Nucleocytoplasmic Transport Proteins

核质细胞转运蛋白
  • 文章类型: Journal Article
    当mRNA在细胞核中被转录和加工时,它们被输出到细胞质中进行翻译。这种输出是由酿酒酵母中的输出受体异二聚体Mex67-Mtr2(人类中的TAP-p15)1,2介导的。有趣的是,许多长非编码RNA(lncRNAs)也离开细胞核,但目前尚不清楚为什么它们会移动到细胞质。在这里,我们显示反义RNA(asRNA)通过解旋酶Dbp2与有义对应物退火来加速mRNA的输出。与单链RNA(ssRNA)相比,这些双链RNA(dsRNA)主导出口,因为它们对出口受体Mex67具有更高的容量和亲和力。这样,asRNAs促进基因表达,这对细胞是有益的。当表达式程序发生变化时,这一点尤其重要。因此,dsRNA的降解,或防止其形成,对细胞有毒。这种机制阐明了asRNA的一般细胞发生并解释了它们的核输出。
    When mRNAs have been transcribed and processed in the nucleus, they are exported to the cytoplasm for translation. This export is mediated by the export receptor heterodimer Mex67-Mtr2 in the yeast Saccharomyces cerevisiae (TAP-p15 in humans)1,2. Interestingly, many long non-coding RNAs (lncRNAs) also leave the nucleus but it is currently unclear why they move to the cytoplasm3. Here we show that antisense RNAs (asRNAs) accelerate mRNA export by annealing with their sense counterparts through the helicase Dbp2. These double-stranded RNAs (dsRNAs) dominate export compared with single-stranded RNAs (ssRNAs) because they have a higher capacity and affinity for the export receptor Mex67. In this way, asRNAs boost gene expression, which is beneficial for cells. This is particularly important when the expression program changes. Consequently, the degradation of dsRNA, or the prevention of its formation, is toxic for cells. This mechanism illuminates the general cellular occurrence of asRNAs and explains their nuclear export.
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  • 文章类型: Journal Article
    SARS-CoV-2(严重急性呼吸综合征冠状病毒2)的非结构蛋白1(Nsp1)是一种毒力因子,靶向多种细胞途径以抑制宿主基因表达和抗病毒反应。然而,各种Nsp1介导的功能的潜在机制及其对SARS-CoV-2毒力的贡献仍不清楚.Nsp1的靶标是mRNA(信使核糖核酸)输出受体NXF1-NXT1,其介导mRNA从细胞核向细胞质的核输出。基于Nsp1的晶体结构,我们在Nsp1表面上产生了突变体,并鉴定了一个酸性N末端贴片,该贴片对于与NXF1-NXT1的相互作用至关重要。可光活化的Nsp1探针揭示NXF1的RNA识别基序(RRM)结构域作为Nsp1的N-末端结合位点。通过突变Nsp1N末端酸性贴片,我们确定了Nsp1的功能分离突变体,该突变体保留了其翻译抑制功能,但实质上失去了与NXF1的相互作用,并恢复了Nsp1介导的mRNA输出抑制.然后,我们在Nsp1N端酸性贴片上产生了重组(r)SARS-CoV-2突变体,发现该表面是促进NXF1结合和抑制宿主mRNA核输出的关键,病毒复制,和体内致病性。因此,这些发现提供了对Nsp1介导的mRNA输出抑制的机制理解,并确定了该途径在SARS-CoV-2毒力中的重要性。
    The nonstructural protein 1 (Nsp1) of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a virulence factor that targets multiple cellular pathways to inhibit host gene expression and antiviral response. However, the underlying mechanisms of the various Nsp1-mediated functions and their contributions to SARS-CoV-2 virulence remain unclear. Among the targets of Nsp1 is the mRNA (messenger ribonucleic acid) export receptor NXF1-NXT1, which mediates nuclear export of mRNAs from the nucleus to the cytoplasm. Based on Nsp1 crystal structure, we generated mutants on Nsp1 surfaces and identified an acidic N-terminal patch that is critical for interaction with NXF1-NXT1. Photoactivatable Nsp1 probe reveals the RNA Recognition Motif (RRM) domain of NXF1 as an Nsp1 N-terminal binding site. By mutating the Nsp1 N-terminal acidic patch, we identified a separation-of-function mutant of Nsp1 that retains its translation inhibitory function but substantially loses its interaction with NXF1 and reverts Nsp1-mediated mRNA export inhibition. We then generated a recombinant (r)SARS-CoV-2 mutant on the Nsp1 N-terminal acidic patch and found that this surface is key to promote NXF1 binding and inhibition of host mRNA nuclear export, viral replication, and pathogenicity in vivo. Thus, these findings provide a mechanistic understanding of Nsp1-mediated mRNA export inhibition and establish the importance of this pathway in the virulence of SARS-CoV-2.
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  • 文章类型: Journal Article
    背景:我们先前的研究表明,姜黄素(CUR)通过降低细胞内总RNAm6A水平来减轻心肌缺血再灌注损伤(MIRI)。然而,机制仍然未知。
    方法:对于缺血再灌注(IR),H9c2细胞在无血清的低血糖(1g/L)培养基和无氧的气体环境中培养6h,然后在补充有10%FBS和21%氧气环境的高血糖(4.5g/L)培养基中培养6h。检查了不同浓度的CUR(5、10和20µM)处理对常规培养和IR处理的H9c2细胞中信号分子的影响。
    结果:CUR治疗显著上调H2S水平,以及胱硫醚γ-裂解酶(CSE)的mRNA和蛋白表达,并下调常规培养并进行IR的H9c2细胞中硫代硫酸盐硫转移酶(TST)和乙基丙二酸脑病1(ETHE1)的mRNA和蛋白质水平。外源性H2S供应(NaHS和GYY4137)显着降低细胞内总RNAm6A水平,和RNAm6A“writers”METTL3和METTL14的表达,并增加了常规培养和经受IR的H9c2细胞中RNAm6A“橡皮擦”FTO的表达。CSE敲低抵消了CUR处理对ROS产生的抑制作用,促进细胞活力,并抑制IR对H9c2细胞的凋亡。
    结论:CUR通过调节H2S水平调节酶的表达和增加内源性H2S水平来减弱MIRI。增加的H2S水平可以调节m6A相关蛋白的表达和细胞内总RNAm6A的水平。
    BACKGROUND: Our previous research shows that Curcumin (CUR) attenuates myocardial ischemia-reperfusion injury (MIRI) by reducing intracellular total RNA m6A levels. However, the mechanism remains unknown.
    METHODS: For ischemia-reperfusion (IR), H9c2 cells were cultured for 6 h in serum-free low-glycemic (1 g/L) medium and a gas environment without oxygen, and then cultured for 6 h in high-glycemic (4.5 g/L) medium supplemented with 10% FBS and a 21% oxygen environment. The effects of different concentrations of CUR (5, 10, and 20 µM) treatments on signaling molecules in conventionally cultured and IR-treated H9c2 cells were examined.
    RESULTS: CUR treatment significantly up-regulated the H2S levels, and the mRNA and protein expression of cystathionine γ-lyase (CSE), and down-regulated the mRNAs and proteins levels of thiosulfate sulfurtransferase (TST) and ethylmalonic encephalopathy 1 (ETHE1) in H9c2 cells conventionally cultured and subjected to IR. Exogenous H2S supply (NaHS and GYY4137) significantly reduced intracellular total RNA m6A levels, and the expression of RNA m6A \"writers\" METTL3 and METTL14, and increased the expression of RNA m6A \"eraser\" FTO in H9c2 cells conventionally cultured and subjected to IR. CSE knockdown counteracted the inhibitory effect of CUR treatment on ROS production, promotion on cell viability, and inhibition on apoptosis of H9c2 cells subjected to IR.
    CONCLUSIONS: CUR attenuates MIRI by regulating the expression of H2S level-regulating enzymes and increasing the endogenous H2S levels. Increased H2S levels could regulate the m6A-related proteins expression and intracellular total RNA m6A levels.
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  • 文章类型: Journal Article
    精确调节核输出对于维持mRNA稳态和影响肿瘤进展至关重要。然而,控制核mRNA输出的机制仍未阐明。在这里,揭示了乳腺癌(BC)中增强的缺氧长非编码RNA(lncRNA前列腺癌相关转录本6(PCAT6)促进m6A修饰的mRNA的核输出,支持乳腺癌干细胞(BCSCs)干性和阿霉素抗性。临床上,低氧PCAT6与恶性BC特征和不良预后相关。机械上,PCAT6充当干扰素刺激基因15(ISG15)和异质核核糖核蛋白A2/B1(hnRNPA2B1)之间的支架,导致hnRNPA2B1的ISG化,从而保护hnRNPA2B1免受泛素化介导的蛋白酶体降解。有趣的是,作为M6A阅读器,hnRNPA2B1通过Aly/REF出口因子(ALYREF)/核RNA出口因子1(NXF1)复合物选择性介导m6A标记的mRNA核出口,这促进了干性相关基因的表达。HnRNPA2B1敲低或mRNA输出抑制可导致保留与干性维持相关的核m6A标记的mRNA,抑制BCSCs自我更新,有效提高阿霉素治疗的疗效。这些发现证明了m6A修饰的mRNA核输出在BC进展中的关键作用,强调抑制m6A标记的mRNA及其核输出是改善癌症化疗的潜在治疗策略。
    Regulating nuclear export precisely is essential for maintaining mRNA homeostasis and impacts tumor progression. However, the mechanisms governing nuclear mRNA export remain poorly elucidated. Herein, it is revealed that the enhanced hypoxic long no-ncoding RNA (lncRNA prostate cancer associated transcript 6 (PCAT6) in breast cancer (BC) promotes the nuclear export of m6A-modified mRNAs, bolstering breast cancer stem cells (BCSCs) stemness and doxorubicin resistance. Clinically, hypoxic PCAT6 correlates with malignant BC features and poor prognosis. Mechanically, PCAT6 functions as a scaffold between interferon-stimulated gene 15 (ISG15) and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1), leading to ISGylation of hnRNPA2B1, thus protecting hnRNPA2B1 from ubiquitination-mediated proteasomal degradation. Interestingly, as an m6A reader, hnRNPA2B1 selectively mediates m6A-tagged mRNAs nuclear export via the Aly/REF export factor (ALYREF)/ nuclear RNA export factor 1 (NXF1) complex, which promotes stemness-related genes expression. HnRNPA2B1 knockdown or mRNA export inhibition can result in the retention of nuclear m6A-tagged mRNA associated with stemness maintenance, which suppresses BCSCs self-renewal and effectively improves the efficacy of doxorubicin therapy. These findings demonstrate the pivotal role of m6A-modified mRNA nuclear export in BC progression, highlighting that the inhibition of m6A-tagged mRNA and its nuclear export is a potential therapeutic strategy for the amelioration of cancer chemotherapy.
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  • 文章类型: Journal Article
    已发现各种遗传变异与过早卵巢功能不全(POI)的临床发作有关。然而,当在体外测量时,变体的功能影响可能难以确定。通过对93例散发性POI患者的全外显子组测序(WES),我们发现了一个错义变体c.623G>A;p。R208H中的EIF4ENIF1基因。使用不同算法对变体进行计算机模拟预测表明,它可能是破坏性变体。我们比较了EIF4ENIF1R208H和Q842P的性质,我们之前报道的POI相关突变体,在体外使用293FT细胞与野生型(WT)蛋白。令人惊讶的是,未观察到亚细胞分布和颗粒形成能力(Q842P)和核输入能力(R208H)的变化,尽管有领域预测证据。由于据报道EIF4ENIF1抑制翻译,我们雇佣了T&Tseq,翻译-转录双组测序方法,在EIF4ENIF1WT和突变体过表达时描述基因表达。EIF4ENIF1WT过表达组表现出比空载体或GFP过表达对照组显著(P<0.0001)更低的翻译效率(TE)。令人惊讶的是,EIF4ENIF1Q842P过表达未能抑制全球翻译,显示总TE显著高于WT组。过表达R208H显著(P<0.0001)降低总TE,而对高TE基因表现出降低的翻译抑制作用(GFP对照组中TE>2)。几个与生育相关的基因,如Q842P组的AMH和R208H组的SERPINE1和THBS1,与WT对照相比,在突变体组中翻译上调,提示EIF4ENIF1突变通过翻译抑制受损引起POI的潜在机制。进一步提出,T&T-seq可以是一种灵敏的评估工具,用于测量许多其他翻译调节基因中变体的功能改变。不仅EIF4ENIF1,有助于消除对遗传变异的临床意义的误解。
    Various genetic variants have been found to be associated with the clinical onset of premature ovarian insufficiency (POI). However, when measured in vitro, the functional influence of the variants can be difficult to determine. By whole-exome sequencing (WES) of 93 patients with sporadic POI, we found a missense variant c.623G > A;p.R208H in the EIF4ENIF1 gene. In silico prediction of the variant using different algorithms suggested it might be a damaging variant. We compared the property of EIF4ENIF1 R208H and Q842P, a POI-related mutant that we reported previously, with wildtype (WT) protein using 293FT cells in vitro. Surprisingly, a change in subcellular distribution and granule forming ability (Q842P) and nuclear import capacity (R208H) was not observed, despite domain prediction evidences. Since EIF4ENIF1 was reported to inhibit translation, we employed T&T-seq, a translation-transcription dual-omics sequencing method, to profile gene expression upon overexpression of EIF4ENIF1 WT and mutants. EIF4ENIF1 WT overexpression group exhibited significantly (P < 0.0001) lower translation efficiency (TE) than empty vector or GFP overexpression control group. Surprisingly, EIF4ENIF1 Q842P overexpression failed to repress global translation, showing an overall TE significantly higher than WT group. Overexpression R208H significantly (P < 0.0001) lowered the overall TE, whereas exhibiting a reduced translation inhibitory effect on high-TE genes (TE > 2 in GFP control group). Several fertility-associated genes, such as AMH in Q842P group and SERPINE1 and THBS1 in R208H group, was translationally up-regulated in mutant groups versus WT control, suggesting a potential mechanism of mutated EIF4ENIF1 causing POI via impaired translation repression. It is further proposed that T&T-seq can be a sensitive evaluation tool for the measurement of functional alteration by variants in many other translational regulator genes, not only EIF4ENIF1, helping to eliminate misinterpretation of clinical significance of genetic variants.
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  • 文章类型: Journal Article
    mRNAs在他们的一生中不断改变他们的蛋白质伴侣,然而,我们对mRNA-蛋白复合物(mRNP)重塑的理解受到缺乏时间数据的限制.这里,我们通过在人类细胞中使用可光活化的核糖核苷进行脉冲代谢标记来提供时间分辨的mRNA相互作用组数据,UVA交联,poly(A)+RNA分离,和质谱。这种纵向方法允许在10个时间点定量超过700个RNA结合蛋白(RBP)。总的来说,mRNA结合的顺序与已知功能一致,亚细胞位置,和分子相互作用。然而,我们还观察到RBPs具有意想不到的动力学:转录-输出(TREX)复合物在核输出因子1(NXF1)结合后转录后募集,挑战转录偶联mRNA输出的当前观点,和衰老mRNPs中普遍存在的应激颗粒蛋白,表明在mRNA生命周期后期的作用。为了系统地识别具有未知功能的mRBP,我们使用机器学习来比较mRNA结合动力学与基因本体论(GO)注释。我们的数据可以按时间顺序进行探索。rna.snu.AC.kr.
    mRNAs continually change their protein partners throughout their lifetimes, yet our understanding of mRNA-protein complex (mRNP) remodeling is limited by a lack of temporal data. Here, we present time-resolved mRNA interactome data by performing pulse metabolic labeling with photoactivatable ribonucleoside in human cells, UVA crosslinking, poly(A)+ RNA isolation, and mass spectrometry. This longitudinal approach allowed the quantification of over 700 RNA binding proteins (RBPs) across ten time points. Overall, the sequential order of mRNA binding aligns well with known functions, subcellular locations, and molecular interactions. However, we also observed RBPs with unexpected dynamics: the transcription-export (TREX) complex recruited posttranscriptionally after nuclear export factor 1 (NXF1) binding, challenging the current view of transcription-coupled mRNA export, and stress granule proteins prevalent in aged mRNPs, indicating roles in late stages of the mRNA life cycle. To systematically identify mRBPs with unknown functions, we employed machine learning to compare mRNA binding dynamics with Gene Ontology (GO) annotations. Our data can be explored at chronology.rna.snu.ac.kr.
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  • 文章类型: Journal Article
    严重急性呼吸系统综合症冠状病毒2(SARS-CoV-2)辅助蛋白Orf6作为干扰素拮抗剂,在某种程度上,通过抑制核输入激活的p-STAT1,干扰素刺激基因的激活剂,和poly(A)RNA的输出。对Orf6的运输调节功能的认识来自以下观察:Orf6与核孔复合物(NPC)成分Rae1和Nup98结合。为了进一步了解Orf6介导的转运抑制的机制,我们研究了Rae1和Nup98的作用。我们表明,单独的Rae1对于支持poly(A)RNA的p-STAT1导入或核输出是不必要的。此外,Rae1的缺失抑制了Orf6的转运抑制活性。我们建议Rae1/Nup98复合体在NPC中战略性地定位Orf6,以改变FG-Nup的相互作用及其支持核运输的能力。此外,我们表明,在SARS-CoV-2感染期间,Rae1是正常病毒蛋白产生所必需的,大概是通过其在支持Orf6功能中的作用。
    The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) accessory protein Orf6 works as an interferon antagonist, in part, by inhibiting the nuclear import activated p-STAT1, an activator of interferon-stimulated genes, and the export of the poly(A) RNA. Insight into the transport regulatory function of Orf6 has come from the observation that Orf6 binds to the nuclear pore complex (NPC) components: Rae1 and Nup98. To gain further insight into the mechanism of Orf6-mediated transport inhibition, we examined the role of Rae1 and Nup98. We show that Rae1 alone is not necessary to support p-STAT1 import or nuclear export of poly(A) RNA. Moreover, the loss of Rae1 suppresses the transport inhibitory activity of Orf6. We propose that the Rae1/Nup98 complex strategically positions Orf6 within the NPC where it alters FG-Nup interactions and their ability to support nuclear transport. In addition, we show that Rae1 is required for normal viral protein production during SARS-CoV-2 infection presumably through its role in supporting Orf6 function.
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  • 文章类型: Journal Article
    目的:本研究的目的是探讨RAE1在胃癌(GC)细胞侵袭和转移中的作用。
    方法:通过逆转录聚合酶链反应(qRT-PCR)和蛋白质印迹(WB)检测GC细胞中RAE1的表达。通过慢病毒转染构建RAE1基因沉默和过表达的细胞模型;迁移,细胞计数检测细胞的侵袭能力,集落形成试验,会愈合试验,以及Transwell入侵和迁移测试。WB分析ERK/MAPK信号通路(ERK1/2,p-ERK1/2,c-Myc)和EMT相关分子(ZEB1,E-cadherin,N-钙黏着蛋白,和Vimentin)。
    结果:RAE1在GC中的表达水平明显高于癌旁组织。RAE1表达升高与GC患者的不良预后相关。与对照组相比,RAE1的击倒,导致增殖的显著抑制,迁移,和GC细胞系的侵袭能力。此外,RAE1敲低导致N-cadherin的表达大幅下降,波形蛋白,ZEB1、p-ERK1/2和c-Myc蛋白,再加上E-cadherin表达的显著增加。通过使用SCH772984抑制ERK/MAPK信号通路,可以有效逆转RAE1在GC细胞中的生物学效应。此外,RAE1敲低显示了对体内GC肿瘤大小的抑制作用。免疫组织化学(IHC)结果显示,与对照组相比,RAE1敲除小鼠中Ki-67的表达显着降低。
    结论:RAE1通过ERK/MAPK通路促进GC细胞迁移和侵袭,是GC治疗的潜在治疗靶点。
    OBJECTIVE: The purpose of this study was to explore the role of RAE1 in the invasion and metastasis of gastric cancer (GC) cells.
    METHODS: RAE1 expression in GC cells was determined by reverse-transcription polymerase chain reaction (qRT-PCR) and Western blotting (WB). Cell models featuring RAE1 gene silencing and overexpression were constructed by lentiviral transfection; The proliferation, migration, and invasion ability of cells were detected by cell counting, colony formation assay, would healing assay, and transwell invasion and migration test. WB analysis of ERK/MAPK signaling pathway (ERK1/2, p-ERK1/2, c-Myc) and EMT-related molecules (ZEB1, E-cadherin, N-cadherin, and Vimentin).
    RESULTS: The expression level of RAE1 in GC was notably higher than in adjacent tissues. Elevated RAE1 expression correlated with an unfavorable prognosis for GC patients. Knockdown of RAE1, as compared to the control group, resulted in a significant inhibition of proliferation, migration, and invasion abilities in GC cell lines. Furthermore, RAE1 knockdown led to a substantial decrease in the expression of N-cadherin, vimentin, ZEB1, p-ERK1/2, and c-Myc proteins, coupled with a marked increase in E-cadherin expression. The biological effects of RAE1 in GC cells were effectively reversed by the inhibition of the ERK/MAPK signaling pathway using SCH772984. Additionally, RAE1 knockdown demonstrated a suppressive effect on GC tumor size in vivo. Immunohistochemistry (IHC) results revealed significantly lower expression of Ki-67 in RAE1 knockout mice compared to the control group.
    CONCLUSIONS: RAE1 promotes GC cell migration and invasion through the ERK/MAPK pathway and is a potential therapeutic target for GC therapy.
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  • 文章类型: Journal Article
    通过核孔复合物输出核mRNA是真核基因表达的重要步骤。尽管参与mRNA转运的因素已经被表征,缺乏对这一过程及其调控的全面机械理解。这里,我们在酵母中使用单RNA成像显示细胞在应激期间利用mRNA滞留来控制mRNA的输出.我们证明,葡萄糖戒断后,必需的RNA结合因子Nab2在细胞核中形成依赖RNA的缩合样结构。这与核孔处DEAD-boxATPaseDbp5的丰度降低相吻合。消耗Dbp5,从而阻断mRNA输出,是必要的,足以触发Nab2冷凝。Nab2缩合状态影响核mRNA积累的程度,可以在体外概括,其中Nab2形成依赖RNA的液滴。我们假设细胞在应激期间使用凝聚来调节mRNA输出和控制基因表达。
    Nuclear mRNA export via nuclear pore complexes is an essential step in eukaryotic gene expression. Although factors involved in mRNA transport have been characterized, a comprehensive mechanistic understanding of this process and its regulation is lacking. Here, we use single-RNA imaging in yeast to show that cells use mRNA retention to control mRNA export during stress. We demonstrate that, upon glucose withdrawal, the essential RNA-binding factor Nab2 forms RNA-dependent condensate-like structures in the nucleus. This coincides with a reduced abundance of the DEAD-box ATPase Dbp5 at the nuclear pore. Depleting Dbp5, and consequently blocking mRNA export, is necessary and sufficient to trigger Nab2 condensation. The state of Nab2 condensation influences the extent of nuclear mRNA accumulation and can be recapitulated in vitro, where Nab2 forms RNA-dependent liquid droplets. We hypothesize that cells use condensation to regulate mRNA export and control gene expression during stress.
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  • 文章类型: Journal Article
    接触抑制增殖(CIP)表示细胞密度依赖性的生长抑制,CIP的丢失代表了癌症的标志。然而,CIP调节基因表达的机制仍知之甚少。染色质是由DNA组成的高度复杂的结构,组蛋白,和反式作用因子(TAF)。TAF蛋白与特定染色体基因座的结合调节基因表达。因此,染色质谱分析对于深入了解CIP的基因表达机制至关重要。在这项研究中,使用与DNA结合的TAFs的改良蛋白质组学,我们发现了一种蛋白质,以细胞密度依赖的方式在细胞核和细胞质之间穿梭。我们确认了TIPARP,PTGES3,CBFB,和SMAD4作为细胞密度依赖性核质穿梭蛋白。在低密度细胞中,这些蛋白质主要存在于细胞核中;然而,一旦达到高密度,他们重新定位到细胞质。鉴于它们在基因调控中的作用,我们的研究结果表明他们参与CIP依赖性TAF。我们还鉴定并表征了对细胞密度变化敏感的潜在开放染色质区域。这些发现为CIP调节染色质结构提供了见解。
    The contact inhibition of proliferation (CIP) denotes the cell density-dependent inhibition of growth, and the loss of CIP represents a hallmark of cancer. However, the mechanism by which CIP regulates gene expression remains poorly understood. Chromatin is a highly complex structure consisting of DNA, histones, and trans-acting factors (TAFs). The binding of TAF proteins to specific chromosomal loci regulates gene expression. Therefore, profiling chromatin is crucial for gaining insight into the gene expression mechanism of CIP. In this study, using modified proteomics of TAFs bound to DNA, we identified a protein that shuttles between the nucleus and cytosol in a cell density-dependent manner. We identified TIPARP, PTGES3, CBFB, and SMAD4 as cell density-dependent nucleocytoplasmic shuttling proteins. In low-density cells, these proteins predominantly reside in the nucleus; however, upon reaching high density, they relocate to the cytosol. Given their established roles in gene regulation, our findings propose their involvement as CIP-dependent TAFs. We also identified and characterized potential open chromatin regions sensitive to changes in cell density. These findings provide insights into the modulation of chromatin structure by CIP.
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