Journal articles on the topic 'RagD'
To see the other types of publications on this topic, follow the link: RagD.
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 journal articles for your research on the topic 'RagD.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
1
Lee, Minji, Jong Hyun Kim, Ina Yoon, Chulho Lee, Mohammad Fallahi Sichani, Jong Soon Kang, Jeonghyun Kang, et al. "Coordination of the leucine-sensing Rag GTPase cycle by leucyl-tRNA synthetase in the mTORC1 signaling pathway." Proceedings of the National Academy of Sciences 115, no. 23 (May 21, 2018): E5279—E5288. http://dx.doi.org/10.1073/pnas.1801287115.
Full textAbstract:
A protein synthesis enzyme, leucyl-tRNA synthetase (LRS), serves as a leucine sensor for the mechanistic target of rapamycin complex 1 (mTORC1), which is a central effector for protein synthesis, metabolism, autophagy, and cell growth. However, its significance in mTORC1 signaling and cancer growth and its functional relationship with other suggested leucine signal mediators are not well-understood. Here we show the kinetics of the Rag GTPase cycle during leucine signaling and that LRS serves as an initiating “ON” switch via GTP hydrolysis of RagD that drives the entire Rag GTPase cycle, whereas Sestrin2 functions as an “OFF” switch by controlling GTP hydrolysis of RagB in the Rag GTPase–mTORC1 axis. The LRS–RagD axis showed a positive correlation with mTORC1 activity in cancer tissues and cells. The GTP–GDP cycle of the RagD–RagB pair, rather than the RagC–RagA pair, is critical for leucine-induced mTORC1 activation. The active RagD–RagB pair can overcome the absence of the RagC–RagA pair, but the opposite is not the case. This work suggests that the GTPase cycle of RagD–RagB coordinated by LRS and Sestrin2 is critical for controlling mTORC1 activation, and thus will extend the current understanding of the amino acid-sensing mechanism.
2
Gollwitzer, Peter, Nina Grützmacher, Sabine Wilhelm, Daniel Kümmel, and Constantinos Demetriades. "A Rag GTPase dimer code defines the regulation of mTORC1 by amino acids." Nature Cell Biology 24, no. 9 (September 2022): 1394–406. http://dx.doi.org/10.1038/s41556-022-00976-y.
Full textAbstract:
AbstractAmino acid availability controls mTORC1 activity via a heterodimeric Rag GTPase complex that functions as a scaffold at the lysosomal surface, bringing together mTORC1 with its activators and effectors. Mammalian cells express four Rag proteins (RagA–D) that form dimers composed of RagA/B bound to RagC/D. Traditionally, the Rag paralogue pairs (RagA/B and RagC/D) are referred to as functionally redundant, with the four dimer combinations used interchangeably in most studies. Here, by using genetically modified cell lines that express single Rag heterodimers, we uncover a Rag dimer code that determines how amino acids regulate mTORC1. First, RagC/D differentially define the substrate specificity downstream of mTORC1, with RagD promoting phosphorylation of its lysosomal substrates TFEB/TFE3, while both Rags are involved in the phosphorylation of non-lysosomal substrates such as S6K. Mechanistically, RagD recruits mTORC1 more potently to lysosomes through increased affinity to the anchoring LAMTOR complex. Furthermore, RagA/B specify the signalling response to amino acid removal, with RagB-expressing cells maintaining lysosomal and active mTORC1 even upon starvation. Overall, our findings reveal key qualitative differences between Rag paralogues in the regulation of mTORC1, and underscore Rag gene duplication and diversification as a potentially impactful event in mammalian evolution.
3
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.
Full textAbstract:
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.
4
Shen, Kuang, and David M. Sabatini. "Ragulator and SLC38A9 activate the Rag GTPases through noncanonical GEF mechanisms." Proceedings of the National Academy of Sciences 115, no. 38 (September 4, 2018): 9545–50. http://dx.doi.org/10.1073/pnas.1811727115.
Full textAbstract:
The mechanistic target of rapamycin complex 1 (mTORC1) growth pathway detects nutrients through a variety of sensors and regulators that converge on the Rag GTPases, which form heterodimers consisting of RagA or RagB tightly bound to RagC or RagD and control the subcellular localization of mTORC1. The Rag heterodimer uses a unique “locking” mechanism to stabilize its active (GTPRagA–RagCGDP) or inactive (GDPRagA–RagCGTP) nucleotide states. The Ragulator complex tethers the Rag heterodimer to the lysosomal surface, and the SLC38A9 transmembrane protein is a lysosomal arginine sensor that upon activation stimulates mTORC1 activity through the Rag GTPases. How Ragulator and SLC38A9 control the nucleotide loading state of the Rag GTPases remains incompletely understood. Here we find that Ragulator and SLC38A9 are each unique guanine exchange factors (GEFs) that collectively push the Rag GTPases toward the active state. Ragulator triggers GTP release from RagC, thus resolving the locked inactivated state of the Rag GTPases. Upon arginine binding, SLC38A9 converts RagA from the GDP- to the GTP-loaded state, and therefore activates the Rag GTPase heterodimer. Altogether, Ragulator and SLC38A9 act on the Rag GTPases to activate the mTORC1 pathway in response to nutrient sufficiency.
5
Mu, Ying, Dongmei Zheng, Cong Wang, Wei Yu, and Xiaonan Zhang. "RagD regulates amino acid mediated-casein synthesis and cell proliferation via mTOR signalling in cow mammary epithelial cells." Journal of Dairy Research 85, no. 2 (May 2018): 204–11. http://dx.doi.org/10.1017/s0022029918000146.
Full textAbstract:
This research paper addresses the hypothesis that RagD is a key signalling factor that regulates amino acid (AA) mediated-casein synthesis and cell proliferation in cow mammary epithelial cells (CMECs). The expression of RagD was analysed at different times during pregnancy and lactation in bovine mammary tissue from dairy cows. We showed that expression of RagD at lactation period was higher (P < 0·05) than that at pregnancy period. When CMECs were treated with methionine (Met) or lysine (Lys), expression of RagD, β-casein (CSN2), mTOR and p-mTOR, and cell proliferation were increased. Further, when CMECs were treated to overexpress RagD, expression of CSN2, mTOR and p-mTOR, and cell proliferation were up-regulated. Furthermore, the increase in expression of CSN2, mTOR and p-mTOR, and cell proliferation in response to Met or Lys supply was inhibited by inhibiting RagD, and those effects were reversed in the overexpression model. When CMECs were treated with RagD overexpression together with mTOR inhibition or conversely with RagD inhibition together with mTOR overexpression, results showed that the increase in expression of CSN2 and cell proliferation in response to RagD overexpression was prevented by inhibiting mTOR, and those effects were reversed by overexpressing mTOR. The interaction of RagD with subunit proteins of mTORC1 was analysed, and the result showed that RagD interacted with Raptor. CMECs were treated with Raptor inhibition, and the result showed that the increase in expression of mTOR and p-mTOR in response to RagD overexpression was inhibited by inhibiting Raptor.In conclusion, our study showed that RagD is an important activation factor of mTORC1 in CMECs, activating AA-mediated casein synthesis and cell proliferation, potentially acting via Raptor.
6
Zhang, Yiwen, Hongrong Hu, Weiwei Liu, Shu-Mei Yan, Yuzhuang Li, Likai Tan, Yingshi Chen, et al. "Amino acids and RagD potentiate mTORC1 activation in CD8+ T cells to confer antitumor immunity." Journal for ImmunoTherapy of Cancer 9, no. 4 (April 2021): e002137. http://dx.doi.org/10.1136/jitc-2020-002137.
Full textAbstract:
BackgroundIn the tumor microenvironment, tumor cells are able to suppress antitumor immunity by competing for essential nutrients, including amino acids. However, whether amino acid depletion modulates the activity of CD8+ tumor-infiltrating lymphocytes (TILs) is unclear.MethodIn this study, we evaluated the roles of amino acids and the Rag complex in regulating mammalian target of rapamycin complex 1 (mTORC1) signaling in CD8+ TILs.ResultsWe discovered that the Rag complex, particularly RagD, was crucial for CD8+ T-cell antitumor immunity. RagD expression was positively correlated with the antitumor response of CD8+ TILs in both murine syngeneic tumor xenografts and clinical human colon cancer samples. On RagD deficiency, CD8+ T cells were rendered more dysfunctional, as demonstrated by attenuation of mTORC1 signaling and reductions in proliferation and cytokine secretion. Amino acids maintained RagD-mediated mTORC1 translocation to the lysosome, thereby achieving maximal mTORC1 activity in CD8+ T cells. Moreover, the limited T-cell access to leucine (LEU), overshadowed by tumor cell amino acid consumption, led to impaired RagD-dependent mTORC1 activity. Finally, combined with antiprogrammed cell death protein 1 antibody, LEU supplementation improved T-cell immunity in MC38 tumor-bearing mice in vivo.ConclusionOur results revealed that robust signaling of amino acids by RagD and downstream mTORC1 signaling were crucial for T-cell receptor-initiated antitumor immunity. The characterization the role of RagD and LEU in nutrient mTORC1 signaling in TILs might suggest potential therapeutic strategies based on the manipulation of RagD and its upstream pathway.
7
Prior, C., P. Mamessier, H. Fukuhara, X. J. Chen, and M. Wesolowski-Louvel. "The hexokinase gene is required for transcriptional regulation of the glucose transporter gene RAG1 in Kluyveromyces lactis." Molecular and Cellular Biology 13, no. 7 (July 1993): 3882–89. http://dx.doi.org/10.1128/mcb.13.7.3882-3889.1993.
Full textAbstract:
The RAG1 gene of Kluyveromyces lactis encodes a low-affinity glucose/fructose transporter. Its transcription is induced by glucose, fructose, and several other sugars. The RAG4, RAG5, and RAG8 genes are trans-acting genes controlling the expression of the RAG1 gene. We report here the characterization of one of these genes, RAG5. The nucleotide sequence of the cloned RAG5 gene indicated that it encodes a protein that is homologous to hexokinases of Saccharomyces cerevisiae. rag5 mutants showed no detectable hexokinase or glucokinase activity, suggesting that the sugar kinase activity encoded by this gene is the only hexokinase in K. lactis. Both high- and low-affinity transport systems of glucose were affected in rag5 mutants. The defect of the low-affinity component was found to be due to a block of transcription of the RAG1 gene by the hexokinase mutation. In vivo complementation of the rag5 mutation by the HXK2 gene of S. cerevisiae and complementation of hxk1 hxk2 mutations of S. cerevisiae by the RAG5 gene showed that RAG5 and HXK2 were equivalent for sugar-phosphorylating activity but that RAG5 could not restore glucose repression in the S. cerevisiae hexokinase mutants.
8
Prior, C., P. Mamessier, H. Fukuhara, X. J. Chen, and M. Wesolowski-Louvel. "The hexokinase gene is required for transcriptional regulation of the glucose transporter gene RAG1 in Kluyveromyces lactis." Molecular and Cellular Biology 13, no. 7 (July 1993): 3882–89. http://dx.doi.org/10.1128/mcb.13.7.3882.
Full textAbstract:
The RAG1 gene of Kluyveromyces lactis encodes a low-affinity glucose/fructose transporter. Its transcription is induced by glucose, fructose, and several other sugars. The RAG4, RAG5, and RAG8 genes are trans-acting genes controlling the expression of the RAG1 gene. We report here the characterization of one of these genes, RAG5. The nucleotide sequence of the cloned RAG5 gene indicated that it encodes a protein that is homologous to hexokinases of Saccharomyces cerevisiae. rag5 mutants showed no detectable hexokinase or glucokinase activity, suggesting that the sugar kinase activity encoded by this gene is the only hexokinase in K. lactis. Both high- and low-affinity transport systems of glucose were affected in rag5 mutants. The defect of the low-affinity component was found to be due to a block of transcription of the RAG1 gene by the hexokinase mutation. In vivo complementation of the rag5 mutation by the HXK2 gene of S. cerevisiae and complementation of hxk1 hxk2 mutations of S. cerevisiae by the RAG5 gene showed that RAG5 and HXK2 were equivalent for sugar-phosphorylating activity but that RAG5 could not restore glucose repression in the S. cerevisiae hexokinase mutants.
9
Kim, Chanwoo, Jinjoo Jung, Truong T. Tung, and Seung Bum Park. "β-Turn mimetic-based stabilizers of protein–protein interactions for the study of the non-canonical roles of leucyl-tRNA synthetase." Chemical Science 7, no. 4 (2016): 2753–61. http://dx.doi.org/10.1039/c5sc03493k.
Full textAbstract:
For the systematic perturbation of protein–protein interactions, we designed and synthesized tetra-substituted hexahydro-4H-pyrazino[2,1-c][1,2,4]triazine-4,7(6H)-diones as β-turn mimetics.5c{3,9}stabilizes the direct interaction between LRS and RagD and activates mTORC1 in living cells.
10
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.
Full textAbstract:
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.
11
Jones, Edith, and Ken Inoki. "Microphthalmia-associated transcription factors activate mTORC1 through RagD GTPase gene expression." Translational Cancer Research 6, S7 (October 2017): S1234—S1238. http://dx.doi.org/10.21037/tcr.2017.09.31.
Full text
12
Di Malta, Chiara, Diletta Siciliano, Alessia Calcagni, Jlenia Monfregola, Simona Punzi, Nunzia Pastore, Andrea N. Eastes, et al. "Transcriptional activation of RagD GTPase controls mTORC1 and promotes cancer growth." Science 356, no. 6343 (June 15, 2017): 1188–92. http://dx.doi.org/10.1126/science.aag2553.
Full text
13
SASAKI, HIDEFUMI, MASAYUKI SHITARA, KEISUKE YOKOTA, YU HIKOSAKA, SATORU MORIYAMA, MOTOKI YANO, and YOSHITAKA FUJII. "RagD gene expression and NRF2 mutations in lung squamous cell carcinomas." Oncology Letters 4, no. 6 (2012): 1167–70. http://dx.doi.org/10.3892/ol.2012.938.
Full text
14
Schlingmann, Karl P., François Jouret, Kuang Shen, Anukrati Nigam, Francisco J. Arjona, Claudia Dafinger, Pascal Houillier, et al. "mTOR-Activating Mutations in RRAGD Are Causative for Kidney Tubulopathy and Cardiomyopathy." Journal of the American Society of Nephrology 32, no. 11 (October 4, 2021): 2885–99. http://dx.doi.org/10.1681/asn.2021030333.
Full textAbstract:
BackgroundOver the last decade, advances in genetic techniques have resulted in the identification of rare hereditary disorders of renal magnesium and salt handling. Nevertheless, approximately 20% of all patients with tubulopathy lack a genetic diagnosis.MethodsWe performed whole-exome and -genome sequencing of a patient cohort with a novel, inherited, salt-losing tubulopathy; hypomagnesemia; and dilated cardiomyopathy. We also conducted subsequent in vitro functional analyses of identified variants of RRAGD, a gene that encodes a small Rag guanosine triphosphatase (GTPase).ResultsIn eight children from unrelated families with a tubulopathy characterized by hypomagnesemia, hypokalemia, salt wasting, and nephrocalcinosis, we identified heterozygous missense variants in RRAGD that mostly occurred de novo. Six of these patients also had dilated cardiomyopathy and three underwent heart transplantation. We identified a heterozygous variant in RRAGD that segregated with the phenotype in eight members of a large family with similar kidney manifestations. The GTPase RagD, encoded by RRAGD, plays a role in mediating amino acid signaling to the mechanistic target of rapamycin complex 1 (mTORC1). RagD expression along the mammalian nephron included the thick ascending limb and the distal convoluted tubule. The identified RRAGD variants were shown to induce a constitutive activation of mTOR signaling in vitro.ConclusionsOur findings establish a novel disease, which we call autosomal dominant kidney hypomagnesemia (ADKH-RRAGD), that combines an electrolyte-losing tubulopathy and dilated cardiomyopathy. The condition is caused by variants in the RRAGD gene, which encodes Rag GTPase D; these variants lead to an activation of mTOR signaling, suggesting a critical role of Rag GTPase D for renal electrolyte handling and cardiac function.
15
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.
Full textAbstract:
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.
16
Nilavar, Namrata M., Mayilaadumveettil Nishana, Amita M. Paranjape, Raghunandan Mahadeva, Rupa Kumari, Bibha Choudhary, and Sathees C. Raghavan. "Znc2 module of RAG1 contributes towards structure-specific nuclease activity of RAGs." Biochemical Journal 477, no. 18 (September 24, 2020): 3567–82. http://dx.doi.org/10.1042/bcj20200361.
Full textAbstract:
Recombination activating genes (RAGs), consisting of RAG1 and RAG2 have ability to perform spatially and temporally regulated DNA recombination in a sequence specific manner. Besides, RAGs also cleave at non-B DNA structures and are thought to contribute towards genomic rearrangements and cancer. The nonamer binding domain of RAG1 binds to the nonamer sequence of the signal sequence during V(D)J recombination. However, deletion of NBD did not affect RAG cleavage on non-B DNA structures. In the present study, we investigated the involvement of other RAG domains when RAGs act as a structure-specific nuclease. Studies using purified central domain (CD) and C-terminal domain (CTD) of the RAG1 showed that CD of RAG1 exhibited high affinity and specific binding to heteroduplex DNA, which was irrespective of the sequence of single-stranded DNA, unlike CTD which showed minimal binding. Furthermore, we show that ZnC2 of RAG1 is crucial for its binding to DNA structures as deletion and point mutations abrogated the binding of CD to heteroduplex DNA. Our results also provide evidence that unlike RAG cleavage on RSS, central domain of RAG1 is sufficient to cleave heteroduplex DNA harbouring pyrimidines, but not purines. Finally, we show that a point mutation in the DDE catalytic motif is sufficient to block the cleavage of CD on heteroduplex DNA. Therefore, in the present study we demonstrate that the while ZnC2 module in central domain of RAG1 is required for binding to non-B DNA structures, active site amino acids are important for RAGs to function as a structure-specific nuclease.
17
Hnatova, Martina, Micheline Wésolowski-Louvel, Guenaëlle Dieppois, Julien Deffaud, and Marc Lemaire. "Characterization of KlGRR1 and SMS1 Genes, Two New Elements of the Glucose Signaling Pathway of Kluyveromyces lactis." Eukaryotic Cell 7, no. 8 (June 13, 2008): 1299–308. http://dx.doi.org/10.1128/ec.00454-07.
Full textAbstract:
ABSTRACT The expression of the major glucose transporter gene, RAG1, is induced by glucose in Kluyveromyces lactis. This regulation involves several pathways, including one that is similar to Snf3/Rgt2-ScRgt1 in Saccharomyces cerevisiae. We have identified missing key components of the K. lactis glucose signaling pathway by comparison to the same pathway of S. cerevisiae. We characterized a new mutation, rag19, which impairs RAG1 regulation. The Rag19 protein is 43% identical to the F-box protein ScGrr1 of S. cerevisiae and is able to complement an Scgrr1 mutation. In the K. lactis genome, we identified a single gene, SMS1 (for similar to Mth1 and Std1), that encodes a protein showing an average of 50% identity with Mth1 and Std1, regulators of the ScRgt1 repressor. The suppression of the rag4 (glucose sensor), rag8 (casein kinase I), and rag19 mutations by the Δsms1 deletion, together with the restoration of RAG1 transcription in the double mutants, demonstrates that Sms1 is a negative regulator of RAG1 expression and is acting downstream of Rag4, Rag8, and Rag19 in the cascade. We report that Sms1 regulates KlRgt1 repressor activity by preventing its phosphorylation in the absence of glucose, and that SMS1 is regulated by glucose, both at the transcriptional and the posttranslational level. Two-hybrid interactions of Sms1 with the glucose sensor and KlRgt1 repressor suggest that Sms1 mediates the glucose signal from the plasma membrane to the nucleus. All of these data demonstrated that Sms1 was the K. lactis homolog of MTH1 and STD1 of S. cerevisiae. Interestingly, MTH1 and STD1 were unable to complement a Δsms1 mutation.
18
Swaminathan, Srividya, Lars Klemm, Eugene Park, Anthony M. Ford, Soo-mi Kweon, Daniel Trageser, Brian Hasselfeld, et al. "Mechanisms of Clonal Evolution of Pre-Leukemic Clones in Childhood Pre-B Acute Lymphoblastic Leukemia." Blood 124, no. 21 (December 6, 2014): 861. http://dx.doi.org/10.1182/blood.v124.21.861.861.
Full textAbstract:
Abstract Background and hypothesis: Childhood pre-B acute lymphoblastic leukemia (ALL) can frequently be retraced to a pre-leukemic clone carrying a prenatally acquired genetic lesion (e.g. ETV6-RUNX1gene rearrangement). After birth, pre-leukemic clones can acquire secondary mutations and, hence, evolve towards overt leukemia. While this concept is well established, the mechanism(s) driving clonal evolution are not known. Epidemiological findings hint to a role of delayed childhood infections and chronic inflammation as etiologic factors of childhood ALL, but do not illuminate mechanism of clonal evolution of pre-leukemic cells. In this study, we demonstrate that cooperation between the AID cytosine deaminase and the RAG1/RAG2 V(D)J recombinase promotes acquisition of secondary genetic lesions that promote progress of pre-leukemic B cell precursors towards full-blown leukemia. Results: The enzymatic activity of RAG1/RAG2 (VDJ recombination) and AID (somatic hypermutation, class-switch recombination) are strictly segregated to early and late stages of B cell development, respectively. While RAG1 and RAG2 are actively expressed at stages of early B cell development (bone marrow and fetal liver) that give rise to pre-B ALL, little is known about the function of AID in early B-lymphopoesis. As the involvement of AID in pre-B leukemic clonal evolution is incumbent on its expression during early stages of B-lymphopoesis, we tested CD19+ pre-B cells isolated from human bone marrow (BM) for indicators of AID activity, namely, somatic hypermutation (SHM) and class switch recombination (CSR). Interestingly, most pre-B cell clones carry rearranged Ig VH region genes that are mutated at low levels (average mutation frequency 26 x 10-3 bp). Likewise, pre-B cells isolated from fetal liver tissues (three donors; 10-19 weeks of gestation) carried Ig VH region genes mutated at low levels (average mutation frequency 14 x 10-3 bp). In addition, about one third of fetal liver pre-B cells had undergone CSR to Cγ3, Cγ1 and Cα regions. These findings highlight the previously unknown function of AID in two important sites of early human B-lymphopoesis. Based on these results, we hypothesized that a specific B cell subset during early pro- and pre-B cell differentiation can concomitantly express both AID and the RAGs and, hence, would be particularly susceptible to clonal evolution of cells that carry a pre-leukemic lesion. Our subsequent studies identified late pre-B cells (Fraction D) as a natural subset of increased genetic vulnerability. Late pre-B cells downregulate IL7 receptor/Stat5 signaling, which enables expression of RAG1 and RAG2 and immunoglobulin light chain gene rearrangement. Loss of IL7 receptor/Stat5 signaling also removes an important safeguard against premature expression of AID. Therefore, late pre-B cells are poised to express AID at high levels in response to inflammatory stimuli (e.g. LPS) in concurrence with RAG1 and RAG2. Studying clonal evolution of patient-derived pre-B ALL cells, we found evidence for concomitant AID and RAG1/RAG2 activity. Further studying a genetic mouse model for pre-leukemic pre-B cells carrying ETV6-RUNX1, we found that repeated exposure to LPS can cause overt leukemia but not in the absence of either AID or Rag1. Additionally, whole exome sequencing of human B cell clones that were engineered to express AID, RAG1/RAG2 alone or in combination revealed that concurrent expression of AID with RAG1/RAG2 dramatically increased the frequency of structural chromosomal lesions. Conclusion: Consistent with epidemiological findings on the etiology of childhood ALL, we conclude that reduced cytokine signaling (here, IL7R) in late pre-B cells renders pre-leukemic clones distinctively vulnerable to genetic lesions that can be acquired in the context of repeated exposure to inflammatory stimuli (e.g. chronic and recurrent infections during childhood). Our results support a role for AID and RAGs cooperation for the generation of secondary lesions in leukemia subgroups that require additional leukemogenic events, and therefore, provide the genetic and molecular basis to support the Delayed Infections Hypothesis for leukemia progression in children. Disclosures No relevant conflicts of interest to declare.
19
Akamatsu, Yoshiko, and Marjorie A. Oettinger. "Distinct Roles of RAG1 and RAG2 in Binding the V(D)J Recombination Signal Sequences." Molecular and Cellular Biology 18, no. 8 (August 1, 1998): 4670–78. http://dx.doi.org/10.1128/mcb.18.8.4670.
Full textAbstract:
ABSTRACT The RAG1 and RAG2 proteins initiate V(D)J recombination by introducing double-strand breaks at the border between a recombination signal sequence (RSS) and a coding segment. To understand the distinct functions of RAG1 and RAG2 in signal recognition, we have compared the DNA binding activities of RAG1 alone and RAG1 plus RAG2 by gel retardation and footprinting analyses. RAG1 exhibits only a three- to fivefold preference for binding DNA containing an RSS over random sequence DNA. Although direct binding of RAG2 by itself was not detected, the presence of both RAG1 and RAG2 results in the formation of a RAG1-RAG2-DNA complex which is more stable and more specific than the RAG1-DNA complex and is active in V(D)J cleavage. These results suggest that biologically effective discrimination between an RSS and nonspecific sequences requires both RAG1 and RAG2. Unlike the binding of RAG1 plus RAG2, RAG1 can bind to DNA in the absence of a divalent metal ion and does not require the presence of coding flank sequence. Footprinting of the RAG1-RAG2 complex with 1,10-phenanthroline-copper and dimethyl sulfate protection reveal that both the heptamer and the nonamer are involved. The nonamer is protected, with extensive protein contacts within the minor groove. Conversely, the heptamer is rendered more accessible to chemical attack, suggesting that binding of RAG1 plus RAG2 distorts the DNA near the coding/signal border.
20
Wu, Zeguang, Narmadha Subramanian, Eva-Maria Jacobsen, Kerstin Laib Sampaio, Johannes van der Merwe, Manfred Hönig, and Thomas Mertens. "NK Cells from RAG- or DCLRE1C-Deficient Patients Inhibit HCMV." Microorganisms 7, no. 11 (November 10, 2019): 546. http://dx.doi.org/10.3390/microorganisms7110546.
Full textAbstract:
The recombination-activating genes (RAGs) and the DNA cross-link repair 1C gene (DCLRE1C) encode the enzymes RAG1, RAG2 and Artemis. They are critical components of the V(D)J recombination machinery. V(D)J recombination is well known as a prerequisite for the development and antigen diversity of T and B cells. New findings suggested that RAG deficiency impacts the cellular fitness and function of murine NK cells. It is not known whether NK cells from severe combined immunodeficiency (SCID) patients with defective RAGs or DCLRE1C (RAGs−/DCLRE1C−-NK) are active against virus infections. Here, we evaluated the anti-HCMV activity of RAGs−/DCLRE1C−-NK cells. NK cells from six SCID patients were functional in inhibiting HCMV transmission between cells in vitro. We also investigated the expansion of HCMV-induced NK cell subset in the RAG- or DCLRE1C-deficient patients. A dynamic expansion of NKG2C+ NK cells in one RAG-2-deficient patient was observed post HCMV acute infection. Our study firstly reveals the antiviral activity of human RAGs−/ DCLRE1C−-NK cells.
21
Shi, Xiaoju, Shirley A. Hanley, Marie-Claire Faray-Kele, Stuart C. Fawell, Joseph Aduse-Opoku, Robert A. Whiley, Michael A. Curtis, and Lucinda M. C. Hall. "The rag Locus of Porphyromonas gingivalis Contributes to Virulence in a Murine Model of Soft Tissue Destruction." Infection and Immunity 75, no. 4 (February 5, 2007): 2071–74. http://dx.doi.org/10.1128/iai.01785-06.
Full textAbstract:
ABSTRACT The rag locus of Porphyromonas gingivalis encodes a putative TonB-dependent outer membrane receptor, RagA, and a 55-kDa immunodominant antigen, RagB. Inactivation of either ragA or ragB prevented expression of both RagA and RagB. Both the ragA and ragB mutants were significantly less virulent than wild-type strains in a murine model of infection.
22
Lima, Raquel, Diana Sousa, Ana Gomes, Nuno Mendes, Rune Matthiesen, Madalena Pedro, Franklim Marques, Madalena Pinto, Emília Sousa, and M. Vasconcelos. "The Antitumor Activity of a Lead Thioxanthone is Associated with Alterations in Cholesterol Localization." Molecules 23, no. 12 (December 12, 2018): 3301. http://dx.doi.org/10.3390/molecules23123301.
Full textAbstract:
The search for novel anticancer small molecules and strategies remains a challenge. Our previous studies have identified TXA1 (1-{[2-(diethylamino)ethyl]amino}-4-propoxy-9H- thioxanthen-9-one) as a hit compound, with in vitro antitumor potential by modulating autophagy and apoptosis in human tumor cell lines. In the present study, the mechanism of action and antitumor potential of the soluble salt of this molecule (TXA1.HCl) was further investigated using in vitro and mouse xenograft tumor models of NSCLC. Our results showed that TXA1.HCl affected steroid biosynthesis, increased RagD expression, and caused abnormal cellular cholesterol localization. In addition, TXA1.HCl treatment presented no toxicity to nude mice and significantly reduced the growth of human NSCLC cells xenografts in mice. Overall, this work provides new insights into the mechanism of action of TXA1, which may be relevant for the development of anticancer therapeutic strategies, which target cholesterol transport.
23
Meru, Nadine, Andreas Jung, Irith Baumann, and Gerald Niedobitek. "Expression of the recombination-activating genes in extrafollicular lymphocytes but no apparent reinduction in germinal center reactions in human tonsils." Blood 99, no. 2 (January 15, 2002): 531–37. http://dx.doi.org/10.1182/blood.v99.2.531.
Full textAbstract:
Abstract V(D)J recombination in lymphocytes is mediated by 2 recombination-activating genes, RAG1 and RAG2,which are expressed during lymphocyte development in bone marrow and thymus. Prompted by studies reporting re-expression of the RAGs in germinal center B cells, the expression of RAGs and terminal deoxynucleotidyl transferase (TdT) in human lymphoid tissues was examined using in situ hybridization and immunohistochemistry, respectively. Here it is shown that RAGs and TdT are not reinduced in germinal center reactions. However, RAG+/TdT+ cells are frequently present in extrafollicular areas of tonsils mainly at the boundary between lymphoid tissue and fibrous scaffold. Phenotypic analyses suggest that these cells are B cells. Finally, it is shown that RAG+/TdT+ cells are found more frequently in tonsils than in other peripheral lymphoid tissues. This may reflect an increased influx of RAG+/TdT+ cells as a result of higher antigenic stimulation at this site. Alternatively, this observation may indicate that the tonsils are an additional site of lymphocyte ontogeny.
24
Nagano, Keiji, Yukitaka Murakami, Kiyoshi Nishikawa, Junpei Sakakibara, Kazuo Shimozato, and Fuminobu Yoshimura. "Characterization of RagA and RagB in Porphyromonas gingivalis: study using gene-deletion mutants." Journal of Medical Microbiology 56, no. 11 (November 1, 2007): 1536–48. http://dx.doi.org/10.1099/jmm.0.47289-0.
Full textAbstract:
The major outer-membrane proteins RagA and RagB of Porphyromonas gingivalis are considered to form a receptor complex functionally linked to TonB. In this study, P. gingivalis mutants with ragA, ragB or both deleted were constructed from strain W83 as the parent to examine the physiological and pathological functions of RagA and RagB. The double-deletion mutant completely lacked both RagA and RagB, whereas the ΔragA mutant reduced RagB expression considerably and the ΔragB mutant produced degraded RagA. Growth of the three mutants in a nutrient-rich medium and synthetic media containing digested protein as a unique nutrient source was similar to that of the parental strain; however, both the ΔragA and ΔragAB mutants exhibited very slow growth in a synthetic medium containing undigested, native protein, and the two mutants tended to lose their viability during experiments, although gingipain (protease) activities were unchanged in the mutants. A mouse model showed that the ΔragB mutant had reduced virulence. Cell-surface labelling with biotin and dextran revealed that both RagA and RagB localized on the outermost cell surface. A cross-linking experiment using wild-type P. gingivalis showed that RagA and RagB were closely associated with each other. Furthermore, co-immunoprecipitation confirmed that RagA and RagB formed a protein–protein complex. These results suggest that physically associated RagA and RagB may stabilize themselves on the cell surface and function as active transporters of large degradation products of protein and in part as a virulence factor.
25
McMahan, C. J., M. J. Sadofsky, and D. G. Schatz. "Definition of a large region of RAG1 that is important for coimmunoprecipitation of RAG2." Journal of Immunology 158, no. 5 (March 1, 1997): 2202–10. http://dx.doi.org/10.4049/jimmunol.158.5.2202.
Full textAbstract:
Abstract Interaction between the RAG1 and RAG2 proteins is probably critical for V(D)J recombination. Using a coimmunoprecipitation assay, we define a large region of RAG1 (amino acids 504-1008) that is sufficient for interaction with RAG2. This region comprises the C-terminal half of the RAG1 protein, and is within the region defined as the recombinationally active core. Deletion of either of two regions of RAG1 (amino acids 504-570 or 850-1008) causes a loss of interaction with RAG2. Loss of coimmunoprecipitation is also seen with RAG1 core proteins containing deletions of smaller stretches of amino acids (amino acids 506-511 or 545-550), emphasizing the importance of this region of RAG1 in forming a complex with RAG2. A variety of other small deletion mutations within the amino acid region 504-1008 also decrease coimmunoprecipitation of RAG2 with RAG1, indicating that much or all of this region is important for complex formation.
26
Kim, Jae Hyun, Kilsoo Jung, Chulho Lee, Doona Song, Kibum Kim, Hee Chan Yoo, Seung Joon Park, et al. "Structure-based modification of pyrazolone derivatives to inhibit mTORC1 by targeting the leucyl-tRNA synthetase-RagD interaction." Bioorganic Chemistry 112 (July 2021): 104907. http://dx.doi.org/10.1016/j.bioorg.2021.104907.
Full text
27
Kondratenko, I. V., O. E. Pashchenko, Y. A. Rodina, M. V. Belevtcev, Den M. Van, and A. A. Bologov. "Clinical and laboratory phenotypes of severe combined immunodeficiencies with mutations in RAG1/RAG2 genes." Russian Journal of Allergy 9, no. 4 (December 15, 2012): 26–32. http://dx.doi.org/10.36691/rja689.
Full textAbstract:
Background. The RAG1 and RAG2 proteins are key players in the V(D)J recombination process leading to the assembly of antigen receptor genes. Defects in RAG1/RAG2 genes are caused to different phenotypes of severe combined immunodeficiencies (SCID). Objective — to determine the clinical and laboratory manifestations in patients with RAG1 / RAG2 mutations from one single center, to identify the phenotype-genotype correlations. Materials and methods. We described 4 children with RAG1 mutations. Diagnosis of SCID was confirmed by criteria’s of European society of immunodeficiencies (ESID). Results. In two patients we observed Omenn syndrome, in 1 — classic T -B -NK + SCID, in 1 — «sof» T +B -NK + SCID. One patient with Omenn syndrome and patient with «soft» SCID had same RAG1 mutations. Conclusions. RAG 1 / RAG2 mutations are caused to severe life-threatening combined immunodeficiency, requiring radical therapy. We found no genotype-phenotype correlations in patients with RAG1 defects.
28
Bailin, Tu, Xianming Mo, and Moshe J. Sadofsky. "A RAG1 and RAG2 Tetramer Complex Is Active in Cleavage in V(D)J Recombination." Molecular and Cellular Biology 19, no. 7 (July 1, 1999): 4664–71. http://dx.doi.org/10.1128/mcb.19.7.4664.
Full textAbstract:
ABSTRACT During V(D)J recombination two proteins, RAG1 and RAG2, assemble as a protein-DNA complex with the appropriate DNA targets containing recombination signal sequences (RSSs). The properties of this complex require a fairly elaborate set of protein-protein and protein-DNA contacts. Here we show that a purified derivative of RAG1, without DNA, exists predominantly as a homodimer. A RAG2 derivative alone has monomer, dimer, and larger forms. The coexpressed RAG1 and RAG2 proteins form a mixed tetramer in solution which contains two molecules of each protein. The same tetramer of RAG1 and RAG2 plus one DNA molecule is the form active in cleavage. Additionally, we show that both DNA products following cleavage can still be held together in a stable protein-DNA complex.
29
Zarrin, A. A., I. Fong, L. Malkin, P. A. Marsden, and N. L. Berinstein. "Cloning and characterization of the human recombination activating gene 1 (RAG1) and RAG2 promoter regions." Journal of Immunology 159, no. 9 (November 1, 1997): 4382–94. http://dx.doi.org/10.4049/jimmunol.159.9.4382.
Full textAbstract:
Abstract Recombination activating gene 1 (RAG1) and RAG2 are the essential and tissue-specific components of V(D)J recombination. We have characterized the genomic organization of the human RAG locus, mapped the transcriptional initiation sites, and partially sequenced and performed functional reporter assays on the 5' flanking regions of human RAG1 and RAG2. Transcription initiation sites were mapped by rapid amplification of 5' cDNA ends, primer extension, and/or RNase protection in normal thymocytes, three pre-B cell lines, and a mature B cell line. A single promoter region was used for RAG1 transcription. In contrast, transcription of RAG2 initiates at two distinct regions of the genome. The 5'-flanking region of the human RAG2 gene is TATA-less; however, there is a GATAA consensus at position -34 with respect to the major transcriptional initiation site of RAG1. Promoter regions of human RAG1 and RAG2 are active in both lymphoid and nonlymphoid cell lines, suggesting that an outside regulatory element is probably involved in the tissue-specific transcriptional regulation of the RAG genes.
30
Naik, Abani Kanta, Aaron T. Byrd, Aaron C. K. Lucander, and Michael S. Krangel. "Hierarchical assembly and disassembly of a transcriptionally active RAG locus in CD4+CD8+ thymocytes." Journal of Experimental Medicine 216, no. 1 (December 13, 2018): 231–43. http://dx.doi.org/10.1084/jem.20181402.
Full textAbstract:
Expression of Rag1 and Rag2 is tightly regulated in developing T cells to mediate TCR gene assembly. Here we have investigated the molecular mechanisms governing the assembly and disassembly of a transcriptionally active RAG locus chromatin hub in CD4+CD8+ thymocytes. Rag1 and Rag2 gene expression in CD4+CD8+ thymocytes depends on Rag1 and Rag2 promoter activation by a distant antisilencer element (ASE). We identify GATA3 and E2A as critical regulators of the ASE, and Runx1 and E2A as critical regulators of the Rag1 promoter. We reveal hierarchical assembly of a transcriptionally active chromatin hub containing the ASE and RAG promoters, with Rag2 recruitment and expression dependent on assembly of a functional ASE–Rag1 framework. Finally, we show that signal-dependent down-regulation of RAG gene expression in CD4+CD8+ thymocytes depends on Ikaros and occurs with disassembly of the RAG locus chromatin hub. Our results provide important new insights into the molecular mechanisms that orchestrate RAG gene expression in developing T cells.
31
Klemm, Lars, Srividya Swaminathan, Elli Papaemmanuil, Anthony M. Ford, Mel Greaves, Rafael Casellas, David Schatz, Michael R. Lieber, and Markus Muschen. "Exposure to Inflammatory Immune Responses As Driver of Clonal Evolution in Childhood Acute Lymphoblastic Leukemia." Blood 126, no. 23 (December 3, 2015): 166. http://dx.doi.org/10.1182/blood.v126.23.166.166.
Full textAbstract:
Abstract Background: Pediatric pre-B acute lymphoblastic leukemia (ALL) may develop from prenatal chromosomal translocations acquired in utero. For instance, the ETV6-RUNX1 gene rearrangement (~25% of childhood ALL) is found in the umbilical cord blood and Guthrie blood spots of 1 in 100 healthy newborns, however, only 1 in 14,000 carriers develop overt leukemia. The molecular mechanisms driving clonal evolution towards overt leukemia were not clear. Rationale: Activation Induced Cytidine Deaminase (AID) and Recombination Activation Genes 1 and 2 (RAG1-RAG2) are genetic modifiers of the immunoglobulin (Ig) genes that are expressed during normal B cell development. Although AID and RAG1/RAG2 are thought to be segregated to early (RAG1/RAG2) and late (AID) stages of B cell development, respectively, we found that the two enzymes can be concurrently expressed during early B-lymphopoiesis in the context of repeated inflammatory stimuli. Results: Our experiments identified transitional pre-B cells as the subset that is particularly vulnerable to concomitant expression of AID and RAG1-RAG2 with earlier B cells being protected by IL7 signaling.Human B cells from children lacking a functional IL-7 receptor (IL-7R) displayed both increased expression and activity of AID concurrently with RAG1-RAG2. These results demonstrated that AID activation in both mouse and human early B cell compartments increases genetic instability. Although concurrent activation of AID and RAG1-RAG2 in patient samples implicated a correlation between the two enzymes in the pathogenesis of leukemia, this as such did not prove that the enzymes causally induce overt leukemogenesis. Therefore, we next evaluated the requirement of AID and RAG1-RAG2 in leukemogenic transformation, and identified a condition that leads to massive activation of these enzymes in a pre-leukemic B cell. Importantly, AID and RAG1-RAG2 expression increased dramatically during inflammatory immune responses (e.g. infection), where both these enzymes diversify the antibody repertoire and improve its affinity to antigens from infectious pathogens. We therefore tested whether the pre-B cell subset that concurrently expresses AID and RAG1-RAG2 can respond to an inflammatory stimulus, such as LPS. We observed that pre-B cells require protection from IL7, which prevents aberrant activation of AID. In the absence of protective IL-7, these pre-B cells acquired responsiveness to LPS and strongly activated AID concurrently with RAG1-RAG2 enzymes. We developed IL7-dependent pre-B cell cultures as a disease model for ETV6-RUNX1 pre-leukemia and tested the role of AID and RAG1 in the progression of pre-leukemic clones. To this end, we expanded ETV6-RUNX1 pre-B cells from wildtype (AID and RAG1 expressing) mice, or from mice lacking AID (Aid-/-Rag1+/+) or RAG1 (Aid+/+Rag1-/-). We then challenged pre-B cell cultures by withdrawal of IL7 (loss of protection) and inflammatory stimuli (LPS) and transplanted pre-B cells that had undergone five cycles of -IL7/LPS challenge. Upon transplanting -IL7/LPS-treated Aid+/+Rag1+/+ or Aid-/-Rag1+/+ or Aid+/+Rag1-/- pre-B cells containing ETV6-RUNX1 into NOD-SCID recipient mice, we observed that loss of either Aid or Rag1 dramatically prolonged the latency and reduced the penetrance of leukemia in transplant recipients. This proved that AID and RAG1-RAG2 causally accelerate clonal evolution of a pre-leukemic B cell towards leukemia. Our findings provide a mechanism by which pre-leukemic clones carrying a prenatal genetic lesion such as ETV6-RUNX1 can evolve through infectious and inflammatory stimuli ultimately leading to full blown leukemia. Conclusion: The impact of inflammatory stimuli on leukemogenesis has been previously implicated in multiple epidemiological studies. For instance, day-care attendance primed the immune system during early childhood and is thought to protect against exacerbation of B cell responses and to prevent collateral damage driving clonal evolution towards leukemia. Although inflammation (LPS stimulation) seems to play a role in accelerating pre-B leukemogenesis in our model, further experiments testing actual infectious pathogens are needed to corroborate this concept. Moreover, it is crucial to test whether leukemogenesis is accelerated in individuals infected with restricted classes of pathogens, not all of which may activate AID in pre-B cells. Disclosures No relevant conflicts of interest to declare.
32
Malshetty, Vidyasagar, Jian Chen, Mary Hanna, and Patricia Cortes. "Role of Pax5 and YY1 in regulation of V(D)J recombination (111.1)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 111.1. http://dx.doi.org/10.4049/jimmunol.188.supp.111.1.
Full textAbstract:
Abstract The infinite variety of antigen-binding receptors originates from the rearrangement of B and T-cell receptor loci in a process known as V(D)J recombination. The initial site-specific DNA cleavage steps of this process are catalyzed by the lymphoid specific proteins RAG1 and RAG2. Deregulation of this process, leads to a spectrum of diseases including immunodeficiency and leukemia. These leukemias are believed to arise from RAG1/2 mediated oncogenic chromosomal translocations. The transcription factor Pax5 is critical for B cell development and is known that, it interacts with RAG1/2 and mediates V to DJ recombination. Pax5 is also been implicated in human B cell malignancies. Another multifunctional transcription factor, YY1, is known to play a role in V to DJ recombination similar to Pax5. We were able to see an interaction of YY1 with RAG1/2 and Pax5, when these proteins were over-expressed in 293T cells. When we performed a plasmid based recombination assay in presence of Pax5, YY1 or both a consistent decrease in recombination levels was observed. To elucidate the mechanism of inhibition, we analyzed the recombination intermediates and found that the initial cleavage step is inhibited by these two factors. In addition, purified Pax5 and YY1 inhibit the in vitro cleavage activity of RAG1/2 recombinase. The expression of a mutant Pax5 increased the RAG recombinase activity, suggesting the importance of these two factors in regulation of RAGs mediated recombination.
33
Sawchuk, Dennis J., Frances Weis-Garcia, Sohail Malik, Eva Besmer, Michael Bustin, Michel C. Nussenzweig, and Patricia Cortes. "V(D)J Recombination: Modulation of RAG1 and RAG2 Cleavage Activity on 12/23 Substrates by Whole Cell Extract and DNA-bending Proteins." Journal of Experimental Medicine 185, no. 11 (June 2, 1997): 2025–32. http://dx.doi.org/10.1084/jem.185.11.2025.
Full textAbstract:
Antigen receptor gene rearrangement is directed by DNA motifs consisting of a conserved heptamer and nonamer separated by a nonconserved spacer of either 12 or 23 base pairs (12 or 23 recombination signal sequences [RSS]). V(D)J recombination requires that the rearranging DNA segments be flanked by RSSs of different spacer lengths, a phenomenon known as the 12/23 rule. Recent studies have shown that this restriction operates at the level of DNA cleavage, which is mediated by the products of the recombination activating genes RAG1 and RAG2. Here, we show that RAG1 and RAG2 are not sufficient for 12/23 dependent cleavage, whereas RAG1 and RAG2 complemented with whole cell extract faithfully recapitulates the 12/23 rule. In addition, HMG box containing proteins HMG1 and HMG2 enhance RAG1- and RAG2-mediated cleavage of substrates containing 23 RSS but not of substrates containing only 12 RSS. These results suggest the existence of a nucleoprotein complex at the cleavage site, consisting of architectural, catalytic, and regulatory components.
34
Hao, Bingtao, Abani Kanta Naik, Akiko Watanabe, Hirokazu Tanaka, Liang Chen, Hunter W. Richards, Motonari Kondo, et al. "An anti-silencer– and SATB1-dependent chromatin hub regulates Rag1 and Rag2 gene expression during thymocyte development." Journal of Experimental Medicine 212, no. 5 (April 6, 2015): 809–24. http://dx.doi.org/10.1084/jem.20142207.
Full textAbstract:
Rag1 and Rag2 gene expression in CD4+CD8+ double-positive (DP) thymocytes depends on the activity of a distant anti-silencer element (ASE) that counteracts the activity of an intergenic silencer. However, the mechanistic basis for ASE activity is unknown. Here, we show that the ASE physically interacts with the distant Rag1 and Rag2 gene promoters in DP thymocytes, bringing the two promoters together to form an active chromatin hub. Moreover, we show that the ASE functions as a classical enhancer that can potently activate these promoters in the absence of the silencer or other locus elements. In thymocytes lacking the chromatin organizer SATB1, we identified a partial defect in Tcra gene rearrangement that was associated with reduced expression of Rag1 and Rag2 at the DP stage. SATB1 binds to the ASE and Rag promoters, facilitating inclusion of Rag2 in the chromatin hub and the loading of RNA polymerase II to both the Rag1 and Rag2 promoters. Our results provide a novel framework for understanding ASE function and demonstrate a novel role for SATB1 as a regulator of Rag locus organization and gene expression in DP thymocytes.
35
Xue, Jeff, Kelly Keys, Aaron McCoy, and Edward Weinstein. "Rag1 and Rag2 Knockout rats to drive xenografts and oncology studies (126.30)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 126.30. http://dx.doi.org/10.4049/jimmunol.188.supp.126.30.
Full textAbstract:
Abstract Rejection of grafted tissues by host immune systems is problematic during xenotransplantation. To help alleviate this and provide models amenable to the xenograft process, we created rats with genetic modifications of genes: Rag1 or Rag2. Rag1 and Rag2 play an important role in the rearrangement and recombination of genes of immunoglobulin and T-cell receptor molecules during the process of VDJ recombination. These rats were created via zinc finger nuclease (ZFN) targeted editing of Sprague-Dawley and F344 rat genomes. ZFN creates a double stranded break at the target locus. This lesion is repaired by the low fidelity repair mechanism non-homologous end-joining which commonly yields out of frame mutations in the gene, leading to non-functional proteins. Phenotypic characterization of these animals occurs when homozygosity is achieved. Rag1 and Rag2 protein ablation was demonstrated by western blot analysis. Flow cytometric analysis of peripheral blood cells in Rag1 KO animals at 4, 10 and 20 weeks demonstrated a significant decrease in mature T and B lymphocytes relative to age-matched wild type rats. Rag2 KO rats demonstrate a non-leaky depletion of mature T and B lymphocytes at 4 and 10 weeks. By employing the ZFN technique, we developed Rag1 and Rag2 KO rats with compromised immunology systems, which we believe will be helpful in the studies of xenografts, cancer, vaccine development, autoimmune, infectious and GVHD diseases.
36
Eastman, Quinn M., Isabelle J. Villey, and David G. Schatz. "Detection of RAG Protein-V(D)J Recombination Signal Interactions Near the Site of DNA Cleavage by UV Cross-Linking." Molecular and Cellular Biology 19, no. 5 (May 1, 1999): 3788–97. http://dx.doi.org/10.1128/mcb.19.5.3788.
Full textAbstract:
ABSTRACT V(D)J recombination is initiated by double-strand cleavage at recombination signal sequences (RSSs). DNA cleavage is mediated by the RAG1 and RAG2 proteins. Recent experiments describing RAG protein-RSS complexes, while defining the interaction of RAG1 with the nonamer, have not assigned contacts immediately adjacent to the site of DNA cleavage to either RAG polypeptide. Here we use UV cross-linking to define sequence- and site-specific interactions between RAG1 protein and both the heptamer element of the RSS and the coding flank DNA. Hence, RAG1-DNA contacts span the site of cleavage. We also detect cross-linking of RAG2 protein to some of the same nucleotides that cross-link to RAG1, indicating that, in the binding complex, both RAG proteins are in close proximity to the site of cleavage. These results suggest how the heptamer element, the recognition surface essential for DNA cleavage, is recognized by the RAG proteins and have implications for the stoichiometry and active site organization of the RAG1-RAG2-RSS complex.
37
De, Pallabi, Mandy M. Peak, and Karla K. Rodgers. "DNA Cleavage Activity of the V(D)J Recombination Protein RAG1 Is Autoregulated." Molecular and Cellular Biology 24, no. 15 (August 1, 2004): 6850–60. http://dx.doi.org/10.1128/mcb.24.15.6850-6860.2004.
Full textAbstract:
ABSTRACT RAG1 and RAG2 catalyze the first DNA cleavage steps in V(D)J recombination. We demonstrate that the isolated central domain of RAG1 has inherent single-stranded (ss) DNA cleavage activity, which does not require, but is enhanced by, RAG2. The central domain, therefore, contains the active-site residues necessary to perform hydrolysis of the DNA phosphodiester backbone. Furthermore, the catalytic activity of this domain on ss DNA is abolished by addition of the C-terminal domain of RAG1. The inhibitory effects of this latter domain are suppressed on substrates containing double-stranded (ds) DNA. Together, the activities of the reconstituted domains on ss versus mixed ds-ss DNA approximate the activity of intact RAG1 in the presence of RAG2. We propose how the combined actions of the RAG1 domains may function in V(D)J recombination and also in aberrant cleavage reactions that may lead to genomic instability in B and T lymphocytes.
38
Fisher, Megan, and Craig Bassing. "Pre-B cells suppress RAG expression in response to DNA double-strand breaks (HEM1P.225)." Journal of Immunology 194, no. 1_Supplement (May 1, 2015): 50.8. http://dx.doi.org/10.4049/jimmunol.194.supp.50.8.
Full textAbstract:
Abstract The ability of the Rag1/Rag2 (RAG) endonuclease to assemble antigen receptor (AgR) genes is essential for adaptive immunity. However, aberrant induction or repair of RAG-induced DNA double strand breaks (DSBs) can lead to oncogenic AgR translocations. We have previously shown that RAG-induced DSBs in pre-B cells activate the ATM kinase to prevent RAG cleavage of the homologous allele, and to inhibit expression of Rag1 and Rag2. These breaks also suppress expression of Gadd45α, which promotes Rag1 and Rag2 transcription. Since DSBs induced by ionizing radiation (IR) signal through ATM to increase Gadd45α expression in all other cell types including mature B cells, these results indicate that RAG DSBs and/or pre-B cells use unique mechanisms to regulate Gadd45α expression. We now demonstrate that IR-induced DSBs in pre-B cells signal through the ATM kinase to down-regulate Gadd45α expression. We also demonstrate that these IR DSBs signal ATM-dependent suppression of Rag1 and Rag2 transcription, without affecting stability of Rag1 or Rag2 mRNA. Because the presence of multiple DSBs in a cell greatly increases the risk of translocation, we hypothesize that pre-B cells have developed a unique developmental stage-specific DSB response to suppress the induction of RAG DSBs in the presence of other DSBs. Our ongoing studies will determine the role of this pre-B cell specific DSB response in suppressing AgR translocations and enforcing mono-allelic assembly of immunoglobulin genes.
39
Gomez, Carlos A., Leon M. Ptaszek, Anna Villa, Fabio Bozzi, Cristina Sobacchi, Edward G. Brooks, Luigi D. Notarangelo, et al. "Mutations in Conserved Regions of the Predicted RAG2 Kelch Repeats Block Initiation of V(D)J Recombination and Result in Primary Immunodeficiencies." Molecular and Cellular Biology 20, no. 15 (August 1, 2000): 5653–64. http://dx.doi.org/10.1128/mcb.20.15.5653-5664.2000.
Full textAbstract:
ABSTRACT The V(D)J recombination reaction is composed of multiple nucleolytic processing steps mediated by the recombination-activating proteins RAG1 and RAG2. Sequence analysis has suggested that RAG2 contains six kelch repeat motifs that are predicted to form a six-bladed β-propeller structure, with the second β-strand of each repeat demonstrating marked conservation both within and between kelch repeat-containing proteins. Here we demonstrate that mutations G95R and ΔI273 within the predicted second β-strand of repeats 2 and 5 of RAG2 lead to immunodeficiency in patients P1 and P2. Green fluorescent protein fusions with the mutant proteins reveal appropriate localization to the nucleus. However, both mutations reduce the capacity of RAG2 to interact with RAG1 and block recombination signal cleavage, therefore implicating a defect in the early steps of the recombination reaction as the basis of the clinical phenotype. The present experiments, performed with an extensive panel of site-directed mutations within each of the six kelch motifs, further support the critical role of both hydrophobic and glycine-rich regions within the second β-strand for RAG1-RAG2 interaction and recombination signal recognition and cleavage. In contrast, multiple mutations within the variable-loop regions of the kelch repeats had either mild or no effects on RAG1-RAG2 interaction and hence on the ability to mediate recombination. In all, the data demonstrate a critical role of the RAG2 kelch repeats for V(D)J recombination and highlight the importance of the conserved elements of the kelch motif.
40
Anandapadamanaban, Madhanagopal, Glenn R. Masson, Olga Perisic, Alex Berndt, Jonathan Kaufman, Chris M. Johnson, Balaji Santhanam, Kacper B. Rogala, David M. Sabatini, and Roger L. Williams. "Architecture of human Rag GTPase heterodimers and their complex with mTORC1." Science 366, no. 6462 (October 10, 2019): 203–10. http://dx.doi.org/10.1126/science.aax3939.
Full textAbstract:
The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag heterodimers is critical for their association with mTORC1. Our cryo–electron microscopy structure of RagA/RagC in complex with mTORC1 shows the details of RagA/RagC binding to the RAPTOR subunit of mTORC1 and explains why only the RagAGTP/RagCGDP nucleotide state binds mTORC1. Previous kinetic studies suggested that GTP binding to one Rag locks the heterodimer to prevent GTP binding to the other. Our crystal structures and dynamics of RagA/RagC show the mechanism for this locking and explain how oncogenic hotspot mutations disrupt this process. In contrast to allosteric activation by RHEB, Rag heterodimer binding does not change mTORC1 conformation and activates mTORC1 by targeting it to lysosomes.
41
Raval, Prafulla, Sushil Kumar, Aleksei N. Kriatchko, and Patrick C. Swanson. "Evidence for Ku70/Ku80 association with full length RAG1 (35.6)." Journal of Immunology 178, no. 1_Supplement (April 1, 2007): S2. http://dx.doi.org/10.4049/jimmunol.178.supp.35.6.
Full textAbstract:
Abstract Antigen receptor genes are assembled by a form of site-specific DNA rearrangement termed V(D)J recombination. This process proceeds through two distinct phases: a cleavage phase in which the RAG1 and RAG2 proteins introduce DNA double-strand breaks at recombination signal sequences (RSSs), and a joining phase in which the resulting DNA breaks are processed and repaired via the non-homologous end-joining (NHEJ) repair pathway. Genetic and biochemical evidence suggest that the RAG proteins play an active role in guiding the repair of DNA breaks introduced during V(D)J recombination to the NHEJ pathway. However, evidence for specific association between the RAG proteins and any of the factors involved in NHEJ remains elusive. Here we present biochemical evidence that two components of the NHEJ pathway, Ku70 and Ku80, interact with full-length RAG1 and can become integrated into a stable RAG-RSS complex assembled with full-length RAG1 and core RAG2, but not core RAG1 (aa 384–1040) and either core or full-length RAG2. Formation of this complex minimally requires residues 211–1040 of RAG1. These results provide a biochemical link between the two phases of V(D)J recombination. This research is supported by National Institutes of Health grant 1R01 AI055599 to P.C.S.
42
Lypaczewski, Patrick, Wen-Wei Zhang, and Greg Matlashewski. "Evidence that a naturally occurring single nucleotide polymorphism in the RagC gene of Leishmania donovani contributes to reduced virulence." PLOS Neglected Tropical Diseases 15, no. 2 (February 23, 2021): e0009079. http://dx.doi.org/10.1371/journal.pntd.0009079.
Full textAbstract:
Leishmaniasis is a widespread neglected tropical disease transmitted by infected sand flies resulting in either benign cutaneous infection or fatal visceral disease. Leishmania donovani is the principal species responsible for visceral leishmaniasis, yet an atypical L. donovani has become attenuated in several countries including Sri Lanka and causes cutaneous leishmaniasis. Previous studies have identified 91 genes altered in the atypical cutaneous L. donovani compared to typical visceral disease associated L. donovani including mutations in the RagC and Raptor genes that are part of the eukaryotic conserved TOR pathway and its upstream sensing pathway. In the present study, we investigate whether the RagC R231C mutation present in atypical cutaneous L. donovani introduced into the virulent L. donovani 1S2D chromosome by CRISPR gene editing could affect virulence for survival in visceral organs. Through bioinformatic analysis, we further investigated the presence of sensing pathway components upstream of TOR in L. donovani including RagC complexing proteins, RagA and Raptor. L. donovani 1S2D edited to express mutant RagC R231C were viable in promastigote but had reduced visceral parasitemia in infected BALB/c mice. The RagC R231C mutant retained the ability to interact with RagA and gene knockout experiments revealed that although the RagA gene was essential, the RagC gene was not essential under promastigote culture conditions but was essential for survival in the liver of experimentally infected mice. These results provide evidence that the TOR associated sensing pathway plays a prominent role in L. donovani visceral disease and the RagC R231C mutation contributed to the atypical pathology of cutaneous L. donovani in Sri Lanka.
43
Mundy, Cynthia L., Nadja Patenge, Adam G. W. Matthews, and Marjorie A. Oettinger. "Assembly of the RAG1/RAG2 Synaptic Complex." Molecular and Cellular Biology 22, no. 1 (January 1, 2002): 69–77. http://dx.doi.org/10.1128/mcb.22.1.69-77.2002.
Full textAbstract:
ABSTRACT Assembly of antigen receptor genes by V(D)J recombination requires the site-specific recognition of two distinct DNA elements differing in the length of the spacer DNA that separates two conserved recognition motifs. Under appropriate conditions, V(D)J cleavage by the purified RAG1/RAG2 recombinase is similarly restricted. Double-strand breakage occurs only when these proteins are bound to a pair of complementary signals in a synaptic complex. We examine here the binding of the RAG proteins to signal sequences and find that the full complement of proteins required for synapsis of two signals and coupled cleavage can assemble on a single signal. This complex, composed of a dimer of RAG2 and at least a trimer of RAG1, remains inactive for double-strand break formation until a second complementary signal is provided. Thus, binding of the second signal activates the complex, possibly by inducing a conformational change. If synaptic complexes are formed similarly in vivo, one signal of a recombining pair may be the preferred site for RAG1/RAG2 assembly.
44
Shockett, Penny E., and David G. Schatz. "DNA Hairpin Opening Mediated by the RAG1 and RAG2 Proteins." Molecular and Cellular Biology 19, no. 6 (June 1, 1999): 4159–66. http://dx.doi.org/10.1128/mcb.19.6.4159.
Full textAbstract:
ABSTRACT The lymphoid cell-specific proteins RAG1 and RAG2 initiate V(D)J recombination by cleaving DNA adjacent to recombination signals, generating blunt signal ends and covalently sealed, hairpin coding ends. A critical next step in the reaction is opening of the hairpins, but the factor(s) responsible has not been identified and had been thought to be a ubiquitous component(s) of the DNA repair machinery. Here we demonstrate that RAG1 and RAG2 possess an intrinsic single-stranded nuclease activity capable of nicking hairpin coding ends at or near the hairpin tip. In Mn2+, a synthetic hairpin is nicked 5 nucleotides (nt) 5′ of the hairpin tip, with more distant sites of nicking suppressed by HMG2. In Mg2+, hairpins generated by V(D)J cleavage are nicked whereas synthetic hairpins are not. Cleavage-generated hairpins are nicked at the tip and predominantly 1 to 2 nt 5′ of the tip. RAG1 and RAG2 may therefore be responsible for initiating the processing of coding ends and for the generation of P nucleotides during V(D)J recombination.
45
Shetty, Keerthi, and David G. Schatz. "Recruitment of RAG1 and RAG2 to Chromatinized DNA during V(D)J Recombination." Molecular and Cellular Biology 35, no. 21 (August 24, 2015): 3701–13. http://dx.doi.org/10.1128/mcb.00219-15.
Full textAbstract:
V(D)J recombination is initiated by the binding of the RAG1 and RAG2 proteins to recombination signal sequences (RSSs) that consist of conserved heptamer and nonamer sequences separated by a spacer of either 12 or 23 bp. Here, we used RAG-inducible pro-B v-Abl cell lines in conjunction with chromatin immunoprecipitation to better understand the protein and RSS requirements for RAG recruitment to chromatin. Using a catalytic mutant form of RAG1 to prevent recombination, we did not observe cooperation between RAG1 and RAG2 in their recruitment to endogenous Jκ gene segments over a 48-h time course. Using retroviral recombination substrates, we found that RAG1 was recruited inefficiently to substrates lacking an RSS or containing a single RSS, better to substrates with two 12-bp RSSs (12RSSs) or two 23-bp RSSs (23RSSs), and more efficiently to a substrate with a 12/23RSS pair. RSS mutagenesis demonstrated a major role for the nonamer element in RAG1 binding, and correspondingly, a cryptic RSS consisting of a repeat of CA dinucleotides, which poorly re-creates the nonamer, was ineffective in recruiting RAG1. Our findings suggest that 12RSS-23RSS cooperation (the “12/23 rule”) is important not only for regulating RAG-mediated DNA cleavage but also for the efficiency of RAG recruitment to chromatin.
46
Lee, Baeck-seung, Joseph D. Dekker, Bum-kyu Lee, Vishwanath R. Iyer, Barry P. Sleckman, Arthur L. Shaffer, Gregory C. Ippolito, and Philip W. Tucker. "The BCL11A Transcription Factor Directly Activates RAG Gene Expression and V(D)J Recombination." Molecular and Cellular Biology 33, no. 9 (February 25, 2013): 1768–81. http://dx.doi.org/10.1128/mcb.00987-12.
Full textAbstract:
Recombination-activating gene 1 protein (RAG1) and RAG2 are critical enzymes for initiating variable-diversity-joining (VDJ) segment recombination, an essential process for antigen receptor expression and lymphocyte development. The transcription factor BCL11A is required for B cell development, but its molecular function(s) in B cell fate specification and commitment is unknown. We show here that the major B cell isoform, BCL11A-XL, binds the RAG1 promoter and Erag enhancer to activate RAG1 and RAG2 transcription in pre-B cells. We employed BCL11A overexpression with recombination substrates in a cultured pre-B cell line as well as Cre recombinase-mediated Bcl11a lox/lox deletion in explanted murine pre-B cells to demonstrate direct consequences of BCL11A/RAG modulation on V(D)J recombination. We conclude that BCL11A is a critical component of a transcriptional network that regulates B cell fate by controlling V(D)J recombination.
47
Swaminathan, Srividya, Lars Klemm, Anthony M. Ford, Klaus Schwarz, Rafael Casellas, Lothar Hennighausen, Huimin Geng, et al. "Cooperation Between Aid and the Rag1/Rag2 V(D)J Recombinase Drives Clonal Evolution of Childhood Acute Lymphoblastic Leukemia." Blood 120, no. 21 (November 16, 2012): 519. http://dx.doi.org/10.1182/blood.v120.21.519.519.
Full textAbstract:
Abstract Abstract 519 Background and Rationale: In many cases, childhood acute lymphoblastic leukemia (ALL) can be retraced to a recurrent genetic lesion in utero which establishes a pre-leukemic clone. The TEL-AML1 fusion gene for instance, arises prenatally and defines the most frequent subtype of childhood ALL. Strikingly, ∼1 of 100 healthy newborns carry a TEL-AML1 pre-leukemic clone, but only less than 1% of these children eventually develop leukemia. Encounter of infectious antigen leads to activation of the mutator enzyme AID in mature B cells. While AID is required for somatic hypermutation and class switching during late stages of B cell development, its pre-mature activation may be deleterious. The underlying questions for this project were: (1) how are B cells safeguarded from pre-mature AID expression during their early development and (2) whether pre-mature AID expression during early B cell development is deleterious in the sense that it promotes the clonal evolution of a pre-leukemic B cell clone in the bone marrow. Results: Studying gene expression in a clinical trial for children with high risk pre-B ALL (COG P9906; n=207), we found that high expression levels of AID at the time of diagnosis is predictive of poor overall survival and a higher frequency of leukemia relapse. These findings suggest that AID may be a contributing factor to the clonal evolution of childhood pre-B ALL. Previous work by Michael Lieber's group proposed cooperation of AID and the V(D)J recombinase Rag1/Rag2 as a key mechanism leading to the acquisition of chromosomal translocations in human B cell malignancies (Tsai et al., 2008). Activity of Rag1/Rag2 V(D)J recombinase and AID is segregated to early and late stages of B cell development, respectively. However, we found that experimental withdrawal of IL7 receptor (IL7R) signaling in pre-B cells not only activates Rag1/Rag2 expression and V(D)J recombinase but also rendered pre-B cells responsive to antigen (LPS) encounter with strong upregulation of AID. We found that upon withdrawal of IL7, transcription of AID and Rag1/Rag2 is activated by the same elements through a Pten/FoxO-dependent pathway. To test whether IL7R signaling also negatively regulates AID activation in human pre-B cells, we performed a comprehensive analysis of human B cell development in bone marrow samples from two children carrying deleterious mutations of the IL7RA and IL2RG genes encoding the two chains of the human IL7R. As opposed to normal human pre-B cells, pre-B cells from IL7RA and IL2RG-mutant patients carried somatically mutated immunoglobulin genes consistent with aberrant expression of AID in these cells. Based on these observations, we propose that Fraction D pre-B cells represent a subset of increased genetic vulnerability owing to concomitant expression of both AID and Rag1/Rag2. To test whether the vulnerability of Fraction D pre-B cells is relevant in the clonal evolution of childhood ALL, we challenged pre-B cells carrying the TEL-AML1 fusion gene with 5 consecutive cycles of IL7 withdrawal (−IL7) and LPS stimulation (+LPS). To distinguish between the potential contribution of AID and Rag1/Rag2 to secondary genetic lesions, -IL7/+LPS-challenges were performed with wildtype pre-B cells, AID−/−, Rag1−/− and AID−/− Rag1−/− double knockout pre-B cells. TEL-AML1-bearing pre-B cells were labeled with firefly luciferase and then 25 million cells were injected into 7 recipient animals per group. While wildtype TEL-AML1 pre-B cells that went through 5 rounds of -IL7/+LPS-challenge caused leukemia in all recipient mice, TEL-AML1 pre-B cells lacking either AID or Rag1 failed to give rise to full-blown leukemia in transplant recipients. Conclusion: While one in 100 newborns carry the TEL-AML1 fusion molecule, the mechanisms that lead to the acquisition of critical secondary genetic lesions are not known. Here, we report a novel, IL7R/Stat5-dependent mechanism by which pre-B cells are rendered non-responsive to LPS-dependent upregulation of AID. We propose that Fraction D pre-B cells represent a subset of increased natural genetic vulnerability in the context of concomitant activativity of AID and Rag1/Rag2. Frequent exposure to infectious antigens (e.g. LPS) in this constellation may propagate clonal evolution towards full-blown leukemia. Disclosures: No relevant conflicts of interest to declare.
48
Oudinet, Chloé, Fatima-Zohra Braikia, Audrey Dauba, and Ahmed Amine Khamlichi. "Recombination may occur in the absence of transcription in the immunoglobulin heavy chain recombination centre." Nucleic Acids Research 48, no. 7 (February 22, 2020): 3553–66. http://dx.doi.org/10.1093/nar/gkaa108.
Full textAbstract:
Abstract Developing B cells undergo V(D)J recombination to generate a vast repertoire of Ig molecules. V(D)J recombination is initiated by the RAG1/RAG2 complex in recombination centres (RCs), where gene segments become accessible to the complex. Whether transcription is the causal factor of accessibility or whether it is a side product of other processes that generate accessibility remains a controversial issue. At the IgH locus, V(D)J recombination is controlled by Eμ enhancer, which directs the transcriptional, epigenetic and recombinational events in the IgH RC. Deletion of Eμ enhancer affects both transcription and recombination, making it difficult to conclude if Eμ controls the two processes through the same or different mechanisms. By using a mouse line carrying a CpG-rich sequence upstream of Eμ enhancer and analyzing transcription and recombination at the single-cell level, we found that recombination could occur in the RC in the absence of detectable transcription, suggesting that Eμ controls transcription and recombination through distinct mechanisms. Moreover, while the normally Eμ-dependent transcription and demethylating activities were impaired, recruitment of chromatin remodeling complexes was unaffected. RAG1 was efficiently recruited, thus compensating for the defective transcription-associated recruitment of RAG2, and providing a mechanistic basis for RAG1/RAG2 assembly to initiate V(D)J recombination.
49
Ji, Yanhong, Alicia J. Little, Joydeep K. Banerjee, Bingtao Hao, Eugene M. Oltz, Michael S. Krangel, and David G. Schatz. "Promoters, enhancers, and transcription target RAG1 binding during V(D)J recombination." Journal of Experimental Medicine 207, no. 13 (November 29, 2010): 2809–16. http://dx.doi.org/10.1084/jem.20101136.
Full textAbstract:
V(D)J recombination assembles antigen receptor genes in a well-defined order during lymphocyte development. This sequential process has long been understood in the context of the accessibility model, which states that V(D)J recombination is regulated by controlling the ability of the recombination machinery to gain access to its chromosomal substrates. Indeed, many features of “open” chromatin correlate with V(D)J recombination, and promoters and enhancers have been strongly implicated in creating a recombinase-accessible configuration in neighboring chromatin. An important prediction of the accessibility model is that cis-elements and transcription control binding of the recombination-activating gene 1 (RAG1) and RAG2 proteins to their DNA targets. However, this prediction has not been tested directly. In this study, we use mutant Tcra and Tcrb alleles to demonstrate that enhancers control RAG1 binding globally at Jα or Dβ/Jβ gene segments, that promoters and transcription direct RAG1 binding locally, and that RAG1 binding can be targeted in the absence of RAG2. These findings reveal important features of the genetic mechanisms that regulate RAG binding and provide a direct confirmation of the accessibility model.
50
Lassoued, Kaiss, Vincent Fuentes, Hussein Gamlouch, Eliane Bissac, Jean-Pierre Marolleau, Jacques Rochette, and Hicham Bouhlal. "Role of the MAPK and PI3-Kinase/Akt Pathways in the Pre-B Cell Receptor (pre-BCR)-Induced NF-κb Activation and Rag1 and Rag2 Down Regulation." Blood 114, no. 22 (November 20, 2009): 2667. http://dx.doi.org/10.1182/blood.v114.22.2667.2667.
Full textAbstract:
Abstract Abstract 2667 Poster Board II-643 The pre-BCR acts as a critical checkpoint in pre-B cell development and might be also involved in leukemogenesis. Using the 697 and Nalm6 human pre-B cell lines, we have previously shown that pre-BCR stimulation resulted in cell cycle progression associated with activation of number of adaptors and signaling pathways including the PI3-Kinase/Akt, Ras/MAPK, AP1 and the canonical NFkB pathway. We have also demonstrated that Src kinases together with Syk played a crucial role in controlling the pre-BCR-associated functions, acting upstream the above-mentioned signaling pathways. Pre-BCR crosslinking also induced down regulation of Rag1 and Rag2 transcription. In this study we aimed to evaluate the role of MAPK and Akt in the pre-BCR-induced NF-kB activation and Rag1/2 down modulation. For this purpose the 697 pre-B cells and normal bone marrow primary pre-B cells were treated with the U0126 and LY294002, and with MEK1/2 and Akt inhibitors, respectively. A dominant negative form of Akt fused to the HIV1 Tat peptide was also used to inhibit the PI3-Kinase/Akt pathway. We bring evidence that LY294002 could alter the pre-BCR-induced NF-kB activation by inhibiting : i) p105 degradation, ii) p50 NF-kB1 nuclear translocation and, iii) the binding of p50 to an oligonucleotide containing a specific consensus sequence. On the contrary, U0126 significantly enhanced p105 degradation, indicating that MAPK and Akt exerted antagonistic effects on the pre-BCR-induced NF-kB activation. Strikingly the baseline levels of Rag1 and Rag2 transcripts were increased in the LY294002 but not the U0126-treated pre-B cells. Futhermore, both inhibitors were shown to induce a strong increase in the expression of Rag1 and Rag2 transcripts upon pre-BCR crosslinking, suggesting that this receptor exerts dual effects on Rag1/2 expression with a predominant negative regulatory component mediated by both PI3-K and MAPK. No changes in the levels of Pax5, E2A, EBF, IFR4, IRF8, FOXO1, FOXO3, Myb, MAZ, LEF1 and SP1 (transcription factors implied in the regulation of Rag1 and Rag2 transcription) were observed in the pre-BCR stimulated or unstimulated-697 cells, treated or not with the MAPK and Akt inhibitors. Our results suggest that the pre-BCR signaling is a complex and tightly self-controlled process, which deregulation might alter cell growth and survival pathways via NF-kB as well as genomic stability trough Rag1/2 expression. Disclosures: No relevant conflicts of interest to declare.