关键词： nerve injury   regeneration   sensory neuron   single-cell RNA-seq   gene expression   dorsal root ganglion   reprogramming   cell identity   axon growth   ATF3  

摘要： Primary somatosensory neurons are specialized to transmit specific types of sensory information through differences in cell size, myelination, and the expression of distinct receptors and ion channels, which together define their transcriptional and functional identity. By profiling sensory ganglia at single-cell resolution, we find that all somatosensory neuronal subtypes undergo a similar transcriptional response to peripheral nerve injury that both promotes axonal regeneration and suppresses cell identity. This transcriptional reprogramming, which is not observed in non-neuronal cells, resolves over a similar time course as target reinnervation and is associated with the restoration of original cell identity. Injury-induced transcriptional reprogramming requires ATF3, a transcription factor that is induced rapidly after injury and necessary for axonal regeneration and functional recovery. Our findings suggest that transcription factors induced early after peripheral nerve injury confer the cellular plasticity required for sensory neurons to transform into a regenerative state.

关键词： breast cancer   endocrine therapy   drug-resistance   ATF3   RPPA  

摘要： Simple Summary Breast cancer is one of the leading causes of death for women worldwide. Patients whose tumors express estrogen receptor alpha (ER alpha) are the vast majority and are mostly treated with targeted endocrine therapy. However, resistance development poses an urgent clinical problem and the mechanisms of this phenomenon are not fully understood. In this study we identified ATF3 as a novel regulator of the response to therapy via rewiring of central signaling processes towards the adaptation to endocrine treatment. Our work adds a new piece to the complex puzzle of the molecular mechanisms at the basis of endocrine therapy resistance and proposes ATF3 and the downstream pathways as putative targets for novel combinatorial treatment strategies. Breast cancer is one of the leading causes of death for women worldwide. Patients whose tumors express Estrogen Receptor alpha account for around 70% of cases and are mostly treated with targeted endocrine therapy. However, depending on the degree of severity of the disease at diagnosis, 10 to 40% of these tumors eventually relapse due to resistance development. Even though recent novel approaches as the combination with CDK4/6 inhibitors increased the overall survival of relapsing patients, this remains relatively short and there is a urgent need to find alternative targetable pathways. In this study we profiled the early phases of the resistance development process to uncover drivers of this phenomenon. Time-resolved analysis revealed that ATF3, a member of the ATF/CREB family of transcription factors, acts as a novel regulator of the response to therapy via rewiring of central signaling processes towards the adaptation to endocrine treatment. ATF3 was found to be essential in controlling crucial processes such as proliferation, cell cycle, and apoptosis during the early response to treatment through the regulation of MAPK/AKT signaling pathways. Its essential role was confirmed in vivo in a mouse model, and eleva

关键词： Peptides and proteins   Cytosine   Genetics   Fluorescence   Chromatography  

摘要： In mammalian cells, 5-methylcytosine (5mC) occurs in genomic double-stranded DNA (dsDNA) and is enzymatically oxidized to 5-hydroxymethylcytosine (ShmC), then to 5-formylcy-tosine (SfC), and finally to 5-carboxylcytosine (ScaC). These cytosine modifications are enriched in regulatory regions of the genome. The effect of these oxidative products on five bZIP dimers (CREB1, ATF2, Zta, ATF3 vertical bar cJun, and cFos vertical bar cJun) binding to five types of dsDNA was measured using protein binding microarrays. The five dsDNAs contain either cytosine in both DNA strands or cytosine in one strand and either 5mC, 5hmC, 5fC, or 5caC in the second strand. Some sequences containing the CEBP half-site GCAA are bound more strongly by all five bZIP domains when dsDNA contains 5fC, 5hmC, or 5fC. dsDNA containing 5caC in some TRE (AP-1)-like sequences, e.g., TGACTAA, is better bound by Zta, ATF3 vertical bar cJun, and cFos vertical bar cJun.

关键词： 中国仓鼠卵巢细胞(CHO)   转录激活因子3(ATF3)   CRISPR/Cas9   DNMT3A  

摘要： 转录激活因子3(ATF3)是一种早期应激反应基因,能够影响细胞增殖、分化、转化和凋亡。中国仓鼠卵巢细胞(CHO)是重组蛋白、单克隆抗体等生物大分子药物生产的主要宿主细胞之一。ATF3能够促进肿瘤细胞的增殖及抗凋亡等作用,理论上可对CHO细胞发挥调节作用。因此本试验对CHO细胞中ATF3的甲基化水平进行深入研究,通过重亚硫酸盐测序法检测CHO-K1细胞中ATF3的甲基化水平,发现ATF3启动子区域的胞嘧啶-磷酸-鸟嘌呤(CpG)甲基化高达90%。为了进一步研究ATF3甲基化的上游调控机制,构建了敲除DNA甲基转移酶3A(DNMT3A)的CHO-K1来研究DNMT3A对ATF3甲基化水平的影响。结果表明,敲除DNMT3A后,CHO-K1细胞中的ATF3甲基化水平降低至0,这充分证明了DNMT3A对ATF3的甲基化的调控作用,为CHO细胞的ATF3基因调控研究以及后续CHO细胞株改造提供了理论依据。

关键词： Nanopillar   Deformation   Cytoskeleton   Physiology   ATF3  

摘要： Architecture of cell correlates with the onset of disease. External factors as mechanical force and geometric constraint trigger physical deformation. In this study, we took advantage of nanopillar-based vertical array as a mechanobiological platform to quantitatively deconstruct the relationship between morphological deformation and cell behavior. Technically, this nanoscale platform enables us to make a comprehensive and detailed illustration on alternation of physiological behavior to mechanical stimuli. Morphologically, we found whole cell architecture and nuclear deformation were highly associated with cytoskeleton rearrangement. Mechanistically, we found that cell deformation induced translocation of activating transcription factor 3 (ATF3) into nucleus. Unraveling the signal responding networks associated with morphological deformation will not only increase our understanding of mechanotransduction, but may also encourage the development of a potential therapeutically target to treat diseases. (c) 2020 Elsevier Ltd. All rights reserved.

关键词： AT/RT   ALDH   Disulfiram   Cisplatin   Combination therapy   ATF3  

摘要： Atypical teratoid/rhabdoid tumor (AT/RT) is the most malignant tumor of the central nervous system that generally occurs in young children. Despite the use of intensive multimodal therapy for AT/RT, the prognosis is still poor. The brain tumor initiating cells in AT/RT cells has been suggested as one of the challenges in AT/RT treatment. These cells have high expression of aldehyde dehydrogenase (ALDH). We investigated the combination effect of the ALDH inhibitor, disulfiram and cisplatin in the treatment of AT/RT cells. Isobologram analysis revealed that the combination therapy synergistically increases AT/RT cell death. The enzyme activity of ALDH AT/RT cells was effectively reduced by the combination therapy. We proposed that the synergistic augmentation occurs, at least partially through an increase in cleaved Poly (ADP-ribose) polymerase (PARP)-dependent apoptosis mediated by activating transcription factor 3 (ATF3). In the AT/RT mouse model, the combination therapy decreased tumor volume and prolonged survival. Immunofluorescence assay in mouse brain tissues were consistent with the expression of ATF3 and cleaved PARP. Our study demonstrates enhanced anti-cancer effect of combination therapy of disulfiram and cisplatin. This combination might provide a viable therapeutic strategy for AT/RT patients.

关键词： Epithelial–mesenchymal transition   Mesenchymal migration  

摘要： Endothelial to mesenchymal transition (EndMT) is an important pathological change in many diseases. Semaphorin7A (Sema7A) has been reported to regulate nerve and vessel homeostasis, but its role in EndMT remains unclear. Here we investigate the effect of Sema7A on EndMT and the underlying mechanism. Sema7A-overexpressed human umbilical vein endothelial cells (Sema7A-HUVECs) were generated and showed lower levels of endothelial cell markers and higher levels of mesenchymal cell markers indicating the occurrence of EndMT. RNA-sequencing analysis showed a total of 1168 upregulated genes and 886 downregulated genes. Among them, most of the molecules associated with EndMT were upregulated in Sema7A-HUVECs. Mechanistically, Sema7A-HUVECs showed a higher TGF-beta 2 expression and activated TGF-beta/Smad Signaling. Importantly, Sema7A overexpression upregulated activating transcription factor 3 (ATF3) that was found to selectively bind the promotor region of TGF-beta 2, but not TGF-beta 1, promoting TGF-beta 2 transcription, which was further confirmed by ATF3-siRNA knockdown approach. Blocking beta 1 integrin, a known Sema7A receptor, alleviated the expression of ATF3, TGF-beta 2, and EndMT in Sema7A-overexpressed HUVECs, implying a role of beta 1 integrin/ATF3/TGF-beta 2 axis in mediating Sema7A-induced EndMT. Using Sema7A-deficient mice and the partial carotid artery ligation (PCL) model, we showed that Sema7A deletion attenuated EndMT induced by blood flow disturbance in vivo. In conclusion, Sema7A promotes TGF-beta 2 secretion by upregulating transcription factor ATF3 in a beta 1 integrin-dependent manner, and thus facilitates EndMT through TGF/Smad signaling, implying Sema7A as a potential therapeutic target for EndMT-related vascular diseases.

关键词： Degradation   Peptides and proteins   Genetics   Rodent models   Biopolymers  

摘要： The Arg/N-degron pathway targets proteins for degradation by recognizing their specific N-terminal residues or, alternatively, their non-N-terminal degrons. In mammals, this pathway is mediated by the UBR1, UBR2, UBR4, and UBR5 E3 ubiquitin ligases, and by the p62 regulator of autophagy. UBR1 and UBR2 are sequelogous, functionally overlapping, and dominate the targeting of Arg/N-degron substrates in examined cell lines. We constructed, here, mouse strains in which the double mutant [UBR1(-/-)UBR2(-/-)] genotype can be induced conditionally, in adult mice. We also constructed human [UBR1(-/-)UBR2(-/-)] HEK293T cell lines that unconditionally lack UBR1/UBR2. ATF3 is a basic leucine zipper transcription factor that regulates hundreds of genes and can act as either a repressor or an activator of transcription. Using the above double-mutant mice and human cells, we found that the levels of endogenous, untagged ATF3 were significantly higher in both of these [UBR1(-/-)UBR2(-/-)] settings than in wild-type cells. We also show, through chase-degradation assays with [UBR1(-/-)UBR2(-/-)] and wild-type human cells, that the Arg/N-degron pathway mediates a large fraction of ATF3 degradation. Furthermore, we used split-ubiquitin and another protein interaction assay to detect the binding of ATF3 to both UBR1 and UBR2, in agreement with the UBR1/UBR2-mediated degradation of endogenous ATF3. Full-length 24 kDa ATF3 binds to similar to 100 kDa fragments of 200 kDa UBR1 and UBR2 but does not bind (in the setting of interaction assays) to full-length UBR1/UBR2. These and other binding patterns, whose mechanics remain to be understood, may signify a conditional (regulated) degradation of ATF3 by the Arg/N-degron pathway.

关键词： activating transcription factor 3   Crohn's disease   inflammatory bowel disease   ulcerative colitis  

摘要： Introduction: Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the intestinal tract. Known types are Crohn's disease (CD) and ulcerative colitis (UC), but their cause remains unclear and there is no convenient biomarker for IBD. The present study aimed to demonstrate an association between the onset of CD and activating transcription factor 3 (ATF3); as a new biomarker, measurement of blood ATF3 mRNA would be useful for distinguishing between CD and UC. Methods: First, in a mouse model of IBD in which damage to the intestinal mucosa was chemically induced with dextran sulfate sodium (DSS), intestinal ATF3 mRNA was evaluated. Next, in human subjects, CD and UC patients, blood ATF3 mRNA and intestinal ATF3 protein production were evaluated. Results: In the mouse model of IBD, intestinal ATF3 mRNA was elevated compared with the control (P < 0.0001). In CD patients, blood ATF3 mRNA was elevated as compared with normal controls (NCs) and UC patients (P < 0.05). In addition, we observed an increase in ATF3 production in the intestinal tract specific to CD. Conclusion: ATF3 is involved in the onset of CD, and blood ATF3 mRNA measurements would be useful for distinguishing it from UC. [ABSTRACT FROM AUTHOR]

关键词： 转录激活因子3   乳腺丝氨酸蛋白酶抑制因子   基质金属蛋白酶2   脑膜瘤  

摘要： 目的探讨转录激活因子3(ATF3)、乳腺丝氨酸蛋白酶抑制因子(maspin)和基质金属蛋白酶2(MMP2)在人脑各病理分级脑膜瘤中的表达和临床意义。方法采用免疫组化染色法、qRT-PCR和Western blot法检测71例脑膜瘤和32例正常脑组织中ATF3、maspin和MMP2的蛋白和mRNA表达情况,分析其在不同病理级别脑膜瘤患者中的可能作用和临床意义。结果 ATF3和MMP2在脑膜瘤中的表达明显高于正常脑组织,maspin在脑膜瘤中的表达明显低于正常脑组织。随着脑膜瘤病理级别的增高,ATF3和MMP2的表达量逐级增高,而maspin表达量逐级递减。ATF3和MMP2的蛋白和mRNA表达与脑膜瘤病理分级呈正相关,maspin蛋白和mRNA表达与脑膜瘤病理分级呈负相关。结论 ATF3、maspin和MMP2可能协同参与了脑膜瘤发生,ATF3、maspin和MMP2可能与脑膜瘤恶性进展紧密相关。