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Ilsley, Melissa, Kevin R. Gillinder, Graham Magor, Merlin Crossley, and Andrew C. Perkins. "Fine-Tuning Erythropoiesis By Competition Between Krüppel-like Factors for Promoters and Enhancers." Blood 128, no. 22 (December 2, 2016): 1036. http://dx.doi.org/10.1182/blood.v128.22.1036.1036.
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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.
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Simmen, R. C. M., J. M. P. Pabona, M. C. Velarde, C. Simmons, O. Rahal, and F. A. Simmen. "The emerging role of Krüppel-like factors in endocrine-responsive cancers of female reproductive tissues." Journal of Endocrinology 204, no. 3 (October 15, 2009): 223–31. http://dx.doi.org/10.1677/joe-09-0329.
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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.
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Fernandez-Zapico, Martin E., Gwen A. Lomberk, Shoichiro Tsuji, Cathrine J. DeMars, Michael R. Bardsley, Yi-Hui Lin, Luciana L. Almada, et al. "A functional family-wide screening of SP/KLF proteins identifies a subset of suppressors of KRAS-mediated cell growth." Biochemical Journal 435, no. 2 (March 29, 2011): 529–37. http://dx.doi.org/10.1042/bj20100773.
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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.
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Ulfhammer, Erik, Pia Larsson, Mia Magnusson, Lena Karlsson, Niklas Bergh, and Sverker Jern. "Dependence of Proximal GC Boxes and Binding Transcription Factors in the Regulation of Basal and Valproic Acid-Induced Expression of t-PA." International Journal of Vascular Medicine 2016 (2016): 1–9. http://dx.doi.org/10.1155/2016/7928681.
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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.
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Funnell, Alister P. W., Christopher A. Maloney, Lucinda J. Thompson, Janelle Keys, Michael Tallack, Andrew C. Perkins, and Merlin Crossley. "Erythroid Krüppel-Like Factor Directly Activates the Basic Krüppel-Like Factor Gene in Erythroid Cells." Molecular and Cellular Biology 27, no. 7 (February 5, 2007): 2777–90. http://dx.doi.org/10.1128/mcb.01658-06.
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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.
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Gillinder, Kevin R., Graham Magor, Charles Bell, Melissa D. Ilsley, Stephen Huang, and Andrew Perkins. "KLF1 Acts As a Pioneer Transcription Factor to Open Chromatin and Facilitate Recruitment of GATA1." Blood 132, Supplement 1 (November 29, 2018): 501. http://dx.doi.org/10.1182/blood-2018-99-119608.
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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.
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Orzechowska-Licari, Emilia J., Joseph F. LaComb, Aisharja Mojumdar, and Agnieszka B. Bialkowska. "SP and KLF Transcription Factors in Cancer Metabolism." International Journal of Molecular Sciences 23, no. 17 (September 1, 2022): 9956. http://dx.doi.org/10.3390/ijms23179956.
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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.
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Hong, Jie, George Stamatoyannopoulos, and Chao-Zhong Song. "Regulation of Globin Gene Expression and Erythroid Differentiation by Sp/KLF Factors." Blood 106, no. 11 (November 16, 2005): 4241. http://dx.doi.org/10.1182/blood.v106.11.4241.4241.
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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.
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Wittner, Jens, Sebastian R. Schulz, Tobit Steinmetz, Andreas R. Hutloff, Adam F. Cunningham, Hans-Martin Jäck, and Wolfgang Schuh. "Krüppel-Like-Factor 2, a new player in mucosal IgA homeostasis." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 235.10. http://dx.doi.org/10.4049/jimmunol.204.supp.235.10.
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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.
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Lei, Lijuan, Minghua Chen, Chenyin Wang, Xinhai Jiang, Yinghong Li, Weizhi Wang, Shunwang Li, et al. "Trichostatin D as a Novel KLF2 Activator Attenuates TNFα-Induced Endothelial Inflammation." International Journal of Molecular Sciences 23, no. 21 (November 3, 2022): 13477. http://dx.doi.org/10.3390/ijms232113477.
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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.
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Suske, Guntram, Elspeth Bruford, and Sjaak Philipsen. "Mammalian SP/KLF transcription factors: Bring in the family." Genomics 85, no. 5 (May 2005): 551–56. http://dx.doi.org/10.1016/j.ygeno.2005.01.005.
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Kim, Chang-Kyung, Ping He, Agnieszka B. Bialkowska, and Vincent W. Yang. "SP and KLF Transcription Factors in Digestive Physiology and Diseases." Gastroenterology 152, no. 8 (June 2017): 1845–75. http://dx.doi.org/10.1053/j.gastro.2017.03.035.
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Presnell, Jason S., Christine E. Schnitzler, and William E. Browne. "KLF/SP Transcription Factor Family Evolution: Expansion, Diversification, and Innovation in Eukaryotes." Genome Biology and Evolution 7, no. 8 (July 30, 2015): 2289–309. http://dx.doi.org/10.1093/gbe/evv141.
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Singh, Parul, Natalia Kunz, Gaelle Le Friec, Luopin Wang, Paul Lavender, Majid Kazemian, and Claudia Kemper. "A novel and human-specific CD46-KLF/SP interaction mediates gene expression required for successful Th1 induction." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 76.13. http://dx.doi.org/10.4049/jimmunol.204.supp.76.13.
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Abstract Signals mediated by autocrine activation of the human-specific complement receptor CD46 during T cell receptor (TCR) stimulation are vital to Th1 induction in human CD4+ T cells, but how exactly CD46 in a molecular level mediates this role is currently undefined. CD46 is expressed in different isoforms that can bear either one of two distinct cytoplasmic tails: CYT-1 or CYT-2. Nuclear translocation of CYT-1 is a critical requirement for the expression of genes coding for nutrient-influx-channels and mTORC1 activity that mediate metabolic adaptations needed for Th1 responses. The lack of a DNA binding domain in CD46-CYT-1 precludes it from acting directly as a transcription factor (TF) and we hence hypothesized that CYT-1 regulates gene expression via direct interaction with specific TF activator and/or repressor complexes. Indeed, CUT&RUN experiments performed using our novel antibody raised against cleaved CYT-1 identified key members of the KLF/SP TFs gene family as potential interacting partners of CD46-CYT-1. Subsequent ELISA and MST experiment confirmed strong, dose-dependent CYT-1/KLF/SP TFs interactions and also demonstrated that CYT-1 fostered KLF/SP TFs binding to appropriate DNA motifs. Genome-wide comparison of the KLF/SP TFs and CYT1 bound genes in T cells revealed their enrichment in crucial basic cell-physiological pathways. Moreover, the CUT&RUN data in conjunction with ATAC-seq analyses indicated that this novel CYT-1/KLF/SP axis may control general chromatin remodeling – a notion we are currently exploring. These data define a novel and critical human-specific pathway of gene regulation and further underpin the vital role of intracellular/autocrine complement in the regulation of normal cellular activity.
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McCulloch, Kyle J., and Kristen M. Koenig. "Krüppel-like factor/specificity protein evolution in the Spiralia and the implications for cephalopod visual system novelties." Proceedings of the Royal Society B: Biological Sciences 287, no. 1937 (October 21, 2020): 20202055. http://dx.doi.org/10.1098/rspb.2020.2055.
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The cephalopod visual system is an exquisite example of convergence in biological complexity. However, we have little understanding of the genetic and molecular mechanisms underpinning its elaboration. The generation of new genetic material is considered a significant contributor to the evolution of biological novelty. We sought to understand if this mechanism may be contributing to cephalopod-specific visual system novelties. Specifically, we identified duplications in the Krüppel-like factor/specificity protein (KLF/SP) sub-family of C2H2 zinc-finger transcription factors in the squid Doryteuthis pealeii . We cloned and analysed gene expression of the KLF/SP family, including two paralogs of the DpSP6-9 gene. These duplicates showed overlapping expression domains but one paralog showed unique expression in the developing squid lens, suggesting a neofunctionalization of DpSP6-9a . To better understand this neofunctionalization, we performed a thorough phylogenetic analysis of SP6-9 orthologues in the Spiralia. We find multiple duplications and losses of the SP6-9 gene throughout spiralian lineages and at least one cephalopod-specific duplication. This work supports the hypothesis that gene duplication and neofunctionalization contribute to novel traits like the cephalopod image-forming eye and to the diversity found within Spiralia.
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Shi, Lewis Zhichang, Jordy Saravia, Hu Zeng, Nishan S. Kalupahana, Clifford S. Guy, Geoffrey Neale, and Hongbo Chi. "Gfi1-Foxo1 axis controls the fidelity of effector gene expression and developmental maturation of thymocytes." Proceedings of the National Academy of Sciences 114, no. 1 (December 19, 2016): E67—E74. http://dx.doi.org/10.1073/pnas.1617669114.
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Double-positive (DP) thymocytes respond to intrathymic T-cell receptor (TCR) signals by undergoing positive selection and lineage differentiation into single-positive (SP) mature cells. Concomitant with these well-characterized events is the acquisition of a mature T-cell gene expression program characterized by the induction of the effector molecules IL-7Rα, S1P1, and CCR7, but the underlying mechanism remains elusive. We report here that transcription repressor Growth factor independent 1 (Gfi1) orchestrates the fidelity of the DP gene expression program and developmental maturation into SP cells. Loss of Gfi1 resulted in premature induction of effector genes and the transcription factors forkhead box protein O1 (Foxo1) and Klf2 in DP thymocytes and the accumulation of postselection intermediate populations and accelerated transition into SP cells. Strikingly, partial loss of Foxo1 function, but not restored survival fitness, rectified the dysregulated gene expression and thymocyte maturation in Gfi1-deficient mice. Our results establish the Gfi1-Foxo1 axis and the transcriptional circuitry that actively maintain DP identity and shape the proper generation of mature T cells.
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Yerra, Veera Ganesh, and Konstantinos Drosatos. "Specificity Proteins (SP) and Krüppel-like Factors (KLF) in Liver Physiology and Pathology." International Journal of Molecular Sciences 24, no. 5 (February 28, 2023): 4682. http://dx.doi.org/10.3390/ijms24054682.
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The liver acts as a central hub that controls several essential physiological processes ranging from metabolism to detoxification of xenobiotics. At the cellular level, these pleiotropic functions are facilitated through transcriptional regulation in hepatocytes. Defects in hepatocyte function and its transcriptional regulatory mechanisms have a detrimental influence on liver function leading to the development of hepatic diseases. In recent years, increased intake of alcohol and western diet also resulted in a significantly increasing number of people predisposed to the incidence of hepatic diseases. Liver diseases constitute one of the serious contributors to global deaths, constituting the cause of approximately two million deaths worldwide. Understanding hepatocyte transcriptional mechanisms and gene regulation is essential to delineate pathophysiology during disease progression. The current review summarizes the contribution of a family of zinc finger family transcription factors, named specificity protein (SP) and Krüppel-like factors (KLF), in physiological hepatocyte functions, as well as how they are involved in the onset and development of hepatic diseases.
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Daftary, Gaurang S., Ye Zheng, Zaid M. Tabbaa, John K. Schoolmeester, Ravi P. Gada, Adrienne L. Grzenda, Angela J. Mathison, Gary L. Keeney, Gwen A. Lomberk, and Raul Urrutia. "A Novel Role of the Sp/KLF Transcription Factor KLF11 in Arresting Progression of Endometriosis." PLoS ONE 8, no. 3 (March 28, 2013): e60165. http://dx.doi.org/10.1371/journal.pone.0060165.
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van Vliet, Jane, Linda A. Crofts, Kate G. R. Quinlan, Robert Czolij, Andrew C. Perkins, and Merlin Crossley. "Human KLF17 is a new member of the Sp/KLF family of transcription factors." Genomics 87, no. 4 (April 2006): 474–82. http://dx.doi.org/10.1016/j.ygeno.2005.12.011.
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Kennedy, Mark W., Ravindra B. Chalamalasetty, Sara Thomas, Robert J. Garriock, Parthav Jailwala, and Terry P. Yamaguchi. "Sp5 and Sp8 recruit β-catenin and Tcf1-Lef1 to select enhancers to activate Wnt target gene transcription." Proceedings of the National Academy of Sciences 113, no. 13 (March 11, 2016): 3545–50. http://dx.doi.org/10.1073/pnas.1519994113.
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The ancient, highly conserved, Wnt signaling pathway regulates cell fate in all metazoans. We have previously shown that combined null mutations of the specificity protein (Sp) 1/Klf-like zinc-finger transcription factors Sp5 and Sp8 (i.e., Sp5/8) result in an embryonic phenotype identical to that observed when core components of the Wnt/β-catenin pathway are mutated; however, their role in Wnt signal transduction is unknown. Here, we show in mouse embryos and differentiating embryonic stem cells that Sp5/8 are gene-specific transcriptional coactivators in the Wnt/β-catenin pathway. Sp5/8 bind directly to GC boxes in Wnt target gene enhancers and to adjacent, or distally positioned, chromatin-bound T-cell factor (Tcf) 1/lymphoid enhancer factor (Lef) 1 to facilitate recruitment of β-catenin to target gene enhancers. Because Sp5 is itself directly activated by Wnt signals, we propose that Sp5 is a Wnt/β-catenin pathway-specific transcripton factor that functions in a feed-forward loop to robustly activate select Wnt target genes.
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Hu, Jie Hong, Patrick Navas, Hua Cao, George Stamatoyannopoulos, and Chao-Zhong Song. "Systematic RNAi Studies on the Role of Sp/KLF Factors in Globin Gene Expression and Erythroid Differentiation." Journal of Molecular Biology 366, no. 4 (March 2007): 1064–73. http://dx.doi.org/10.1016/j.jmb.2006.12.047.
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Alpy, Fabien, Anne Boulay, Christel Moog-Lutz, Kumari L. Andarawewa, Sébastien Degot, Isabelle Stoll, Marie-Christine Rio, and Catherine Tomasetto. "Metastatic lymph node 64 (MLN64), a gene overexpressed in breast cancers, is regulated by Sp/KLF transcription factors." Oncogene 22, no. 24 (June 2003): 3770–80. http://dx.doi.org/10.1038/sj.onc.1206500.
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Sharma, Archna, Qinghua Chen, Trang Nguyen, Qing Yu, and Jyoti Sen. "T Cell Factor-1 and beta-catenin regulate the development of memory-like CD8 thymocytes (110.6)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 110.6. http://dx.doi.org/10.4049/jimmunol.188.supp.110.6.
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Abstract In addition to conventional alpha-beta T cells, recent studies have demonstrated the presence of a subset of CD8 single positive (SP) mature thymocytes, referred to as memory-like or innate-like thymocytes that acquire effector functions in the thymus as a result of the maturation process. These cells have been described in mice with mutations in KLF2, Itk, CBP genes and SLAM family members and in non-mutant Balb/c mice in response to IL-4 produced by thymocytes. Here, we demonstrate that enhanced function of transcription factors T Cell Factor (TCF)-1 and beta-catenin regulate the frequency of IL-4 producing thymocytes that promote the generation of Eomes-expressing memory-like CD8 thymocytes in trans. By contrast, TCF1-deficient mice show a paucity of these cells demonstrating an essential requirement of TCF1 for the generation of Eomes-expressing memory-like CD8 thymocytes. Generation of TCF1 and beta-catenin-dependent memory-like CD8 thymocytes requires the expression of IL-4 and IL-4 receptor. CD8 memory-like T cells migrate to the periphery and are functional. Thus, TCF1 and beta-catenin regulate the generation of memory-like CD8 T cells in the thymus in a non-cell intrinsic manner.
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Truty, M. J., M. E. Fernandez-Zapico, and R. Urrutia. "COMPREHENSIVE ANALYSIS OF THE SP/KLF FAMILY OF TRANSCRIPTION FACTORS IN THE TGF-BETA SIGNALING IN PANCREATIC CANCER CELLS." Pancreas 31, no. 4 (November 2005): 474. http://dx.doi.org/10.1097/01.mpa.0000193781.42217.51.
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Daftary, Gaurang S., Gwen A. Lomberk, Navtej S. Buttar, Thomas W. Allen, Adrienne Grzenda, Jinsan Zhang, Ye Zheng, et al. "Detailed Structural-Functional Analysis of the Krüppel-like Factor 16 (KLF16) Transcription Factor Reveals Novel Mechanisms for Silencing Sp/KLF Sites Involved in Metabolism and Endocrinology." Journal of Biological Chemistry 287, no. 10 (December 27, 2011): 7010–25. http://dx.doi.org/10.1074/jbc.m111.266007.
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Zheng, Ye, Zaid M. Tabbaa, Zaraq Khan, John K. Schoolmeester, Sherif El-Nashar, Abimbola Famuyide, Gary L. Keeney, and Gaurang S. Daftary. "Epigenetic Regulation of Uterine Biology by Transcription Factor KLF11 via Posttranslational Histone Deacetylation of Cytochrome p450 Metabolic Enzymes." Endocrinology 155, no. 11 (November 1, 2014): 4507–20. http://dx.doi.org/10.1210/en.2014-1139.
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Abstract Endocrine regulation of uterine biology is critical for embryo receptivity and human reproduction. Uterine endometrium depends on extrinsic sex steroid input and hence likely has mechanisms that enable adaptation to hormonal variation. Emerging evidence suggests that sex steroid bioavailability in the endometrium is determined by adjusting their metabolic rate and fate via regulation of cytochrome (CYP) p450 enzymes. The CYP enzymes are targeted by ubiquitously expressed Sp/Krüppel-like (Sp/KLF) transcription factors. Specifically, KLF11 is highly expressed in reproductive tissues, regulates an array of endocrine/metabolic pathways via epigenetic histone-based mechanisms and, when aberrantly expressed, is associated with diabetes and reproductive tract diseases, such as leiomyoma and endometriosis. Using KLF11 as a model to investigate epigenetic regulation of endometrial first-pass metabolism, we evaluated the expression of a comprehensive array of metabolic enzymes in Ishikawa cells. KLF11 repressed most endometrial CYP enzymes. To characterize KLF11-recruited epigenetic regulatory mechanisms, we focused on the estrogen-metabolizing enzyme CYP3A4. KLF11 expression declined in secretory phase endometrial epithelium associated with increased CYP3A4 expression. Additionally, KLF11 bound to CYP3A4 promoter GC elements and thereby repressed promoter, message, protein as well as enzymatic function. This repression was epigenetically mediated, because KLF11 colocalized with and recruited the corepressor SIN3A/histone deacetylase resulting in selective deacetylation of the CYP3A4 promoter. Repression was reversed by a mutation in KLF11 that abrogated cofactor recruitment and binding. This repression was also pharmacologically reversible with an histone deacetylase inhibitor. Pharmacological alteration of endometrial metabolism could have long-term translational implications on human reproduction and uterine disease.
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Utami, Trianna W., Keiko Miyoshi, Hiroko Hagita, Ryna Dwi Yanuaryska, Taigo Horiguchi, and Takafumi Noma. "Possible Linkage of SP6 Transcriptional Activity with Amelogenesis by Protein Stabilization." Journal of Biomedicine and Biotechnology 2011 (2011): 1–10. http://dx.doi.org/10.1155/2011/320987.
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Ameloblasts produce enamel matrix proteins such as amelogenin, ameloblastin, and amelotin during tooth development. The molecular mechanisms of ameloblast differentiation (amelogenesis) are currently not well understood. SP6 is a transcription factor of the Sp/KLF family that was recently found to regulate cell proliferation in a cell-type-specific manner.Sp6-deficient mice demonstrate characteristic tooth anomalies such as delayed eruption of the incisors and supernumerary teeth with disorganized amelogenesis. However, it remains unclear howSp6controls amelogenesis. In this study, we used SP6 high producer cells to identify SP6 target genes. Based on the observations that long-term culture of SP6 high producer cells reduced SP6 protein expression but notSp6mRNA expression, we found that SP6 is short lived and specifically degraded through a proteasome pathway. We established anin vitroinducible SP6 expression system coupled with siRNA knockdown and found a possible linkage between SP6 and amelogenesis through the regulation ofamelotinandRock1gene expression by microarray analysis. Our findings suggest that the regulation of SP6 protein stability is one of the crucial steps in amelogenesis.
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Setton, Emily V. W., and Prashant P. Sharma. "Cooption of an appendage-patterning gene cassette in the head segmentation of arachnids." Proceedings of the National Academy of Sciences 115, no. 15 (March 26, 2018): E3491—E3500. http://dx.doi.org/10.1073/pnas.1720193115.
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The jointed appendages of arthropods have facilitated the spectacular diversity and success of this phylum. Key to the regulation of appendage outgrowth is the Krüppel-like factor (KLF)/specificity protein (Sp) family of zinc finger transcription factors. In the fruit fly, Drosophila melanogaster, the Sp6-9 homolog is activated by Wnt-1/wingless (wg) and establishes ventral appendage (leg) fate. Subsequently, Sp6-9 maintains expression of the axial patterning gene Distal-less (Dll), which promotes limb outgrowth. Intriguingly, in spiders, Dll has been reported to have a derived role as a segmentation gap gene, but the evolutionary origin and regulation of this function are not understood because functional investigations of the appendage-patterning regulatory network are restricted to insects. We tested the evolutionary conservation of the ancestral appendage-patterning network of arthropods with a functional approach in the spider. RNAi-mediated knockdown of the spider Sp6-9 ortholog resulted in diminution or loss of Dll expression and truncation of appendages, as well as loss of the two body segments specified by the early Dll function. In reciprocal experiments, Dll is shown not to be required for Sp6-9 expression. Knockdown of arrow (Wnt-1 coreceptor) disrupted segmentation and appendage development but did not affect the early Sp6-9 expression domain. Ectopic appendages generated in the spider “abdomen” by knockdown of the Hox gene Antennapedia-1 (Antp-1) expressed Sp6-9 comparably to wild-type walking legs. Our results support (i) the evolutionary conservation of an appendage-patterning regulatory network that includes canonical Wnt signaling, Sp6-9, and Dll and (ii) the cooption of the Sp6-9/Dll regulatory cassette in arachnid head segmentation.
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van Ouwerkerk, Antoinette F., Fernanda M. Bosada, Karel van Duijvenboden, Arjan C. Houweling, Koen T. Scholman, Vincent Wakker, Cornelis P. Allaart, et al. "Patient-Specific TBX5-G125R Variant Induces Profound Transcriptional Deregulation and Atrial Dysfunction." Circulation 145, no. 8 (February 22, 2022): 606–19. http://dx.doi.org/10.1161/circulationaha.121.054347.
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Background: The pathogenic missense variant p.G125R in TBX5 (T-box transcription factor 5) causes Holt–Oram syndrome (also known as hand–heart syndrome) and early onset of atrial fibrillation. Revealing how an altered key developmental transcription factor modulates cardiac physiology in vivo will provide unique insights into the mechanisms underlying atrial fibrillation in these patients. Methods: We analyzed ECGs of an extended family pedigree of Holt–Oram syndrome patients. Next, we introduced the TBX5-p.G125R variant in the mouse genome ( Tbx5 G125R ) and performed electrophysiologic analyses (ECG, optical mapping, patch clamp, intracellular calcium measurements), transcriptomics (single-nuclei and tissue RNA sequencing), and epigenetic profiling (assay for transposase-accessible chromatin using sequencing, H3K27ac [histone H3 lysine 27 acetylation] CUT&RUN [cleavage under targets and release under nuclease sequencing]). Results: We discovered high incidence of atrial extra systoles and atrioventricular conduction disturbances in Holt–Oram syndrome patients. Tbx5 G125R/+ mice were morphologically unaffected and displayed variable RR intervals, atrial extra systoles, and susceptibility to atrial fibrillation, reminiscent of TBX5-p.G125R patients. Atrial conduction velocity was not affected but systolic and diastolic intracellular calcium concentrations were decreased and action potentials were prolonged in isolated cardiomyocytes of Tbx5 G125R/+ mice compared with controls. Transcriptional profiling of atria revealed the most profound transcriptional changes in cardiomyocytes versus other cell types, and identified over a thousand coding and noncoding transcripts that were differentially expressed. Epigenetic profiling uncovered thousands of TBX5-p.G125R-sensitive, putative regulatory elements (including enhancers) that gained accessibility in atrial cardiomyocytes. The majority of sites with increased accessibility were occupied by Tbx5. The small group of sites with reduced accessibility was enriched for DNA-binding motifs of members of the SP (specificity protein) and KLF (Krüppel-like factor) families of transcription factors. These data show that Tbx5-p.G125R induces changes in regulatory element activity, alters transcriptional regulation, and changes cardiomyocyte behavior, possibly caused by altered DNA binding and cooperativity properties. Conclusions: Our data reveal that a disease-causing missense variant in TBX5 induces profound changes in the atrial transcriptional regulatory network and epigenetic state in vivo, leading to arrhythmia reminiscent of those seen in human TBX5-p.G125R variant carriers.
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Zhang, Yachao, Jieqiong Yang, Shijian Lv, Dong-Qin Zhao, Zi-Jiang Chen, Wei-Ping Li, and Cong Zhang. "Downregulation of decidual SP1 and P300 is associated with severe preeclampsia." Journal of Molecular Endocrinology 60, no. 2 (February 2018): 133–43. http://dx.doi.org/10.1530/jme-17-0180.
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Preeclampsia (PE) is a pregnancy-induced disorder characterized by hypertension and proteinuria after 20 weeks of gestation, affecting 5–7% of pregnancies worldwide. So far, the etiology of PE remains poorly understood. Abnormal decidualization is thought to contribute to the development of PE. SP1 belongs to the Sp/KLF superfamily and can recruit P300 to regulate the transcription of several genes. SP1 is also very important for decidualization as it enhances the expression of tissue factor. In this study, we investigated the expression of SP1 and P300 in deciduae and their relationship with PE. A total of 42 decidua samples were collected, of which 21 were from normal pregnant (NP) and 21 from severe PE. SP1 and P300 expression in deciduae and the levels of SP1 and P300 in cultured human endometrial stromal cells (hESCs) and primary hESCs during decidualization were determined. To further investigate the role of SP1 and P300 in human decidualization, RNA interference was used to silence SP1 and P300 in hESCs and primary hESCs. The following results were obtained. We found that the expressions of SP1 and P300 were reduced in decidual tissues with PE compared to those from NP. In thein vitromodel of induction of decidualization, we found an increase in bothSP1andP300levels. Silencing ofSP1andP300resulted in abnormal decidualization and a significant reduction of decidualization markers such as insulin-like growth factor-binding protein1 and prolactin. Furthermore, the expression of vascular endothelial growth factor was also decreased uponSP1andP300silencing. Similar results were observed in primary hESCs. Our results suggest that SP1 and P300 play an important role during decidualization. Dysfunction of SP1 and P300 leads to impaired decidualization and might contribute to PE.
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Nishimura, Toshinobu, Shin Kaneko, Yoko Tajima, Naoya Takayama, Ai Tachikawa-Kawana, Koji Eto, and Hiromitsu Nakauchi. "In Vitro Generation of Mature T Lymphocytes From Human Ips Cells and Genetic Analysis of TCR Gene Rearrangements." Blood 118, no. 21 (November 18, 2011): 2984. http://dx.doi.org/10.1182/blood.v118.21.2984.2984.
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Abstract Abstract 2984 T lymphocytes play central roles in cellular immunity, exerting their proliferative and effector activities when they recognize antigens via T-cell receptors (TCRs) in HLA-restricted and antigen-specific manner. Adoptive cell transfer therapy (ACT), the administration of ex vivo-activated and -expanded autologous tumor-reactive T lymphocytes, is currently one of the effective methods for immunotherapy, especially for treatment of metastatic solid tumors including melanoma. However, the successful applications of this method are currently limited for tumor therapies. To broaden the range of the application of ACT, we endeavored to develop easier method to obtain cells that carry antigen-specific TCR genes. For the purpose, generation of induced pluripotent stem (iPS) cells from an antigen-reactive single T lymphocyte is attractive and rewarding way. iPS cells have a capacity for unlimited self-renewal while maintaining pluripotency. These features may enable us to induce an unlimited number of T lymphocytes, especially high proliferative naïve / central memory-type T lymphocytes, showing reactivity to specific antigens. If they retain properties of naïve T lymphocytes, they may proliferate for a longer period and achieve better therapeutic effects than their peripheral blood counterparts expanded in vitro. Peripheral T lymphocytes were isolated from healthy volunteers. Then reprogramming factors (OCT4, SOX2, KLF4, and c-MYC) were transduced into fresh or frozen / thawed T lymphocytes. T lymphocyte-derived iPS-like colonies were observed within 3 weeks and they were isolated and clonally expanded. They exhibited standard ES-like morphology, cell surface marker expression, alkaline phosphatase activity, as well as differentiation potential into various tissues related to all three germ layers. Human TCRs are encoded in four genes (TCRA, TCRB, TCRG, TCRD), which should be genetically assembled in an irreversible manner during T-lymphocyte development. This feature allowed us to retrospectively confirm that the iPS cells were generated from T lymphocyte. The TCR gene rearrangements encoded in an iPS colony were clonal for all iPS lines, indicating that each iPS colony was derived from a single T lymphocyte. Sequence analyses of TCR genes revealed whether the rearrangements were productive, and the productivity might promise the conservation of TCR genes rearrangement during the reprogramming process. Next, we tried to re-differentiate T lymphocyte derived-iPS (T-iPS) cells into T cells by co-culturing them with murine stromal cell layers (OP9 and OP9-DL1). T-cell differentiation was evidenced by the expression of T-cell markers, such as CD5, CD7, CD27, CD4, CD8, TCR α β and CD3. We obtained 33.5 ± 17.9% CD4+ CD8+ double positive (DP) cells, 6.51 ± 5.40% CD4+ CD8− single positive (SP) cells and 3.80 ± 1.28% CD4− CD8+ SP cells. They could be activated via TCR stimulation, and produce cytokines as functionally matured T lymphocytes do. The re-differentiation efficiency of T-iPS cells was higher than those of other pluripotent stem cells, such as embryonic stem (ES) cells, fibroblasts derived-iPS cells, or cord blood derived-iPS cells. Transcribed TCR mRNA sequences in re-differentiated T cells were analyzed, and they were revealed to be identical to that engraved in the pre-differentiated T-iPS cells genome in CD4+ CD8+ DP phase. However, fully matured CD4+ CD8− or CD4− CD8+ SP phase cells had several TCRA gene rearrangement patterns distinct from the original T-iPS cell's. On the other hand, TCRB gene maintained identity with the original. The variance of the sequences, especially antigen-recognition site sequences, indicated that the antigen-specificity in the original T lymphocyte might be converted during DP to SP transition process in vitro. These data indicate that functionally matured T cells were generated by re-differentiating T-iPS cells in vitro, and that re-assemble of TCRA genes could take place during SP T cell maturation process. In order to fulfill the T-iPS-mediated immunotherapy, we need to overcome the obstacle of further TCRA gene rearrangements. We think the solution lies in refinement of the re-differentiation method for controlling the expression of RAG1 and RAG2 recombinases or for inhibiting their activities. Disclosures: No relevant conflicts of interest to declare.
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Lin, Kuanyin Karen, Lara Rossi, and Margaret A. Goodell. "CD81 Is Essential for HSC Self-Renewal through Suppressing Proliferation." Blood 112, no. 11 (November 16, 2008): 76. http://dx.doi.org/10.1182/blood.v112.11.76.76.
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Abstract Hematopoietic stem cells (HSCs) comprise only ~0.02% of the whole bone marrow cells but possess the capacity to extensively proliferate in order to restore hematopoietic homeostasis. Under homeostasis, HSCs are relatively quiescent with a slow cell cycle progression rate. However, upon stimulation, HSCs are able to promptly proliferate and undergo self-renewal to regenerate HSCs as daughter cells. While regulatory mechanisms involved in cell cycle progression are well characterized to be essential for HSC self-renewal, the mechanisms that facilitate the return of proliferating HSC to their quiescent state have been largely overlooked. The expression of CD81 (also called TAPA-1), a transmembrane protein that belongs to the Tetraspanin family, has been found associated with HSC proliferation. While CD81 is normally absent on HSC, it becomes markedly upregulated during HSC proliferation (Figure 1). To understand the function of CD81 in regenerating HSCs, we utilized a murine stem cell retroviral vector to deliver genes into 5-FU treated bone marrow progenitors to test the effect of enforced CD81 overexpression on HSC. The CD81-transduced proliferating progenitors were found to give rise to an increased number of phenotypically-defined HSC (SP-KLS) without significantly affecting the homeostasis in peripheral organs. In addition, we also characterized the HSCs from CD81 knock-out mice. We discovered that CD81-null HSC failed to engraft in peripheral blood of secondary recipients in serial transplantation assays (Figure 2), suggesting a role of CD81 in preserving a functional HSC compartment during proliferation-induced stress. When investigating further, we discovered that CD81 is a cell cycle suppressor for HSC, as the CD81KO HSCs are delayed in returning quiescence. In addition, clustering of CD81 on the HSC cell membrane using a monoclonal antibody rapidly induced a quiescent phenotype. This was found to be associated with an altered phosphorylation level of AKT, an inhibitor of the transcription factor FOXO1a and FOXO3a, which have been reported to be essential for HSC self-renewal through suppressing HSC proliferation. Taken together, these results demonstrate an essential role of CD81 in HSC self-renewal, and a novel mechanism that advances quiescence from a proliferating state. Figure 1. CD81 expression is upregulated at the time when HSCs (SPKLS) are proliferating in response to 5FU stimulation, a chemotheraputic agent that induces HSC to proliferate. The expression of CD81 is found at a background level in quiescent stages (5FU-Day0 and 5FU-Day11), and is upregulated during proliferation stages (starting 5FU-Day2) Figure 1. CD81 expression is upregulated at the time when HSCs (SPKLS) are proliferating in response to 5FU stimulation, a chemotheraputic agent that induces HSC to proliferate. The expression of CD81 is found at a background level in quiescent stages (5FU-Day0 and 5FU-Day11), and is upregulated during proliferation stages (starting 5FU-Day2) Figure 2. CD8KO HSCs fail to engraft in the secondary competitive transplantation assay, indicating a self-renewal defect. In this assay, 300 donor-derived HSCs (CD45.2 SPKLS) were purified from the primary recipients and transplanted along with 2×105 competitors into lethally irradiated mice (**p<0.01). Figure 2. CD8KO HSCs fail to engraft in the secondary competitive transplantation assay, indicating a self-renewal defect. In this assay, 300 donor-derived HSCs (CD45.2 SPKLS) were purified from the primary recipients and transplanted along with 2×105 competitors into lethally irradiated mice (**p<0.01).
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Hu, Kang, Qing-Kang Zheng, Rui-Jie Ma, Chao Ma, Zhi-Gang Sun, and Nan Zhang. "Krüppel-Like Factor 6 Splice Variant 1: An Oncogenic Transcription Factor Involved in the Progression of Multiple Malignant Tumors." Frontiers in Cell and Developmental Biology 9 (March 18, 2021). http://dx.doi.org/10.3389/fcell.2021.661731.
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Krüppel-like factor 6 (KLF6) is one of the most studied members of the specificity protein/Krüppel-like factor (SP/KLF) transcription factor family. It has a typical zinc finger structure and plays a pivotal role in regulating the biological processes of cells. Recently, it has been considered to play a role in combatting cancer. Krüppel-like factor 6 splice variant 1 (KLF6-SV1), being one of the alternative KLF6 splicing isoforms, participates in tumor occurrence and development and has the potential to become a new target for molecular targeted therapy, although its action mechanism remains to be determined. The purpose of this article is to provide a comprehensive and systematic review of the important role of KLF6-SV1 in human malignant tumors to provide novel insights for oncotherapy.
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Helbing, Thomas, Jennifer Heinke, Franziska Volkmar, Leonie Wehofsits, Kim-Miriam Baar, Philipp Diehl, Christoph Bode, and Cam Patterson. "Abstract 403: BMPER Is an Angiogenic Modulator that is Regulated by KLF-15 and FoxO3A and Controls Bone Morphogentic Protein Activity." Circulation 118, suppl_18 (October 28, 2008). http://dx.doi.org/10.1161/circ.118.suppl_18.s_298.
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BMPER (bone morphogenetic protein [BMP] endothelial precursor cell derived regulator) is an extracellular protein, that interacts with BMPs and thereby modulates BMP dependent vasculogenesis and angiogenesis. Our previous observations suggest a complex regulation of BMPER expression. During embryogenesis BMPER is expressed at the time and at sites of vasculogenesis, whereas in the adult organism it is expressed in heart, lung and skin. Methods and Results: We have cloned the mouse BMPER promoter and appropriate deletion constructs into pGL3 to regulate luciferase expression. As predicted in silicio, we found that Sp1 and Sp1-like transcription factors such as the krueppel-like factors (KLFs) regulate BMPER transcription. KLF-15 resulted in a 4.5 fold upregulation. Accordingly, BMPER expression was inhibited by the Sp-1/SP-1 like inhibitor mitramycin A. Site specific mutation of a proximal KLF-15 binding site reduced the effect of KLF-15 on BMPER expression. Along the same lines, knock down of KLF-15 in HUVEC by siRNA reduced BMPER expression. The transactivating effect of KLF-15 could be competed away by coexpression of Sp-1 suggesting that both factors may compete for the same binding site in the BMPER promoter. In EMSA, an oligo representing a well characterized KLF-15 binding site in the AceCs2 promoter but not an oligo encoding for a NFkappa-B site competed with the oligo coding for the KLF-15 site in the BMPER promoter. In contrast FoxO3A, a member of the FoxO family of transcription factors, serves as an inhibitor of BMPER expression, as shown by gain and lack of FoxO3A experiments. Additionally, we found that BMPER stimulates angiogenesis in a BMP-4 dependent manner in several in vitro and in vivo assays. Vice versa, BMPER is necessary for BMP-4 to exert is angiogenic activity on endothelial cells. Conclusion: BMPER is upregulated by KLF-15 and inhibited by FoxO3a. BMPER has angiogenic activity and is a key modulator of the BMP pathway.
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Meng, Guoliang, Liping Xie, and Yong Ji. "Abstract 72: Hydrogen Sulfide Prevents Myocardial Hypertrophy in a Klf5-dependent Manner." Circulation Research 117, suppl_1 (July 17, 2015). http://dx.doi.org/10.1161/res.117.suppl_1.72.
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Rationale: H 2 S is a gasotransmitter that regulates multiple cardiovascular functions. Krüppel-like transcription factor (KLF) exerts diverse functions in the cardiovascular system. Objectives: The aim of present study was to investigate the effect of hydrogen sulfide (H 2 S) on myocardial hypertrophy. Methods and results: Myocardial samples of 22 patients with left ventricle hypertrophy were collected and underwent histological and molecular biological analysis. Spontaneously hypertensive rats (SHR) and neonatal rat cardiomyocytes were studied for functional and signaling response to GYY4137, a H 2 S-releasing compound. Expression of cystathionine -lyase (CSE), a main enzyme for H 2 S generation in human heart, decreased in human hypertrophic myocardium, while KLF5 expression increased. In SHR treated with GYY4137 for 4 weeks, myocardial hypertrophy was inhibited as evidenced by improvement in cardiac structural parameters, heart mass index, size of cardiac myocytes and expression of atrial natriuretic peptide (ANP). Levels of oxidative stress and phosphorylation of mitogen-activated protein kinases were also decreased after H 2 S treatment. H 2 S diminished expression of the KLF5 in myocardium of SHR and in neonatal rat cardiomyocytes rendered hypertrophy by angiotensin II (Ang II). H 2 S also inhibited ANP promoter activity and ANP expression in Ang II-induced neonatal rat cardiomyocyte hypertrophy, and these effects were suppressed by KLF5 knockdown. KLF5 promoter activity was increased by Ang II stimulation, and this was reversed by H 2 S. H 2 S also decreased activity of specificity protein-1 (SP-1) binding to the KLF5 promoter and attenuated KLF5 nuclear translocation by Ang II stimulation. Conclusion: H 2 S attenuated myocardial hypertrophy, which might be related to inhibiting oxidative stress and decreasing ANP transcription activity in a KLF5-dependent manner.
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Schmidt, Arne, Maximilian Fuchs, Stevan D. Stojanović, Chunguang Liang, Kevin Schmidt, Mira Jung, Ke Xiao, et al. "Deciphering Pro-angiogenic Transcription Factor Profiles in Hypoxic Human Endothelial Cells by Combined Bioinformatics and in vitro Modeling." Frontiers in Cardiovascular Medicine 9 (June 17, 2022). http://dx.doi.org/10.3389/fcvm.2022.877450.
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BackgroundConstant supply of oxygen is crucial for multicellular tissue homeostasis and energy metabolism in cardiac tissue. As a first response to acute hypoxia, endothelial cells (ECs) promote recruitment and adherence of immune cells to the dysbalanced EC barrier by releasing inflammatory mediators and growth factors, whereas chronic hypoxia leads to the activation of a transcription factor (TF) battery, that potently induces expression of growth factors and cytokines including platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). We report a hypoxia-minded, targeted bioinformatics approach aiming to identify and validate TFs that regulate angiogenic signaling.ResultsA comprehensive RNA-Seq dataset derived from human ECs subjected to normoxic or hypoxic conditions was selected to identify significantly regulated genes based on (i) fold change (normoxia vs. hypoxia) and (ii) relative abundancy. Transcriptional regulation of this gene set was confirmed via qPCR in validation experiments where HUVECs were subjected to hypoxic conditions for 24 h. Screening the promoter and upstream regulatory elements of these genes identified two TFs, KLF5 and SP1, both with a potential binding site within these regions of selected target genes. In vitro, siRNA experiments confirmed SP1- and KLF5-mediated regulation of identified hypoxia-sensitive endothelial genes. Next to angiogenic signaling, we also validated the impact of TFs on inflammatory signaling, both key events in hypoxic sensing. Both TFs impacted on inflammatory signaling since endogenous repression led to increased NF-κB signaling. Additionally, SP1 silencing eventuated decreased angiogenic properties in terms of proliferation and tube formation.ConclusionBy detailed in silico analysis of promoter region and upstream regulatory elements for a list of hypoxia-sensitive genes, our bioinformatics approach identified putative binding sites for TFs of SP or KLF family in vitro. This strategy helped to identify TFs functionally involved in human angiogenic signaling and therefore serves as a base for identifying novel RNA-based drug entities in a therapeutic setting of vascularization.
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Finco, Timothy Scott, Victoria E. Hamilton, Shivani J. Patel, Cindy Zheng, and Geri E. Justice. "Regulation of the human LAT gene by Ets and Sp/KLF transcription factors." FASEB Journal 20, no. 4 (March 2006). http://dx.doi.org/10.1096/fasebj.20.4.a82-a.
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Ilsley, Melissa D., Stephen Huang, Graham W. Magor, Michael J. Landsberg, Kevin R. Gillinder, and Andrew C. Perkins. "Corrupted DNA-binding specificity and ectopic transcription underpin dominant neomorphic mutations in KLF/SP transcription factors." BMC Genomics 20, no. 1 (May 24, 2019). http://dx.doi.org/10.1186/s12864-019-5805-z.
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mukund, kavitha, and Shankar Subramaniam. "Signed Differential Co‐Expression Network Analysis Suggests Differential Regulation of SP/KLF Family of Transcription Factors in Dilated Cardiomyopathy." FASEB Journal 32, S1 (April 2018). http://dx.doi.org/10.1096/fasebj.2018.32.1_supplement.803.5.
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Gura, Megan A., Soňa Relovská, Kimberly M. Abt, Kimberly A. Seymour, Tong Wu, Haskan Kaya, James M. A. Turner, Thomas G. Fazzio, and Richard N. Freiman. "TAF4b transcription networks regulating early oocyte differentiation." Development 149, no. 3 (February 1, 2022). http://dx.doi.org/10.1242/dev.200074.
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ABSTRACT Establishment of a healthy ovarian reserve is contingent upon numerous regulatory pathways during embryogenesis. Previously, mice lacking TBP-associated factor 4b (Taf4b) were shown to exhibit a diminished ovarian reserve. However, potential oocyte-intrinsic functions of TAF4b have not been examined. Here, we use a combination of gene expression profiling and chromatin mapping to characterize TAF4b-dependent gene regulatory networks in mouse oocytes. We find that Taf4b-deficient oocytes display inappropriate expression of meiotic, chromatin modification/organization, and X-linked genes. Furthermore, dysregulated genes in Taf4b-deficient oocytes exhibit an unexpected amount of overlap with dysregulated genes in oocytes from XO female mice, a mouse model of Turner Syndrome. Using Cleavage Under Targets and Release Using Nuclease (CUT&RUN), we observed TAF4b enrichment at genes involved in chromatin remodeling and DNA repair, some of which are differentially expressed in Taf4b-deficient oocytes. Interestingly, TAF4b target genes were enriched for Sp/Klf family and NFY target motifs rather than TATA-box motifs, suggesting an alternative mode of promoter interaction. Together, our data connect several gene regulatory nodes that contribute to the precise development of the mammalian ovarian reserve.
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Houser, Josef, Kristina Jendruchova, Andrea Knight, and Martin Piskacek. "The NFkB activation domain is 14-amino-acid-long variant of the 9aaTAD." Biochemical Journal, February 24, 2023. http://dx.doi.org/10.1042/bcj20220605.
Full textAbstract:
The nine-amino-acid TransActivation Domains (9aaTAD) was identified in numerous transcription factors including Gal4, p53, E2A, MLL, c-Myc, N-Myc, and also in SP, KLF and SOX families. Most of the 9aaTAD domains interact with the KIX domain of transcription mediators MED15 and CBP to activate transcription. The NFkB activation domain occupied the same position on the KIX domain as the 9aaTADs of MLL, E2A and p53. Binding of the KIX domain is established by the two-point interaction involving 9aaTAD positions p3-4 and p6-7. The NFkB primary binding region (position p3-4) is almost identical to MLL and E2A, but secondary NFkB binding region differs by the position and engages the distal NFkB region p10-11. Thus, the NFkB activation domain is 5 amino acids longer than the other 9aaTADs. The NFkB activation domain includes an additional region, which we called the Omichinski Insert extending activation domain length to 14 amino acids. By deletion, we demonstrated that Omichinski Insert is an entirely non-essential part of NFkB activation domain. In summary, we recognized the NFkB activation domain as prolonged 9aaTAD conserved in evolution from humans to amphibians.
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Koike, Nobuyuki, Jun Sugimoto, Motonori Okabe, Kenichi Arai, Makiko Nogami, Hiroshi Okudera, and Toshiko Yoshida. "Distribution of Amniotic Stem Cells in Human Term Amnion Membrane." Microscopy, September 18, 2021. http://dx.doi.org/10.1093/jmicro/dfab035.
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Abstract Amnion membrane studies related to miscarriage have been conducted in the field of obstetrics and gynecology. However, the distribution of stem cells within the amnion, and the differences in the properties of each type of stem cells are still not well understood. We address this gap in knowledge in the present study where we morphologically classified, the amnion membrane, and we clarified the distribution of stem cells here to identify functionally different amniotic membrane-derived stem cells. The amnion is composed of the chorion frondosum region [umbilical cord -adjacent amnion (area A) and the placenta-covered amnion surrounding the umbilical cord (area B)] as well as the reflected amnion (area C). We found that human amnion epithelial stem cells (HAEC) that strongly express stem cell markers were abundant in region A. In addition to having the surface markers TRA-1-60, Tra-1-81, SSEA4 and SSEA3, HAEC are OCT-3/4 positive and have alkalinephosphatase activity. Human amniotic mesenchymal stem cells (HAMC) expressed CD73, and were found in region A and B, the expression of BCRP which is related to isolate stem cells as called SP population cells. Other cells that expressed the undifferentiated transcription factors KLF-A, OCTA, Oct3/4, c-MYC, and Sox2 were diffusely distributed in region C. These data suggest that different types of stem cells exist each functional region. Thus, understanding the distribution of the subclasses of stem cells would allow for the efficient harvest of suitable HAE and HAM stem cells for disease.
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Lu, Tianyuan, and Jessica C. Mar. "Investigating transcriptome-wide sex dimorphism by multi-level analysis of single-cell RNA sequencing data in ten mouse cell types." Biology of Sex Differences 11, no. 1 (November 5, 2020). http://dx.doi.org/10.1186/s13293-020-00335-2.
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Abstract Background It is a long established fact that sex is an important factor that influences the transcriptional regulatory processes of an organism. However, understanding sex-based differences in gene expression has been limited because existing studies typically sequence and analyze bulk tissue from female or male individuals. Such analyses average cell-specific gene expression levels where cell-to-cell variation can easily be concealed. We therefore sought to utilize data generated by the rapidly developing single cell RNA sequencing (scRNA-seq) technology to explore sex dimorphism and its functional consequences at the single cell level. Methods Our study included scRNA-seq data of ten well-defined cell types from the brain and heart of female and male young adult mice in the publicly available tissue atlas dataset, Tabula Muris. We combined standard differential expression analysis with the identification of differential distributions in single cell transcriptomes to test for sex-based gene expression differences in each cell type. The marker genes that had sex-specific inter-cellular changes in gene expression formed the basis for further characterization of the cellular functions that were differentially regulated between the female and male cells. We also inferred activities of transcription factor-driven gene regulatory networks by leveraging knowledge of multidimensional protein-to-genome and protein-to-protein interactions and analyzed pathways that were potential modulators of sex differentiation and dimorphism. Results For each cell type in this study, we identified marker genes with significantly different mean expression levels or inter-cellular distribution characteristics between female and male cells. These marker genes were enriched in pathways that were closely related to the biological functions of each cell type. We also identified sub-cell types that possibly carry out distinct biological functions that displayed discrepancies between female and male cells. Additionally, we found that while genes under differential transcriptional regulation exhibited strong cell type specificity, six core transcription factor families responsible for most sex-dimorphic transcriptional regulation activities were conserved across the cell types, including ASCL2, EGR, GABPA, KLF/SP, RXRα, and ZF. Conclusions We explored novel gene expression-based biomarkers, functional cell group compositions, and transcriptional regulatory networks associated with sex dimorphism with a novel computational pipeline. Our findings indicated that sex dimorphism might be widespread across the transcriptomes of cell types, cell type-specific, and impactful for regulating cellular activities.