Добірка наукової літератури з теми "Sp/KLFs factor"

Abstract Krüppel-like factors (KLF) are a group of 17 transcription factors with highly conserved DNA-binding domains that contain three C-terminal C2H2-type zinc fingers and a variable N-terminal domain responsible for recruiting cofactors 1. KLFs participate in diverse roles in stem cell renewal, early patterning, organogenesis and tissue homeostasis. Krüppel-like factor 1 (KLF1) is an erythroid-specific KLF responsible for coordinating many aspects of terminal erythroid differentiation 2. It functions as a transcriptional activator by recruiting cofactors such as p300 and chromatin modifiers such as Brg1 via N-terminal transactivation domains 3. Krüppel-like factor 3 (KLF3) acts as a transcriptional repressor via recruitment of C-terminal binding proteins 4. In erythropoiesis, KLF1 directly activates KLF3 via an erythroid-specific promoter 5. Some KLF1 target genes are upregulated in Klf3-/- fetal liver suggesting possible competition between the two factors for promoter/enhancer occupancy. We generated three independent clones of the erythroid cell line, J2E, by retroviral transduction of a tamoxifen-inducible version of Klf3 (Klf3-ERTM) as previously described 6. Using next-generation sequencing of newly synthesised RNA (4sU-labeling), we show KLF3 induction leads to immediate repression of a set of ~580 genes; a subset of these (54) are also directly induced by KLF1 in K1-ER cells, suggesting antagonistic regulation. Indeed, ChIP-seq revealed KLF1 and KLF3 bind many of the same regulatory sites within the erythroid cell genome. KLF3 also binds an independent set of promoters which are not bound by KLF1, suggesting it also plays a KLF1-independent role in maintenance of gene repression. By de novo motif discovery we confirm KLF3 binds preferably to a extended CACCC motif, R-CCM-CRC-CCN, so the DNA-binding specificity in vivo is indistinguishable from the KLF1 binding specificity 7, and is independent of co-operating DNA-binding proteins or cofactors. Using Q-PCR of KLF1 ChIPed DNA in J2E-Klf3ER cells, we show that overexpression of KLF3 directly displaces KLF1 from many key target sites such as the E2f2 enhancer and this leads to down regulation of gene expression. This is the first proof that KLF1 and KLF3 directly compete for key promoters and enhancers which drive erythroid cell proliferation and differentiation. We propose KLF3 acts to 'fine-tune' transcription in erythropoiesis by repressing genes activated by KLF1 and that this negative feedback system is necessary for precise control over the generation of erythrocytes. It also works independently of KLF1 perhaps via competition for binding with other KLF/SP factors. References: 1. van Vliet J, Crofts LA, Quinlan KG, Czolij R, Perkins AC, Crossley M. Human KLF17 is a new member of the Sp/KLF family of transcription factors. Genomics. 2006;87(4):474-482. 2. Tallack MR, Magor GW, Dartigues B, et al. Novel roles for KLF1 in erythropoiesis revealed by mRNA-seq. Genome Res. 2012. 3. Perkins A, Xu X, Higgs DR, et al. "Kruppeling" erythropoiesis: an unexpected broad spectrum of human red blood cell disorders due to KLF1 variants unveiled by genomic sequencing. Blood. 2016. 4. Dewi V, Kwok A, Lee S, et al. Phosphorylation of Kruppel-like factor 3 (KLF3/BKLF) and C-terminal binding protein 2 (CtBP2) by homeodomain-interacting protein kinase 2 (HIPK2) modulates KLF3 DNA binding and activity. J Biol Chem. 2015;290(13):8591-8605. 5. Funnell AP, Maloney CA, Thompson LJ, et al. Erythroid Kruppel-like factor directly activates the basic Kruppel-like factor gene in erythroid cells. Mol Cell Biol. 2007;27(7):2777-2790. 6. Coghill E, Eccleston S, Fox V, et al. Erythroid Kruppel-like factor (EKLF) coordinates erythroid cell proliferation and hemoglobinization in cell lines derived from EKLF null mice. Blood. 2001;97(6):1861-1868. 7. Tallack MR, Whitington T, Yuen WS, et al. A global role for KLF1 in erythropoiesis revealed by ChIP-seq in primary erythroid cells. Genome Res. 2010;20(8):1052-1063. Disclosures Perkins: Novartis Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria.

Krüppel-like factors (KLFs), of which there are currently 17 known protein members, belong to the specificity protein (Sp) family of transcription factors and are characterized by the presence of Cys2/His2 zinc finger motifs in their carboxy-terminal domains that confer preferential binding to GC/GT-rich sequences in gene promoter and enhancer regions. While previously regarded to simply function as silencers of Sp1 transactivity, many KLFs are now shown to be relevant to human cancers by their newly identified abilities to mediate crosstalk with signaling pathways involved in the control of cell proliferation, apoptosis, migration, and differentiation. Several KLFs act as tumor suppressors and/or oncogenes under distinct cellular contexts, underscoring their prognostic potential for cancer survival and outcome. Recent studies suggest that a number of KLFs can influence steroid hormone signaling through transcriptional networks involving steroid hormone receptors and members of the nuclear receptor family of transcription factors. Since inappropriate sensitivity or resistance to steroid hormone actions underlies endocrine-related malignancies, we consider the intriguing possibility that dysregulation of expression and/or activity of KLF members is linked to the pathogenesis of endometrial and breast cancers. In this review, we focus on recently described mechanisms of actions of several KLFs (KLF4, KLF5, KLF6, and KLF9) in cancers of the mammary gland and uterus. We suggest that understanding the mode of actions of KLFs and their functional networks may lead to the development of novel therapeutics to improve current prospects for cancer prevention and cure.

SP/KLF (Specificity protein/Krüppel-like factor) transcription factors comprise an emerging group of proteins that may behave as tumour suppressors. Incidentally, many cancers that display alterations in certain KLF proteins are also associated with a high incidence of KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue) mutations. Therefore in the present paper we investigate whether SP/KLF proteins suppress KRAS-mediated cell growth, and more importantly, the potential mechanisms underlying these effects. Using a comprehensive family-wide screening of the 24 SP/KLF members, we discovered that SP5, SP8, KLF2, KLF3, KLF4, KLF11, KLF13, KLF14, KLF15 and KLF16 inhibit cellular growth and suppress transformation mediated by oncogenic KRAS. Each protein in this subset of SP/KLF members individually inhibits BrdU (5-bromo-2-deoxyuridine) incorporation in KRAS oncogenic-mutant cancer cells. SP5, KLF3, KLF11, KLF13, KLF14 and KLF16 also increase apoptosis in these cells. Using KLF11 as a representative model for mechanistic studies, we demonstrate that this protein inhibits the ability of cancer cells to form both colonies in soft agar and tumour growth in vivo. Molecular studies demonstrate that these effects of KLF11 are mediated, at least in part, through silencing cyclin A via binding to its promoter and leading to cell-cycle arrest in S-phase. Interestingly, similar to KLF11, KLF14 and KLF16 mechanistically share the ability to modulate the expression of cyclin A. Collectively, the present study stringently defines a distinct subset of SP/KLF proteins that impairs KRAS-mediated cell growth, and that mechanistically some members of this subset accomplish this, at least in part, through regulation of the cyclin A promoter.

Objective.Endothelial tissue-type plasminogen activator (t-PA) release is a pivotal response to protect the circulation from occluding thrombosis. We have shown that the t-PA gene is epigenetically regulated and greatly induced by the histone deacetylase (HDAC) inhibitor valproic acid (VPA). We now investigated involvement of known t-PA promoter regulatory elements and evaluated dependence of potential interacting transcription factors/cofactors.Methods.A reporter vector with an insert, separately mutated at either the t-PA promoter CRE or GC box II or GC box III elements, was transfected into HT-1080 and HUVECs and challenged with VPA. HUVECs were targeted with siRNA against histone acetyl transferases (HAT) and selected transcription factors from the Sp/KLF family.Results.An intact VPA-response was observed with CRE mutated constructs, whereas mutation of GC boxes II and III reduced the magnitude of the induction by 54 and 79% in HT-1080 and 49 and 50% in HUVECs, respectively. An attenuated induction of t-PA mRNA was observed after Sp2, Sp4, and KLF5 depletion. KLF2 and p300 (HAT) were identified as positive regulators of basal t-PA expression and Sp4 and KLF9 as repressors.Conclusion.VPA-induced t-PA expression is dependent on the proximal GC boxes in the t-PA promoter and may involve interactions with Sp2, Sp4, and KLF5.

ABSTRACT The Sp/Krüppel-like factor (Sp/Klf) family is comprised of around 25 zinc finger transcription factors that recognize CACCC boxes and GC-rich elements. We have investigated basic Krüppel-like factor (Bklf/Klf3) and show that in erythroid tissues its expression is highly dependent on another family member, erythroid Krüppel-like factor (Eklf/Klf1). We observe that Bklf mRNA is significantly reduced in erythroid tissues from Eklf-null murine embryos. We find that Bklf is driven primarily by two promoters, a ubiquitously active GC-rich upstream promoter, 1a, and an erythroid downstream promoter, 1b. Transcripts from the two promoters encode identical proteins. Interestingly, both the ubiquitous and the erythroid promoter are dependent on Eklf in erythroid cells. Eklf also activates both promoters in transient assays. Experiments utilizing an inducible form of Eklf demonstrate activation of the endogenous Bklf gene in the presence of an inhibitor of protein synthesis. The kinetics of activation are also consistent with Bklf being a direct Eklf target. Chromatin immunoprecipitation assays confirm that Eklf associates with both Bklf promoters. Eklf is typically an activator of transcription, whereas Bklf is noted as a repressor. Our results support the hypothesis that feedback cross-regulation occurs within the Sp/Klf family in vivo.

Abstract Only a small subset of transcription factors (TFs) can act as pioneer factors; i.e. those that can 'open' otherwise 'closed' chromatin to facilitate assembly of TF complexes and co-factors to enable transcription. The KLF/SP family of TFs bind to a 9-10 bp consensus motif in DNA to activate or repress target gene expression. We have studied the potential for KLF1, which is essential for erythropoiesis, to provide a pioneering function in erythroid progentior cells. Previous ChIP-seq studies have shown KLF1 binds a few thousand enhancers and promoters to activate erythroid cell gene expression 1. It often binds near to other key erythroid TFs such as GATA1 and SCL/TAL1, so is likely to work in concert with them in some contexts. We have employed an inducible stable KLF1-ERTM construct to rescue gene expression and differentiation of Klf1-/- erythroid cell lines 2. We employed ChIP-seq, ATAC-seq and DNAse1 HS to show KLF1 can bind to closed sites in chromatin and induce an open state. We show this is essential for recruitment of the settler transcription, GATA1, at certain co-bound sites but not others. This pioneering function occurs at ~300 key erythroid enhancers and super-enhancers such the one at -26kb in the a-globin LCR and one within the body of the E2f2 gene 3 but rarely at promoters. We further show that two different neomorphic mutations in the KLF1 DNA-binding domain lead to ectopic pioneering (opening of closed chromatin) and aberrant gene activation 4. We generated a series of N-terminal deletions in KLF1 and employed ATAC-seq to map the domain/s within KLF1 responsible for the pioneering activity and show it is distinct from DNA-binding activity. The domain is responsible for bromodomain protein recruitment, the likely effector of chromatin remodelling. We have also examined whether KLF3, which acts as a transcription repressor via recruitment of the co-repressor, CtBP2, can force the closure of otherwise open chromatin 5. We find it cannot. Rather, KLF3 (and likely other members of this subclade) works via active recruitment of co-repressors rather than rendering chromatin inaccessible. This likely enables rapid reactivation of pioneered enhancers without the need to reprogram chromatin. This work has broad implications for how the KLF/SP family of TFs work in vivo to reprogram cells and direct differentiation. We will present data for such activity in non-erythroid cell systems. References:Tallack MR, Whitington T, Yuen WS, et al. A global role for KLF1 in erythropoiesis revealed by ChIP-seq in primary erythroid cells. Genome Res. 2010;20(8):1052-1063.Coghill E, Eccleston S, Fox V, et al. Erythroid Kruppel-like factor (EKLF) coordinates erythroid cell proliferation and hemoglobinization in cell lines derived from EKLF null mice. Blood. 2001;97(6):1861-1868.Tallack MR, Keys JR, Humbert PO, Perkins AC. EKLF/KLF1 controls cell cycle entry via direct regulation of E2f2. J Biol Chem. 2009;284(31):20966-20974.Gillinder KR, Ilsley MD, Nebor D, et al. Promiscuous DNA-binding of a mutant zinc finger protein corrupts the transcriptome and diminishes cell viability. Nucleic Acids Res. 2017;45(3):1130-1143.Turner J, Crossley M. Cloning and characterization of mCtBP2, a co-repressor that associates with basic Kruppel-like factor and other mammalian transcriptional regulators. Embo J. 1998;17(17):5129-5140. Disclosures Perkins: Novartis Oncology: Honoraria.

Tumor development and progression depend on reprogramming of signaling pathways that regulate cell metabolism. Alterations to various metabolic pathways such as glycolysis, oxidative phosphorylation, lipid metabolism, and hexosamine biosynthesis pathway are crucial to sustain increased redox, bioenergetic, and biosynthesis demands of a tumor cell. Transcription factors (oncogenes and tumor suppressors) play crucial roles in modulating these alterations, and their functions are tethered to major metabolic pathways under homeostatic conditions and disease initiation and advancement. Specificity proteins (SPs) and Krüppel-like factors (KLFs) are closely related transcription factors characterized by three highly conserved zinc fingers domains that interact with DNA. Studies have demonstrated that SP and KLF transcription factors are expressed in various tissues and regulate diverse processes such as proliferation, differentiation, apoptosis, inflammation, and tumorigenesis. This review highlights the role of SP and KLF transcription factors in the metabolism of various cancers and their impact on tumorigenesis. A better understanding of the role and underlying mechanisms governing the metabolic changes during tumorigenesis could provide new therapeutic opportunities for cancer treatment.

Abstract Sp/Krüppel-like factor (KLF) family of proteins are characterized by the presence of three highly homologous Cys2His2 type zinc-fingers near the C-terminus that bind GC/CACCC boxes, which are one of the most common regulatory elements found in promoters of many cellular and viral genes. Currently, more than 20 members have been identified in the family. This family of factors plays important roles in cell growth, differentiation, development and homeostasis by regulating the expression of their target genes. The GC and GT/CACCC boxes in the globin gene promoters and the beta globin locus control region play an important role in the tissue- and developmental stage- specific expression of globin genes. We have carried out extensive studies to identify the KLF factors that regulate gamma globin expression and erythroid differentiation. Gene expression analysis revealed that most of the Sp/KLF factors are expressed, albeit at variable levels, in human fetal liver and adult blood cells. To determine the role of the Sp/KLF factors in gamma globin expression and erythroid differentiation, functional studies using systematic RNAi to knockdown selected Sp/KLF factors were performed. We used a lentiviral mediated siRNA expression system for specific silencing selected Sp/KLF factors. Effective knockdown of Sp/KLF factors was achieved as judged by a 70–90% decrease in their mRNA levels in the cells. Analyses of globin gene expression showed that the knockdown of some Sp/KLF factors resulted in changes in globin gene expression in K562 cells. We also observed that knockdown of specific Sp/KLF factors resulted in erythroid differentiation of K562 cells. These results suggest that specific Sp/KLF factors may play a role in regulation of globin gene expression and erythroid differentiation.

Abstract Krüppel-Like-Factor 2 (KLF2) is a transcription factor that controls organ development, differentiation and trafficking of cells. In the immune system, KLF2 fosters the egress of T lymphocytes from the thymus via S1PR1 and promotes quiescent states of B lymphocytes as well as homing of antigen-specific IgG plasmablasts (PB) and plasma cells (PC) to the bone marrow (BM) via the α4β7 receptor. To investigate the PC-specific role of KLF2, we analyzed CD138+/TACI+ PB/PC subpopulations and isotype changes in various organs such as spleen (SP), BM, gut associated lymphoid tissues (GALT) and blood of KLF2-deficient mice in comparison to their mb1cre+ KLF2wt/wt controls. Therefore, FACS and Elispot analyses showed a striking reduction of IgA+ PB/PC in SP, BM and blood of non-immunized mice. Elisa and multiplex data revealed a strong reduction in serum IgA as well as (s)IgA in the feces of KLF2-deficient mice. However, frequencies of IgA+ PB/PC were not changed in GALT but total PB/PC accumulated in mesenteric lymph nodes (mLN) and Peyer’s Patches. In addition, IgA secretion of these cells was not effected. Based on these data, we conclude that the observed IgA-deficiency in KLF2-deficient mice can in part be explained by impaired egress of class switched PB/PC from their organ of generation to survival niches in the bone marrow and gut by controlling the expression of integrins. To address the consequences of a dysregulated PB/PC migration during infection, we are analyzing a GALT-dependent immunization with recombinant Flagellin which is known to trigger a systemic IgG as well as an mucosal IgA response. Furthermore, we are identifying KLF2 target genes that control PC egress and trafficking by RNAseq of IgA+ PB/PC from the mLN of KLF2-deficient mice.

Krüppel-like factor 2 (KLF2) is an atherosclerotic protective transcription factor that maintains endothelial cell homeostasis through its anti-inflammatory, anti-oxidant, and antithrombotic properties. The aim of this study was to discover KLF2 activators from microbial secondary metabolites and explore their potential molecular mechanisms. By using a high-throughput screening model based on a KLF2 promoter luciferase reporter assay, column chromatography, electrospray ionization mass spectrometry (ESI-MS), and nuclear magnetic resonance (NMR) spectra, trichostatin D (TSD) was isolated from the rice fermentation of Streptomyces sp. CPCC203909 and identified as a novel KLF2 activator. Real-time-quantitative polymerase chain reaction (RT-qPCR) results showed that TSD upregulated the mRNA level of KLF2 in endothelial cells. Functional assays showed that TSD attenuated monocyte adhesion to endothelial cells, decreased vascular cell adhesion protein 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) expression, and exhibited an anti-inflammatory effect in tumor necrosis factor alpha (TNFα)-induced endothelial cells. We further demonstrated through siRNA and western blot assays that the effects of TSD on monocyte adhesion and inflammation in endothelial cells were partly dependent on upregulating KLF2 expression and then inhibiting the NOD-like receptor protein 3 (NLRP3)/Caspase-1/interleukin-1beta (IL-1β) signaling pathway. Furthermore, histone deacetylase (HDAC) overexpression and molecular docking analysis results showed that TSD upregulated KLF2 expression by inhibiting HDAC 4, 5, and 7 activities. Taken together, TSD was isolated from the fermentation of Streptomyces sp. CPCC203909 and first reported as a potential activator of KLF2 in this study. Furthermore, TSD upregulated KLF2 expression by inhibiting HDAC 4, 5, and 7 and attenuated endothelial inflammation via regulation of the KLF2/NLRP3/Caspase-1/IL-1β signaling pathway.