KLF4 Research Articles

Non‐invasively derived human induced mesenchymal stem cells demonstrate an enhanced protection of cardiomyocytes from injuries

BackgroundMesenchymal stem are multipotent stem cells used for treating various diseases including cardiomyopathy. However, their heterogeneity, limited proliferation and low retention in the transplanted tissue restricts their application for cell therapy. Recently, induced pluripotent stem cells (iPSCs)‐derived mesenchymal stem cells (iMSCs) have created an exciting source of cells for autologous therapy.ObjectiveIn this study, we have compared the therapeutic potential of non‐invasively derived iMSCs with adult umbilical cord MSCs (hereafter referred as MSCs) for the protection of cardiomyocytes from injury.Methods and ResultsThe urinary epithelial cells obtained from adult human urine were safely reprogrammed into iPSCs using a mixture of mRNA of pluripotency genes (Oct4, Nanog, Sox2, Klf4, cMyc and Lin28) along with a cocktail of miRNAs. The generated iPSCs were subsequently differentiated into iMSCs, which had the capacity to differentiate into osteocytes, chondrocytes and adipocytes. Our comparative qRT‐PCR and Western blot analysis showed that the mesenchymal‐specific CD73, CD90 and CD105 gene transcripts and proteins were highly expressed in both iMSCs and MSCs. But the iMSCs maintained their mesenchymal markers even at a late passage (P18), during which the MSCs started losing their characteristics. Scratch assay and transwell migration assay demonstrated that iMSCs had a greater wound healing property compared to MSCs. Furthermore, the cardioprotective properties of iMSCs and their exosomes (obtained from the conditioned media by precipitation method) were evaluated using human skin fibroblast‐derived induced‐cardiomyocytes, treated with 50 µg of exosomes 1 hr before subjecting them to either 24 hrs of angiotensin II treatment (10 µM) or 6 hrs of 1% hypoxia and 24 hrs of reoxygenation. In comparison with MSCs, iMSCs and their exosomes elicited an enhanced cardiomyocyte protection studied through measurement of mitochondrial membrane potentials (JC1 dye), intracellular reactive oxygen species (CM‐H2DCFDA), in situ cell death by apoptosis, and qRT‐PCR expression of survival genes. Further studies are underway to evaluate the specific role of exosomal RNA and protein in mediating the cardioprotection.ConclusionIn this study, we have demonstrated a non‐invasive method of generating iMSCs for regenerative therapy. This homogeneous and highly proliferative iMSCs may provide an alternative source of cells or cell‐free exosomes for treating ischemic cardiomyopathy.

Identification and Validation of Reference Genes for Gene Expression Analysis in Monochamus saltuarius Under Bursaphelenchus xylophilus Treatment.

A special mutual relationship exists between the pine wood nematode (PWN) Bursaphelenchus xylophilus and its vector beetles of genus Monochamus, which enables PWN to spread, at the same time provides longhorned beetles with more weak hosts. PWN are attracted to the pupal chambers and then carried inside the trachea of beetle adults, which is a necessary part to complete the B. xylophilus infection cycle. The growth and immune responses of the vector beetle will affect this carrying process, however, they were rarely studied in Monochamus saltuarius. Real-time quantitative polymerase chain reaction (RT-qPCR), one of the most common methods for quantitative gene expression analysis, was performed to explore the key genes and pathways involved in the growth, development and immune responses of M. saltuarius at different developmental stages associated with infection of PWN and PWN treatment conditions. To enhance the accuracy of RT-qPCR data, the expression of target genes needs to be normalized with reference genes, which are stably expressed under varied experimental conditions. In our study, the stability of 14 candidate reference genes in M. saltuarius samples at different developmental stages associated with infection of PWN or PWN treatment conditions was evaluated using delta Ct, geNorm, NormFinder, BestKeeper and RefFinder algorithms. Moreover, KLF gene was used to validate the stability of the selected reference genes. Under experimental conditions of this study, RPL7 and TER were suitable reference genes at different developmental stages associated with infection of PWN. RPL7 and RPS5 were considered the most stable reference genes in the pupae treated with PWN. RPS5 and SNX6 could be used as reference genes in the adults treated with PWN. RPL7, EF1-γ, and RPS5 could be used as stable reference genes in all the samples. This work is the first to evaluate reference genes in M. saltuarius, laying a foundation for further gene expression experimental procedures and understanding the phoretic relationship between M. saltuarius and B. xylophilus.

Insulin-like Growth Factor I Couples Metabolism with Circadian Activity through Hypothalamic Orexin Neurons.

Uncoupling of metabolism and circadian activity is associated with an increased risk of a wide spectrum of pathologies. Recently, insulin and the closely related insulin-like growth factor I (IGF-I) were shown to entrain feeding patterns with circadian rhythms. Both hormones act centrally to modulate peripheral glucose metabolism; however, whereas central targets of insulin actions are intensely scrutinized, those mediating the actions of IGF-I remain less defined. We recently showed that IGF-I targets orexin neurons in the lateral hypothalamus, and now we evaluated whether IGF-I modulates orexin neurons to align circadian rhythms with metabolism. Mice with disrupted IGF-IR activity in orexin neurons (Firoc mice) showed sexually dimorphic alterations in daily glucose rhythms and feeding activity patterns which preceded the appearance of metabolic disturbances. Thus, Firoc males developed hyperglycemia and glucose intolerance, while females developed obesity. Since IGF-I directly modulates orexin levels and hepatic expression of KLF genes involved in circadian and metabolic entrainment in an orexin-dependent manner, it seems that IGF-I entrains metabolism and circadian rhythms by modulating the activity of orexin neurons.

Transcription factors KLF15 and PPARδ cooperatively orchestrate genome-wide regulation of lipid metabolism in skeletal muscle

Skeletal muscle dynamically regulates systemic nutrient homeostasis through transcriptional adaptations to physiological cues. In response to changes in the metabolic environment (e.g., alterations in circulating glucose or lipid levels), networks of transcription factors and coregulators are recruited to specific genomic loci to fine-tune homeostatic gene regulation. Elucidating these mechanisms is of particular interest as these gene regulatory pathways can serve as potential targets to treat metabolic disease. The zinc-finger transcription factor Krüppel-like factor 15 (KLF15) is a critical regulator of metabolic homeostasis; however, its genome-wide distribution in skeletal muscle has not been previously identified. Here, we characterize the KLF15 cistrome in vivo in skeletal muscle and find that the majority of KLF15 binding is localized to distal intergenic regions and associated with genes related to circadian rhythmicity and lipid metabolism. We also identify critical interdependence between KLF15 and the nuclear receptor PPARδ in the regulation of lipid metabolic gene programs. We further demonstrate that KLF15 and PPARδ colocalize genome-wide, physically interact, and are dependent on one another to exert their transcriptional effects on target genes. These findings reveal that skeletal muscle KLF15 plays a critical role in metabolic adaptation through its direct actions on target genes and interactions with other nodal transcription factors such as PPARδ.

Multiplexed genome regulation in vivo with hyper-efficient Cas12a.

Multiplexed modulation of endogenous genes is crucial for sophisticated gene therapy and cell engineering. CRISPR-Cas12a systems enable versatile multiple genomic loci targeting by processing numerous crRNAs from a single transcript, however, their low efficiency has hindered applications in vivo. Through structure-guided protein engineering, we develop a hyper-efficient LbCas12a variant, termed hyperCas12a, with its catalytically dead version hyperdCas12a showing significantly enhanced efficacy for gene activation, particularly at low crRNA conditions. We demonstrate that hyperdCas12a has minimal off-target effects compared to the wildtype system and exhibits enhanced activity for gene editing and repression. Delivery of the hyperdCas12a-activator and a single crRNA array simultaneously activating endogenous Oct4, Sox2, and Klf4 genes in the retina of postnatal mice alters the differentiation of retinal progenitor cells. The hyperCas12a system offers a versatile in vivo tool for a broad range of gene modulation and gene therapy applications.

Congenital Dyserythropoietic Anemia Type IV with Kruppel-Like Factor 1 E325K Mutation in a Preterm Neonate

Background: Clinical, pathologic, and genetic heterogeneity is a challenge in identifying and classifying congenital dyserythropoietic anemia (CDA). CDA type IV, the rarest CDA with only 11 reported cases, results from KLF1 gene mutation. Clinical Description: A male preterm neonate presented with jaundice, anemia, pulmonary hypertension and hepatosplenomegaly in the immediate postnatal period, requiring multiple red blood cell transfusions. Management and Outcome: The workup for non-immune haemolytic anemia including red blood cell structural and enzymatic studies and were normal, with peripheral blood smear showing multiple polychromatic cells and numerous nucleated red blood cells including binucleate ones and fetal haemoglobin of 91.2%. Genetic testing revealed KLF1 E325K mutation suggestive of CDA type IV, though parental testing was normal, suggesting de novo mutation. The infant has been receiving packed RBC transfusion every three to four weeks initially and then every two months. The baby is now of twelve months of age, and receives oral vitamin B12 and folic acid supplementation for ineffective erythropoiesis. Though his weight is in the 3rd centile for age and height, he has been developmentally normal. Conclusions: Our report, the first description of a CDA type IV diagnosis in the neonatal period, adds to the limited knowledge of this disorder, which we also comprehensively review. The report highlights the phenotype of the disorder and the importance of neonatal genetic testing in a case of transfusion dependent anemia, having ruled out other causes.

WDR82-binding long noncoding RNA lncEry controls mouse erythroid differentiation and maturation.

Hematopoietic differentiation is controlled by both genetic and epigenetic regulators. Long noncoding RNAs (lncRNAs) have been demonstrated to be important for normal hematopoiesis, but their function in erythropoiesis needs to be further explored. We profiled the transcriptomes of 16 murine hematopoietic cell populations by deep RNA sequencing and identified a novel lncRNA, Gm15915, that was highly expressed in erythroid-related progenitors and erythrocytes. For this reason, we named it lncEry. We also identified a novel lncEry isoform, which was the principal transcript that has not been reported before. lncEry depletion impaired erythropoiesis, indicating the important role of the lncRNA in regulating erythroid differentiation and maturation. Mechanistically, we found that lncEry interacted with WD repeat-containing protein 82 (WDR82) to promote the transcription of Klf1 and globin genes and thus control the early and late stages of erythropoiesis, respectively. These findings identified lncEry as an important player in the transcriptional regulation of erythropoiesis.

KLF15 cistromes reveal a hepatocyte pathway governing plasma corticosteroid transport and systemic inflammation.

Circulating corticosteroids orchestrate stress adaptation, including inhibition of inflammation. While pathways governing corticosteroid biosynthesis and intracellular signaling are well understood, less is known about mechanisms controlling plasma corticosteroid transport. Here, we show that hepatocyte KLF15 (Kruppel-like factor 15) controls plasma corticosteroid transport and inflammatory responses through direct transcriptional activation of Serpina6, which encodes corticosteroid-binding globulin (CBG). Klf15-deficient mice have profoundly low CBG, reduced plasma corticosteroid binding capacity, and heightened mortality during inflammatory stress. These defects are completely rescued by reconstituting CBG, supporting that KLF15 works primarily through CBG to control plasma corticosterone homeostasis. To understand transcriptional mechanisms, we generated the first KLF15 cistromes using newly engineered Klf153xFLAG mice. Unexpectedly, liver KLF15 is predominantly promoter enriched, including Serpina6, where it binds a palindromic GC-rich motif, opens chromatin, and transactivates genes with minimal associated direct gene repression. Overall, we provide critical mechanistic insight into KLF15 function and identify a hepatocyte-intrinsic transcriptional module that potently regulates systemic corticosteroid transport and inflammation.

Differentiation and molecular characterization of endothelial progenitor and vascular smooth muscle cells from induced pluripotent stem cells.

Pluripotent stem cells have been used by various researchers to differentiate and characterize endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) for the clinical treatment of vascular injuries. Studies continue to differentiate and characterize the cells with higher vascularization potential and low risk of malignant transformation to the recipient. Unlike previous studies, this research aimed to differentiate induced pluripotent stem (iPS) cells into endothelial progenitor cells (EPCs) and VSMCs using a step-wise technique. This was achieved by elucidating the spatio-temporal expressions of the stage-specific genes and proteins during the differentiation process. The presence of highly expressed oncogenes in iPS cells was also investigated during the differentiation period. Induced PS cells were differentiated into lateral mesoderm cells (Flk1+). The Flk1+ populations were isolated on day 5.5 of the mesodermal differentiation period. Flk1+ cells were further differentiated into EPCs and VSMCs using VEGF165 and platelet-derived growth factor-BB (PDGF-BB), respectively, and then characterized using gene expression levels, immunocytochemistry (ICC), and western blot (WB) methods. During the differentiation steps, the expression levels of the marker genes and proto-oncogenic Myc and Klf4 genes were simultaneously studied. The optimal time for the isolation of Flk1+ cells was on day 5.5. EPCs and VSMCs were differentiated from Flk1+ cells and characterized with EPC-specific markers, including Kdr, Pecam1, CD133, Cdh5, Efnb2, Vcam1; and VSMC-specific markers, including Acta2, Cnn1, Des, and Myh11. Differentiated cells were validated based on their temporal gene expressions, protein synthesis, and localization at certain time points. Significant decreases in Myc and Klf4 gene expression levels were observed during the EPCs and VSMC differentiation period. EPCs and VSMCs were successfully differentiated from iPS cells and characterized by gene expression levels, ICC, and WB. We observed significant decreases in oncogene expression levels in the differentiated EPCs and VSMCs. In terms of safety, the described methodology provided a better safety margin. EPCs and VSMC obtained using this method may be good candidates for transplantation and vascular regeneration.

Identification of key genes in pathogenesis of placental insufficiency intrauterine growth restriction

BackgroundIntrauterine growth restriction (IUGR) is defined as a fetus that fails to achieve its genetically determined growth potential. The exact molecular mechanisms of placental insufficiency IUGR pathogenesis are a little known. Our goal was to identify key genes and gene co-expression modules related to placental insufficiency IUGR.MethodsWe used weighted gene co-expression network analysis (WGCNA) and protein-protein interaction (PPI) network analysis to examine the IUGR dataset GSE114691 from NCBI Gene Expression Omnibus. Core modules and hub nodes of the protein-protein interaction network were identified. A gene network was constructed and genes were classified by WGCNA into different modules. The validation of potential key genes was carried out using additional datasets (GSE12216 and GSE24129).ResultsWe identified in GSE114691 539 down regulated genes and 751 up regulated genes in placental tissues characteristic of placental insufficiency IUGR compared with non-IUGR, and defined 76 genes as hub nodes in the protein-protein interaction network. Genes in the key modules of the WGCNA network were most closely associated with placental insufficiency IUGR and significantly enriched in biological process such as cellular metabolic process and macromolecule metabolic process. We identified as key genes TGFB1, LEP, ENG, ITGA5, STAT5A, LYN, GATA3, FPR1, TGFB2, CEBPB, KLF4, FLT1, and PNPLA2. The RNA expression levels of ENG and LEP, as biomarkers, were validated.ConclusionA holistic gene expression profile of placental insufficiency IUGR has been generated and the key genes ENG and LEP has potential to serve as circulating diagnosis biomarkers and therapeutic targets for placental insufficiency IUGR.

Reduced Gene Expression and Genetic Variation in Kruppel-Like Factor 15 Are Associated with Left Ventricular Hypertrophy and All-Cause Mortality in Patients with Aortic Stenosis

Kruppel-like factor 15 (KLF15) is expressed in the heart and is a repressor of cardiac hypertrophy. We previously reported the KLF15 single nucleotide polymorphism (SNP) rs9838915 A allele is associated with left ventricular hypertrophy (LVH) and adverse outcomes in diabetes. As LVH is prevalent in aortic stenosis (AS), in this study we investigate myocardial KLF15 gene expression in patients with AS, determine if rs9838915 and LVH predict mortality, and perform functional studies to elucidate mechanisms.

Chemotherapy-induced adenosine A2B receptor expression mediates epigenetic regulation of pluripotency factors and promotes breast cancer stemness.

Rationale: Triple-negative breast cancer (TNBC) is characterized by its unique molecular profile, aggressive nature and lack of targeted therapy. Chemotherapy induces expression of pluripotency factors and mediates an active induction of breast cancer stem cells (BCSCs) in TNBC, which potentiates the risk of tumor recurrence and metastasis and increases patient mortality. Adenosine receptor 2B (A2BR) expression and activation of its downstream signaling pathway has been implied to promote breast cancer metastasis. This study is to investigate the role of A2BR in the regulation of chemotherapy-induced BCSC enrichment.Methods: We generated shRNA-mediated A2BR knockdown subclones in TNBC cell lines and evaluated the effect on the BCSC phenotype by Aldefluor and mammosphere assays in vitro. We performed chromatin immunoprecipitation (ChIP) assay to investigate recruitment of transcription factor FOXO3 and histone modification enzymes KDM6A and p300 to the regulatory regions of pluripotency factors, as well as levels of histone modification marks H3K27ac and H3K27me3 on these regions. We employed both xenograft model and genetically engineered, autochthonous breast cancer model to evaluate the effect of A2BR on chemotherapy-induced BCSC enrichment in vivo.Results: We demonstrated that chemotherapy increased protein level of A2BR, which contributed to chemotherapy-induced pluripotency factor expression and BCSC enrichment in TNBC. A2BR mediated activation of p38 MAPK and nuclear translocation of chromatin remodeling factor SMARCD3, which interacted and recruited histone demethylase KDM6A and histone acetyltransferase p300 specifically to the pluripotency factor genes NANOG, SOX2 and KLF4. Recruitment of KDM6A and p300 decreased histone H3K27me3 and increases H3K27ac marks, and increased transcription factor FOXO3 binding to NANOG, SOX2 and KLF4 genes, leading to transcriptional activation of these genes and BCSC specification. Genetic or pharmacological inhibition of A2BR blocked chemotherapy-mediated epigenetic activation of pluripotency factor genes and BCSC enrichment in vitro and in vivo, and delayed tumor recurrence after chemotherapy was discontinued.Conclusion: Chemotherapy-induced A2BR expression mediates epigenetic activation of pluripotency factors and promotes breast cancer stemness. Targeting A2BR in combination with chemotherapy may block BCSC enrichment and improve outcome in TNBC.

Performance of KLF4 and Wnt1 genes expression fluctuations in tumoral and margin tissues of colorectal cancer in an Iranian population

Introduction and objectiveColorectal cancer is the third leading cause of death in the world. The disease can be prevented and treated with early diagnosis. The KLF4 and Wnt1 genes, in their natural state, participate in cell growth, proliferation, and differentiation by activating a variety of transcription factors and regulating the expression of some genes. This study aimed to investigate the expression of KLF4 and Wnt1 in tumoral and margin tissues of colorectal cancer and also normal tissues in an Iranian population. Materials and methodsIn this case study, 30 specimens of tumoral tissues of colorectal cancer and 30 margin tissues or non-neoplastic counterpart tissues, as well as 20 samples of normal colorectal tissues were collected from patients referred to the Surgery room of Razi Hospital in Rasht, Iran, 2019. The Real-Time PCR technique was used to evaluate the expression of genes. SPSS software and ANOVA statistical tests, t-test, regression were used to analyze the data. P-value values of 0.05 were considered significant. ResultsThe expression of KLF4 gene in tumoral tissues was reduced (p = 0.01) in comparison to margin tissues but had no significant relationship with tumor characteristics. The expression of Wnt1 gene in tumoral specimens was significantly increased (p = 0.001). Also, a significant relationship was observed between Wnt1 gene expression and tumor stage (p = 0.001) and tumor grade (p = 0.003). ConclusionThe data showed that there is a significant relationship between Wnt1 and KLF4 gene expression of colorectal cancer and may be used in diagnosis and treatment.

KLF15 overexpression in myocytes fails to ameliorate ALS-related pathology or extend the lifespan of SOD1G93A mice

Amyotrophic Lateral Sclerosis (ALS) is a currently incurable disease that causes progressive motor neuron loss, paralysis and death. Skeletal muscle pathology occurs early during the course of ALS. It is characterized by impaired mitochondrial biogenesis, metabolic dysfunction and deterioration of the neuromuscular junction (NMJ), the synapse through which motor neurons communicate with muscles. Therefore, a better understanding of the molecules that underlie this pathology may lead to therapies that slow motor neuron loss and delay ALS progression. Kruppel Like Factor 15 (KLF15) has been identified as a transcription factor that activates alternative metabolic pathways and NMJ maintenance factors, including Fibroblast Growth Factor Binding Protein 1 (FGFBP1), in skeletal myocytes. In this capacity, KLF15 has been shown to play a protective role in Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA), however its role in ALS has not been evaluated. Here, we examined whether muscle-specific KLF15 overexpression promotes the health of skeletal muscles and NMJs in the SOD1G93A ALS mouse model. We show that muscle-specific KLF15 overexpression did not elicit a significant beneficial effect on skeletal muscle atrophy, NMJ health, motor function, or survival in SOD1G93A ALS mice. Our findings suggest that, unlike in mouse models of DMD and SMA, KLF15 overexpression has a minimal impact on ALS disease progression in SOD1G93A mice.

Activation of PKG-CREB-KLF15 by melatonin attenuates Angiotensin II-induced vulnerability to atrial fibrillation via enhancing branched-chain amino acids catabolism

Mitochondrial reactive oxygen species (ROS) damage and atrial remodeling serve as the crucial substrates for the genesis of atrial fibrillation (AF). Branched-chain amino acids (BCAAs) catabolic defect plays critical roles in multiple cardiovascular diseases. However, the alteration of atrial BCAA catabolism and its role in AF remain largely unknown. This study aimed to explore the role of BCAA catabolism in the pathogenesis of AF and to further evaluate the therapeutic effect of melatonin with a focus on protein kinase G (PKG)-cAMP response element binding protein (CREB)-Krüppel-like factor 15 (KLF15) signaling. We found that angiotensin II-treated atria exhibited significantly elevated BCAA level, reduced BCAA catabolic enzyme activity, increased AF vulnerability, aggravated atrial electrical and structural remodeling, and enhanced mitochondrial ROS damage. These deleterious effects were attenuated by melatonin co-administration while exacerbated by BCAA oral supplementation. Melatonin treatment ameliorated BCAA-induced atrial damage and reversed BCAA-induced down-regulation of atrial PKGIα expression, CREB phosphorylation as well as KLF15 expression. However, inhibition of PKG partly abolished melatonin-induced beneficial actions. In summary, these data demonstrated that atrial BCAA catabolic defect contributed to the pathogenesis of AF by aggravating tissue fibrosis and mitochondrial ROS damage. Melatonin treatment ameliorated Ang II-induced atrial structural as well as electrical remodeling by activating PKG-CREB-KLF15. The present study reveals additional mechanisms contributing to AF genesis and highlights the opportunity of a novel therapy for AF by targeting BCAA catabolism. Melatonin may serve as a potential therapeutic agent for AF intervention.

Erythropoietic properties of human induced pluripotent stem cells-derived red blood cells in immunodeficient mice.

Transfusion of red blood cells (RBCs) is a life-saving intervention for anemic patients. Human induced pluripotent stem cells (iPSC) have the capability to expand and differentiate into RBCs (iPSC-RBCs). Here we developed a murine model to investigate the in vivo properties of human iPSC-RBCs. iPSC lines were produced from human peripheral blood mononuclear cells by transient expression of plasmids containing OCT4, SOX2, MYC, KLF4, and BCL-XL genes. Human iPSC-RBCs were generated in culture supplemented with human platelet lysate, and were CD34- CD235a+ CD233+ CD49dlow CD71low ; about 13% of iPSC-RBCs were enucleated before transfusion. Systemic administration of clodronate liposomes (CL) and cobra venom factor (CVF) to NOD scid gamma (NSG) mice markedly promoted the circulatory survival of human iPSC-RBCs following transfusion. While iPSC-RBCs progressively decreased with time, 90% of circulating iPSC-RBCs were enucleated 1 day after transfusion (CD235a+ CD233+ CD49d- CD71- ). Surprisingly, human iPSC-RBCs reappeared in the peripheral circulation at 3 weeks after transfusion at levels more than 8-fold higher than at 1h after transfusion. Moreover, a substantial portion of the transfused nucleated iPSC-RBCs preferentially homed to the bone marrow, and were detectable at 24 days after transfusion. These results suggest that nucleated human iPSC-derived cells that homed to the bone marrow of NSG mice retained the capability to complete differentiation into enucleated erythrocytes and egress the bone marrow into peripheral blood. The results offer a new model using human peripheral blood-derived iPSC and CL/CVF-treated NSG mice to investigate the development and circulation of human erythroid cells in vivo.

Comparative Genomic Characterization of Buffalo Fibronectin Type III Domain Proteins: Exploring the Novel Role of FNDC5/Irisin as a Ligand of Gonadal Receptors.

Simple SummaryA total of 29 fibronectin type III domain (FN-III) protein genes from the buffalo genome were detected and characterized. The molecular and evolutionary analysis demonstrated the well-conserved nature of FN-III proteins with a variety of stable to unstable, hydrophobic to hydrophilic, and thermostable to thermo-unstable properties. For the first time, we predicted the binding scores and interface residues of FNDC5/irisin as a ligand for six representative receptors, having a functional role in energy homeostasis, and significant involvement in folliculogenesis, and spermatogenesis in buffalo.FN-III proteins are widely distributed in mammals and are usually involved in cellular growth, differentiation, and adhesion. The FNDC5/irisin regulates energy metabolism and is present in different tissues (liver, brain, etc.). The present study aimed to investigate the physiochemical characteristics and the evolution of FN-III proteins and FNDC5/irisin as a ligand targeting the gonadal receptors including androgen (AR), DDB1 and CUL4 associated factor 6 (DCAF6), estrogen-related receptor β (ERR-β), estrogen-related receptor γ (ERR-γ), Krüppel-like factor 15 (KLF15), and nuclear receptor subfamily 3 group C member 1 (NR3C1). Moreover, the putative role of irisin in folliculogenesis and spermatogenesis was also elucidated. We presented the molecular structure and function of 29 FN-III genes widely distributed in the buffalo genome. Phylogenetic analysis, motif, and conserved domain pattern demonstrated the evolutionary well-conserved nature of FN-III proteins with a variety of stable to unstable, hydrophobic to hydrophilic, and thermostable to thermo-unstable properties. The comparative structural configuration of FNDC5 revealed amino acid variations but still the FNDC5 structure of humans, buffalo, and cattle was quite similar to each other. For the first time, we predicted the binding scores and interface residues of FNDC5/irisin as a ligand for six representative receptors having a functional role in energy homeostasis, and a significant involvement in folliculogenesis and spermatogenesis in buffalo.

RNA-seq of buffalo fibroblasts over-expressed pluripotent-related genes to investigate characteristics of its preliminarily reprogrammed stage

Induced pluripotent stem cells (iPSCs) can enhance the efficiency of buffalo genetic improvements because of their differentiation potential and proliferation ability, which are similar to those of embryonic stem cells. However, very few studies have focussed on buffalo iPSCs, and a stable induction system has not been established for buffalo somatic cell reprogramming. In this study, we constructed a PiggyBac transposon vector co-expressing buffalo OCT4, C-MYC, KLF4 and SOX2 genes (PB_OMKS) separated by the nucleotide sequence of three 2A peptides and established the buffalo foetal skin fibroblast (BFSF) cell line BFSF_OMKS. RNA-seq technology and bioinformatics analysis methods were mainly employed to perform a transcriptome analysis between BFSF and BFSF_OMKS. The results revealed that over-expression of OCT4, C-MYC, KLF4 and SOX2 in BFSFs led to the activation of reprogramming-related LIF, activin, BMP4, SMAD1/5/9 and Wnt signals. These results increased our understanding of buffalo somatic cell reprogramming mechanisms and could provide a possible theory for the selection of small-molecule cocktails to promote reprogramming.

Stress Induced Transcription Factors Transactivate the Herpes Simplex Virus 1 Infected Cell Protein 27 (ICP27) Transcriptional Enhancer.

Following acute infection, herpes simplex virus 1 (HSV-1) establishes lifelong latency in neurons, including sensory neurons within trigeminal ganglia. During latency, lytic cycle viral gene expression is silenced. However, stressful stimuli can trigger reactivation from latency. The viral tegument protein, VP-16, transactivates all immediate early (IE) promoters during productive infection. Conversely, cellular factors are expected to trigger viral gene expression during early stages of reactivation from latency and in non-neuronal cells that do not support high levels of productive infection. The glucocorticoid receptor (GR), synthetic corticosteroid dexamethasone, and certain stress-induced transcription factors cooperatively transactivate infected cell protein 0 (ICP0) and ICP4 promoters. Since ICP27 protein expression is required for productive infection, we hypothesized that the ICP27 promoter is transactivated by stress-induced transcription factors. New studies have demonstrated that ICP27 enhancer sequences were transactivated by GR and Krüppel-like factor 15 (KLF15). Mutation of a consensus Sp1 binding site within ICP27 enhancer sequences impaired transactivation by GR and KLF15. Chromatin immunoprecipitation studies have demonstrated that GR and KLF15 occupy ICP27 promoter sequences during productive infection. Cells transfected with an ICP27 enhancer fragment revealed the GR and KLF15 occupancy of ICP27 enhancer sequences required the intact Sp1 binding site. Notably, GR and KLF15 form a feed-forward transcription loop in response to stress, suggesting these cellular factors promote viral replication following stressful stimuli.

FoxO-KLF15 pathway switches the flow of macronutrients under the control of insulin

SummaryKLF15 is a transcription factor that plays an important role in the activation of gluconeogenesis from amino acids as well as the suppression of lipogenesis from glucose. Here we identified the transcription start site of liver-specific KLF15 transcript and showed that FoxO1/3 transcriptionally regulates Klf15 gene expression by directly binding to the liver-specific Klf15 promoter. To achieve this, we performed a precise in vivo promoter analysis combined with the genome-wide transcription-factor-screening method “TFEL scan”, using our original Transcription Factor Expression Library (TFEL), which covers nearly all the transcription factors in the mouse genome. Hepatic Klf15 expression is significantly increased via FoxOs by attenuating insulin signaling. Furthermore, FoxOs elevate the expression levels of amino acid catabolic enzymes and suppress SREBP-1c via KLF15, resulting in accelerated amino acid breakdown and suppressed lipogenesis during fasting. Thus, the FoxO-KLF15 pathway contributes to switching the macronutrient flow in the liver under the control of insulin.