Transcription Factor Gene Expression Research Articles

#2949 Diverse effect of STAT3 inhibition on murine primary tubular epithelial cells

Abstract Background and Aims The incidence of chronic kidney diseases increases worldwide, and kidney fibrosis is the final common pathway regardless of the etiology. TGF-β plays a crucial role in the pathogenesis, affecting pro-fibrotic genes and transcription factors. STAT3 (signal transducer and activator of transcription 3) acts via the JAK/STAT signaling. Inhibition of STAT3 in animal studies was shown to attenuate the progression of kidney fibrosis. Yet, the effect of STAT3 inhibition on renal tubular epithelial cells has not been elucidated. We aimed to investigate in vitro the impact of pharmacological STAT3 inhibition with NSC74859 (NSC) and STATTIC on the expression of fibrosis associated genes of primary tubular epithelial cells. Method Primary tubular epithelial cells from kidneys of C57Bl6/J mice were isolated and used in our studies. Cells (P10-P15) were seeded on gelatin-coated 6-well culture plates (100000 cells/well) and subjected to treatment with 10 ng/ml TGF-β, 100 µM NSC, 10 µM STATTIC or vehicle (n = 3/group) as follows: Control; NSC; STATTIC; TGF-β; TGF-β+NSC; TGF-β+STATTIC. After 24 h treatment, mRNA and protein expressions were analyzed. Statistical evaluation was performed using Kruskal-Wallis test. Results In cells not treated with TGF-β, neither NSC nor STATTIC treatment affected the expression of Tgfb1 (TGF-β) and Acta2 (SMA) genes compared to controls (100%), but reduced Col1a1 expression to 58% and 54%, respectively (p < 0.05). NSC reduced to 54%, while STATTIC tended to increase (169%) the gene expression of Egr2 transcription factor. TGF-β treatment induced STAT3 phosphorylation by 45% accompanied by a fibrotic response and epithelial-to-mesenchymal transition (EMT) as indicated by significantly increased Tgfb1, Acta2, Egr2 and Col1a1 (p < 0.05). NSC treatment did not inhibit the TGF-β-induced Tgfb1 upregulation (178%), but markedly, although not significantly, reduced the expression of Acta2 (100%), Egr2 (98%) and Col1a1 (114%) in the TGF-β+NSC group. Interestingly, although STATTIC treatment dramatically inhibited STAT3 expression (38%, p < 0.05) it significantly enhanced the TGF-β-induced overexpression of Tgfb1 (210%, p < 0.01) and Egr2 (307%, p < 0.05), while markedly reduced Acta2 (64%, p < 0.001) and Col1a1 (81%, p < 0.01) in the TGF-β+STATTIC group. Conclusion Our results indicate that in vitro inhibition of STAT3 using two inhibitors with same mode of action lead to diverse responses in primary murine tubular epithelial cells, partially suppressing EMT markers but inducing pro-fibrotic EGR2. Further studies are needed to clarify the underlying mechanisms.

ALTERED MERISTEM PROGRAM1 sustains cellular differentiation by limiting HD-ZIP III transcription factor gene expression.

Plants show remarkable developmental and regenerative plasticity through the sustained activity of stem cells in meristems. Under certain conditions, pluripotency can even be re-established in cells that have already entered differentiation. Mutation of the putative carboxypeptidase ALTERED MERISTEM PROGRAM1 (AMP1) in Arabidopsis (Arabidopsis thaliana) causes a set of hypertrophic phenotypes, indicating a defect in the suppression of pluripotency. A role of AMP1 in the miRNA-mediated inhibition of translation has previously been reported, however, how this activity is related to its developmental functions is unclear. Here we examined the functional interaction between AMP1 and the Class III homeodomain-leucine zipper (HD-ZIP III) transcription factors, which are miRNA-controlled determinants of shoot meristem specification. We found that the HD-ZIP III transcriptional output is enhanced in the amp1 mutant and that plant lines with increased HD-ZIP III activity not only developed amp1 mutant-like phenotypes but also showed a synergistic genetic interaction with the mutant. Conversely, the reduction of HD-ZIP III function suppressed the shoot hypertrophy defects of the amp1 mutant. We further provide evidence that the expression domains of HD-ZIP III family members are expanded in the amp1 mutant and that this misexpression occurs at the transcriptional level and does not involve the function of miRNA165/166. Finally, amp1 mutant-specific phenotypes cannot be mimicked by a general inhibition of miRNA function in the AMP1 expression domain. These findings lead us to a model in which AMP1 restricts cellular pluripotency upstream of HD-ZIP III proteins and this control appears to be not directly mediated by the canonical miRNA pathway.

Genome-Wide Analysis of Transcription Factor R2R3-MYB Gene Family and Gene Expression Profiles during Anthocyanin Synthesis in Common Walnut (Juglans regia L.).

The R2R3-MYB gene family, encoding plant transcriptional regulators, participates in many metabolic pathways of plant physiology and development, including flavonoid metabolism and anthocyanin synthesis. This study proceeded as follows: the JrR2R3-MYB gene family was analyzed genome-wide, and the family members were identified and characterized using the high-quality walnut reference genome "Chandler 2.0". All 204 JrR2R3-MYBs were established and categorized into 30 subgroups via phylogenetic analysis. JrR2R3-MYBs were unevenly distributed over 16 chromosomes. Most JrR2R3-MYBs had similar structures and conservative motifs. The cis-acting elements exhibit multiple functions of JrR2R3-MYBs such as light response, metabolite response, and stress response. We found that the expansion of JrR2R3-MYBs was mainly caused by WGD or segmental duplication events. Ka/Ks analysis indicated that these genes were in a state of negative purifying selection. Transcriptome results suggested that JrR2R3-MYBs were widely entangled in the process of walnut organ development and differentially expressed in different colored varieties of walnuts. Subsequently, we identified 17 differentially expressed JrR2R3-MYBs, 9 of which may regulate anthocyanin biosynthesis based on the results of a phylogenetic analysis. These genes were present in greater expression levels in 'Zijing' leaves than in 'Lvling' leaves, as revealed by the results of qRT-PCR experiments. These results contributed to the elucidation of the functions of JrR2R3-MYBs in walnut coloration. Collectively, this work provides a foundation for exploring the functional characteristics of the JrR2R3-MYBs in walnuts and improving the nutritional value and appearance quality of walnuts.

Ubiquitin ligase VvPUB26 in grapevine promotes proanthocyanidin synthesis and resistance to powdery mildew.

Proanthocyanidins (PAs) are an important group of flavonoids that contribute to astringency, color, and flavor in grape (Vitis vinifera) and wines. They also play a crucial role in enhancing plant resistance to various stresses. However, the underlying regulatory mechanism governing PAs biosynthesis, particularly in relation to conferring resistance to powdery mildew, has not been extensively explored. This study focused on identifying a key player in PAs biosynthesis, namely the plant U-box (PUB) E3 ubiquitin ligase VvPUB26. We discovered that overexpression of VvPUB26 in grape leads to a significant increase in PAs content, whereas interfering with VvPUB26 has the opposite effect. Additionally, our findings demonstrated that overexpression of VvPUB26 in transgenic grapevines enhances defense against powdery mildew, while interfering with VvPUB26 results in increased susceptibility to the pathogen. Interestingly, we observed that VvPUB26 interacts with the WRKY transcription factor VvWRKY24, thereby facilitating ubiquitination and degradation processes. Through RNA-Seq analysis, we found that VvWRKY24 primarily participates in secondary metabolites biosynthesis, metabolic pathways, and plant-pathogen interaction. Notably, VvWRKY24 directly interacts with the promoters of dihydroflavonol-4-reductase (DFR) and leucoanthocyanidin reductase (LAR) to inhibit PAs biosynthesis. Meanwhile, VvWRKY24 also influences the expression of MYB transcription factor genes related to PAs synthesis. In conclusion, our results unveil a regulatory module involving VvPUB26-VvWRKY24-VvDFR/VvLAR that plays a fundamental role in governing PAs biosynthesis in grapevines. These findings enhance our understanding of the relationship between PAs biosynthesis and defense mechanisms against powdery mildew.

The Role of Cytokinins and Abscisic Acid in the Growth, Development and Virulence of the Pathogenic Fungus Stagonospora nodorum (Berk.).

Cytokinins (CKs) and abscisic acid (ABA) play an important role in the life of both plants and pathogenic fungi. However, the role of CKs and ABA in the regulation of fungal growth, development and virulence has not been sufficiently studied. We compared the ability of two virulent isolates (SnB and Sn9MN-3A) and one avirulent isolate (Sn4VD) of the pathogenic fungus Stagonospora nodorum Berk. to synthesize three groups of hormones (CKs, ABA and auxins) and studied the effect of exogenous ABA and zeatin on the growth, sporulation and gene expression of necrotrophic effectors (NEs) and transcription factors (TFs) in them. Various isolates of S. nodorum synthesized different amounts of CKs, ABA and indoleacetic acid. Using exogenous ABA and zeatin, we proved that the effect of these hormones on the growth and sporulation of S. nodorum isolates can be opposite, depends on both the genotype of the isolate and on the concentration of the hormone and is carried out through the regulation of carbohydrate metabolism. ABA and zeatin regulated the expression of fungal TF and NE genes, but correlation analysis of these parameters showed that this effect depended on the genotype of the isolate. This study will contribute to our understanding of the role of the hormones ABA and CKs in the biology of the fungal pathogen S. nodorum.

Transcriptomics Analysis Identifies the Decline in the AT2 Stem Cell Niche in Aged Human Lungs.

Aging poses a global public health challenge, which is linked to the rise of age-related lung diseases. The precise understanding of the molecular and genetic changes in the aging lung that elevate the risk of acute and chronic lung diseases remains incomplete. Alveolar type II (AT2) cells are stem cells that maintain epithelial homeostasis and repair the lung after injury. AT2 progenitor function decreases with aging. The maintenance of AT2 function requires niche support from other cell types, but little has been done to characterize alveolar alterations with aging in the AT2 niche. To systematically profile the genetic changes associated with age, we present a single-cell transcriptional atlas comprising nearly half a million cells from the healthy lungs of human subjects spanning various ages, sexes, and smoking statuses. Most annotated cell lineages in aged lungs exhibit dysregulated genetic programs. Specifically, the aged alveolar epithelial (AT2) cells demonstrate loss of epithelial identities, heightened inflammaging characterized by increased expression of AP-1 transcription factor and chemokine genes, and significantly increased cellular senescence. Furthermore, the aged mesenchymal cells display a remarkable decrease in Collagen and Elastin transcription and a loss of support to epithelial cell stemness. The decline of the AT2 niche is further exacerbated by a dysregulated genetic program in macrophages and dysregulated communications between AT2 and macrophages in aged human lungs. These findings highlight the dysregulations observed in both AT2 stem cells and their supportive niche cells, potentially contributing to the increased susceptibility of aged populations to lung diseases.

Potential molecular mechanism of photosynthesis regulation by PeMPK7 in poplar under para-hydroxybenzoic acid stress

Due to continuous plantation of poplar, its growth and biomass accumulation may be negatively affected by the accumulation of allelochemicals such as para-hydroxybenzoic acid (pHBA) in soil. As photosynthesis is the most fundamental process in plants, it can be negatively impacted by pHBA stress. Therefore, it is crucial to improve photosynthetic capacity under pHBA stress to facilitate poplar plant growth. The mitogen-activated protein kinase (MAPK) cascade pathway is widely involved in environmental stress responses in plants. However, the regulation mechanisms of photosynthesis-related pathways by MAPK pathway genes under pHBA stress are still unclear. In this study, through transcriptome analysis and weighted gene co-expression network analysis, we observed that PeMPK7 overexpression in poplar can regulate the expression of photosynthesis-related genes and transcription factor genes, namely, WRKY1, WRKY33, and ERF3, during the early stage of pHBA stress. In addition, PeMPK7 can improve photosynthesis in poplar under long-term pHBA stress. Moreover, yeast two-hybrid and pull-down assays confirmed the interaction between PeMPK7 and PeMKK7/10. Based on these results, a schematic diagram of the pathways involved in the regulation of photosynthesis by PeMPK7 was constructed. This study provided novel insights into the molecular mechanisms of regulation of pHBA stress via MAPK cascade pathway.

Arbuscular mycorrhizal fungi enhanced resistance to low-temperature weak-light stress in snapdragon (Antirrhinum majus L.) through physiological and transcriptomic responses.

Low temperature (LT) and weak light (WL) seriously affects the yield and quality of snapdragon in winter greenhouse. Arbuscular mycorrhizal fungi (AMF) exert positive role in regulating growth and enhancing abiotic stress tolerance in plants. Nevertheless, the molecular mechanisms by AMF improve the LT combined with WL (LTWL) tolerance in snapdragon remain mostly unknown. We compared the differences in root configuration, osmoregulatory substances, enzymatic and non-enzymatic antioxidant enzyme defense systems and transcriptome between AMF-inoculated and control groups under LT, WL, low light, and LTWL conditions. Our analysis showed that inoculation with AMF effectively alleviated the inhibition caused by LTWL stress on snapdragon root development, and significantly enhanced the contents of soluble sugars, soluble proteins, proline, thereby maintaining the osmotic adjustment of snapdragon. In addition, AMF alleviated reactive oxygen species damage by elevating the contents of AsA, and GSH, and the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR). RNA-seq analysis revealed that AMF regulated the expression of genes related to photosynthesis (photosystem I related proteins, photosystem II related proteins, chlorophyll a/b binding protein), active oxygen metabolism (POD, Fe-SOD, and iron/ascorbate family oxidoreductase), plant hormone synthesis (ARF5 and ARF16) and stress-related transcription factors gene (bHLH112, WRKY72, MYB86, WRKY53, WRKY6, and WRKY26) under LTWL stress. We concluded that mycorrhizal snapdragon promotes root development and LTWL tolerance by accumulation of osmoregulatory substances, activation of enzymatic and non-enzymatic antioxidant defense systems, and induction expression of transcription factor genes and auxin synthesis related genes. This study provides a theoretical basis for AMF in promoting the production of greenhouse plants in winter.

Molecular phenotyping of small cell lung cancer using targeted cfDNA profiling of transcriptional regulatory regions.

We report an approach for cancer phenotyping based on targeted sequencing of cell-free DNA (cfDNA) for small cell lung cancer (SCLC). In SCLC, differential activation of transcription factors (TFs), such as ASCL1, NEUROD1, POU2F3, and REST defines molecular subtypes. We designed a targeted capture panel that identifies chromatin organization signatures at 1535 TF binding sites and 13,240 gene transcription start sites and detects exonic mutations in 842 genes. Sequencing of cfDNA from SCLC patient-derived xenograft models captured TF activity and gene expression and revealed individual highly informative loci. Prediction models of ASCL1 and NEUROD1 activity using informative loci achieved areas under the receiver operating characteristic curve (AUCs) from 0.84 to 0.88 in patients with SCLC. As non-SCLC (NSCLC) often transforms to SCLC following targeted therapy, we applied our framework to distinguish NSCLC from SCLC and achieved an AUC of 0.99. Our approach shows promising utility for SCLC subtyping and transformation monitoring, with potential applicability to diverse tumor types.

Exploring the immune-modulating properties of boswellic acid in experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a condition where the central nervous system loses its myelin coating due to autoimmune inflammation. The experimental autoimmune encephalomyelitis (EAE) simulates some aspects of human MS. Boswellic acids are natural compounds derived from frankincense extract, known for their anti-inflammatory properties. The purpose of this research was to investigate therapeutic potential of boswellic acids. Mice were divided into three groups: low-dose (LD), high-dose (HD), and control groups (CTRL). Following EAE induction, the mice received daily doses of boswellic acid for 25 days. Brain tissue damage, clinical symptoms, and levels of TGF-β, IFN-γ, and IL-17 cytokines in cell cultured supernatant of lymphocytes were assessed. Gene expression of transcription factors in brain was measured using real-time PCR. The levels of brain demyelination were significantly lower in the treatment groups compared to the CTRL group. Boswellic acid reduced the severity and duration of EAE symptoms. Furthermore, boswellic acid decreased the amounts of IFN-γ and IL-17, also the expression of T-bet and ROR-γt in brain. On the contrary, it increased the levels of TGF-β and the expression FoxP3 and GATA3. Our findings suggest that boswellic acids possess therapeutic potential for EAE by modulating the immune response and reducing inflammation.

Genome-wide characterization and expression of Oryza sativa AP2 transcription factor genes associated with the metabolism of mesotrione

BackgroundThe APETALA2 (AP2) transcription factor (TF) superfamily, one of the largest gene families in plants, plays an essential role in regulating plant growth and their stress responses. However, the role of AP2 in rice under pesticide stress remains unclear. To investigate the characteristics and functions of the rice AP2 gene family under pesticide stress, the expression of 105 AP2-coding genes and 26 AP2 differentially expressed genes (DEGs) were identified in mesotrione (MTR)-treated rice transcriptome datasets.ResultsThree subfamilies of the AP2 gene family (AP2/ERF, RAV, and soloists) were identified using sequence alignment and phylogenetic analysis. Chromosome location analysis revealed that the 26 rice AP2 DEGs were unevenly distributed on 10 of the 12 rice chromosomes, and segmental duplication contributed to the expansion of Oryza sativa AP2 (OsAP2) gene family. Collinearity analyses demonstrated that rice AP2 genes displayed 16 orthologous gene pairs, and 12 and 26 orthologous gene pairs were shared of Arabidopsis and soybean, respectively. In addition, rice AP2 genes featured various gene structures, cis-elements, motif compositions, and conserved domains that allowed them to encode genes that elicit biotic and abiotic stress responses. An analysis of docking between MTR and six AP2 DEGs revealed amino acid residues involved in MTR binding. Quantitative reverse transcription–polymerase chain reaction verified that several AP2 genes were preferentially expressed during MTR-induced stress. The roles of OsAP2 proteins in MTR metabolism were further supported by protein–protein interaction network analysis, which illustrated how these proteins interact with target proteins.ConclusionThe initial findings of this study define the features of the OsAP2 superfamily and offer important tools for functional analyses of OsAP2 genes implicated in the metabolism of MTR.Graphical

In silico genome-wide analysis of homeodomain-leucine zipper transcription factors in Cannabis sativa L

HD-Zip (Homeodomain-Leucine Zipper) is a family of transcription factors unique to higher plants and plays a vital role in plant growth and development. Increasing research results show that HD-Zip transcription factors are widely involved in many life processes in plants. However, the HD-Zip transcription factor for cannabis, a valuable crop, has not yet been identified. The sequence characteristics, chromosome localization, system evolution, conservative motif, gene structure, and gene expression of the HD-Zip transcription factor in the cannabis genome were systematically studied. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to verify its function. The results showed that cannabis contained 33 HD-Zip gene members. The number of amino acids is 136–849aa, the isoelectric point is 4.54–9.04, and the molecular weight is 23264.32–93147.87Da. Many cis-acting elements are corresponding to hormone and abiotic stress in the HD-Zip family promoter area of cannabis. Sequencing of the transcriptome at 5 tissue sites of hemp, stems, leaves, bracts, and seeds showed similar levels of expression of 33 members of the HD-Zip gene family at 5 tissue sites. Bioinformatics results show that HD-Zip expression is tissue-specific and may be influenced by hormones and environmental factors. This lays a foundation for further research on the gene function of HD-Zip.

Decoding Non-coding Variants: Recent Approaches to Studying Their Role in Gene Regulation and Human Diseases.

Genome-wide association studies (GWAS) have mapped over 90% of disease- and quantitative-trait-associated variants within the non-coding genome. Non-coding regulatory DNA (e.g., promoters and enhancers) and RNA (e.g., 5' and 3' UTRs and splice sites) are essential in regulating temporal and tissue-specific gene expressions. Non-coding variants can potentially impact the phenotype of an organism by altering the molecular recognition of the cis-regulatory elements, leading to gene dysregulation. However, determining causality between non-coding variants, gene regulation, and human disease has remained challenging. Experimental and computational methods have been developed to understand the molecular mechanism involved in non-coding variant interference at the transcriptional and post-transcriptional levels. This review discusses recent approaches to evaluating disease-associated single-nucleotide variants (SNVs) and determines their impact on transcription factor (TF) binding, gene expression, chromatin conformation, post-transcriptional regulation, and translation.

Unraveling the Impact of Blood Transfusion on Transcription Factors Regulating T Helper 1, 2, 17 and Regulatory T cells.

T helper 1 (TH1) and TH2 lymphocytes are the most important components of the immune system affected by blood transfusion. This study aimed`` to evaluate the effect of blood transfusion on gene expression of transcription factors related to the development of TH1, TH2, TH17 and regulatory T cells (Tregs). In this cross-sectional study, 20 patients diagnosed with abdominal aortic aneurysms requiring surgical repair were studied from January 2018 to August 2020. We utilized real-time PCR to evaluate the expression of transcription factor genes associated with TH1, TH2, TH17, and Treg, namely T-box-expressed-in-T-cells (T-bet), GATA-binding protein 3 (GATA-3), retinoid-related orphan receptor (RORγt), and fork head box protein 3 (Foxp3), respectively. The sampling occurred before anesthesia, 24- and 72 hours post-transfusion, and at the time of discharge. The results showed that the T-bet gene expression, compared to the time before transfusion, was significantly decreased 24 hours after blood transfusion and upon discharge while GATA3 genes exhibited a significant reduction both 24 and 72 hours after the transfusion, as compared to the pre-transfusion levels and the time of patient discharge. The Foxp3 gene demonstrated an increase at all study stages, with a notable surge, particularly 72 hours after red blood cell (RBC) transfusion. Conversely, the expression of RORγt gene, consistently decreased throughout all stages of the study. RBC transfusion in abdominal aortic aneurysm patients altered the balance of transcription gene expression of TH1, TH2, TH17, and Treg cells.

Up-regulation of the IL-1 signaling pathway enhances gene expression of transcription factors (Jun, Junb, Jund, Fos, and Fosb)

This paper focuses on the experiment method to find out the relationship between the IL-1 signaling pathway and the transcription factors; we use 5xFAD mice with targeted deletion of Bace-1 in microglia,We conjecture that the up-regulation of the IL-1 signaling pathway enhances gene expression of transcription factors (Jun, Junb, Jund, Fos, and Fosb), which probably increases the transition microglial signature. The specifically targeted inhibition of BACE-1 in microglia will likely offer a superior therapeutic alternative for improving AD outcomes, circumventing the safety concerns associated with neuronal deletion or global inhibition of BACE-1.

Gene regulatory patterning codes in early cell fate specification of the C. elegans embryo.

Pattern formation originates during embryogenesis by a series of symmetry-breaking steps throughout an expanding cell lineage. In Drosophila, classic work has shown that segmentation in the embryo is established by morphogens within a syncytium, and the subsequent action of the gap, pair-rule, and segment polarity genes. This classic model however does not translate directly to species that lack a syncytium - such as Caenorhabditis elegans - where cell fate is specified by cell-autonomous cell lineage programs and their inter-signaling. Previous single-cell RNA-Seq studies in C. elegans have analyzed cells from a mixed suspension of cells from many embryos to study late differentiation stages, or individual early stage embryos to study early gene expression in the embryo. To study the intermediate stages of early and late gastrulation (28- to 102-cells stages) missed by these approaches, here we determine the transcriptomes of the 1- to 102-cell stage to identify 119 embryonic cell states during cell fate specification, including 'equivalence-group' cell identities. We find that gene expression programs are modular according to the sub-cell lineages, each establishing a set of stripes by combinations of transcription factor gene expression across the anterior-posterior axis. In particular, expression of the homeodomain genes establishes a comprehensive lineage-specific positioning system throughout the embryo beginning at the 28-cell stage. Moreover, we find that genes that segment the entire embryo in Drosophila have orthologs in C. elegans that exhibit sub-lineage-specific expression. These results suggest that the C. elegans embryo is patterned by a juxtaposition of distinct lineage-specific gene regulatory programs each with a unique encoding of cell location and fate. This use of homologous gene regulatory patterning codes suggests a deep homology of cell fate specification programs across diverse modes of development.

Regulus infers signed regulatory relations from few samples' information using discretization and likelihood constraints.

Transcriptional regulation is performed by transcription factors (TF) binding to DNA in context-dependent regulatory regions and determines the activation or inhibition of gene expression. Current methods of transcriptional regulatory circuits inference, based on one or all of TF, regions and genes activity measurements require a large number of samples for ranking the candidate TF-gene regulation relations and rarely predict whether they are activations or inhibitions. We hypothesize that transcriptional regulatory circuits can be inferred from fewer samples by (1) fully integrating information on TF binding, gene expression and regulatory regions accessibility, (2) reducing data complexity and (3) using biology-based likelihood constraints to determine the global consistency between a candidate TF-gene relation and patterns of genes expressions and region activations, as well as qualify regulations as activations or inhibitions. We introduce Regulus, a method which computes TF-gene relations from gene expressions, regulatory region activities and TF binding sites data, together with the genomic locations of all entities. After aggregating gene expressions and region activities into patterns, data are integrated into a RDF (Resource Description Framework) endpoint. A dedicated SPARQL (SPARQL Protocol and RDF Query Language) query retrieves all potential relations between expressed TF and genes involving active regulatory regions. These TF-region-gene relations are then filtered using biological likelihood constraints allowing to qualify them as activation or inhibition. Regulus provides signed relations consistent with public databases and, when applied to biological data, identifies both known and potential new regulators. Regulus is devoted to context-specific transcriptional circuits inference in human settings where samples are scarce and cell populations are closely related, using discretization into patterns and likelihood reasoning to decipher the most robust regulatory relations.

Viable tendon neotissue from adult adipose-derived multipotent stromal cells.

Background: Tendon healing is frequently prolonged, unpredictable, and results in poor tissue quality. Neotissue formed by adult multipotent stromal cells has the potential to guide healthy tendon tissue formation. Objectives: The objective of this study was to characterize tendon neotissue generated by equine adult adipose-derived multipotent stromal cells (ASCs) on collagen type I (COLI) templates under 10% strain in a novel bioreactor. The tested hypothesis was that ASCs assume a tendon progenitor cell-like morphology, express tendon-related genes, and produce more organized extracellular matrix (ECM) in tenogenic versus stromal medium with perfusion and centrifugal fluid motion. Methods: Equine ASCs on COLI sponge cylinders were cultured in stromal or tenogenic medium within bioreactors during combined perfusion and centrifugal fluid motion for 7, 14, or 21days under 10% strain. Viable cell distribution and number, tendon-related gene expression, and micro- and ultra-structure were evaluated with calcein-AM/EthD-1 staining, resazurin reduction, RT-PCR, and light, transmission, and scanning electron microscopy. Fibromodulin was localized with immunohistochemistry. Cell number and gene expression were compared between culture media and among culture periods (p < 0.05). Results: Viable cells were distributed throughout constructs for up to 21days of culture, and cell numbers were higher in tenogenic medium. Individual cells had a round or rhomboid shape with scant ECM in stromal medium in contrast to clusters of parallel, elongated cells surrounded by highly organized ECM in tenogenic medium after 21days of culture. Transcription factor, extracellular matrix, and mature tendon gene expression profiles confirmed ASC differentiation to a tendon progenitor-like cell in tenogenic medium. Construct micro- and ultra-structure were consistent with tendon neotissue and fibromodulin was present in the ECM after culture in tenogenic medium. Conclusion: Long-term culture in custom bioreactors with combined perfusion and centrifugal tenogenic medium circulation supports differentiation of equine adult ASCs into tendon progenitor-like cells capable of neotissue formation.

Trisomy 12 compromises the mesendodermal differentiation propensity of human pluripotent stem cells

Trisomy 12 is one of the most frequent chromosomal abnormalities in cultured human pluripotent stem cells (hPSCs). Although potential oncogenic properties and augmented cell cycle caused by trisomy 12 have been reported, the consequences of trisomy 12 in terms of cell differentiation, which is the basis for regenerative medicine, drug development, and developmental biology studies, have not yet been investigated. Here, we report that trisomy 12 compromises the mesendodermal differentiation of hPSCs. We identified sublines of hPSCs carrying trisomy 12 after their prolonged culture. Transcriptome analysis revealed that these hPSC sublines carried abnormal gene expression patterns in specific signaling pathways in addition to cancer-related cell cycle pathways. These hPSC sublines showed a lower propensity for mesendodermal differentiation in embryoid bodies cultured in a serum-free medium. BMP4-induced exit from the self-renewal state was impaired in the trisomy 12 hPSC sublines, with less upregulation of key transcription factor gene expression. As a consequence, the differentiation efficiency of hematopoietic and hepatic lineages was also impaired in the trisomy 12 hPSC sublines. We reveal that trisomy 12 disrupts the genome-wide expression patterns that are required for proper mesendodermal differentiation.

TFAP2 paralogs regulate midfacial development in part through a conserved ALX genetic pathway.

Cranial neural crest development is governed by positional gene regulatory networks (GRNs). Fine-tuning of the GRN components underlies facial shape variation, yet how those networks in the midface are connected and activated remain poorly understood. Here, we show that concerted inactivation of Tfap2a and Tfap2b in the murine neural crest, even during the late migratory phase, results in a midfacial cleft and skeletal abnormalities. Bulk and single-cell RNA-seq profiling reveal that loss of both TFAP2 family members dysregulates numerous midface GRN components involved in midface morphogenesis, patterning and differentiation. Notably, Alx1, Alx3 and Alx4 (ALX) transcript levels are reduced, whereas ChIP-seq analyses suggest TFAP2 family members directly and positively regulate ALX gene expression. Tfap2a, Tfap2b and ALX co-expression in midfacial neural crest cells of both mouse and zebrafish implies conservation of this regulatory axis across vertebrates. Consistent with this notion, tfap2a zebrafish mutants present with abnormal alx3 expression patterns, Tfap2a binds ALX loci and tfap2a-alx3 genetic interactions are observed. Together, these data demonstrate TFAP2 paralogs regulate vertebrate midfacial development in part by activating expression of ALX transcription factor genes.