scholarly journals Mutations in the transmembrane and juxtamembrane domains enhance IL27R transforming activity

2011 ◽  
Vol 438 (1) ◽  
pp. 155-164 ◽  
Author(s):  
Que T. Lambert ◽  
Anuradha Pradhan ◽  
J. Devon Roll ◽  
Gary W. Reuther
Keyword(s):  
Transmembrane Domain ◽  
Jak2 V617f ◽  
Lymphoid Cells ◽  
Receptor Expression ◽  
Janus Kinase ◽  
Cellular Growth ◽  
Wild Type ◽  
Juxtamembrane Region ◽  
Transforming Activity ◽  
Haemopoietic Cells

Cytokines and their receptors regulate haemopoiesis by controlling cellular growth, survival and differentiation. Thus it is not surprising that mutations of cytokine receptors contribute to the formation of haemopoietic disorders, including cancer. We recently identified transforming properties of IL27R, the ligand-binding component of the receptor for interleukin-27. Although wild-type IL27R exhibits transforming properties in haemopoietic cells, in the present study we set out to determine if the transforming activity of IL27R could be enhanced by mutation. We identified three mutations of IL27R that enhance its transforming activity. One of these mutations is a phenylalanine to cysteine mutation at residue 523 (F523C) in the transmembrane domain of the receptor. The two other mutations identified involve deletions of amino acids in the cytoplasmic juxtamembrane region of the receptor. Expression of each of these mutant IL27R proteins led to rapid cytokine-independent transformation in haemopoietic cells. Moreover, the rate of transformation induced by these mutants was significantly greater than that induced by wild-type IL27R. Expression of these IL27R mutants also induced enhanced activation of JAK (Janus kinase)/STAT (signal transducer and activator of transcription) signalling compared with wild-type. An activating deletion mutation of IL27R enhanced homodimerization of the receptor by a mechanism that may involve disulfide bonding. These transforming IL27R mutants displayed equal or greater transforming activity than bona fide haemopoietic oncogenes such as BCR–ABL (breakpoint cluster region–Abelson murine leukaemia viral oncogene homologue) and JAK2-V617F. Since IL27R is expressed on haemopoietic stem cells, lymphoid cells and myeloid cells, including acute myeloid leukaemia blast cells, mutation of this receptor has the potential to contribute to a variety of haemopoietic neoplasms.

Preferential Inhibition of An Activated Form of Janus Kinase 2 (JAK2) by a Novel JAK2 Inhibitor, NS-018

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4107-4107 ◽  
Author(s):  
Yohei Nakaya ◽  
Haruna Naito ◽  
Junko Homan ◽  
Seishi Sugahara ◽  
Tatsuya Horio ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Jak2 V617f ◽  
Janus Kinase ◽  
Wild Type ◽  
X Ray ◽  
Jak2 Inhibitor ◽  
Colony Forming Units ◽  
Ic50 Value ◽  
Marrow Cells

Abstract Abstract 4107 A somatic point mutation of Janus Kinase 2 (JAK2) tyrosine kinase (JAK2 V617F) has been shown to occur at a high frequency in myeloproliferative neoplasm (MPN) patients. JAK2 V617F is a constitutively activated kinase that activates the JAK/STAT signaling pathway and dysregulates cell growth and function. These findings suggest that the inhibition of aberrant JAK2 activation has a therapeutic benefit. Our novel JAK2 inhibitor, NS-018, is highly active against JAK2 with an IC50 value of less than 1 nM, and it has 30–50-fold selectivities for JAK2 over other JAK-family kinases such as JAK1, JAK3 and Tyk2. We determined the X-ray structure of JAK2 in complex with NS-018. An Asp-Phe-Gly (DFG) motif is located at the N-terminus of the activation loop and regulates ATP binding. The resolved X-ray structure showed that NS-018 bound to JAK2 in the “DFG-in” active conformation. A molecular modeling study indicated that NS-018 would hardly bind to JAK2 in the “DFG-out” inactive conformation. In accordance with the structural analysis, NS-018 preferentially suppressed the growth of bone-marrow cells expressing activated JAK2. Thus, NS-018 reduced in a dose-dependent manner the number of erythroid colony-forming units (CFU-E) derived from bone-marrow cells taken from JAK2 V617F transgenic mice, but had only a limited effect on the number of colonies from wild-type mice (Figure A). NS-018 had no effect on the number of granulocyte-macrophage colony-forming units (CFU-GM) from either mouse strain. Furthermore, NS-018 showed potent antiproliferative activity against Ba/F3 cells expressing JAK2 V617F with an IC50 value of <100 nM but showed only minimal cytotoxicity against most other hematopoietic and non-hematopoietic cell lines (IC50 >3 μ M). In a mouse Ba/F3-JAK2 V617F leukemia model, NS-018 significantly prolonged survival during repeated oral administrations at 6.25 mg/kg bid and reduced splenomegaly at doses as low as 1.5 mg/kg bid. NS-018 was well tolerated at dosages of more than 100 mg/kg bid. In conclusion, NS-018 is a potent JAK2 inhibitor which preferentially inhibits an activated form of JAK2 and has potent in vitro and in vivo efficiency in preclinical studies. NS-018 is expected to be suitable for the treatment of MPN caused by aberrant JAK2 activation and its effectiveness will be verified by early-phase clinical investigations in the near future. JAK2 V617F preferential inhibition of erythrocyte colony growth Bone-marrow cells were collected from femurs of JAK2 V617F transgenic mice and same-strain BDF1 wild-type mice. (a) To detect CFU-E colonies, cells were treated with NS-018 in semisolid methylcellulose containing erythropoietin (EPO) and cell clusters were counted after incubation for two days. (b) To detect CFU-GM colonies, cells were treated with NS-018 in semisolid methylcellulose containing EPO, interleukin-3 (IL-3), IL-6 and stem cell factor and colonies were counted on day 7. Disclosures: Nakaya: Nippon Shinyaku Co., Ltd: Employment. Naito:Nippon Shinyaku Co., Ltd: Employment. Homan:Nippon Shinyaku Co., Ltd: Employment. Sugahara:Nippon Shinyaku Co., Ltd: Employment. Horio:Nippon Shinyaku Co., Ltd: Employment. Niwa:Nippon Shinyaku Co., Ltd: Employment. Shimoda:Nippon Shinyaku Co., Ltd: Research Funding.

Fusion of the erythropoietin receptor and the Friend spleen focus-forming virus gp55 glycoprotein transforms a factor-dependent hematopoietic cell line

1993 ◽  
Vol 13 (2) ◽  
pp. 739-748
Author(s):  
M O Showers ◽  
J C DeMartino ◽  
Y Saito ◽  
A D D'Andrea
Keyword(s):  
Cell Line ◽  
Fusion Proteins ◽  
Transmembrane Domain ◽  
Specific Interaction ◽  
Erythropoietin Receptor ◽  
Hematopoietic Cell ◽  
Wild Type ◽  
Hematopoietic Cell Line ◽  
Transforming Activity ◽  
Fusion Polypeptides

The Friend spleen focus-forming virus (SFFV) gp55 glycoprotein binds to the erythropoietin receptor (EPO-R), causing constitutive receptor signaling and the first stage of Friend erythroleukemia. We have used three independent strategies to further define this transforming molecular interaction. First, using a retroviral selection strategy, we have isolated the cDNAs encoding three fusion polypeptides containing regions of both EPO-R and gp55. These fusion proteins, like full-length gp55, transformed the Ba/F3 factor-dependent hematopoietic cell line and localized the transforming activity of gp55 to its transmembrane domain. Second, we have isolated a mutant of gp55 (F-gp55-M1) which binds, but fails to activate, EPO-R. We have compared the transforming activity of this gp55 mutant with the EPO-R-gp55 fusion proteins and with other variants of gp55, including wild-type polycythemia Friend gp55 and Rauscher gp55. All of the fusion polypeptides and mutant gp55 polypeptides were expressed at comparable levels, and all coimmunoprecipitated with wild-type EPO-R, but only the Friend gp55 and the EPO-R-gp55 fusion proteins constitutively activated wild-type EPO-R. Third, we have examined the specificity of the EPO-R-gp55 interaction by comparing the differential activation of murine and human EPO-R by gp55. Wild-type gp55 had a highly specific interaction with murine EPO-R; gp55 bound, but did not activate, human EPO-R.

Activation of JAK2-V617F by Heterodimeric Receptor Components.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2955-2955
Author(s):  
Anuradha R. Pradhan ◽  
Que T. Lambert ◽  
Lori N. Griner ◽  
Gary W. Reuther
Keyword(s):  
Hematopoietic Cells ◽  
Jak2 V617f ◽  
Receptor Expression ◽  
Janus Kinase ◽  
Cytokine Receptor ◽  
Altered Expression ◽  
Receptor Alpha ◽  
Interleukin 3 ◽  
Mutational Activation ◽  
Interacting Domain

Abstract Abstract 2955 Poster Board II-931 Mutational activation of Janus Kinase 2 (JAK2) is an important etiologic factor for the development of myeloproliferative neoplasms (MPNs). JAK2 is mutated in nearly all patients with polycythemia vera and about half of patients with essential thrombocythemia and primary myelofibrosis. The most prevalent mutation of JAK2 is valine 617 mutated to phenylalanine (V617F). The normal function of JAK2 is to interact with and become activated by cytokine receptors following their interaction with ligand. Interestingly, while JAK2-V617F is considered a constitutively activated kinase, there is evidence that it still requires interaction with a cytokine receptor to elicit its transforming signal. When expressed at less than or near endogenous JAK2 levels in hematopoietic cells, JAK2-V617F requires co-expression of a homodimeric receptor in order to become activated, transduce downstream signals, and induce transformation. When expressed at high levels in hematopoietic cells, co-expression of a homodimeric cytokine receptor is not needed. However, a functional cytokine receptor interacting domain is still required, suggesting that even when expressed at high levels JAK2-V617F requires interaction with a receptor. Also, a functional cytokine receptor interacting domain in JAK2-V617F is required for it to induce MPN-like disease in mouse models. The ability of cytokine receptor expression to activate JAK2-V617F has focused on homodimeric receptors. In this study we demonstrate that single components of heterodimeric receptors can also activate JAK2-V617F. Expression of interleukin-27 receptor alpha (IL27Ra), the ligand-binding component of the IL-27 receptor, enhances phosphorylation of JAK2-V617F on tyrosines 1007 and 1008. This activation of JAK2 also leads to tyrosine phosphorylation of signal transducers and activators of transcription-5 (STAT5), a main downstream effector of JAK2 activation. We obtain similar results when we utilize interleukin-12 receptor beta 1 (IL12RB1), a receptor belonging to the same family as IL27Ra. To extend these studies to other heterodimeric cytokine receptor components, we utilized the components of the interleukin-3 receptor, interleukin-3 receptor alpha (IL3Ra) and the beta common chain, which is also utilized in the receptors for interleukin-5 and granulocyte macrophage colony stimulating factor. Expression of each of these receptor subunits activates JAK2-V617F as well as STAT5. Importantly, we demonstrate that expression of IL27Ra can functionally replace the expression of a homodimeric cytokine receptor to support the activation of JAK2-V617F in BaF3 cells as well as promote the transforming activity of JAK2-V617F in these cytokine dependent hematopoietic cells. Interestingly, while IL12RB1 expression activates JAK2-V617F in 293T cells, it is not capable of enhancing the transforming signals of JAK2-V617F in BaF3 cells. Expression of IL3Ra or the beta common chain in BaF3 cells also enhances the ability of JAK2-V617F to transform these cells to cytokine independence. However, this enhancement is not immediate as it only becomes evident at later time points. Together our data demonstrate that in addition to homodimeric receptors, some heterodimeric receptor components may contribute to JAK2-V617F activation. It should also be considered that such receptors may play a role in JAK2-V617F-negative MPNs, perhaps through altered expression or activating receptor mutations, analogous to mutated thrombopoietin receptor proteins that play a role in the development of disease in a fraction of these MPN patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Fusion of the erythropoietin receptor and the Friend spleen focus-forming virus gp55 glycoprotein transforms a factor-dependent hematopoietic cell line.

1993 ◽  
Vol 13 (2) ◽  
pp. 739-748 ◽  
Author(s):  
M O Showers ◽  
J C DeMartino ◽  
Y Saito ◽  
A D D'Andrea
Keyword(s):  
Cell Line ◽  
Fusion Proteins ◽  
Transmembrane Domain ◽  
Specific Interaction ◽  
Erythropoietin Receptor ◽  
Hematopoietic Cell ◽  
Wild Type ◽  
Hematopoietic Cell Line ◽  
Transforming Activity ◽  
Fusion Polypeptides

The Friend spleen focus-forming virus (SFFV) gp55 glycoprotein binds to the erythropoietin receptor (EPO-R), causing constitutive receptor signaling and the first stage of Friend erythroleukemia. We have used three independent strategies to further define this transforming molecular interaction. First, using a retroviral selection strategy, we have isolated the cDNAs encoding three fusion polypeptides containing regions of both EPO-R and gp55. These fusion proteins, like full-length gp55, transformed the Ba/F3 factor-dependent hematopoietic cell line and localized the transforming activity of gp55 to its transmembrane domain. Second, we have isolated a mutant of gp55 (F-gp55-M1) which binds, but fails to activate, EPO-R. We have compared the transforming activity of this gp55 mutant with the EPO-R-gp55 fusion proteins and with other variants of gp55, including wild-type polycythemia Friend gp55 and Rauscher gp55. All of the fusion polypeptides and mutant gp55 polypeptides were expressed at comparable levels, and all coimmunoprecipitated with wild-type EPO-R, but only the Friend gp55 and the EPO-R-gp55 fusion proteins constitutively activated wild-type EPO-R. Third, we have examined the specificity of the EPO-R-gp55 interaction by comparing the differential activation of murine and human EPO-R by gp55. Wild-type gp55 had a highly specific interaction with murine EPO-R; gp55 bound, but did not activate, human EPO-R.

JAK2 V617F Mutation Is Infrequent in “5q-Syndrome” and 5q-Associated MDS.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4833-4833
Author(s):  
Ling Zhang ◽  
Saskia Gueller ◽  
Sophie Raynaud ◽  
Phillip H. Koeffler ◽  
Stephen Lee
Keyword(s):  
Bone Marrow ◽  
Chromosomal Abnormalities ◽  
Bone Marrow Aspiration ◽  
K562 Cells ◽  
Jak2 V617f ◽  
Janus Kinase ◽  
Jak2 V617f Mutation ◽  
Wild Type ◽  
Jak2 Mutation ◽  
V617f Mutation

Abstract Background: V617F mutation in Janus Kinase 2 (JAK2) gene has been found in chronic myeloproliferative disorders (MPD) including polycythemia vera (90%), essential thrombocythemia and chronic idiopathic myelofibrosis (30–50%), and occasionally in myelodysplastic syndromes (MDS). “5q- Syndrome” is a MDS that shares features with MPD and characterized by an atypical megakaryocytic hyperplasia in bone marrow and usually thrombocytosis in peripheral blood. The most common deleted region for this syndrome is 5q13.3q33.1. An interstitial deletion with variable proximal (5q12-14) and distal (5q31-33) breakpoints has been found in other MDS with/without additional chromosomal abnormalities beyond “5q- Syndrome”. To date, JAK2 mutation was detected in 6/97(6.2%) of patients having diagnosis of MDS with “5q- Syndrome”. Design: In our study 21 MDS patients (10 with “5q- Syndrome” and 11 MDS with isolated or complex 5q-) whose diagnosis by both bone marrow aspiration/biopsy and conventional chromosomal analysis were confirmed. Materials and Method: Primers were created to amplify a 460 bp fragment containing the site of JAK2 V617F mutation. Forty-five cycles of PCR were performed at an annealing temperature of 57°C. Resulting PCR product was digested with 2 U BsaXI for 16 hours and with an additional 2 U BsaXI for another 16 hours at 37°C, then analyzed on a 2% agarose gel. The mutant allele remained undigested whereas the wild-type allele was digested into 241 bp, 189 bp and 30 bp fragments. All experiments included a positive (HEL cells) and negative (K562 cells) control. Results: PCR results showed clear wild type PCR patterns in all 21 cases. Conclusion: No JAK2 mutations were detected in 21 patients either with “5q- Syndrome” or other 5q- associated MDS suggesting that JAK2 mutations are infrequent in these MDS patients.

Activating PP2A Tumor Suppressor Activity as a Potential Therapy for Treating Jak2 V617F-Driven Myeloproliferative Disorders.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3548-3548
Author(s):  
Joshua J. Oaks ◽  
Ramasamy Santhanam ◽  
Roger Briesewitz ◽  
Danilo Perrotti
Keyword(s):  
Bone Marrow ◽  
Tumor Suppressor ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Jak2 V617f ◽  
Janus Kinase ◽  
Wild Type ◽  
Semisolid Medium ◽  
Pp2a Activity ◽  
Marrow Cells

Abstract Polycythemia vera (PV) is a myeloproliferative disorder (MPD) caused by a valine to phenylalanine mutation in the inhibitory JH2 “pseudokinase” domain of the signaling protein Janus Kinase 2 (Jak2). While Jak2 has several known regulators (e.g. SOCS1, SOCS3, PP2A, and SHP-1), the role played by these signaling molecules in the development of PV is still largely unclear. One of these regulators, the tumor suppressor phosphatase PP2A, has been found functionally inactivated in different hematologic myeloid and lymphoid malignancies characterized by the expression of constitutively activated oncogenic tyrosine kinases (e.g. BCR/ABL). To investigate the role of PP2A in the pathogenesis of Jak2 V617F+ MPDs and the potential therapeutic relevance of PP2A activating drugs (e.g. FTY720), we determined the effects of wild type and V617F Jak2 expression on PP2A activity and assessed the molecular and biological effects of FTY720 in hematopoietic precursor cell lines and/or primary lineage-negative bone marrow cells engineered to expressed either wild type or V617F Jak2 protein. Herein we report that PP2A activity is significantly reduced by about 82% and 78% (P&lt; 0.01) in mJak2 V617F-transduced growth factor-dependent and erythropoietin receptor-expressing 32Dcl3 (32D-EPO) and Ba/F3 cells, respectively, compared to MigR1-transduced controls. Furthermore, addition of the PP2A activator FTY720 (2.5μM) for 10 hours restored PP2A activity to 66% and 75% respectively compared to that of MigR1 controls. Mechanistically, we demonstrated that inactivation of PP2A was due to constitutive Jak2 activity. In fact, treatment of V617F Jak2-expressing cells with Jak inhibitor I (1μM; 10 hours) restored PP2A activity to 80% of controls in 32Dcl3 cells, while 600nM was sufficient to restore activity to 108% of controls in Ba/f3 cells. Likewise, transduction of murine lineage-negative bone marrow cells with wild-type Jak2 produced a 62% reduction in PP2A activity (P&lt;0.01), while expression of the Jak2 V617F mutant produced a 97% reduction (P&lt; 0.01). Moreover, as we previously reported for BCR/ABL, pharmacologic restoration of PP2A activity by treatment of Ba/F3 or 32D-EPO cells with the PP2A activator FTY720 (2.5μM for 10 hours) led to reduced expression and dephosphorylation of wild type and V617F Jak2. This, in turn, resulted in a 60% reduction in the colony forming ability of IL-3 cultured cells overexpressing wild type Jak2, while a 94% suppression of colony formation was evident in Jak2 V617-expressing cells maintained in the absence of cytokines. Moreover, addition of IL-3 to the FTY720-containing semisolid medium restored viability to levels similar to those of FTY720-treated wild type Jak2-expressing cells (70% suppression of colony formation upon treatment), indicating that restoration of PP2A activity counteracts cytokine-independent pathways triggered by the V617F Jak2 mutated tyrosine kinase. Altogether, these results suggest that inactivation of PP2A is essential for Jak2 (V617F mutant included) -driven cell/proliferation and survival and that pharmacologic activation of PP2A might represent a potential avenue for treatment of PV and, perhaps, of other MPDs characterized by the expression of a mutated Jak2 kinase.

scholarly journals Four novel RET germline variants in exons 8 and 11 display an oncogenic potential in vitro

2010 ◽  
Vol 162 (4) ◽  
pp. 771-777 ◽  
Author(s):  
Marina Muzza ◽  
Daniela Cordella ◽  
Johny Bombled ◽  
Brigitte Bressac-de Paillerets ◽  
Fabiana Guizzardi ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Focus Formation ◽  
Oncogenic Potential ◽  
Mutant Proteins ◽  
Activating Mutations ◽  
Juxtamembrane Region ◽  
Erk Phosphorylation ◽  
Transforming Activity ◽  
Exon 11

ContextMost germline-activating mutations of the RET proto-oncogene associated with inherited medullary thyroid cancer (MTC) are localized in exons 10, 11 and 13–15. Four novel RET variants, located in the extracellular domain (p.A510V, p.E511K and p.C531R) coded by exon 8 and in the intracellular juxtamembrane region (p.K666N) coded by exon 11, were identified on the leukocyte DNA from apparently sporadic cases.MethodsPlasmids carrying Ret9-wild-type (Ret9-WT), Ret9-C634R and all Ret9 variants were transfected, and the phosphorylation levels of RET and ERK were evaluated by western blot analyses. The transforming potentials were assessed by the focus formation assay.ResultsThe p.A510V, p.E511K and p.C531R variants were found to generate RET and ERK phosphorylation levels and to have a transforming activity higher than that of Ret9-WT variant, but lower than that of Ret9-C634R variant. Differently, the p.K666N variant, located immediately downstream of the transmembrane domain, and involving a conserved residue, displayed high kinase and transforming activities. Computational analysis predicted non-conservative alterations in the mutant proteins consistent with putative modifications of the receptor conformation.ConclusionsThe molecular analyses revealed an oncogenic potential for all the novel germline RET variants. Therefore, the prevalence of exon 8 genomic variations with an oncogenic potential may be higher than previously thought, and the analysis of this exon should be considered after the exclusion of mutations in the classical hotspots. In addition, on the basis of these functional data, it is advisable to extend the genetic screening to all the first-degree relatives of the MTC patients, and to perform a strict follow-up of familial carriers.

scholarly journals STAT5b Is Required for GH-Induced Liver Igf-I Gene Expression

Endocrinology ◽  
2001 ◽  
Vol 142 (9) ◽  
pp. 3836-3841 ◽  
Author(s):  
Helen W. Davey ◽  
Tao Xie ◽  
Michael J. McLachlan ◽  
Richard J. Wilkins ◽  
David J. Waxman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Intracellular Signaling ◽  
Normal Liver ◽  
Receptor Expression ◽  
Janus Kinase ◽  
Sexually Dimorphic ◽  
Wild Type ◽  
Gh Secretion ◽  
Igf I ◽  
I Gene

Abstract Although the increased expression of Igf-I in liver in response to GH is well characterized, the intracellular signaling pathways that mediate this effect have not been identified. Intracellular signaling molecules belonging to the Janus kinase-signal transducer and activator of transcription 5b (JAK2-STAT5b) pathway are activated by GH and have previously been shown to be required for sexually dimorphic body growth and the expression of liver cytochrome P450 proteins known to be regulated by the gender-specific temporal patterns of pituitary GH secretion. Here, we evaluate the role of STAT5b in GH activation of Igf-I by monitoring the induction of Igf-I mRNA in livers of wild-type and Stat5b−/−mice stimulated with exogenous pulses of GH. GH induced the expression of liver Igf-I mRNA in hypophysectomized male wild-type, but not in hypophysectomized male Stat5b−/− mice, although theStat5b−/− mice exhibit both normal liver GH receptor expression and strong GH induction of Cytokine-inducible SH2 protein (Cis), which is believed to contribute to the down-regulation of GH-induced liver STAT5b signaling. Thus, STAT5b plays an important and specific role in liver Igf-I gene expression.

scholarly journals Ectopic Overexpression of Histone H3K4 Methyltransferase CsSDG36 from Tea Plant Decreases Hyperosmotic Stress Tolerance in Arabidopsis thaliana

2021 ◽  
Vol 22 (10) ◽  
pp. 5064
Author(s):  
Qinghua Chen ◽  
Linghui Guo ◽  
Yanwen Yuan ◽  
Shuangling Hu ◽  
Fei Guo ◽  
...  
Keyword(s):  
Drought Stress ◽  
Transmembrane Domain ◽  
Tea Plant ◽  
Microtubule Assembly ◽  
Pcr Analysis ◽  
Drought Response ◽  
Stomatal Development ◽  
Wild Type ◽  
Over Expression ◽  
Histone H3k4

Histone methylation plays an important regulatory role in the drought response of many plants, but its regulatory mechanism in the drought response of the tea plant remains poorly understood. Here, drought stress was shown to induce lower relative water content and significantly downregulate the methylations of histone H3K4 in the tea plant. Based on our previous analysis of the SET Domain Group (SDG) gene family, the full-length coding sequence (CDS) of CsSDG36 was cloned from the tea cultivar ‘Fuding Dabaicha’. Bioinformatics analysis showed that the open reading frame (ORF) of the CsSDG36 gene was 3138 bp, encoding 1045 amino acids and containing the conserved structural domains of PWWP, PHD, SET and PostSET. The CsSDG36 protein showed a close relationship to AtATX4 of the TRX subfamily, with a molecular weight of 118,249.89 Da, and a theoretical isoelectric point of 8.87, belonging to a hydrophilic protein without a transmembrane domain, probably located on the nucleus. The expression of CsSDG36 was not detected in the wild type, while it was clearly detected in the over-expression lines of Arabidopsis. Compared with the wild type, the over-expression lines exhibited lower hyperosmotic resistance by accelerating plant water loss, increasing reactive oxygen species (ROS) pressure, and increasing leaf stomatal density. RNA-seq analysis suggested that the CsSDG36 overexpression caused the differential expression of genes related to chromatin assembly, microtubule assembly, and leaf stomatal development pathways. qRT-PCR analysis revealed the significant down-regulation of stomatal development-related genes (BASL, SBT1.2(SDD1), EPF2, TCX3, CHAL, TMM, SPCH, ERL1, and EPFL9) in the overexpression lines. This study provides a novel sight on the function of histone methyltransferase CsSDG36 under drought stress.

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