonsiella spp. foi mais frequente nos animais incluídos no grau IV, entretanto não é possível afirmar que essa bactéria esteja relacionada à gravidade das lesões.
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Kadauke, Stephan, Amy E. Campbell, Aaron J. Stonestrom, Deepti P. Jain, Ross C. Hardison, and Gerd A. Blobel. "GATA1 and the BET Family Protein Brd3 Form a Mitotic Bookmarking Complex." Blood 120, no. 21 (November 16, 2012): 282. http://dx.doi.org/10.1182/blood.v120.21.282.282.
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Abstract Abstract 282 Erythroid-specific transcription patterns are maintained throughout cell division. During mitosis, transcription is silenced globally. This raises the question whether mechanisms are in place that ensure the spatially and temporally correct reassembly of transcriptional regulators and thus maintain lineage fidelity. We recently found that, in contrast to most nuclear regulators, the master hematopoietic regulator GATA1 remains associated at a subset of its targets within mitotic chromosomes in erythroid cells (Kadauke et al., Cell 2012). GATA1 appears to function by creating an epigenetic “bookmark” to facilitate timely post-mitotic transcription reactivation of its mitotic target genes. GATA1 is acetylated at two lysine-rich domains near its zinc finger domains. We recently discovered that acetylated GATA1 recruits the double bromodomain protein Brd3 to erythroid target genes (Lamonica et al., PNAS 2011). Brd3 interacts with acetylated GATA1 via its first bromodomain, and Brd3 recruitment to GATA1 target sites is critically required for induction of terminal erythroid target genes such as α- and β-globin. Notably, Brd3 belongs to a family of proteins (called the BET family) of which two members (Brd2 and Brd4) are known to be retained on mitotic chromosomes. We now find by immunofluorescence and live cell confocal imaging that Brd3 globally binds to mitotic chromosomes. ChIP-seq experiments demonstrate a high degree of co-localization of Brd3 and GATA1 genome-wide both in interphase and in mitosis. We further demonstrate that GATA1 directly recruits Brd3 to mitotic GATA1 target sites. Transient mitosis-specific disruption of the Brd3-GATA1 interaction using the small molecule BET bromodomain inhibitor JQ1 removed Brd3, but not GATA1, from mitotic binding sites and led to a profound delay in the reactivation of GATA1-bookmarked genes. This suggests that Brd3 is an integral component of GATA1's bookmarking function. In concert, these studies support a requirement of mitotic bookmarking by a GATA1/Brd3 complex for the propagation of lineage-specific transcription programs in dividing erythroid cells. Disclosures: No relevant conflicts of interest to declare.
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Kaminaga, Chihiro, Shumpei Mizuta, Tomoya Minami, Kasumi Oda, Haruka Fujita, Keiji Matsui, Ruri Ishino, Akiko Sumitomo, Norinaga Urahama, and Mitsuhiro Ito. "CoCoA/CCAR1 Pair-Mediated Recruitment of Mediator Complex Indicates Novel Pathway for the Function of GATA1 in Erythroid Differentiation." Blood 118, no. 21 (November 18, 2011): 1302. http://dx.doi.org/10.1182/blood.v118.21.1302.1302.
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Abstract Abstract 1302 The mammalian multi-protein complex Mediator, originally identified by ourselves as a nuclear receptor-specific coactivator complex, is a phylogenetically-conserved subcomplex of the RNA polymerase II holoenzyme and serves as an end-point integrator of diverse intracellular signals and transcriptional activators. The 220-kDa Mediator subunit MED1 is a specific coactivator not only for nuclear receptors but for GATA family activators, and serves as a GATA1-specific coactivator that is essential for optimal GATA1-mediated erythropoiesis. In this study, we show a novel nuclear signaling pathway for MED1 action in GATA1-induced transcriptional activation during erythroid differentiation. First, we identified the amino acid residues 681–715 of human MED1 (MED1(aa.681-715)) to be responsible for the direct interaction with GATA1. When MED1 in K562 human erythroleukemic cells was knocked down during hemin-induced erythroid differentiation, the erythroid differentiation was significantly attenuated as assessed by an erythroid differentiation score defined by the number of cells positive for benzidine staining, and the expressions of the GATA1-targeted and erythroid differentiation marker genes, β-globin, γ-globin, PBGD and ALAS-E, were prominently attenuated. However, overexpressions of the N-terminal MED1 truncations without and with nuclear receptor recognition motifs, MED1(aa.1–602) and MED1(aa.1–703), respectively, but neither of which could bind to GATA1 (above), prominently enhanced erythroid differentiation of K562 cells. Luciferase reporter assays by using the human γ-globin promoter and Med1−/− mouse embryonic fibroblasts (MEFs) showed that these N-terminal MED1 truncations rescued GATA1-mediated transactivation, indicating that MED1(a.a.1–602) served as the functional interaction surface for GATA1. Hence, a putative bypass for GATA1-MED1 pathway appears to exist, and is expected to interact with the N-terminus of MED1. As a candidate bypass system, we tested both the recently reported bypass molecule for a nuclear post-activator signaling, CCAR1, and its partner coactivator CoCoA. CCAR1 was reported by others to bypass the estrogen receptor-mediated transactivation by a simultaneous binding of CCAR1 with the estrogen receptor and the N-terminus of MED1. Functionally, serial luciferase reporter assays by using the γ-globin promoter and MEFs demonstrated cooperative transactivation by combinations of GATA1, CCAR1, CoCoA and/or the N-terminus of MED1, but the transactivation mediated by the N-terminus of MED1 was not as prominent as the one mediated by the full-length MED1. An overexperssion of CCAR1 or CoCoA in K562 cells prominently enhanced both the GATA1-mediated erythroid differentiation and the expressions of the GATA1-targeted genes. Next, the mechanisms underlying the CCAR1- and CoCoA-mediated GATA1 functions were analyzed by serial GST-pulldown and mammalian two-hybrid assays, and the following results were obtained. (i) The N-terminus of CCAR1 interacted with the C-terminus of CoCoA. (ii) The N-terminus of MED1 interacted with both the N- and C-termini of CCAR1. (iii) While the N-terminal zinc-finger domain of human GATA1 (GATA1(a.a.204–228)) is known to bind to the well-known GATA1 partner FOG1, intriguingly, the C-terminal zinc-finger domain of GATA1 (GATA1(a.a.258–272)) interacted with all three of the following cofactors; MED1 (MED1(aa.681–715)), CCAR1 (at the C-terminus) and CoCoA (at both the N- and C-termini). The affinity of CoCoA to bind to GATA1 appeared to be a little higher than the other. Thus, the GATA1(a.a.258-272) zinc finger appears to serve as a docking surface for multiple coactivating proteins, where both MED1 and CoCoA/CCAR1 pair can interact, probably in a competitive manner, or perhaps simultaneously. Here, both CoCoA/CCAR1 as a pair and CCAR1 by itself can serve as a bypass. Finally, ChIP assays of hemin-treated K562 cells showed that GATA1, CCAR1/CoCoA and MED1 were all recruited onto the γ-globin promoter during transactivation. Taken together, besides a direct interaction between GATA1 and MED1, the CoCoA/CCAR1 pair appears to relay the GATA1 signal to MED1. The multiple modes of mechanisms for transcription mediated by the GATA1-MED1 axis might contribute to a fine tuning of the GATA1 function, not only during erythropoiesis but also in other GATA1-mediated homeostasis events, within a living animal. Disclosures: No relevant conflicts of interest to declare.
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Doty, Raymond T., Xiaowei Yan, Christopher Lausted, Adam D. Munday, Zhantao Yang, Danielle Yi, Neda Jabbari, et al. "Single-cell analyses demonstrate that a heme–GATA1 feedback loop regulates red cell differentiation." Blood 133, no. 5 (January 31, 2019): 457–69. http://dx.doi.org/10.1182/blood-2018-05-850412.
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Abstract Erythropoiesis is the complex, dynamic, and tightly regulated process that generates all mature red blood cells. To understand this process, we mapped the developmental trajectories of progenitors from wild-type, erythropoietin-treated, and Flvcr1-deleted mice at single-cell resolution. Importantly, we linked the quantity of each cell’s surface proteins to its total transcriptome, which is a novel method. Deletion of Flvcr1 results in high levels of intracellular heme, allowing us to identify heme-regulated circuitry. Our studies demonstrate that in early erythroid cells (CD71+Ter119neg-lo), heme increases ribosomal protein transcripts, suggesting that heme, in addition to upregulating globin transcription and translation, guarantees ample ribosomes for globin synthesis. In later erythroid cells (CD71+Ter119lo-hi), heme decreases GATA1, GATA1-target gene, and mitotic spindle gene expression. These changes occur quickly. For example, in confirmatory studies using human marrow erythroid cells, ribosomal protein transcripts and proteins increase, and GATA1 transcript and protein decrease, within 15 to 30 minutes of amplifying endogenous heme synthesis with aminolevulinic acid. Because GATA1 initiates heme synthesis, GATA1 and heme together direct red cell maturation, and heme stops GATA1 synthesis, our observations reveal a GATA1–heme autoregulatory loop and implicate GATA1 and heme as the comaster regulators of the normal erythroid differentiation program. In addition, as excessive heme could amplify ribosomal protein imbalance, prematurely lower GATA1, and impede mitosis, these data may help explain the ineffective (early termination of) erythropoiesis in Diamond Blackfan anemia and del(5q) myelodysplasia, disorders with excessive heme in colony-forming unit-erythroid/proerythroblasts, explain why these anemias are macrocytic, and show why children with GATA1 mutations have DBA-like clinical phenotypes.
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Davis, Teresa A., Samer El-Kadi, Agus Suryawan, and Marta Fiorotto. "356 Meal feeding compared with continuous feeding enhances insulin and amino acid signaling to translation initiation in skeletal muscle of pigs." Journal of Animal Science 97, Supplement_3 (December 2019): 127–28. http://dx.doi.org/10.1093/jas/skz258.261.
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Abstract Objectives: Meal feeding enhances skeletal muscle protein synthesis and lean growth more than continuous feeding in piglets. This enhanced muscle protein synthesis with meal feeding is associated with increased activation of mTORC1-dependent translation initiation. The mechanism underlying this response is unknown. We aimed to identify insulin and amino acid signaling components involved in the enhanced lean growth that results from meal feeding vs. continuous feeding in term-born pigs. Methods: Newborn piglets were fed for 21 d an equal amount of sow milk replacer (12.8 g protein and 155 kcal/(kg BW.d)) by gastrostomy tube either as intermittent bolus meals every 4 h (MEAL) or by continuous infusion (CON). After 21 d, gastrocnemius muscle was collected from CON, and before (MEAL-0) or 60 min after a meal (MEAL-60). Components of the insulin and amino acid signaling pathways up- and downstream of mTORC1 that regulate protein translation were measured. Results: Phosphorylation of AKT and TCS2 was greater in MEAL-60 than in MEAL-0 and CON (P < 0.05). The association of Sestrin2 with GATOR2 was similar in CON and MEAL-0 but was lower in MEAL-60 (P < 0.05). The abundances of RagA-mTOR, RagC-mTOR, and Rheb-mTOR, but not CASTOR1-GATOR2, complexes were higher in MEAL-60 than in CON and MEAL-0 (P < 0.05). The phosphorylation of S6K1 and 4EBP1 was higher in MEAL-60 than CON and MEAL-0 (P < 0.05). The abundances of Sestrin2, GATOR2, CASTOR1, RagA, RagC, and Rheb and the phosphorylation of eIF2alpha, eEF2, ERK1/2 and AMPK were unaffected by treatments. Conclusions: Our results demonstrate that the enhanced rate of skeletal muscle protein synthesis and lean growth with meal feeding compared with continuous feeding are due to the enhanced activation of both insulin and amino acid signaling pathways that result in the greater stimulation of translation initiation. Support: NIH HD085573, USDA CRIS 6250-51000-055, NIH HD072891, USDA NIFA 2013-67015-20438.
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Masselli, Elena, Lilian Varricchio, Barbara Ghinassi, Carolyn Whitsett, Patricia A. Shi, and Anna Rita F. Migliaccio. "Class IIa HDAC Inhibitors Reduce HDAC1 Activity by off-Target Effects Which Reduce GATA1 Expression In Human Erythroblasts Expanded Ex-Vivo." Blood 116, no. 21 (November 19, 2010): 4780. http://dx.doi.org/10.1182/blood.v116.21.4780.4780.
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Abstract Abstract 4780 Histone deacetylation maintains chromatin in a condensed configuration preventing gene expression in eukaryotic cells. The deacetylation reaction is catalyzed by enzymes of the histone deacetylase (HDAC) superfamily, which perform their functions as multiprotein complexes including at least 2 HDAC isoforms, DNA docking factors (transcription factors and methyl-binding proteins) and protein kinases (PKC and Erk). The well established role of HDACs in gene silencing has suggested studies to identify HDAC inhibitors (HDACi) that, by re-activating γ-globin expression, might treat the anemia due to insufficient β-globin expression (Cao et al Blood 103:701, 2004). Over the years several HDACi have been documented to induce γ-globin expression in human erythroid cultures, adult baboons, and β-thalassemia and sickle cell patients. Among those, Class I HDACi, and in particular those that inhibit HDAC3, appear to be more potent as γ-globin gene activators (Mankidy et al, Blood 108:3179, 2006). We have recently identified two new HDACi (compound 9 and 24) which both improved maturation and reactivated γ-globin expression in β°-thalassemic erythroblasts in vitro (Mai et al Mol Pharmacol 72:111, 2007). Compound 24 inhibits both class I (HDAC1 ID50 =0.2 μ M) and class IIa (HDAC4 ID50=0.6 μ M) HDAC. Compound 9 is a class IIa specific inhibitor (HDAC4 ID50=20 μ M) and does not affect HDAC1 activity but is a more potent γ-globin inducer than compound 24. This observation suggests that HDACi may also affect HDAC activity through indirect effects which alter overall complex activity. To clarify possible off-target effects of Class II and Class I/IIa inhibitors and their consequences for erythroid maturation, we analysed expression and activity of different HDAC isoforms during maturation of normal human erythroblasts in vitro at baseline and with treatment with compounds 9 and 24. The proteins studied included GATA1 (the major transcription regulator of erythroid maturation), p21/p27kip1, two cyclin D dependent kinase inhibitors which favor maturation, Caspase 3 (the protease which specifically cleaves GATA1) and Erk (a component of the HDAC complex). During normal erythroid maturation (without HDACi), all the HDAC isoforms were expressed at the mRNA and protein levels. Immunoprecipitation studies followed by determination of HDAC activity indicated that the activities which changed most during maturation are those of HDAC1 (class I), increased by 2-fold, and HDAC5 (class IIa), decreased by 2-fold. In addition, co-immunoprecipitation studies revealed an increase in the association between HDAC1 and GATA1 with erythroid maturation. Changes in the expression of key regulatory proteins were observed with normal erythroid maturation: activation of Caspase 3 decreased with resultant increase in GATA-1, and phosphorylation of pErk decreased while expression of p21 and p27 increased. With exposure to increasing HDACi concentrations (0.2, 2 and 6 μ M), there were class-specific, concentration-dependent alterations in protein expression: compound 9 (Class IIa inhibitor) induced Caspase 3 activation and reduced GATA1 content, while compound 24 decreased Caspase3 activation and greatly increased GATA1 content. In addition, compound 9 did not induce Erk phosphorylation and decreased p21 expression, while compound 24 did induce Erk phosphorylation and inhibited p27 expression (see figure). These results confirm the hypothesis that, in addition to class I inhibitors that directly inhibit class I HDAC, class II HDACi can also affect class I HDAC activity, through indirect effects that involve other components of the complex (repression of GATA1 expression and decrease of Erk phosphorylation). Disclosures: No relevant conflicts of interest to declare.
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Kelly, Soady, Gaëtan Juban, Ludovic Lhermitte, Elena Karkoulia, John Strouboulis, Irene Roberts, and Paresh Vyas. "Cellular and Molecular Basis of Mutant Haemopoietic Transcription Factor GATA1s." Blood 124, no. 21 (December 6, 2014): 607. http://dx.doi.org/10.1182/blood.v124.21.607.607.
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Abstract Down Syndrome (DS) (Trisomy 21 – T21) is a common constitutional aneuploidy. Neonates and children with DS have a 150-fold increased risk of developing Acute Myeloid Leukaemia (DS-ML), characterized by a differentiation arrest of immature megakaryocyte-erythroid cells. In virtually all DS-ML patients, somatic mutations in the gene encoding the megakaryocyte-erythroid transcription factor GATA1 are acquired during fetal life leading to the production of a N-terminal truncated form of the GATA1 protein (GATA1s). We, and others, have previously shown that this N-terminal domain is necessary to prevent excessive megakaryocytic proliferation. However, the mechanisms by which GATA1, but not GATA1s, restrains megakaryocytes proliferation are unclear. To gain mechanistic insight, we generated knock-in murine ES cell models expressing biotinylated forms of either full length GATA1 or GATA1s protein. We established large scale in vitro differentiation assays to interrogate embryonic-fetal megakaryocyte differentiation (adapted from Nishikii et al., 2008. J Exp Med; 205 (8) : 1917-27; Figure 1) to define the normal megakaryocytic differentiation pathway in GATA1-expressing cells. ES cells were differentiated into embryoid bodies (EB), which were disaggregated at D6 and CD41+c-kit+ cells were cultured on OP9 feeder layers with TPO, IL6 and IL11. Detailed examination of the differentiation kinetics of populations including FACS-sorting of specific populations followed by reculture, showed complex differentiation pathways as wild type cells differentiated into both megakaryocyte and non-megakaryocyte fates. By contrast, GATA1s-expressing cells principally differentiated into megakaryocyte fate. In addition, as immature CD41+ haemopoietic cells differentiate into the megakaryocyte lineage they lose c-kit expression and CD41 expression increases (Figure 2). In the GATA1s-expressing cells compared to GATA1-expressing cells, there is marked accumulation (5 to 10-fold) of a specific immature megakaryocyte CD41++c-kit+ population that is partially blocked in differentiation (Gate R6). Cell cycle analysis shows an increase in cells in S-phase specifically in this population in GATA1s-expressing cells compared to normal cells (44% vs 27%) together with a decrease in apoptosis (5% vs 11%). To determine GATA1s direct and indirect target genes, we performed ChIP-sequencing and RNA-sequencing. RNA-sequencing of GATA1- and GATA1s-expressing CD41+ populations at D12 showed around 4500 differentially expressed genes (at a p-value of 0.05). Given the differences in cell cycle, it is noteworthy that cyclin D3 and cdk6 were expressed 1.7-fold and 1.5-fold higher in GATA1s- expressing cells. Chromatin in cis-regulatory regions of both genes was bound by GATA1 and GATA1s. Chemical inhibition of the Cyclin D3:Cdk4/6 complex reduced proliferation and induced partial differentiation of mutant cells, suggesting a role of this complex in regulating GATA1s-induced proliferation and differentiation inhibition. Knock-down and overexpression experiments to further test the role of Cyclin D:Cdk4/6 complex in both wild-type and mutant cells are in progress. Taken together, these results suggest that GATA1s alters cell cycle at a specific stage in megakaryocyte differentiation causing partial differentiation arrest and that this is mediated by altered expression and function of a Cyclin D3:Cdk4/6 complex. These results may have more general implications of how mutant transcription factors cause differentiation arrest in leukemia. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Drissen, Roy, Boris Guyot, William Wood, Catherine Porcher, and Paresh Vyas. "Characterisation Erythroid-Specific Cis-Elements Regulating the Key Transcription Factor GATA1." Blood 108, no. 11 (November 16, 2006): 1167. http://dx.doi.org/10.1182/blood.v108.11.1167.1167.
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Abstract The critical myeloid transcription factor GATA1 can specify erythroid cells at expense of granulocytes/macrophages (GM) from the common myeloid progenitor (CMP). In red cells sustained GATA1 expression is required for terminal maturation. Conversely, GATA1 expression has to be extinguished to allow GM differentiation. Therefore, one component in dissecting myeloid specification will be to define how GATA1 expression is regulated. The level of GATA1 is mainly controlled transcriptionally. Here, we show that murine(m) GATA1 mRNA rises 6-fold as CMPs differentiate to MEPs (megakaryocyte-erythroid progenitor) and another 4-fold as MEPs differentiate to Ter119+ erythroid cells. As a step towards understanding the molecular basis of erythroid-specific GATA1 expression, we have been characterising GATA1 cis-elements. Previously, we and others showed that an upstream enhancer (mHS−3.5) is required to direct erythroid-specific GATA1 expression in cooperation with sequences near the mGata1 gene (IE promoter and intron element mHS+3.5) in transgenic mice. Though a mGata1 transgene regulated by mHS−3.5-IE-mHS+3.5 grossly rescues erythropoiesis in GATA1 knock out mice, germ line deletion of mHS−3.5 leaves red cell GATA1 expression unaffected. This suggests other cis-elements in the mGata1 locus can substitute for mHS−3.5 in red cells. Recently, we identified an erythroid-specific DNase I hypersensitive site (DHS), mHS-25, with enhancer activity in erythroid cell lines and where the chromatin associated with it is hyperacetylated at histone H3/H4. We now demonstrate by fine DHS mapping that mHS-25 is more complex, being composed of two adjacent DHSs ~500 bp apart (mHS-25 and mHS-26). These DHSs are present only in primary red cells and not other primary cells. Chromatin immunoprecipitation (ChiP) shows that H3 and H4 associated with both sites is hyperacetylated only in Ter119+ cells. We tested mHS25/6 function in mice transgenic for a mGata1-LacZ reporter construct regulated by mHS-25/6-IE-mHS+3.5. β-galactosidase expression was quantitated in myeloid lineages by FACS analysis using lineage-specific antibodies and the β-galactosidase substrate FDG. 6 out of 7 F0 transgenic embryos expressed β-galactosidase in 5–23% of fetal liver Ter119+ cells. FDG staining was not detected in CD61+Mac1- megakaryocytes or Mac-1+ macrophages. <0.2% of Ter119+ cells stained with FDG when mHS-25/6 was not present. Erythroid-specific reporter gene expression was confirmed in bone marrow samples in 2 independent lines of mice transgenic for this reporter construct. ChiP analysis demonstrated that GATA1, SCL, E2A, the SCL-interacting protein Ldb1 and LMO2 bind in vivo to mHS-25/6 only in Ter119+ cells (and not primary megakaryocytes or neutrophils). Previously, we and others have shown that these transcription factors exist in a multi-protein complex to activate gene expression. Taken together, these findings suggest mHS-25/6 is an erythroid-specific GATA1 enhancer in primary murine cells. Finally, to begin to understand the relative roles of mHS−3.5 and mHS-25/6 during erythroid differentiation, we show by DHS mapping and ChiP analysis that in the multi-potential myeloid cell line FDCP-mix, only mHS−3.5 is present (but not mHS-25/6) and that it binds GATA1/SCL/E2A/Ldb1/LMO2 in vivo. Later in Ter119+ cells both mHS−3.5 and mHS-25/6 are seen with GATA1/SCL/E2A/Ldb1/LMO2 detected at these sites. This suggests a hierarchical utilisation of these two mGata1 cis-elements during erythroid differentiation.
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Wang, Yuhuan, Ronghua Meng, Vincent Hayes, Rudy Fuentes, Xiang Yu, Charles S. Abrams, Harry F. G. Heijnen, Gerd A. Blobel, Michael S. Marks, and Mortimer Poncz. "Pleiotropic platelet defects in mice with disrupted FOG1-NuRD interaction." Blood 118, no. 23 (December 1, 2011): 6183–91. http://dx.doi.org/10.1182/blood-2011-06-363580.
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Abstract Understanding platelet biology has been aided by studies of mice with mutations in key megakaryocytic transcription factors. We have shown that point mutations in the GATA1 cofactor FOG1 that disrupt binding to the nucleosome remodeling and deacetylase (NuRD) complex have erythroid and megakaryocyte lineages defects. Mice that are homozygous for a FOG1 point mutation (ki/ki), which ablates FOG1-NuRD interactions, have platelets that display a gray platelet syndrome (GPS)–like macrothrombocytopenia. These platelets have few α-granules and an increased number of lysosomal-like vacuoles on electron microscopy, reminiscent of the platelet in patients with GATA1-related X-linked GPS. Here we further characterized the platelet defect in ki/ki mice. We found markedly deficient levels of P-selectin protein limited to megakaryocytes and platelets. Other α-granule proteins were expressed at normal levels and were appropriately localized to α-granule–like structures. Treatment of ki/ki platelets with thrombin failed to stimulate Akt phosphorylation, resulting in poor granule secretion and platelet aggregation. These studies show that disruption of the GATA1/FOG1/NuRD transcriptional system results in a complex, pleiotropic platelet defect beyond GPS-like macrothrombocytopenia and suggest that this transcriptional complex regulates not only megakaryopoiesis but also α-granule generation and signaling pathways required for granule secretion.
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Cheng, Yang, Jiadong Cai, Yuanyuan Fu, Congjing Feng, Yue Hao, and Youheng Wei. "Royal jelly attenuates metabolic defects in a Drosophila mutant with elevated TORC1 activity." Biology Open 9, no. 11 (October 9, 2020): bio054999. http://dx.doi.org/10.1242/bio.054999.
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ABSTRACTTarget of rapamycin complex 1 (TORC1) is a master regulator of cell metabolism, and its dysregulation has been linked to an array of pathologies, including cancer and age-related diseases. Nprl3, a component of GTPase-activating protein towards Rags complex 1 (GATOR1), inhibits TORC1 activity under nutrient scarcity status. The nprl3 mutant exhibits some metabolic defects due to hyper TORC1 activity in Drosophila. Royal jelly (RJ) is a honeybee-secreted product and plays an essential role in caste differentiation that requires TORC1 activity. RJ is also used as a health-benefit food for its potential roles on antioxidant and anti-aging. In this study, nprl3-mutant flies were used to measure the effect of RJ on metabolic modulation. Interestingly, RJ feeding significantly increased survival and decreased TORC1 activity in the nprl3 mutant. RJ feeding also ameliorated the abnormal reactive oxygen species (ROS) levels and energy status in the nprl3 mutant. The proteins in RJ were characterized to be the essential components in increasing nprl3 mutant viability. These findings suggest that RJ modulates some metabolic defects associated with elevated TORC1 activity and that the nprl3-mutant fly might be a useful tool for investigating the bioactive components of RJ in vivo.
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Hasegawa, Atsushi, Hiroshi Kaneko, Daishi Ishihara, Masahiro Nakamura, Akira Watanabe, Cecelia D. Trainor, Yamamoto Masayuki, and Ritsuko Shimizu. "GATA1 Changes DNA-Binding Fashion in a Binding-Site-Specific Manner and Alters Transcriptional Activity during Erythropoiesis." Blood 126, no. 23 (December 3, 2015): 3584. http://dx.doi.org/10.1182/blood.v126.23.3584.3584.
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Abstract GATA1 is a transcription factor that coordinately regulates multiple target genes during the development and differentiation of erythroid and megakaryocytic lineages through binding to GATA motif (A/T)GATA(A/G). GATA1 has four functional domains, i.e., two transactivation domains reside in amino- and carboxyl- terminus, which transactivate GATA1 target genes redundantly and/or cooperatively, and two zinc-finger domains in the middle of the protein. The two zinc finger domains of GATA1 have been characterized extensively and their links to human diseases have also been identified. Carboxyl-terminal side zinc (C)-finger is essential for the DNA binding of GATA1, whereas amino-terminal side zinc (N)-finger retains insufficient binding activity to the GATA motifs by itself, but contributes to stabilize the binding of C-finger to a double GATA site arranged in a palindromic manner. Of note, while this two-finger structure is conserved in six distinct vertebrate GATA factors, there exist GATA factors with single zinc finger in non-vertebrates, indicating that only the C-finger and following basic tail region are evolutionary conserved in both vertebrate and non-vertebrate GATA factors. In our transgenic rescue analyses, GATA1 lacking the N-finger (ΔNF-GATA1) supports, if not completely, the erythropoiesis in mice, but mice without C-finger (ΔCF-GATA1) die in utero showing similar phenotype to the mice with complete loss-of-GATA1-function. Therefore, roles that the N-finger plays have been assumed to be evolutionally acquired features during molecular evolution. In this study, we have examined GATA-motif configuration-specific modulation of GATA1 function by using composite GATA elements in which two GATA motifs aligned side-by-side, either tandem or palindromic. We have defined changes in the GATA1 binding and transactivation activity in accordance with the arrangement of cis -acting GATA motifs. While GATA1 binds to Single-GATA in a monovalent way via C-finger without the influence of N-finger, the N-finger appears to contribute to specific bivalent binding of GATA1 to Pal-GATA, i.e., the N- and C-fingers in a single GATA1 molecule individually bind to two GATA motifs aligned in a palindromic orientation. Showing very good agreement with the human case analyses, the transgenic expression of G1R216Q that lacks N-finger-DNA interaction potential hardly rescues the GATA1-deficient mice due to defects in definitive erythropoiesis, indicating that roles owed by R216 residue are vital for the GATA1 activity in vivo. The N-finger also contributes to GATA1 homodimer formation, which is a prerequisite for two GATA1 binding to two GATA motifs aligned in a tandem orientation. Each GATA1 C-finger in the dimeric GATA1 protein binds to each GATA motif in Tandem-GATA. In this regard, we previously found in a transgenic complementation rescue assay that mutant GATA1 molecule G13KA, which lacks the dimerization potential but possesses most of the other N- and C-finger functions, hardly rescues the GATA1-deficient mice from embryonic lethality, indicating that the GATA1 dimerization is important to attain full GATA1 activity. We surmise based on these observations that the configuration of cis -acting GATA motifs located in the regulatory regions of the GATA1 target genes critically influences the DNA-binding of GATA1 and controls transcription of the genes. Disclosures No relevant conflicts of interest to declare.