scholarly journals SINHCAF/FAM60A links SIN3A function to the hypoxia response and its levels are predictive of cancer patient survival

2017 ◽  
Author(s):  
John Biddlestone ◽  
Michael Batie ◽  
Alena Shmakova ◽  
Daniel Bandarra ◽  
Elena V. Knatko ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Patient Survival ◽  
Cell Signalling ◽  
Complex Function ◽  
Mrna Levels ◽  
Hypoxia Response ◽  
Repressor Complex ◽  
Chromatin Immunoprecipitation Sequencing ◽  
Mrna And Protein Expression

AbstractThe SIN3A-HDAC complex is a master transcriptional repressor, required for development but often deregulated in disease. Here, we report that the recently identified new component of this complex, SINHCAF/FAM60A, links the SIN3A-HDAC co-repressor complex function to the hypoxia response. SINHCAF Chromatin Immunoprecipitation-sequencing and gene expression analysis reveal a signature associated with the activation of the hypoxia response. We show that SINHCAF specifically repress HIF 2α mRNA and protein expression resulting in functional cellular changes in in-vitro angiogenesis, and proliferation. Analysis of patient datasets demonstrates that SINHCAF and HIF 2α mRNA levels are inversely correlated and predict contrasting outcomes for patient survival in both colon and lung cancer. This relationship is also observed in a mouse model of colon cancer, indicating an evolutionary conserved mechanism. Our analysis reveals an unexpected link between SINHCAF and cancer cell signalling via regulation of the hypoxia response that is predictive of poor patient outcome.

scholarly journals SINHCAF/FAM60A and SIN3A specifically repress HIF-2α expression

2018 ◽  
Vol 475 (12) ◽  
pp. 2073-2090 ◽  
Author(s):  
John Biddlestone ◽  
Michael Batie ◽  
Daniel Bandarra ◽  
Ivan Munoz ◽  
Sonia Rocha
Keyword(s):  
Transcription Factor ◽  
Complex Function ◽  
Hypoxia Response ◽  
Specificity Protein 1 ◽  
Cellular Changes ◽  
Repressor Complex ◽  
Mrna And Protein Expression ◽  
Specificity Protein ◽  
In Vitro Angiogenesis

The SIN3A–HDAC (histone deacetylase) complex is a master transcriptional repressor, required for development but often deregulated in disease. Here, we report that the recently identified new component of this complex, SINHCAF (SIN3A and HDAC-associated factor)/FAM60A (family of homology 60A), links the SIN3A–HDAC co-repressor complex function to the hypoxia response. We show that SINHCAF specifically represses HIF-2α mRNA and protein expression, via its interaction with the transcription factor SP1 (specificity protein 1) and recruitment of HDAC1 to the HIF-2α promoter. SINHCAF control over HIF-2α results in functional cellular changes in in vitro angiogenesis and viability. Our analysis reveals an unexpected link between SINHCAF and the regulation of the hypoxia response.

scholarly journals Partnership between epigenetic reader BRD4 and transcription factor CEBPD

2020 ◽  
Author(s):  
Qingwei Wang ◽  
Mengxue Zhang ◽  
Go Urabe ◽  
Bowen Wang ◽  
Hatice Gulcin Ozer ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Histone Acetylation ◽  
Chromatin Immunoprecipitation ◽  
State Transition ◽  
Immune Cell ◽  
Neointimal Hyperplasia ◽  
Mrna Levels ◽  
Enhancer Binding Protein ◽  
Chromatin Immunoprecipitation Sequencing

AbstractVascular smooth muscle cell (SMC) state/phenotype transitions underlie neointimal hyperplasia (IH) predisposing to cardiovascular diseases. Bromodomain protein BRD4 is a histone acetylation reader and enhancer mark that co-activates transcription elongation. CCAAT enhancer binding protein delta (CEBPD) is a transcription factor typically studied in adipogenesis and immune cell differentiation. Here we investigated the association between BRD4 and CEBPD in SMC state transition.Chromatin immunoprecipitation sequencing (ChIPseq) showed enrichment of BRD4 and histone acetylation (H3K27ac) at Cebpd and enhancer in rat carotid arteries undergoing IH. In vitro, BRD4 silencing with siRNA reduced SMC expression of CEBPD. Bromodomain-1 but not bromodoamin-2 accounted for this BRD4 function. Endogenous BRD4 co-IP’ed with CEBPD; Cebpd promoter and enhancer DNA fragments co-IP’ed with CEBPD or endogenous BRD4 (ChIP-qPCR). These co-IPs were abolished by the BRD4 bromodomain blocker JQ1. TNFα upregulated both BRD4 and CEBPD. Silencing CEBPD averted TNFα-induced inflammatory SMC state transition (heightened IL-1β, IL6, and MCP-1 mRNA levels), so did JQ1. CEBPD overexpression increased PDGFRα preferentially over PDGFRβ; so did TNFα, and JQ1 abolished TNFα’s effect.Our data reveal a BRD4/CEBPD partnership that promotes CEBPD’s own transcription and inflammatory SMC state transition, thus shedding new light on epigenetic reader and transcription factor cooperative actions in SMC pathobiology.

scholarly journals The nucleoid-associated protein Gbn binds to GATC sequences and affects sporulation and antibiotic production in Streptomyces

2021 ◽  
Author(s):  
Chao Du ◽  
Joost Willemse ◽  
Amanda M. Erkelens ◽  
Victor J. Carrion ◽  
Remus T. Dame ◽  
...  
Keyword(s):  
Dna Binding ◽  
Chromatin Immunoprecipitation ◽  
Antibiotic Production ◽  
Consensus Sequence ◽  
Associated Proteins ◽  
Chromatin Immunoprecipitation Sequencing ◽  
Number Of Binding Sites ◽  
Trna Editing ◽  
Editing Enzyme

ABSTRACTBacterial chromosome structure is organized by a diverse group of proteins collectively called nucleoid-associated proteins (NAPs). Many NAPs have been studied in detail in Streptomyces, including Lsr2, HupA, HupS, and sIHF. Here, we show that SCO1839 represents a novel family of small NAPs unique to Actinobacteria and recognizes a consensus sequence consisting of GATC followed by (A/T)T. The protein was designated Gbn for GATC-binding NAP. Chromatin immunoprecipitation sequencing (ChIP-Seq) detected more than 2800 binding regions, encompassing some 3600 GATCWT motifs, which comprise 55% of all motifs in the S. coelicolor genome. DNA binding of Gbn in vitro increased DNA stiffness but not compaction, suggesting a role in regulation rather than chromosome organization. Despite the huge number of binding sites, the DNA binding profiles were nearly identical during vegetative and aerial growth. The exceptions were SCO1311 and SCOt32, for a tRNA editing enzyme and a tRNA that recognises the rare leucine codon CUA, respectively, which were nearly exclusively bound during vegetative growth. Deletion of gbn led to pleiotropic alterations in developmental timing, morphogenesis and antibiotic production. Taken together, our data show that Gbn is a highly pleiotropic NAP that impacts growth and development in streptomycetes.

scholarly journals The β-catenin/TCF-4 pathway regulates the expression of OPN in human osteoarthritic chondrocytes

2020 ◽  
Author(s):  
Jian Tian ◽  
Shu-Guang Gao ◽  
Yu-Sheng Li ◽  
Chao Cheng ◽  
Zhen-Han Deng ◽  
...  
Keyword(s):  
Cartilage Destruction ◽  
Transient Transfection ◽  
Human Chondrocytes ◽  
Mrna Levels ◽  
Osteoarthritic Chondrocytes ◽  
Mrna And Protein Expression ◽  
Oa Chondrocytes ◽  
Canonical Wnt ◽  
Dickkopf 1

Abstract Background: Cartilage destruction is the main characteristic of osteoarthritis (OA), and osteopontin (OPN) is elevated in OA articular cartilage; however, the reason for the increased OPN level is not determined. In addition, Wnt/β-catenin signaling participates in the progression of OA. The aim of the present study was to evaluate whether canonical Wnt signaling could regulate the expression of OPN in human chondrocytes in vitro.Methods: Human chondrocytes were cultured in vitro, and we first assayed the mRNA levels of OPN and β-catenin in chondrocytes. Next, we performed transient transfection of TCF 4 shRNA into chondrocytes to inhibit TCF 4 expression and explore changes in the OPN level. Then, the Wnt/β-catenin signaling inhibitor Dickkopf-1 (Dkk-1) was incubated with chondrocytes, and we assayed the changes in β-catenin and OPN.Results: Our results showed that the expression of both β-catenin and OPN was increased in OA chondrocytes, but there were no correlations between β-catenin and OPN expression. TCF4 shRNA downregulated the expression of TCF 4 and OPN in chondrocytes, while after treatment with rDKK-1 at a concentration of 400 ng/ml for 24 h, the mRNA and protein expression of both β-catenin and OPN was significantly decreased in chondrocytes.Conclusions: Elevated OPN expression might be regulated by the β-catenin/TCF-4 pathway, and the Wnt/β-catenin inhibitor DKK1 could inhibit the expression of β-catenin and OPN in OA chondrocytes.

scholarly journals GRO-alpha in normal and pathological thyroid tissues and its regulation in thyroid-derived cells

2001 ◽  
Vol 170 (3) ◽  
pp. 513-520 ◽  
Author(s):  
G Aust ◽  
M Steinert ◽  
C Boltze ◽  
S Kiessling ◽  
C Simchen
Keyword(s):  
T Cells ◽  
Mrna Level ◽  
Thyroid Tissue ◽  
Thyroid Peroxidase ◽  
Mrna Levels ◽  
Carcinoma Cell Lines ◽  
Leukocytic Infiltration ◽  
Cellular Source ◽  
Mrna And Protein Expression

Thyroid glands affected by Graves' disease (GD) show striking leukocytic infiltration, mainly by T-cells. The mechanisms by which the various leukocytes are maintained in the thyroid are unknown. Growth-regulated oncogene-alpha (GRO-alpha) in interaction with its receptor CXCR2 is a chemoattractant for both T-cells and neutrophils and may be one of the chemokines involved in the cell maintenance. GRO-alpha and CD18 mRNA as a marker of leukocytic infiltration were quantified in thyroid tissue using competitive RT-PCR. We found very high GRO-alpha mRNA levels in all thyroid tissues. In GD patients (n=16), the GRO-alpha mRNA did not correlate with the CD18 mRNA level or thyroid peroxidase and TSH-receptor antibodies in patients' sera. In thyroid autonomy (n=10), the GRO-alpha mRNA levels were significantly lower in autonomous single adenomas compared with the corresponding normal tissue. In order to define the cellular source of GRO-alpha mRNA and protein, we examined various thyroid-derived cells. Thyrocytes, thyroid-derived leukocytes and fibroblasts showed basal GRO-alpha mRNA and protein expression, which was remarkably upregulated by different stimuli in vitro. The expression of GRO-alpha by thyroid carcinoma cell lines confirms that thyrocytes may actually produce GRO-alpha. As shown by flow cytometry and immunohistology, CD68+ monocytes/macrophages are the only cell population strongly expressing CXCR2 in the thyroid.

scholarly journals N-Acetylcysteine Inhibits Lipids Production in Mature Adipocytes through the Inhibition of Peroxisome Proliferator-Activated Receptor 

2020 ◽  
pp. 17-29
Author(s):  
Daniela Soto ◽  
Claudia Martini ◽  
Evelyn Frontera ◽  
Laura Montaldo ◽  
Maria C. Vila ◽  
...  
Keyword(s):  
In Vitro Study ◽  
Nutritional Supplement ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Peroxisome Proliferator Activated Receptor ◽  
Mrna And Protein Expression ◽  
Species Production ◽  
Lipids Production

Aims: Reports regarding the effects of antioxidants in obesity have been contradictory. Antioxidant N-acetylcysteine is usually considered a nutritional supplement. Our aim is to evaluate bioactivity of N-acetylcysteine (NAC) on mature adipocytes, which is a close model to in vivo condition. Study Design: In vitro study. Place and Duration of Study: Department of Basic Science (Universidad Nacional de Lujan), Department of Chemical Biology (Universidad de Buenos Aires), CONICET – INEDES and CONICET – IQUIBICEN, between March 2017 and June 2019. Methodology: We evaluated the bioactivity of different concentrations of NAC for 5 days (0.01 mM to 5 mM) on fully differentiated 3T3-L1 cells (mature adipocytes). Results: We demonstrated that NAC treatment was not toxic to mature adipocytes. Only 5mM NAC inhibited reactive oxygen species production. 5 mM NAC treatment resulted in a 60% decrease in cellular triglycerides content and inhibited 70% cholesterol accumulation.  We also determined the mRNA and protein expression levels of Peroxisome Proliferator-Activated Receptor g as well as, mRNA levels of lipid protein Perilipin in NAC treated adipocytes; we observed that 5mM NAC treatment caused nearly 30% decrease in the expression of these parameters. Conclusion: These results suggest that NAC could avoid lipid accumulation in mature adipocytes; the antioxidant NAC could be beneficial in obesity treatment.

scholarly journals The β-catenin/TCF-4 pathway regulates the expression of OPN in human osteoarthritic chondrocytes

2020 ◽  
Author(s):  
Jian Tian ◽  
Shu-Guang Gao ◽  
Yu-Sheng Li ◽  
Chao Cheng ◽  
Zhen-Han Deng ◽  
...  
Keyword(s):  
Cartilage Destruction ◽  
Transient Transfection ◽  
Human Chondrocytes ◽  
Mrna Levels ◽  
In Vitro Methods ◽  
Osteoarthritic Chondrocytes ◽  
Mrna And Protein Expression ◽  
Oa Chondrocytes ◽  
Canonical Wnt

Abstract Background Cartilage destruction is the main characteristic of osteoarthritis (OA), and osteopontin (OPN) is elevated in OA articular cartilage; however, the reason for the increased OPN level is not determined. In addition, Wnt/β-catenin signaling participates in the progression of OA. The aim of the present study was to evaluate whether canonical Wnt signaling could regulate the expression of OPN in human chondrocytes in vitro. Methods Human chondrocytes were cultured in vitro, and we first assayed the mRNA levels of OPN and β-catenin in chondrocytes. Next, we performed transient transfection of TCF 4 shRNA into chondrocytes to inhibit TCF 4 expression and explore changes in the OPN level. Then, the Wnt/β-catenin signaling inhibitor dinkkopf-1 (Dkk-1) was incubated with chondrocytes, and we assayed the changes in β-catenin and OPN. Results Our results showed that the expression of both β-catenin and OPN was increased in OA chondrocytes, but there were no correlations between β-catenin and OPN expression. TCF4 shRNA downregulated the expression of TCF 4 and OPN in chondrocytes, while after treatment with rDKK-1 at a concentration of 400 ng/ml for 24 h, the mRNA and protein expression of both β-catenin and OPN was significantly decreased in chondrocytes. Conclusions Elevated OPN expression might be regulated by the β-catenin/TCF-4 pathway, and the Wnt/β-catenin inhibitor DKK1 could inhibit the expression of β-catenin and OPN in OA chondrocytes.

scholarly journals Enhancer regulation for induced WNT3A expression during neuronal regeneration

2019 ◽  
Author(s):  
Chu-Yuan Chang ◽  
Jui-Hung Hung ◽  
Ching-Chih Wu ◽  
Min-Zong Liang ◽  
Pei-Yuan Huang ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Cortical Neurons ◽  
Intranasal Administration ◽  
Neuronal Regeneration ◽  
Topological Transformation ◽  
Chromatin Immunoprecipitation Sequencing ◽  
Positive Effect ◽  
Enhancer Regulation

AbstractThe treatment of traumatic brain injury (TBI) is limited by a lack of knowledge about the mechanisms underlying neuronal regeneration. WNT family members have been implicated in neurogenesis and aberrant WNT signaling has been associated with neurodegenerative diseases. The current study compared the expression of WNT genes during regeneration of injured cortical neurons. Recombinant WNT3A showed positive effect in promoting neuronal regeneration via in vitro and in vivo TBI models. Intranasal administration of WNT3A protein to TBI mice increased NeuN+ cells compared to control mice as well as retained motor function based on behavior analysis. Since TBI is known to reprogram the epigenome, chromatin immunoprecipitation-sequencing of histone H3K27ac and H3K4me3 was performed to address the transcriptional regulation of WNT3A during neuronal regeneration. We predicted, characterized and proposed that a histone H3K4me1-marked enhancer may undergo topological transformation to regulate the WNT3A gene expression.

scholarly journals Prior hypoxia exposure enhances murine microglial inflammatory gene expression in vitro without concomitant H3K4me3 enrichment

2020 ◽  
Author(s):  
Elizabeth A. Kiernan ◽  
Andrea C. Ewald ◽  
Jonathan N. Ouellette ◽  
Tao Wang ◽  
Avtar Roopra ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chromatin Immunoprecipitation ◽  
Sleep Disordered Breathing ◽  
Inflammatory Responses ◽  
Hypoxic Exposure ◽  
Inflammatory Gene Expression ◽  
Inflammatory Gene ◽  
Hypoxia Exposure ◽  
Chromatin Immunoprecipitation Sequencing

ABSTRACTHypoxia is a component of multiple disorders, including stroke and sleep-disordered breathing, that often precede or are comorbid with neurodegenerative diseases. However, little is known about how hypoxia affects the ability of microglia, resident CNS macrophages, to respond to subsequent inflammatory challenges that are often present during neurodegenerative processes. We therefore tested the hypothesis that hypoxia would enhance or “prime” microglial pro-inflammatory gene expression in response to a later inflammatory challenge without programmatically increasing basal levels of pro-inflammatory cytokine expression. To test this, we pre-exposed immortalized N9 and primary microglia to hypoxia (1% O2) for 16 hrs and then challenged them with pro-inflammatory lipopolysaccharide (LPS) either immediately or 3-6 days following hypoxic exposure. We used RNA sequencing coupled with chromatin immunoprecipitation sequencing to analyze primed microglial inflammatory gene expression and modifications to histone H3 lysine 4 trimethylation (H3K4me3) at the promoters of primed genes. We found that microglia exhibited enhanced responses to LPS 3 days and 6 days post-hypoxia. Surprisingly however, the majority of primed genes were not enriched for H3K4me3 acutely following hypoxia exposure. Using the bioinformatics tool MAGICTRICKS and reversible pharmacological inhibition, we found that primed genes required the transcriptional activities of NF-ĸB. These findings provide evidence that hypoxia pre-exposure could lead to persistent and aberrant inflammatory responses in the context of CNS disorders.

scholarly journals The β-catenin/TCF-4 pathway regulates the expression of OPN in human osteoarthritic chondrocytes

2020 ◽  
Author(s):  
Jian Tian ◽  
Shu-Guang Gao ◽  
Yu-Sheng Li ◽  
Chao Cheng ◽  
Zhen-Han Deng ◽  
...  
Keyword(s):  
Cartilage Destruction ◽  
Human Chondrocytes ◽  
Mrna Levels ◽  
In Vitro Methods ◽  
Osteoarthritic Chondrocytes ◽  
Mrna And Protein Expression ◽  
Oa Chondrocytes ◽  
Canonical Wnt ◽  
Dickkopf 1

Abstract Background Cartilage destruction is the main characteristic of osteoarthritis (OA), and osteopontin (OPN) is elevated in OA articular cartilage; however, the reason for the increased OPN level is not determined. In addition, Wnt/β-catenin signaling participates in the progression of OA. The aim of the present study was to evaluate whether canonical Wnt signaling could regulate the expression of OPN in human chondrocytes in vitro. Methods Human chondrocytes were cultured in vitro, and we first assayed the mRNA levels of OPN and β-catenin in chondrocytes. Next, we performed transient transfection of TCF 4 shRNA into chondrocytes to inhibit TCF 4 expression and explore changes in the OPN level. Then, the Wnt/β-catenin signaling inhibitor Dickkopf-1 (Dkk-1) was incubated with chondrocytes, and we assayed the changes in β-catenin and OPN. Results Our results showed that the expression of both β-catenin and OPN was increased in OA chondrocytes, but there were no correlations between β-catenin and OPN expression. TCF4 shRNA downregulated the expression of TCF 4 and OPN in chondrocytes, while after treatment with rDKK-1 at a concentration of 400 ng/ml for 24 h, the mRNA and protein expression of both β-catenin and OPN was significantly decreased in chondrocytes. Conclusions Elevated OPN expression might be regulated by the β-catenin/TCF-4 pathway, and the Wnt/β-catenin inhibitor DKK1 could inhibit the expression of β-catenin and OPN in OA chondrocytes.

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