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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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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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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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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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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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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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Baldassari, Sara, Laura Licchetta, Paolo Tinuper, Francesca Bisulli, and Tommaso Pippucci. "GATOR1 complex: the common genetic actor in focal epilepsies." Journal of Medical Genetics 53, no. 8 (May 19, 2016): 503–10. http://dx.doi.org/10.1136/jmedgenet-2016-103883.
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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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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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Korenke, Georg-Christoph, Marlene Eggert, Holger Thiele, Peter Nürnberg, Thomas Sander, and Ortrud K. Steinlein. "Nocturnal frontal lobe epilepsy caused by a mutation in the GATOR1 complex geneNPRL3." Epilepsia 57, no. 3 (January 20, 2016): e60-e63. http://dx.doi.org/10.1111/epi.13307.
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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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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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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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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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Dawson, Ruby E., Alvaro F. Nieto Guil, Louise J. Robertson, Sandra G. Piltz, James N. Hughes, and Paul Q. Thomas. "Functional screening of GATOR1 complex variants reveals a role for mTORC1 deregulation in FCD and focal epilepsy." Neurobiology of Disease 134 (February 2020): 104640. http://dx.doi.org/10.1016/j.nbd.2019.104640.
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Sahly, Ahmed N., Robyn Whitney, Gregory Costain, Vann Chau, Hiroshi Otsubo, Ayako Ochi, Elizabeth J. Donner, et al. "Epilepsy surgery outcomes in patients with GATOR1 gene complex variants: Report of new cases and review of literature." Seizure 107 (April 2023): 13–20. http://dx.doi.org/10.1016/j.seizure.2023.03.004.
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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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Imanaga, Hiroshi, Yuichiro Semba, Kensuke Sasaki, Kiyoko Miyata, Takuji Yamauchi, Tatsuya Terasaki, Fumihiko Nakao, et al. "A Genome-Wide CRISPR-Cas9 Screen Reveals GATOR1 Complex Is a Critical Regulator of Glucocorticoid Sensitivity in B-Cell Precursor Acute Lymphoblastic Leukemia." Blood 140, Supplement 1 (November 15, 2022): 5979. http://dx.doi.org/10.1182/blood-2022-164647.
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Becchetti, Andrea, Laura Clara Grandi, Giulia Colombo, Simone Meneghini, and Alida Amadeo. "Nicotinic Receptors in Sleep-Related Hypermotor Epilepsy: Pathophysiology and Pharmacology." Brain Sciences 10, no. 12 (November 25, 2020): 907. http://dx.doi.org/10.3390/brainsci10120907.
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Sleep-related hypermotor epilepsy (SHE) is characterized by hyperkinetic focal seizures, mainly arising in the neocortex during non-rapid eye movements (NREM) sleep. The familial form is autosomal dominant SHE (ADSHE), which can be caused by mutations in genes encoding subunits of the neuronal nicotinic acetylcholine receptor (nAChR), Na+-gated K+ channels, as well as non-channel signaling proteins, such as components of the gap activity toward rags 1 (GATOR1) macromolecular complex. The causative genes may have different roles in developing and mature brains. Under this respect, nicotinic receptors are paradigmatic, as different pathophysiological roles are exerted by distinct nAChR subunits in adult and developing brains. The widest evidence concerns α4 and β2 subunits. These participate in heteromeric nAChRs that are major modulators of excitability in mature neocortical circuits as well as regulate postnatal synaptogenesis. However, growing evidence implicates mutant α2 subunits in ADSHE, which poses interpretive difficulties as very little is known about the function of α2-containing (α2*) nAChRs in the human brain. Planning rational therapy must consider that pharmacological treatment could have different effects on synaptic maturation and adult excitability. We discuss recent attempts towards precision medicine in the mature brain and possible approaches to target developmental stages. These issues have general relevance in epilepsy treatment, as the pathogenesis of genetic epilepsies is increasingly recognized to involve developmental alterations.
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Sharma, Vijendra, Rapita Sood, Danning Lou, Tzu-Yu Hung, Maxime Lévesque, Yelin Han, Jeremy Y. Levett, et al. "4E-BP2–dependent translation in parvalbumin neurons controls epileptic seizure threshold." Proceedings of the National Academy of Sciences 118, no. 15 (April 5, 2021): e2025522118. http://dx.doi.org/10.1073/pnas.2025522118.
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The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) integrates multiple signals to regulate critical cellular processes such as mRNA translation, lipid biogenesis, and autophagy. Germline and somatic mutations in mTOR and genes upstream of mTORC1, such as PTEN, TSC1/2, AKT3, PIK3CA, and components of GATOR1 and KICSTOR complexes, are associated with various epileptic disorders. Increased mTORC1 activity is linked to the pathophysiology of epilepsy in both humans and animal models, and mTORC1 inhibition suppresses epileptogenesis in humans with tuberous sclerosis and animal models with elevated mTORC1 activity. However, the role of mTORC1-dependent translation and the neuronal cell types mediating the effect of enhanced mTORC1 activity in seizures remain unknown. The eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and 2 (4E-BP2) are translational repressors downstream of mTORC1. Here we show that the ablation of 4E-BP2, but not 4E-BP1, in mice increases the sensitivity to pentylenetetrazole (PTZ)- and kainic acid (KA)–induced seizures. We demonstrate that the deletion of 4E-BP2 in inhibitory, but not excitatory neurons, causes an increase in the susceptibility to PTZ-induced seizures. Moreover, mice lacking 4E-BP2 in parvalbumin, but not somatostatin or VIP inhibitory neurons exhibit a lowered threshold for seizure induction and reduced number of parvalbumin neurons. A mouse model harboring a human PIK3CA mutation that enhances the activity of the PI3K-AKT pathway (Pik3caH1047R-Pvalb) selectively in parvalbumin neurons shows susceptibility to PTZ-induced seizures. Our data identify 4E-BP2 as a regulator of epileptogenesis and highlight the central role of increased mTORC1-dependent translation in parvalbumin neurons in the pathophysiology of epilepsy.
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Nabavi Nouri, Maryam, Lama Alandijani, Kalene van Engelen, Soumitra Tole, Emilie Lalonde, and Tugce B. Balci. "From Alpha-Thalassemia Trait to NPRL3-Related Epilepsy: A Genomic Diagnostic Odyssey." Genes 15, no. 7 (June 25, 2024): 836. http://dx.doi.org/10.3390/genes15070836.
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Introduction: The NPRL3 gene is a critical component of the GATOR1 complex, which negatively regulates the mTORC1 pathway, essential for neurogenesis and brain development. Located on chromosome 16p13.3, NPRL3 is situated near the α-globin gene cluster. Haploinsufficiency of NPRL3, either by deletion or a pathogenic variant, is associated with a variable phenotype of focal epilepsy, with or without malformations of cortical development, with known decreased penetrance. Case Description: This work details the diagnostic odyssey of a neurotypical 10-year-old boy who presented at age 2 with unusual nocturnal episodes and a history of microcytic anemia, as well as a review of the existing literature on NPRL3-related epilepsy, with an emphasis on individuals with deletions who also present with α-thalassemia trait. The proband’s episodes were mistaken for gastroesophageal reflux disease for several years. He had molecular testing for his α-thalassemia trait and was noted to carry a deletion encompassing the regulatory region of the α-thalassemia gene cluster. Following the onset of overt focal motor seizures, genetic testing revealed a heterozygous loss of NPRL3, within a 106 kb microdeletion on chromosome 16p13.3, inherited from his mother. This deletion encompassed the entire NPRL3 gene, which overlaps the regulatory region of the α-globin gene cluster, giving him the dual diagnosis of NPRL3-related epilepsy and α-thalassemia trait. Brain imaging postprocessing showed left hippocampal sclerosis and mid-posterior para-hippocampal focal cortical dysplasia, leading to the consideration of epilepsy surgery. Conclusions: This case underscores the necessity of early and comprehensive genetic assessments in children with epilepsy accompanied by systemic features, even in the absence of a family history of epilepsy or a developmental delay. Recognizing phenotypic overlaps is crucial to avoid diagnostic delays. Our findings also highlight the impact of disruptions in regulatory regions in genetic disorders: any individual with full gene deletion of NPRL3 would have, at a minimum, α-thalassemia trait, due to the presence of the major regulatory element of α-globin genes overlapping the gene’s introns.
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Lemke, Johannes R. "Commentary: GATOR Complex-Associated Epilepsies." Epilepsia 58, no. 7 (June 14, 2017): 1121–22. http://dx.doi.org/10.1111/epi.13789.
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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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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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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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Parmigiani, Anita, Aida Nourbakhsh, Boxiao Ding, Wei Wang, Young Chul Kim, Konstantin Akopiants, Kun-Liang Guan, Michael Karin, and Andrei V. Budanov. "Sestrins Inhibit mTORC1 Kinase Activation through the GATOR Complex." Cell Reports 9, no. 4 (November 2014): 1281–91. http://dx.doi.org/10.1016/j.celrep.2014.10.019.
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Steiner, Laurie. "Helping GATA1 make complex decisions." Blood 139, no. 24 (June 16, 2022): 3457–59. http://dx.doi.org/10.1182/blood.2022016347.
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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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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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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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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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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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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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Weckhuysen, Sarah, Elise Marsan, Virginie Lambrecq, Cécile Marchal, Mélanie Morin-Brureau, Isabelle An-Gourfinkel, Michel Baulac, et al. "Involvement of GATOR complex genes in familial focal epilepsies and focal cortical dysplasia." Epilepsia 57, no. 6 (May 13, 2016): 994–1003. http://dx.doi.org/10.1111/epi.13391.
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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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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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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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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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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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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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Mishima, Yuta, Satoru Miyagi, Atsunori Saraya, Masamitsu Negishi, Mitsuhiro Endoh, Naoto Yamaguchi, Issay Kitabayashi, Haruhiko Koseki, and Atsushi Iwama. "The Hbo1-Brd1/Brpf2 HAT Complex Is Required for Erythropoiesis In Fetal Liver." Blood 116, no. 21 (November 19, 2010): 3872. http://dx.doi.org/10.1182/blood.v116.21.3872.3872.
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Abstract Abstract 3872 Bromodomain-containing protein 1 (Brd1, initially designated as BR140-LIKE; BRL) contains a bromodomain, two plant homology domain (PHD) zinc fingers, and a proline-tryptophan-tryptophan-proline (PWWP) domain, three types of modules characteristic of chromatin regulators. Recently, BRD1 appeared to belong to the BRPF family which includes BRPF1, BRD1/BRPF2, and BRPF3. Among them, BRPF1 is known to be a subunit of the MOZ H3 histone acetyltransferase (HAT) complex. BRD1 has been proposed to be additional subunit of the MOZ H3 HAT complex on the analogy of BRPF1. However, its molecular function remains elusive. To elucidate the biological functions of BRD1, we generated Brd1-null mice and found that they die in utero. Brd1-/- embryos were alive and recovered at nearly the expected Mendelian ratio at 12.5 days postcoitum (dpc) but died by 15.5 dpc. Brd1-/- embryos at 12.5 dpc were pale and the cell number of fetal livers, in which fetal hematopoiesis occurs, was decreased to about 20% of the control. Cytological analysis revealed that Brd1-/- fetal livers had profoundly fewer erythroblasts at maturation stages beyond proerythroblasts compared to wild-type fetal livers. Flow cytometric analysis of Brd1-/- fetal livers revealed a significant accumulation of CD71+Ter119- proerythroblasts and a reduction in CD71+Ter119+ and CD71-Ter119+ maturating erythroblasts. A drastic increase in AnnexinV+ apoptotic cells was detected in the CD71+Ter119+ and CD71-Ter119- cell fractions in Brd1-/- fetal livers. These findings suggested that severe anemia caused by compromised differentiation and/or survival of erythroblasts accounts for embryonic lethality of Brd1-/- embryos. To understand the mechanism underlying defective erythropoiesis in Brd1-null embryos, we performed biochemical analyses and found that Brd1 bridges the HAT, HBO1 but not MOZ, and its activator protein, ING4, to form an enzymatically active HAT complex. Forced expression of Brd1 promoted erythroid differentiation of K562 cells, while Brpf1, which preferentially binds to MOZ, had no significant effect. Correspondingly, depletion of Hbo1 by Hbo1 knockdown perturbed erythroid differentiation of mouse fetal liver progenitors. Of note, the level of global acetylation of histone H3 at lysine 14 (H3K14) was specifically decreased in Brd1-deficient erythroblasts. These results collectively implied that acetylation of H3K14 catalyzed by the Hbo1-Brd1 complex has a crucial role in fetal liver erythropoiesis. To identify the downstream targets for the HBO1-BRD1 complex, we performed the ChIP-on-chip analysis in K562 cells and found that BRD1 and HBO1 largely co-localize on the genome, especially on the promoters of erythroid transcription factor genes. ChIP analysis revealed that acetylation of H3K14 at the promoters of erythroid transcription factor genes, including Gata1, Gata2, Tal1, Stat5a, and ETO2, were profoundly diminished in the Brd1-deficient erythroblasts. Among these target genes, we focused on Gata1, which plays a central role in erythropoiesis, and carried out complementation experiments with Gata1 using a Gata1 retrovirus. Exogenous Gata1, but not Bcl-xL, efficiently improved proliferative capacity and survival of Brd1-deficient erythroid progenitors and also restored, at least partially, their impaired differentiation. These results clearly showed that the Hbo1-Brd1 complex is required for the acetylation of H3K14 at the promoters of erythroid transcription factor genes, thereby is crucial for erythropoiesis in fetal liver. 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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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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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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Wagenblast, Elvin, Olga I. Gan, Maria Azkanaz, Sabrina A. Smith, Joana Araújo, Lorien Shakib, Jessica L. McLeod, et al. "Understanding Pre-Leukemia in Trisomy 21 Human HSC and Modeling Progression Towards Down Syndrome Associated Leukemia Using CRISPR/Cas9 at Single Cell Resolution." Blood 134, Supplement_1 (November 13, 2019): 2531. http://dx.doi.org/10.1182/blood-2019-129259.
Full textAbstract:
Leukemia is the most common cancer in children. Sequencing data from identical twins suggests that the first genetic alterations in childhood leukemia occur in utero. Children with Down syndrome (Trisomy 21, T21) have an increased risk of childhood leukemia. In 30% of newborns with Down syndrome, a transient pre-leukemia disease occurs, which is characterized by a clonal proliferation of immature megakaryocytes carrying somatic mutations in the GATA1 transcription factor. These acquired GATA1 mutations lead to the expression of an N-terminal truncated protein (GATA1-Short). In 20% of the cases, acute megakaryoblastic leukemia (AMKL) evolves from the pre-leukemia by acquisition of additional genetic mutations in the transient leukemia clone, predominantly in genes of the cohesin complex. It is hypothesized that this represents a multi-step process of leukemogenesis with three distinct genetic events: T21, GATA1-Short and additional cohesin mutations. Yet, it remains unclear how an extra copy of chromosome 21 predisposes towards leukemia, the mechanisms of leukemic transformation and the interplay between each genetic component. Therefore, we wanted to establish a tractable human model system to investigate the initiation and evolution of transient leukemia and AMKL using CRISPR/Cas9 genome editing in primary human hematopoietic stem cells (HSCs). To model the initiation of Down syndrome associated pre-leukemia, we utilized both neonatal cord blood and fetal liver derived LT-HSCs and other progenitor populations to express either the short or long isoform of GATA1 (GATA1-Short or GATA1-Long). This was carried out using an improved methodology that permits the in vitro and in vivo functional interrogation of CRISPR/Cas9 edited human LT-HSCs at the single cell level (Wagenblast et al., bioRxiv 609040). Importantly, in this case, expression of either GATA1 isoform remained under the regulatory control of the endogenous promoter. Culture of single LT-HSC, short-term (ST-HSC) and myelo-erythroid progenitors (MEP) revealed a drastic shift towards megakaryocytic lineage output upon exclusive expression of GATA1-Short compared to control or GATA1-Long, regardless of the developmental source of the derived cells. To investigate the functional consequences of exclusive GATA1-Short expression in LT-HSCs in vivo, we performed near-clonal xenotransplantation assays in NSG and NSGW41 mice. Strikingly, GATA1-Short edited LT-HSCs injected mice displayed a higher percentage of human CD41+CD45- megakaryocytic lineage derived cells and a decrease in human GlyA+CD45- erythroid cells compared to control. Morphological analysis revealed more immature forms of erythroid cells and fewer enucleated erythrocytes in GATA1-Short edited LT-HSCs injected mice. In order to add an additional genetic determinant to our model, we utilized T21 fetal liver derived LT-HSCs. Un-manipulated T21 LT-HSCs and other progenitor populations showed a bias towards erythroid, myeloid and megakaryocytic lineages at the expense of lymphoid fates. In vitro, the combination of T21 and CRISPR/Cas9-mediated GATA1-Short in LT-HSCs led to an increase in megakaryocytic lineage output, while decreasing erythroid output. This phenotype was similar to what was observed in normal karyotype fetal liver derived LT-HSCs. However, near clonal transplantation of GATA1-Short edited T21 LT-HSCs in NSG mice generated exclusive CD33+ myeloid grafts with disproportionate high levels of CD41+CD45- megakaryocytic lineage derived cells compared to T21 control. In addition a distinct CD34+CD41+CD71+CD45+ population was present. Thus, this phenotype is reminiscent of Down Syndrome associated transient leukemia. In summary, by using an improved CRISPR/Cas9 single cell methodology we show how GATA1 regulates lineage fate in normal and T21 LT-HSCs and other progenitor populations. Importantly, we show for the first time a humanized mouse model of Down syndrome associated transient leukemia, which was induced from T21 human fetal liver derived LT-HSCs engineered to express GATA1-Short. Current studies focus on adding additional mutations of the cohesin complex to progress transient leukemia to AMKL. Disclosures No relevant conflicts of interest to declare.
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Campbell, Amy E., Lorna Wilkinson-White, Joel P. Mackay, Jacqueline M. Matthews, and Gerd A. Blobel. "Dissecting the Molecular Pathways That Underlie Disease-Causing GATA1 Mutations." Blood 120, no. 21 (November 16, 2012): 3439. http://dx.doi.org/10.1182/blood.v120.21.3439.3439.
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Abstract Abstract 3439 Missense mutations in the gene encoding hematopoietic transcription factor GATA1 cause congenital anemias and/or thrombocytopenias. All seven reported mutations give rise to amino acid substitutions within the amino-terminal zinc finger (NF), but produce a range of phenotypes. The clinical severity depends on the site and type of substitution, and different substitutions of the same residue can produce disparate phenotypes. We combined structural, biochemical, in vivo conditional rescue approaches, and genomic analyses to systematically characterize all known GATA1 mutations with the goal of determining how they alter GATA1 function to result in disease. Introducing mutant forms of GATA1 into GATA1-null erythroid or bipotential erythromegakaryocytic cell lines essentially recapitulated patient phenotypes. The V205M, G208S, G208R, and D218Y mutations severely impaired both erythroid and megakaryocyte maturation, while the R216Q, R216W, and D218G mutations had only a mild effect on the maturation of these lineages. Global differentiation defects were reproduced at the level of individual GATA1 target genes. Moreover, the former mutants greatly impaired both the transcriptional activation and repression functions of GATA1, while the latter moderately impaired gene activation but had no effect on repression. It had been suggested previously that GATA1 mutations could be categorized into two classes, those that impair binding of the NF to the essential GATA1 cofactor FOG1 (V205M, G208S, G208R) and those that diminish binding of the NF to DNA (R216Q and R216W). The impact of the final two mutations (D218G and D218Y) remained uncertain, as this residue is not part of any known interaction face. Our work led to the following novel conclusions: Binding studies using isothermal titration calorimetry (ITC) and chromatin immunoprecipitation (ChIP) produced concurrent results showing that the V205M, G208S, G208R, and D218Y mutations diminish the GATA1-FOG1 interaction in vitro and FOG1 recruitment to GATA1 target genes in vivo. Interestingly, in contrast to D218Y, D218G did not affect FOG1 binding in vitro or in vivo. Furthermore, G208S had a less pronounced impact on FOG1 binding than the other three mutations, thus correlating the severity of the clinical presentation with the degree of FOG1 disruption. This confirms and extends previous work linking impaired FOG1 binding to the disease phenotypes associated with this class of mutations.ITC showed that R216Q and R216W disrupt DNA binding in vitro, consistent with previous in vitro studies. However, remarkably, ChIP assays revealed that neither mutation impaired in vivo GATA1 target site occupancy at any examined simple or palindromic GATA elements, suggesting that failure to bind DNA does not account for the associated clinical phenotypes.Notably, the R216Q and D218G mutations selectively diminished recruitment of Tal1/SCL without affecting the interaction with FOG1 or DNA. This implicates for the first time the Tal1/SCL complex in the pathogenesis of disorders caused by GATA1 mutations. Since the Tal1/SCL complex functions mostly during GATA1 gene activation, this also explains the observation that these GATA1 mutants largely retain their ability to repress transcription. Moreover, changes in the gene expression profiles of R216Q and D218G expressing cells are highly correlated with each other but clearly distinct from the gene expression changes associated with different substitutions at the same residues (R216W or D218Y), revealing a specific subset of genes that are most sensitive to disruption of the GATA1-Tal1/SCL interaction.An unexpected finding from our studies is that different substitutions of the same residue can disrupt binding to distinct cofactors (e.g. D218G impairs Tal1/SCL binding while D218Y impairs FOG1 binding), thus accounting for variable disease presentation. In concert, our work on GATA1 mutations in their native environment reveals critical new insights not obtainable from in vitro studies. This highlights the usefulness of gene complementation studies in the relevant lineages for the dissection of transcription pathways to better understand and ultimately diagnose and treat hematologic disease. Disclosures: No relevant conflicts of interest to declare.
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Chen, Jian, Yue Li, Fouad Yousif, Sagi Abelson, Sanaz Manteghi, John D. McPherson, and Johann K. Hitzler. "Postnatally Acquired Mutations Underlie the Progression of Transient Leukemia to Myeloid Leukemia of Down Syndrome." Blood 132, Supplement 1 (November 29, 2018): 442. http://dx.doi.org/10.1182/blood-2018-99-117198.
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Abstract INTRODUCTION . Transient Leukemia (TL; also termed Transient Myeloproliferative disorder, TMD, and Transient Abnormal Myelopoiesis, TAM) occurs in 10-30% of newborns with Down syndrome (DS). Approx. 20% of infants with TL go on to develop acute myeloid leukemia of DS (ML-DS), typically within the first four years of life. Somatic, clone-specific mutations of GATA1 are found both in the blasts of TL and ML-DS, are concordant within the same individual and thought to function as initiating event in the development of ML-DS. In contrast, additional mutations of cohesin complex and related genes (e.g. RAD21, STAG2, CTCF), epigenetic regulators (e. g. EZH2) and signal transducers (e.g. within RAS, JAK signaling pathways) have been identified only in ML-DS blasts and are thought to cooperate with mutant GATA1 in the progression from TL to ML-DS. It is not known whether these cooperating mutations already mark a minor subclone of TL blasts at birth - allowing, at least in principle, a genetic risk stratification of TL - or are acquired postnatally during the first four years of life. OBJECTIVES . We tested the functional impact of impaired function of cohesin complex genes, CTCF and EZH2 on the progression of TL to ML-DS. We asked if mutations representing putative genetic progression events were already detectable at birth in a minor clone of TL blasts or were acquired postnatally (during the first four years of life). METHODS. The spectrum of GATA1 and cooperating mutations was determined by whole exome sequencing in fractions of TL and ML-DS blasts sorted from blood and bone marrow samples of five patients who had successively developed both disorders including one with a relapse of ML-DS. Corresponding normal T lymphocyte fractions of each patient at the stage of TL and ML-DS served as controls. Numbers of blasts harboring specific mutations were quantified by digital droplet PCR (BioRad, Inc.). Primary TL cells were transduced with lentivirus encoding shRNA (pLVX-shRNA, Clontech, Inc.) to suppress expression of cohesin complex genes, CTCF and EZH2 and intrafemurally injected into 8 week old NSG recipient mice. Engraftment in the bone marrow was assessed 8 weeks later by flow cytometry and GATA1 mutational analysis and compared to TL cells transduced with control vector. RESULTS. TL blasts harbored fewer mutations than those of ML-DS. GATA1 mutations were concordant in TL and ML-DS blasts in the same patient, consistent with development of ML-DS from subclone of TL. Knockdown of RAD21 expression in primary TL blasts, mimicking loss of function mutation of a cohesin complex gene, resulted in significantly increased engraftment of transduced cells in xenograft recipients compared to controls. This finding is consistent with RAD21 loss of function mutations playing the role of a progression event. Mutations of cohesin complex genes (SMC1A, STAG2, RAD21), NRAS and other putative cooperating mutations (with mutant GATA1) were not detectable in any sample of primary TL blasts by either whole exome sequencing or digital droplet PCR. The same result was obtained with control T lymphocytes sorted from TL samples. ML-DS blasts in one case were oligo-clonal with regard to cohesin complex gene mutations. Relapse in this patient arose from a minor clone as defined by cohesin complex gene mutations; mutations of NRAS, KNASL1 and SMC1A were present in ML-DS blasts but absent at relapse. CONCLUSIONS . Increased engraftment of TL cells with suppressed RAD21 expression is consistent with a model in which RAD21 loss of function mutations function as a progression event in the development of ML-DS. Absence of detectable cohesin complex gene mutations and other putative cooperating events in TL blasts suggests these mutations are acquired during the first four years of life and do not mark a minor clone of TL blasts present at birth. Genomic screening of TL blasts at birth therefore is unlikely to predict the risk for development of ML-DS. Disclosures No relevant conflicts of interest to declare.
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Chiu, Sung K., Stephanie L. Orive, Mitchell J. Moon, Jesslyn Saw, Sarah Ellis, Benjamin T. Kile, Yizhou Huang, et al. "Shared roles for Scl and Lyl1 in murine platelet production and function." Blood 134, no. 10 (September 5, 2019): 826–35. http://dx.doi.org/10.1182/blood.2019896175.
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Abstract The stem cell leukemia (Scl or Tal1) protein forms part of a multimeric transcription factor complex required for normal megakaryopoiesis. However, unlike other members of this complex such as Gata1, Fli1, and Runx1, mutations of Scl have not been observed as a cause of inherited thrombocytopenia. We postulated that functional redundancy with its closely related family member, lymphoblastic leukemia 1 (Lyl1) might explain this observation. To determine whether Lyl1 can substitute for Scl in megakaryopoiesis, we examined the platelet phenotype of mice lacking 1 or both factors in megakaryocytes. Conditional Scl knockout (KO) mice crossed with transgenic mice expressing Cre recombinase under the control of the mouse platelet factor 4 (Pf4) promoter generated megakaryocytes with markedly reduced but not absent Scl. These Pf4Sclc-KO mice had mild thrombocytopenia and subtle defects in platelet aggregation. However, Pf4Sclc-KO mice generated on an Lyl1-null background (double knockout [DKO] mice) had severe macrothrombocytopenia, abnormal megakaryocyte morphology, defective pro-platelet formation, and markedly impaired platelet aggregation. DKO megakaryocytes, but not single-knockout megakaryocytes, had reduced expression of Gata1, Fli1, Nfe2, and many other genes that cause inherited thrombocytopenia. These gene expression changes were significantly associated with shared Scl and Lyl1 E-box binding sites that were also enriched for Gata1, Ets, and Runx1 motifs. Thus, Scl and Lyl1 share functional roles in platelet production by regulating expression of partner proteins including Gata1. We propose that this functional redundancy provides one explanation for the absence of Scl and Lyl1 mutations in inherited thrombocytopenia.
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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.
Full textAbstract:
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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Gregory, Gregory D., Annarita Miccio, Alexey Bersenev, Yuhuan Wang, Wei Hong, Zhe Zhang, Mortimer Poncz, Wei Tong, and Gerd A. Blobel. "FOG1 requires NuRD to promote hematopoiesis and maintain lineage fidelity within the megakaryocytic-erythroid compartment." Blood 115, no. 11 (March 18, 2010): 2156–66. http://dx.doi.org/10.1182/blood-2009-10-251280.
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Abstract Nuclear factors regulate the development of complex tissues by promoting the formation of one cell lineage over another. The cofactor FOG1 interacts with transcription factors GATA1 and GATA2 to control erythroid and megakaryocyte (MK) differentiation. In contrast, FOG1 antagonizes the ability of GATA factors to promote mast cell (MC) development. Normal FOG1 function in late-stage erythroid cells and MK requires interaction with the chromatin remodeling complex NuRD. Here, we report that mice in which the FOG1/NuRD interaction is disrupted (Fogki/ki) produce MK-erythroid progenitors that give rise to significantly fewer and less mature MK and erythroid colonies in vitro while retaining multilineage capacity, capable of generating MCs and other myeloid lineage cells. Gene expression profiling of Fogki/ki MK-erythroid progenitors revealed inappropriate expression of several MC-specific genes. Strikingly, aberrant MC gene expression persisted in mature Fogki/ki MK and erythroid progeny. Using a GATA1-dependent committed erythroid cell line, select MC genes were found to be occupied by NuRD, suggesting a direct mechanism of repression. Together, these observations suggest that a simple heritable silencing mechanism is insufficient to permanently repress MC genes. Instead, the continuous presence of GATA1, FOG1, and NuRD is required to maintain lineage fidelity throughout MK-erythroid ontogeny.