An alfalfa MYB-like transcriptional factor MsMYBH positively regulates alfalfa seedling drought resistance and undergoes MsWAV3-mediated degradation

Drought is a major threat to alfalfa(Medicago sativa L.)production.The discovery of important alfalfa genes regulating drought response will facilitate breeding for drought-resistant alfalfa cultivars.Here,we report a genome-wide association study of drought resistance in alfalfa.We identified and functionally characterized an MYB-like transcription factor gene(MsMYBH),which increases the drought resistance in alfalfa.Compared with the wild-types,the biomass and forage quality were enhanced in MsMYBH overexpressed plants.Combined RNA-seq,proteomics and chromatin immunoprecipitation analysis showed that MsMYBH can directly bind to the promoters of MsMCP1,MsMCP2,MsPRX1A and MsCARCAB to improve their expression.The outcomes of such interactions include better water balance,high photosynthetic efficiency and scavenge excess H_(2)O_(2)in response to drought.Furthermore,an E3 ubiquitin ligase(MsWAV3)was found to induce MsMYBH degradation under long-term drought,via the 26S proteasome pathway.Furthermore,variable-number tandem repeats in MsMYBH promoter were characterized among a collection of germplasms,and the variation is associated with promoter activity.Collectively,our findings shed light on the functions of MsMYBH and provide a pivotal gene that could be leveraged for breeding drought-resistant alfalfa.This discovery also offers new insights into the mechanisms of drought resistance in alfalfa.

MdDGK3-like as a negative regulator participates in ALA-induced PP2AC to promote stomatal opening in apple leaves

5-Aminolevulinic acid(ALA)can inhibit abscisic acid(ABA)-induced stomatal closure.However,the molecular mechanism is unclear.In this study,we found that ALA upregulated the MdPP2AC expression and PP2A activity in the apple(Malus domestica Borkh.cv.‘Fuji’)leaves.With the promoter of MdPP2AC as bait,a diacylglycerol kinase MdDGK3-like was selected by the Yeast One Hybrid(Y1H)from the cDNA library of the epidermis of apple leaves treated by exogenous ALA.Additional to binding the promoter of MdPP2AC,MdDGK3-like was found to inhibit the transcription activity of MdPP2AC promoter,while ALA significantly eliminated the role of MdDGK3-like.In tobacco leaves,MdDGK3-like was localized in the nucleus of stomatal guard cells.Therefore,MdDGK3-like might act as a transcription factor negatively regulating MdPP2AC expression and causing stomatal closure.To further identify MdDGK3-like functions,several transiently transgenic apple leaves(including overexpression and interference)were established.The results revealed that overexpression of MdDGK3-like promoted stomatal closure by increasing Ca^(2+)and H_(2)O_(2)and decreasing flavonol levels in the guard cells.Conversely,MdDGK3-like(i)led the stomatal opening with lower levels of Ca^(2+)and H_(2)O_(2)but higher flavonols.Based on these,we proposed a new hypothesis that ALA up-regulated MdPP2AC expression via negatively regulating the expression of MdDGK3-like to up-regulate PP2A expression and the enzyme activity,which improved the stomatal aperture.Since it was the first time that MdDGK3-like was showed to act as a transcription factor,the proposed model provided a new insight onto the mechanisms of ALA-induced stomatal opening.

浸润性膀胱癌组织中E2F3表达与CD8^+ T细胞浸润数量的相关性分析

目的探讨膀胱癌组织中E2F转录因子3(E2F3)表达与CD8^+T细胞浸润的特点及其与膀胱癌恶性生物学行为之间的相关性。方法采用免疫组织化学染色法检测110例不同病理分期、分级的膀胱癌组织和相应正常膀胱组织中E2F3的表达情况及CD8^+T细胞浸润特征,Western blot法检测82例膀胱癌组织及相应正常膀胱组织中E2F3、CD8蛋白水平,直线相关分析方法分析E2F3表达与CD8^+T细胞浸润的相关性。结果膀胱癌组织中E2F3高表达,膀胱癌组织中E2F3表达随肿瘤分期、分级增加逐步增高,而相应肿瘤内CD8^+T细胞浸润数目逐步减少,E2F3高表达组肿瘤内CD8^+T细胞浸润数目显著低于E2F3低表达组。E2F3阳性率及CD8^+T细胞的浸润数量减少与肿瘤病理分期、分级、肿瘤大小、伴有淋巴结转移或远处转移等肿瘤临床侵袭性指标密切相关。相关性分析显示肿瘤E2F3表达水平与肿瘤内CD8^+T细胞浸润数目呈负相关。生存分析表明E2F3高表达患者术后5年预后更差。结论膀胱癌组织中E2F3水平与肿瘤侵袭性关系密切,与肿瘤内CD8^+T细胞浸润数量呈负相关。

维生素E对胃癌患者血清中GATA-3、T-bet及血脂的影响

目的观察维生素E对胃癌患者血清GATA-3、T-bet及血脂的影响。方法选取我院2010年8月—2013年4月收治的经胃镜活检证实为胃腺癌的男性患者22例,随机分为维生素E组(n=10)和对照组(n=12),维生素E组口服维生素E(100 mg/d)14 d,对照组不做任何处理,观察两组治疗前后血清中GATA-3、T-bet的浓度及甘油三酯(TG)、总胆固醇(TC)的变化情况。结果治疗后,维生素E组TC、TG、GATA-3较治疗前降低,T-bet表达量升高,差异均有统计学意义(P均<0.05);治疗后维生素E组血清TC、TG水平及GATA-3表达量明显低于对照组,T-bet表达量明显高于对照组,差异均有统计学意义(P均<0.05)。结论维生素E能很好地激活胃癌患者机体细胞免疫机制,并降低脂质过氧化损伤。

STAT3通过ZEB1调节卵巢癌细胞SKOV-3中E-cadherin表达的研究

目的:探讨卵巢癌细胞SKOV-3中信号转导与转录激活因子3(STAT 3)对上皮细胞钙黏蛋白(Ecadherin)表达的调控。方法:构建STAT 3过表达载体或缺失表达载体,并转染SKOV-3细胞,分别用Western blotting和RT-PCR方法检测STAT 3 mRNA及蛋白质水平的变化,确定细胞模型的建立。Western blotting检测构建的细胞模型中p-STAT 3水平与E-cadherin水平的变化,RT-PCR检测STAT 3过表达或缺失对ZEB1和ZEB2 mRNA水平的影响。同时构建ZEB1 siRNA与pCDNA3.1-STAT 3共转染细胞,Western blotting检测ZEB1对p-STAT 3诱导的E-cadherin水平下调的影响。结果:STAT 3过表达/缺失载体的SKOV-3细胞中E-cadherin蛋白质水平显著下调/上调;STAT 3过表达能显著上调ZEB1 mRNA水平,但对ZEB2 mRNA无显著影响;ZEB1 siRNA转染能逆转STAT 3过表达诱导的E-cadherin的水平下降。结论:卵巢癌细胞SKOV-3中,STAT 3能通过ZEB1调节E-cadherin的表达,在卵巢癌的发生发展中起重要作用。

Bexarotene improves motor function after spinal cord injury in mice

Spinal cord injury is a challenge in orthopedics because it causes irreversible damage to the central nervous system.Therefore,early treatment to prevent lesion expansion is crucial for the management of patients with spinal cord injury.Bexarotene,a type of retinoid,exerts therapeutic effects on patients with cutaneous T-cell lymphoma and Parkinson's disease.Bexarotene has been proven to promote autophagy,but it has not been used in the treatment of spinal cord injury.To investigate the effects of bexarotene on spinal cord injury,we established a mouse model of T11–T12 spinal cord contusion and performed daily intraperitoneal injection of bexarotene for 5 consecutive days.We found that bexarotene effectively reduced the deposition of collagen and the number of pathological neurons in the injured spinal cord,increased the number of synapses of nerve cells,reduced oxidative stress,inhibited pyroptosis,promoted the recovery of motor function,and reduced death.Inhibition of autophagy with 3-methyladenine reversed the effects of bexarotene on spinal cord injury.Bexarotene enhanced the nuclear translocation of transcription factor E3,which further activated AMP-activated protein kinase-S-phase kinase-associated protein 2-coactivator-associated arginine methyltransferase 1 and AMP-activated protein kinase-mammalian target of rapamycin signaling pathways.Intravenous injection of transcription factor E3 sh RNA or intraperitoneal injection of compound C,an AMP-activated protein kinase blocker,inhibited the effects of bexarotene.These findings suggest that bexarotene regulates nuclear translocation of transcription factor E3 through the AMP-activated protein kinase-Sphase kinase-associated protein 2-coactivator-associated arginine methyltransferase 1 and AMP-activated protein kinase-mammalian target of rapamycin signal pathways,promotes autophagy,decreases reactive oxygen species level,inhibits pyroptosis,and improves motor function after spinal cord injury.

Two VOZ transcription factors link an E3 ligase and an NLR immune receptor to modulate immunity in rice

Nucleotide-binding leucine-rich repeat(NLR)proteins play critical roles in plant immunity.However,how NLRs are regulated and activate defense signaling is not fully understood.The rice(Oryza sativa)NLR receptor Piz-t confers broad-spectrum resistance to the fungal pathogen Magnaporthe oryzae and the RING-type E3 ligase AVRPIZ-T INTERACTING PROTEIN 10(APIP10)negatively regulates Piz-t accumulation.In this study,we found that APIP10 interacts with two rice transcription factors,VASCULAR PLANT ONEZINC FINGER 1(OsVOZ1)and OsVOZ2,and promotes their degradation through the 26S proteasome pathway.OsVOZ1 displays transcriptional repression activity while OsVOZ2 confers transcriptional activation activity in planta.The osvoz1 and osvoz2 single mutants display modest but opposite M.oryzae resistance in the non-Piz-t background.However,the osvoz1 osvoz2 double mutant exhibits strong dwarfism and cell death,and silencing of both genes via RNA interference also leads to dwarfism,mild cell death,and enhanced resistance to M.oryzae in the non-Piz-t background.Both OsVOZ1 and OsVOZ2 interact with Piz-t.Double silencing of OsVOZ1 and OsVOZ2 in the Piz-t background decreases Piz-t protein accumulation and transcription,reactive oxygen species-dependent cell death,and resistance to M.oryzae containing AvrPiz-t.Taken together,these results indicate that OsVOZ1 and OsVOZ2 negatively regulate basal defense but contribute positively to Piz-t-mediated immunity.

原发性肝癌RUNX3基因启动子区甲基化及其意义

目的检测原发性肝癌(hepatocellular carcinomas,HCC)中人RUNT相关转录因子3(human runt-related tran-scription factor 3,RUNX3)基因启动子的甲基化情况,探讨其与患者临床特征的关系。方法收集95例HCC患者的肿瘤标本及相应的癌旁肝组织、20例正常肝组织,95例HCC患者中合并肝硬化患者57例,按照肝硬化Child-Pugh肝功能分级标准,将57例患者分为A级36例,B级11例,C级10例,运用甲基化特异性PCR检测RUNX3基因启动子CpG岛甲基化状态,分析RUNX3甲基化情况与患者临床特征的关系。结果 95例HCC组织中,45.3%(43/95)存在RUNX3基因CpG岛的异常甲基化,癌旁肝组织中有9.5%(9/95)存在异常甲基化,而正常肝组织中未检测到RUNX3基因CpG岛的异常甲基化;RUNX3基因异常甲基化在HCC组织中的发生率与癌旁组织及正常肝组织比较差异有统计学意义(P<0.01),RUNX3基因CpG岛甲基化与患者肝硬化、门脉癌栓关系密切(P<0.05)。肝硬化Child-Pugh肝功能C级组RUNX3启动子甲基化水平明显高于A级组和B级组(P<0.05)。结论 HCC存在RUNX3基因CpG岛异常甲基化,CpG岛的甲基化可能是导致其基因表达降低的主要原因之一,并与患者肝硬化、门脉癌栓密切相关。

转移相关基因3、锌指转录因子Snaill、E-钙黏素在胆道闭锁患儿肝脏中的表达及相互关系

目的探讨胆道闭锁（biliaryatresia，BA）患儿肝组织中转移相关基因3（MTA3）、锌指转录因子Snaill、E-钙黏素（E-cadherin）三者的表达、相关性及其在胆道闭锁发生、发展中的作用。方法选取2012年2月至2013年9月河北医科大学第二医院小儿外科收治的21例胆道闭锁、17例胆总管囊肿及10例肝破裂患儿的肝组织活检标本，相应分为胆道闭锁（BA）组、胆总管囊肿（CCC）组及正常肝（NL）组。分别应用免疫组织化学法、逆转录一多聚酶链反应从蛋白和基因mRNA水平检测对比三组肝组织中MTA3、Snaill、E—cadherin表达情况。结果在蛋白水平及mRNA水平BA组肝组织中Snaill呈高表达，MTA3、E—cadherin均呈低表达，MTA3和Snaill的表达呈负相关（r=-0．671，P〈0．05；r=一0．862，P〈0．05），Snaill和E—cadherin的表达呈负相关（r=-0．571，P〈0．05；r=-0．715，P〈0．05），MTA3和E．cadherin的表达呈正相关（r=0．671，P〈0．05；r=0．752，P〈0．05）。结论肝组织中MTA3减少，可能会降低对Snaill的抑制，进一步抑制E-cadherin的表达，促使胆管上皮EMT发生，导致BA的发生与发展。

STAT3和ZEB1在食管鳞癌中的表达及其与放疗敏感性的关系

[目的]研究STAT3和ZEB1在食管鳞癌组织中的表达及其与放疗敏感性的关系。[方法 ]采用免疫组化法分别检测100例食管癌组织中STAT3和ZEB1的表达情况,并分析其与放疗敏感性的关系。[结果]STAT3和ZEB1的阳性表达率分别为66.0%(66/100)和64.0%(64/100)。低分化以及临床分期为T_3+T_4的组织STAT3表达阳性率分别为85.7%(30/35)、82.9%(42/51),均显著高于中、高分化以及临床分期为T_1+T_2组织中的55.4%(36/65)、49.0%(24/49);低分化以及临床分期为T_3+T_4的组织ZEB1表达阳性率分别为85.7%(30/35)、76.5%(39/51),均显著高于中、高分化以及临床分期为T_1+T_2组织中的52.3%(34/65)、51.0%(25/49),差异均有统计学意义(P<0.05)。放疗有效组的STAT3、ZEB1阳性率均显著低于放疗无效组,差异均有统计学意义(59.2%vs 87.5%,P=0.011;55.3%vs 91.7%,P=0.001)。多因素Logistic回归分析结果显示T分期是影响放疗疗效的独立因素(P=0.001)。食管癌组织中STAT3与ZEB1的表达呈正相关(r=0.341,P=0.001)。[结论 ]STAT3和ZEB1蛋白表达与食管癌放疗的敏感性呈负相关,二者可能可作为预测食管癌患者的放射敏感性的分子标志物。

Identification of Arabidopsis MYB56 as a Nove Substrate for CRL3BPM E3 Ligases

Controlled stability of proteins is a highly efficient mechanism to direct diverse processes in living cells. A key regulatory system for protein stability is given by the ubiquitin proteasome pathway, which uses E3 ligases to mark specific proteins for degradation. In this work, MYB56 is identified as a novel target of a CULLIN3 （CUL3）-based E3 ligase. Its stability depends on the presence of MATH-BTB/POZ （BPM） proteins, which function as substrate adaptors to the E3 ligase. Genetic studies have indicated that MYB56 is a negative regulator of flowering, while BPMs positively affect this developmental program. The interaction between BPMs and MYB56 occurs at the promoter of FLOWERING LOCUS T （FT）, a key regulator in initiating flowering in Arabidopsis, and results in instability of MYB56. Overall the work establishes MYB transcription factors as substrates of BPM proteins, and provides novel information on components that participate in controlling flowering time in plants.

MYB106 is a negative regulator and a substrate for CRL3^(BPM) E3 ligase in regulating flowering time in Arabidopsis thaliana

Flowering time is crucial for successful reproduction in plants, the onset and progression of which are strictly controlled. However, flowering time is a complex and environmentally responsive history trait and the underlying mechanisms still need to be fully characterized. Post-translational regulation of the activities of transcription factors(TFs) is a dynamic and essential mechanism for plant growth and development. CRL3 BPME3 ligase is a CULLIN3-based E3 ligase involved in orchestrating protein stability via the ubiquitin proteasome pathway. Our study shows that the mutation of MYB106 induced early flowering phenotype while over-expression of MYB106 delayed Arabidopsis flowering. Transcriptome analysis of myb106 mutants reveals 257 differentially expressed genes between wild type and myb106-1 mutants, including Flowering Locus T(FT) which is related to flowering time. Moreover, in vitro electrophoretic mobility shift assays(EMSA), in vivo chromatin immunoprecipitation quantitative polymerase chain reaction(ChIP-q PCR) assays and dual luciferase assays demonstrate that MYB106 directly binds to the promoter of FT to suppress its expression. Furthermore, we confirm that MYB106 interacts with BPM proteins which are further identified by CRL3 BPME3 ligases as the substrate. Taken together, we have identified MYB106 as a negative regulator in the control of flowering time and a new substrate for CRL3 BPM E3 ligases in Arabidopsis.

TGF-β1-regulated miR-3691-3p targets E2F3 and PRDM1 to inhibit prostate cancer progression

Transforming growth factor-β1(TGF-β1)acts as a tumor promoter in advanced prostate cancer(PCa).We speculated that microRNAs(miRNAs)that are inhibited by TGF-β1 might exert anti-tumor effects.To assess this,we identified several miRNAs downregulated by TGF-β1 in PCa cell lines and selected miR-3691-3p for detailed analysis as a candidate anti-oncogene miRNA.miR-3691-3p was expressed at significantly lower levels in human PCa tissue compared with paired benign prostatic hyperplasia tissue,and its expression level correlated inversely with aggressive clinical pathological features.Overexpression of miR-3691-3p in PCa cell lines inhibited proliferation,migration,and invasion,and promoted apoptosis.The miR-3691-3p target genes E2F transcription factor 3(E2F3)and PR domain containing 1,with ZNF domain(PRDM1)were upregulated in miR-3691-3p-overexpressing PCa cells,and silencing of E2F3 or PRDM1 suppressed PCa cell proliferation,migration,and invasion.Treatment of mice bearing PCa xenografts with a miR-3691-3p agomir inhibited tumor growth and promoted tumor cell apoptosis.Consistent with the negative regulation of E2F3 and PRDM1 by miR-3691-3p,both proteins were overexpressed in clinical PCa specimens compared with noncancerous prostate tissue.Our results indicate that TGF-β1-regulated miR-3691-3p acts as an anti-oncogene in PCa by downregulating E2F3 and PRDM1.These results provide novel insights into the mechanisms by which TGF-β1 contributes to the progression of PCa.

Total Saponins of Rubus Parvifolius L.Exhibited Anti-Leukemia Effect in vivo through STAT3 and eIF4E Signaling Pathways

Objective： To investigate the anti-leukemia effect of total saponins of Rubus parvifo/ius L. （TSRP） on K562 cell xenografts in nude mice and the mechanisms of action. Methods： The K562 cell xenografts in nude mice were established, and then randomly divided into 5 groups, the control group, the cytosine arabinoside group（Am-c） and 3 TSRP groups （20, 40 and 100 mg/kg）. The tumor volume and mass of each group of nude mice were measured and the anti-tumor rates of TSRP were calculated subsequently. The apoptosis status of tumor cells was detected by hematoxylin-eosin （HE） and terminal dexynucleotidyl transferase （TdT）-mediated dUTP nick end labeling （TUNEL） staining analysis. Finally, the activities of apoptosis related signaling of signal transducer and activator of transcription 3 （STAT3）, eukaryotic initiation factor 4E （eIF4E） and B-cell lymphoma-2 （bcl-2） were determined with immunohistochemistry tests. Results： Subcutaneous injection of K562 cells induced tumor formation in nude mice, and the TSRP treated group showed a significant inhibitory effect on tumor formation. The nude mice treated with TSRP showed a significant decrease in tumor growth rate and tumor weight in comparison to the control group （all P〈0.05）. The HE staining and TUNEL assay showed that TSRP induced cell death by apoptosis. The immunohistochemical assay showed down-regulation of the bcl-2 gene in the TSRP treated cells. The phosphorylation levels of elF4E and STAT3 were decreased obviously after the treatment of TSRP. Conclusion： TSRP had an excellent tumor-suppressing effect on K562 cells in the nude mice xenograft model, suggesting that TSPR can be developed as a promising anti-chronic myeloide leukemia drug.

The kinase OsSK41/OsGSK5 negatively regulates amylose content in rice endosperm by affecting the interaction between OsEBP89 and OsBP5

Amylose content(AC) is the main factor determining the palatability, viscosity, transparency, and digestibility of rice(Oryza sativa)grains. AC in rice grains is mainly controlled by different alleles of the Waxy(Wx) gene. The AP2/EREBP transcription factor OsEBP89 interacts with the MYC-like protein OsBP5 to synergistically regulate the expression of Wx.Here, we determined that the GLYCOGEN SYNTHASE KINASE 5(OsGSK5, also named SHAGGY-like kinase 41 [OsSK41]) inhibits the transcriptional activation activity of OsEBP89 in rice grains during amylose biosynthesis. The loss of OsSK41 function enhanced Wx expression and increased AC in rice grains. By contrast, the loss of function of OsEBP89 reduced Wx expression and decreased AC in rice grains. OsSK41 interacts with OsEBP89 and phosphorylates four of its sites(Thr-28,Thr-30, Ser-238, and Thr-257), which makes OsEBP89 unstable and attenuates its interaction with OsBP5. Wx promoter activity was relatively weak when regulated by the phosphomimicvariantOsEBP89E–OsBP5but relatively strong when regulated by the nonphosphorylatable variant OsEBP89A–OsBP5.Therefore, OsSK41-mediated phosphorylation of OsEBP89 represents an additional layer of complexity in the regulation of amylose biosynthesis during rice grain development. In addition, our findings provide four possible sites for regulating rice grain AC via precise gene editing.

淋巴瘤细胞膜P糖蛋白、信号转导与转录激活因子3及bcl-2表达水平与化疗耐药的相关性

目的探讨淋巴瘤细胞膜P糖蛋白（P—gP）、细胞胞内信号转导与转录激活因子3（STAT3）、bcl-2、白细胞介素（IL）-6及IL-10表达水平与淋巴瘤患者化疗耐药的相关性。方法对疑诊淋巴瘤的18例患者，手术活检淋巴结，应用流式细胞术（FCM）测定细胞膜P—gP、胞内STAT3、抗凋亡蛋白bcl-2、细胞因子IL-6及IL-10表达水平，前瞻性研究与化疗疗效的关系。其中，化疗耐药组10例，化疗敏感组8例，以10例炎性淋巴结增生为正常对照。结果化疗耐药组P—gP和bcl-2表达水平均高于化疗敏感组（P=0．01和P=0．039），而STAT3、IL-6及IL-10表达水平差异无统计学意义（P〉0．05）。淋巴瘤细胞膜P—gP表达水平高于炎性淋巴结增生（P=0．01），STAT3表达水平明显低于炎性淋巴结增生（P=0．04），淋巴瘤与炎性淋巴结增生bcl-2、IL-6及IL-10表达水平差异无统计学意义（P〉0．05）。结论bcl-2、P—gP表达水平的高低与恶性淋巴瘤化疗疗效密切相关，STAT3参与了淋巴瘤细胞的信号转导，是否参与了多药耐药信号转导尚不能肯定，而胞内IL-6、IL-10的表达与化疗疗效未见相关。

LJbiquitin--Proteasome System in ABA Signaling： From Perception to Action

Protein post-translational modification （PTM） by ubiquitination has been observed during many aspects of plant growth, development, and stress responses. The ubiquitin-proteasome system precisely regulates phytohormone signaling by affecting protein activity, localization, assembly, and interaction ability. Absci- sic acid （ABA） is a major phytohormone, and plays important roles in plants under normal or stressed growth conditions. The ABA signaling pathway is composed of phosphatases, kinases, transcription fac- tors, and membrane ion channels. It has been reported that multiple ABA signaling transducers are sub- jected to the regulations by ubiquitination. In particular, recent studies have identified different types of E3 ligases that mediate ubiquitination of ABA receptors in different cell compartments. This review focuses on modulation of these components by monoubiquitination or polyubiquitination that occurs in the plasma membrane, endomembranes, and from the cytosol to the nucleus; this implies the existence of retrograde and trafficking processes that are regulated by ubiquitination in ABA signaling. A number of single-unit E3 ligases, components of multi-subunit E3 ligases, E2s, and specific subunits of the 26S proteasome involved in ABA signal regulation are discussed. Dissecting the precise functions of ubiquitination in the ABA pathway may help us understand key factors in the signaling of other phytohormones regulated by ubiqui- tination and other types of PTMs.

E2F4 regulates the cell cycle and DNA replication in the silkworm,Bombyx mori

The E2F family of transcription factors is crucial for cell cycle progression and cell fate decisions.Although E2Fs have been widely studied in mammals,there have been few studies performed in insects.Here,we determined the function of E2F4 in the silkworm,Bombyx mori.We demonstrate that E2F proteins are highly conserved among species from lower animals to higher mammals.Overexpression of the BmE2F4 gene led to cell cycle arrest in the G1 phase,whereas interfering with the BmE2F4 mRNA led to accumulation of cells in the S phase.These results indicate that BmE2F4 is important in cell cycle regulation.We also demonstrate that the BmE2F4 gene is involved in DNA replication of BmN-SWU1 cells and DNA synthesis in the silk gland.Furthermore,we identified a protein called Bm14-3-3ζthat can interact with BmE2F4 and allow it to localize in the nucleus.Overexpression of the Bm14-3-3ζgene led to cell cycle arrest in the G1 phase,while knocking down the gene increased the proportion of cells in S phase.These findings provide important insights into the function of E2F transcription factors and increase our understanding of their involvement in cell cycle regulation.