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The Stat1 α C-terminus can act as an independent TAD. ( A ) Schematic view of two forms of Stat1 generated by alternative splicing: Stat1 α and β . Domain structures are according to Chen et al . (1998). Y, Tyr701; S, Ser727; TAD, transcription activation domain. ( B ) Sequences of Stat1 α C 711–750 with mutations of Ser727 and Leu724. Underlined sequence is the conserved XPMSP box (Wen et al ., 1995). ( C ) Effects of mutations of Ser727 and Leu724 on transcription activation by Stat1 α . WT, wild-type; SA, Ser727 to alanine; SD, Ser727 to aspartic acid; LA, Leu724 to alanine. ( D ) The Stat1 α C-terminus is an independent TAD. Transient transfections were performed in U3A cells with the 3xLy6E–GAS luciferase reporter (Wen et al ., 1995) for (C) and 5xGAL4DB luciferase reporter for (D). Twenty-four h after transfection, cells were left untreated or treated with IFN- γ for 6 h and harvested for luciferase assays. Results shown are the mean Ϯ standard deviation of 3–6 experiments.
Source publication
Stat1alpha is a latent cytoplasmic transcription factor activated in response to interferon-gamma (IFN-gamma). The C-terminal 38 amino acids of Stat1alpha are required to trigger transcription and therefore may possibly serve as a transcription activation domain (TAD). Here we show that the C-terminus of Stat1alpha is an independent TAD which can i...
Contexts in source publication
Context 1
... Stat1 proteins containing point mutations of these residues ( Figure 1A and B) were analyzed by transient transfection assays in U3A cells which lack endogenous Stat1 (Muller et al., 1993). Equal amounts of these Stat1 mutant proteins were expressed (data not shown). ...
Context 2
... amounts of these Stat1 mutant proteins were expressed (data not shown). Expression of Stat1β did not produce significant levels of reporter activity ( Figure 1C). Stat1α induced an ~15-fold increase in reporter activity when the cells were treated with IFN-γ. ...
Context 3
... constructs encoding the DNA-binding domain of GAL4 fused to the wild-type residues 711-750 of Stat1α or to mutant versions of these residues ( Figure 1B) were prepared to test the capacity of these residues to act 6964 independently as a TAD. GAL4 fusion proteins containing the wild-type C-terminal region of Stat1α (residues 711- 750; Figure 1B) activated transcription 60-fold compared with the GAL4 DNA-binding domain alone, indicating that the C-terminal domain of Stat1α can function inde- pendently as a TAD ( Figure 1D). ...
Context 4
... constructs encoding the DNA-binding domain of GAL4 fused to the wild-type residues 711-750 of Stat1α or to mutant versions of these residues ( Figure 1B) were prepared to test the capacity of these residues to act 6964 independently as a TAD. GAL4 fusion proteins containing the wild-type C-terminal region of Stat1α (residues 711- 750; Figure 1B) activated transcription 60-fold compared with the GAL4 DNA-binding domain alone, indicating that the C-terminal domain of Stat1α can function inde- pendently as a TAD ( Figure 1D). Untreated cells gave similar reporter activity as treated cells probably due to the constitutive nuclear localization of the GAL4 fusion proteins ( Nelson and Silver, 1989) and a constitutive level of phosphorylation on Ser727 when cells are grown in serum-containing medium. ...
Context 5
... constructs encoding the DNA-binding domain of GAL4 fused to the wild-type residues 711-750 of Stat1α or to mutant versions of these residues ( Figure 1B) were prepared to test the capacity of these residues to act 6964 independently as a TAD. GAL4 fusion proteins containing the wild-type C-terminal region of Stat1α (residues 711- 750; Figure 1B) activated transcription 60-fold compared with the GAL4 DNA-binding domain alone, indicating that the C-terminal domain of Stat1α can function inde- pendently as a TAD ( Figure 1D). Untreated cells gave similar reporter activity as treated cells probably due to the constitutive nuclear localization of the GAL4 fusion proteins ( Nelson and Silver, 1989) and a constitutive level of phosphorylation on Ser727 when cells are grown in serum-containing medium. ...
Context 6
... increase of total MCM5 in these cells enhanced the IFN-γ induced reporter activity ~4-fold in cells containing wild-type Stat1α, but not in cells containing Stat1β. In Stat1αSA cells, IFN-γ treatment induced a transcriptional activity ~20% that of the wild-type Stat1α (Wen et al., 1995) ( Figure 1C). However, this activity was not enhanced by the over-expression of exogenous MCM5 ( Figure 5B, top panel). ...
Context 7
... blot analyses were performed with nuclear extracts from cells collected at different time points during the release from G 2 /M block. Corresponding to 0 h in Figure 6A, cells at G 2 /M block contained low levels of nuclear MCM5 ( Figure 6B, lane 3) compared with asynchronously growing cells ( Figure 6B, lanes 1 and 2). Cells within 3 h of release ( Figure 6B, lanes 4 and 5, corresponding to the 3 h point sample in Figure 6A) also contained low levels of nuclear MCM5. ...
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... releasing for more than 6 h, the level of nuclear MCM5 decreased ( Figure 6B, lanes 9-12, corresponding to the 9 h point sample in Figure 6A). Cells released for 24 h ( Figure 6B, lane 13) contained similar levels of nuclear MCM5 to that of the asynchron- ously growing cells ( Figure 6B, lanes 1 and 2). Except for the G 2 /M blocked cells and cells within the first hour of release which contained a lower level of tyrosine phosphorylated Stat1α, cells at different time points were capable of inducing tyrosine phosphorylation on Stat1α in response to a 15-min treatment with IFN-γ to similar levels ( Figure 6B, bottom panel). ...
Citations
... For instance, it has been shown that the MCM2 subunit interacts with the C-terminal domain of RNA polymerase II necessary for establishing protein-protein interactions throughout transcription and downstream processes 149 . Additionally, two studies have demonstrated that MCM5 interacts with the STAT1alpha transcription factor, which is required for the expression of interferon-gamma-responsive genes during the immune response 150,151 . Nevertheless, future studies will be needed to uncover the functional significance of these interactions and their impact on transcription machinery. ...
Accurate and complete replication of genetic information is a fundamental process of every cell division. The replication licensing is the first essential step that lays the foundation for error-free genome duplication. During licensing, minichromosome maintenance protein complexes, the molecular motors of DNA replication, are loaded to genomic sites called replication origins. The correct quantity and functioning of licensed origins are necessary to prevent genome instability associated with severe diseases, including cancer. Here, we delve into recent discoveries that shed light on the novel functions of licensed origins, the pathways necessary for their proper maintenance, and their implications for cancer therapies.
... For example, MCM3 interacts with Keap1, resulting in an antioxidant response (Tamberg et al., 2018). The interaction between STAT1 and MCM5 is required for IFNγ-induced transcriptional activation (Zhang et al., 1998). MCM6 interacts with RPA, Rb, Timeless/Tipin, and Claspin for efficient replication initiation (Komata et al., 2009;Numata et al., 2010). ...
Maintaining genomic integrity is critical for sustaining individual animals and passing on the genome to subsequent generations. Several enzymes, such as DNA helicases and DNA polymerases, are involved in maintaining genomic integrity by unwinding and synthesizing the genome, respectively. Indeed, several human diseases that arise caused by deficiencies in these enzymes have long been known. In this review, the author presents the DNA helicases associated with human diseases discovered to date using recent analyses, including exome sequences. Since several mouse models that reflect these human diseases have been developed and reported, this study also summarizes the current knowledge regarding the outcomes of DNA helicase deficiencies in humans and mice and discusses possible mechanisms by which DNA helicases maintain genomic integrity in mammals. It also highlights specific diseases that demonstrate mammalian resilience, in which, despite the presence of genomic instability, patients and mouse models have lifespans comparable to those of the general population if they do not develop cancers; finally, this study discusses future directions for therapeutic applications in humans that can be explored using these mouse models.
... MCM5 is a helicase with an ATPase activity involved in DNA replication; it interacts with the TAD region of STAT1, enhancing the transcription induced by STAT1. The interaction between STAT1 and MCM5 requires two specific residues in MCM5: R732 and K734 [42]. Mutations in these residues do not allow interaction between STAT1 and MCM5 in response to IFNγ [43]. ...
The signal transducer and activator of transcription (STAT) 1 protein plays a key role in the immune response against viruses and other pathogens by transducing, in the nucleus, the signal from type I, type II and type III IFNs. STAT1 activates the transcription of hundreds of genes, some of which have been well characterized for their antiviral properties. STAT1 gene deletion in mice and complete STAT1 deficiency in humans both cause rapid death from severe infections. STAT1 plays a key role in the immunoglobulin class-switch recombination through the upregulation of T-bet; it also plays a key role in the production of T-bet+ memory B cells that contribute to tissue-resident humoral memory by mounting an IgG response during re-infection. Considering the key role of STAT1 in the antiviral immune response, many viruses, including dangerous viruses such as Ebola and SARS-CoV-2, have developed different mechanisms to inhibit this transcription factor. The search for drugs capable of targeting the viral proteins implicated in both viral replication and IFN/STAT1 inhibition is important for the treatment of the most dangerous viral infections and for future viral pandemics, as shown by the clinical results obtained with Paxlovid in patients infected with SARS-CoV-2.
... STAT3 transcriptional activity is also subject to other key post-translational modifications beyond p-Y705. S727 phosphorylation, usually mediated by ERK or other kinases, is generally believed to lead to a stronger realization of STAT3 transcriptional activity [25,26]. Acetylation, methylation, and SUMOylation of STAT3 are also regular events which have recently garnered more attention and which we will discuss in later sections [26,27]. ...
Simple Summary
Lung adenocarcinomas with mutations in the K-ras gene are hard to target pharmacologically and highly lethal. As a result, there is a need to identify other therapeutic targets that influence K-ras oncogenesis. One contender is STAT3, a transcription factor that is associated with K-ras mutations and aids tumor development and progression through tumor cell intrinsic and extrinsic mechanisms. In this review, we summarize the lung epithelial and infiltrating immune cells that express STAT3, the roles of STAT3 in K-ras mutant lung adenocarcinoma, and therapies that may be able to target STAT3.
Abstract
Worldwide, lung cancer, particularly K-ras mutant lung adenocarcinoma (KM-LUAD), is the leading cause of cancer mortality because of its high incidence and low cure rate. To treat and prevent KM-LUAD, there is an urgent unmet need for alternative strategies targeting downstream effectors of K-ras and/or its cooperating pathways. Tumor-promoting inflammation, an enabling hallmark of cancer, strongly participates in the development and progression of KM-LUAD. However, our knowledge of the dynamic inflammatory mechanisms, immunomodulatory pathways, and cell-specific molecular signals mediating K-ras-induced lung tumorigenesis is substantially deficient. Nevertheless, within this signaling complexity, an inflammatory pathway is emerging as a druggable target: signal transducer and activator of transcription 3 (STAT3). Here, we review the cell type-specific functions of STAT3 in the pathogenesis and progression of KM-LUAD that could serve as a new target for personalized preventive and therapeutic intervention for this intractable form of lung cancer.
... Meanwhile, phosphatase-dependent STAT1 dephosphorylation constitutes an important negative regulatory event that is central for titrating the IFN response [119,120]. At the transcriptional level, the transcriptional factor ISGF3, a complex that includes IRF9 and STAT1/2, binds to ISREs within the promoter regions of ISGs [121], recruiting various chromatin remodeling factors [122], transcriptional coactivators [123], and corepressors [124], which can either promote or inhibit ISG transcription [120]. In addition to these mechanisms, recent studies utilizing sequencing and proteomics technologies have defined antiviral effectors that are IFN-stimulated in a non-classical way, which have additional consequences of IFN stimulation [56,125]. ...
A key characteristic of Human immunodeficiency virus type 1 (HIV-1) infection is the generation of latent viral reservoirs, which have been associated with chronic immune activation and sustained inflammation. Macrophages play a protagonist role in this context since they are persistently infected while being a major effector of the innate immune response through the generation of type-I interferons (type I IFN) and IFN-stimulated genes (ISGs). The balance in the IFN signaling and the ISG induction is critical to promote a successful HIV-1 infection. Classically, the IFNs response is fine-tuned by opposing promotive and suppressive signals. In this context, it was described that HIV-1-infected macrophages can also synthesize some antiviral effector ISGs and, positive and negative regulators of the IFN/ISG signaling. Recently, epitranscriptomic regulatory mechanisms were described, being the N6-methylation (m6A) modification on mRNAs one of the most relevant. The epitranscriptomic regulation can affect not only IFN/ISG signaling, but also type I IFN expression, and viral fitness through modifications to HIV-1 RNA. Thus, the establishment of replication-competent latent HIV-1 infected macrophages may be due to non-classical mechanisms of type I IFN that modulate the activation of the IFN/ISG signaling network.
... Finally, there may be additional functions mediated by the CMG helicase that contribute to the NK cell phenotype in these patients. MCM5 binds directly to the STAT1α promoter and mediates direct transcriptional control of IFN-γ response genes (52). Alternatively, activation of DDR pathway-associated genes in murine NK cells, including Chk2 and Atm, is mediated by RAG recombinase expression. ...
... Finally, there may be additional functions mediated by the CMG helicase that contribute to the NK cell phenotype in these patients. MCM5 binds directly to the STAT1α promoter and mediates direct transcriptional control of IFN-γ response genes (52). Alternatively, activation of DDR pathway-associated genes in murine NK cells, including Chk2 and Atm, is mediated by RAG recombinase expression. ...
Human natural killer cell deficiency (NKD) arises from inborn errors of immunity that lead to impaired NK cell development, function, or both. Through the understanding of the biological perturbations in individuals with NKD, requirements for the generation of terminally mature functional innate effector cells can be elucidated. Here, we report a cause of NKD resulting from compound heterozygous mutations in minichromosomal maintenance complex member 10 (MCM10) that impaired NK cell maturation in a child with fatal susceptibility to CMV. MCM10 has not been previously associated with monogenic disease and plays a critical role in the activation and function of the eukaryotic DNA replisome. Through evaluation of patient primary fibroblasts, modeling patient mutations in fibroblast cell lines, and MCM10 knockdown in human NK cell lines, we have shown that loss of MCM10 function leads to impaired cell cycle progression and induction of DNA damage-response pathways. By modeling MCM10 deficiency in primary NK cell precursors, including patient-derived induced pluripotent stem cells, we further demonstrated that MCM10 is required for NK cell terminal maturation and acquisition of immunological system function. Together, these data define MCM10 as an NKD gene and provide biological insight into the requirement for the DNA replisome in human NK cell maturation and function.
... 6a). Serine phosphorylation of STAT1 is known to be crucial for maximal transcriptional activation and STAT1 interaction with transcriptional cofactors [30][31][32]. Studies have shown that serine phosphorylation of STAT1 promotes apoptosis. ...
T-cell protein tyrosine phosphatase (TC-PTP), encoded by Ptpn2, has been shown to function as a tumor suppressor during skin carcinogenesis. In the current study, we generated a novel epidermal-specific TC-PTP-overexpressing (K5HA.Ptpn2) mouse model to show that TC-PTP contributes to the attenuation of chemically induced skin carcinogenesis through the synergistic regulation of STAT1, STAT3, STAT5, and PI3K/AKT signaling. We found overexpression of TC-PTP increased epidermal sensitivity to DMBA-induced apoptosis and it decreased TPA-mediated hyperproliferation, coinciding with reduced epidermal thickness. Inhibition of STAT1, STAT3, STAT5, or AKT reversed the effects of TC-PTP overexpression on epidermal survival and proliferation. Mice overexpressing TC-PTP in the epidermis developed significantly reduced numbers of tumors during skin carcinogenesis and presented a prolonged latency of tumor initiation. Examination of human papillomas and squamous cell carcinomas (SCCs) revealed that TC-PTP expression was significantly reduced and TC-PTP expression was inversely correlated with the increased grade of SCCs. Our findings demonstrate that TC-PTP is a potential therapeutic target for the prevention of human skin cancer given that it is a major negative regulator of oncogenic signaling.
... Finally, there may be additional functions mediated by the CMG helicase that contribute to the NK cell phenotype in these patients. MCM5 binds directly to the STAT1α promoter and mediates direct transcriptional control of IFNγ response genes (52). Careful genetic and functional analyses of the effect of these mutations on gene expression throughout NK cell development will be important for definitively elucidating the nature of these deficiencies on NK cell maturation and function. ...
Human natural killer cell deficiency (NKD) arises from inborn errors of immunity that lead to impaired NK cell development, function or both. Through the understanding of the biological perturbations in individuals with NKD, requirements for the generation of terminally mature functional innate effector cells can be elucidated. Here we report a novel cause of NKD resulting from compound heterozygous mutations in MCM10 that impaired NK cell maturation in a child with fatal susceptibility to CMV. MCM10 has not been previously associated with monogenic disease and plays a critical role in the activation and function of the eukaryotic DNA replisome. By modeling MCM10 deficiency in human NK cell lines and primary NK cell precursors, we demonstrate that MCM10 is required for NK cell terminal maturation and acquisition of immunological system function.
... We confirmed that miR-486 is sufficient to increase Stat1 protein levels by immunoblotting on total protein extracted from treated mouse hearts ( Figure 5C). Since phosphorylation of Stat1 is key for the activity of the proteins and transcription of target genes (28,29), we performed immunoblot analysis for p-Stat1 (Ser727). Indeed, miR-486-treated hearts displayed increased p-Stat1 (Ser727) compared with scramble-treated (BlockIT) controls. ...
Perturbations in biomechanical stimuli during cardiac development contribute to congenital cardiac defects such as Hypoplastic Left Heart Syndrome (HLHS). This study sought to identify stretch-responsive pathways involved in cardiac development. microRNA (miRNA)-Sequencing identified miR-486 as being increased in cardiomyocytes exposed to cyclic stretch in vitro (63%, p<0.002). The right ventricles of HLHS patients experience increased stretch and have a trend towards higher miR-486 levels 4.9-fold (p=0.08). Sheep RVs dilated from excessive pulmonary blood flow have 60% more miR-486 vs. control RVs (p<0.05). The left ventricles of newborn mice treated with miR-486 mimic are 16.9%-24.6% larger (p<0.01) and display 2.48 fold increase in cardiomyocyte proliferation (p<0.01). miR-486 treatment decreases FoxO1 and Smad signaling, while increasing the protein levels of Stat1. Stat1 associates with Gata4 and Serum Response Factor (Srf), two key cardiac transcription factors whose protein levels increase in response to miR-486. This is the first report of a stretch-responsive miRNA that increases the growth of the ventricle in vivo.
