Study of expression analysis of SIRT4 and the coordinate regulation of bovine adipocyte differentiation by SIRT4 and its transcription factors

Sirtuins, NAD+-dependent deacylases and ADP-ribosyltransferases, are critical regulators of metabolism involved in many biological processes, and are involved in mediating adaptive responses to the cellular environment. SIRT4 is a mitochondrial sirtuin and has been shown to play a critical role in maintaining insulin secretion and glucose homeostasis. As a regulator of lipid homeostasis, SIRT4 can repress fatty acid oxidation and promote lipid anabolism in nutrient-replete conditions. Using real-time quantitative PCR (qPCR) to explore the molecular mechanisms of transcriptional regulation of bovine SIRT4 during adipocyte differentiation, we found that bovine SIRT4 is expressed at high levels in bovine subcutaneous adipose tissue. SIRT4 knockdown led to decreased expression of adipogenic differentiation marker genes during adipocyte differentiation. The core promoter of bovine SIRT4 was identified in the −402/−60 bp region of the cloned 2-kb fragment containing the 5′-regulatory region. Binding sites were identified in this region for E2F transcription factor-1 (E2F1), CCAAT/enhancer-binding protein β (CEBPβ), homeobox A5 (HOXA5), interferon regulatory factor 4 (IRF4), paired box 4 (PAX4), and cAMP responsive element-binding protein 1 (CREB1) by using Electrophoretic mobility shift assay (EMSA) and luciferase reporter gene assay. We also found that E2F1, CEBPβ, and HOXA5 transcriptionally activate SIRT4 expression, whereas, IRF4, PAX4, and CREB1 transcriptionally repress SIRT4 expression. We further verified that SIRT4 knockdown could affect the ability of these transcription factors (TFs) to regulate the differentiation of bovine adipocytes. In conclusion, our results shed light on the mechanisms underlying the transcriptional regulation of SIRT4 expression in bovine adipocytes.

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Recruitment of histone deacetylase 4 by transcription factors represses interleukin-5 transcription

Biochemical Journal ◽

10.1042/bj20061085 ◽

2006 ◽

Vol 400 (3) ◽

pp. 439-448 ◽

Cited By ~ 33

Author(s):

Songyan Han ◽

Jun Lu ◽

Yu Zhang ◽

Cao Cheng ◽

Liping Han ◽

...

Keyword(s):

Transcriptional Regulation ◽

Transcription Factors ◽

Histone Deacetylase ◽

Molecular Mechanisms ◽

Binding Protein ◽

Critical Role ◽

Allergic Diseases ◽

Activated T Cells ◽

Interleukin 5 ◽

Histone Deacetylase 4

The critical role of IL-5 (interleukin-5) in eosinophilic inflammation implicates it as a therapeutic target for allergic diseases. The aim of the present study was to elucidate the molecular basis for the involvement of reversible histone acetylation in IL-5 transcriptional regulation. We provide evidence that HDAC4 (histone deacetylase 4) and p300, a known HAT (histone acetyltransferase), reversibly controlled the activity of the IL-5 promoter in vivo and in vitro, with a concurrent alteration of histone H3 acetylation status at the promoter regions. The nucleo-cytoplasmic shuttling of HDAC4 was shown to play an important role in the suppressive function of HDAC4 in IL-5 gene expression. Point mutation and reporter ChIP (chromatin immunoprecipitation) studies determined that the four transcription factors binding on the IL-5 promoter, i.e. C/EBPβ (CAAT/enhancer-binding protein β), GATA3 (GATA binding protein 3), NFAT (nuclear factor of activated T cells) and YY1 (Yin and Yang 1), were essential for the recruitment of HDAC4. Consistent with these observations, HDAC4 was found to form protein complexes with GATA3 and YY1, and to co-exist in the nuclei with GATA3. We propose that the unique regulatory mechanism of IL-5 gene transcription involves the reversible histone modification catalysed by HDAC4 and p300, which are recruited by the transcription factors. The dynamic balance in IL-5 transcriptional regulation is achieved through interactions among HATs/HDACs, histones and transcription factors. These data contribute to understanding the molecular mechanisms of IL-5 regulation, which is crucial to the development of new therapeutic strategies for IL-5-related allergic diseases.

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Understanding Adipocyte Differentiation

Physiological Reviews ◽

10.1152/physrev.1998.78.3.783 ◽

1998 ◽

Vol 78 (3) ◽

pp. 783-809 ◽

Cited By ~ 1498

Author(s):

FRANCINE M. GREGOIRE ◽

CYNTHIA M. SMAS ◽

HEI SOOK SUL

Keyword(s):

Adipose Tissue ◽

Transcription Factors ◽

Transcriptional Activation ◽

Molecular Mechanisms ◽

Adipocyte Differentiation ◽

Binding Protein ◽

Critical Role ◽

Tissue Growth ◽

Fatty Acid Binding ◽

Ppar Γ

Gregoire, Francine M., Cynthia M. Smas, and Hei Sook Sul. Understanding Adipocyte Differentiation. Physiol. Rev. 78: 783–809, 1998. — The adipocyte plays a critical role in energy balance. Adipose tissue growth involves an increase in adipocyte size and the formation of new adipocytes from precursor cells. For the last 20 years, the cellular and molecular mechanisms of adipocyte differentiation have been extensively studied using preadipocyte culture systems. Committed preadipocytes undergo growth arrest and subsequent terminal differentiation into adipocytes. This is accompanied by a dramatic increase in expression of adipocyte genes including adipocyte fatty acid binding protein and lipid-metabolizing enzymes. Characterization of regulatory regions of adipose-specific genes has led to the identification of the transcription factors peroxisome proliferator-activated receptor-γ (PPAR-γ) and CCAAT/enhancer binding protein (C/EBP), which play a key role in the complex transcriptional cascade during adipocyte differentiation. Growth and differentiation of preadipocytes is controlled by communication between individual cells or between cells and the extracellular environment. Various hormones and growth factors that affect adipocyte differentiation in a positive or negative manner have been identified. In addition, components involved in cell-cell or cell-matrix interactions such as preadipocyte factor-1 and extracellular matrix proteins are also pivotal in regulating the differentiation process. Identification of these molecules has yielded clues to the biochemical pathways that ultimately result in transcriptional activation via PPAR-γ and C/EBP. Studies on the regulation of the these transcription factors and the mode of action of various agents that influence adipocyte differentiation will reveal the physiological and pathophysiological mechanisms underlying adipose tissue development.

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Function and Transcriptional Regulation of Bovine TORC2 Gene in Adipocytes: Roles of C/EBP, XBP1, INSM1 and ZNF263

International Journal of Molecular Sciences ◽

10.3390/ijms20184338 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4338 ◽

Cited By ~ 12

Author(s):

Khan ◽

Raza ◽

Junjvlieke ◽

Xiaoyu ◽

Garcia ◽

...

Keyword(s):

Transcription Start Site ◽

Promoter Region ◽

Binding Protein ◽

Cell Counting ◽

Luciferase Reporter ◽

Marker Genes ◽

Start Site ◽

Transcription Start ◽

Bovine Adipocytes

The TORC2 gene is a member of the transducer of the regulated cyclic adenosine monophosphate (cAMP) response element binding protein gene family, which plays a key role in metabolism and adipogenesis. In the present study, we confirmed the role of TORC2 in bovine preadipocyte proliferation through cell cycle staining flow cytometry, cell counting assay, 5-ethynyl-2′-deoxyuridine staining (EdU), and mRNA and protein expression analysis of proliferation-related marker genes. In addition, Oil red O staining analysis, immunofluorescence of adiponectin, mRNA and protein level expression of lipid related marker genes confirmed the role of TORC2 in the regulation of bovine adipocyte differentiation. Furthermore, the transcription start site and sub-cellular localization of the TORC2 gene was identified in bovine adipocytes. To investigate the underlying regulatory mechanism of the bovine TORC2, we cloned a 1990 bp of the 5' untranslated region (5′UTR) promoter region into a luciferase reporter vector and seven vector fragments were constructed through serial deletion of the 5′UTR flanking region. The core promoter region of the TORC2 gene was identified at location −314 to −69 bp upstream of the transcription start site. Based on the results of the transcriptional activities of the promoter vector fragments, luciferase activities of mutated fragments and siRNAs interference, four transcription factors (CCAAT/enhancer-binding protein C/BEP, X-box binding protein 1 XBP1, Insulinoma-associated 1 INSM1, and Zinc finger protein 263 ZNF263) were identified as the transcriptional regulators of TORC2 gene. These findings were further confirmed through Electrophoretic Mobility Shift Assay (EMSA) within nuclear extracts of bovine adipocytes. Furthermore, we also identified that C/EBP, XBP1, INSM1 and ZNF263 regulate TORC2 gene as activators in the promoter region. We can conclude that TORC2 gene is potentially a positive regulator of adipogenesis. These findings will not only provide an insight for the improvement of intramuscular fat in cattle, but will enhance our understanding regarding therapeutic intervention of metabolic syndrome and obesity in public health as well.

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Characterization of the rat intestinal Fc receptor (FcRn) promoter: transcriptional regulation of FcRn gene by the Sp family of transcription factors

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00131.2003 ◽

2004 ◽

Vol 286 (6) ◽

pp. G922-G931 ◽

Cited By ~ 12

Author(s):

Lingling Jiang ◽

Jiafang Wang ◽

R. Sergio Solorzano-Vargas ◽

Hugh V. Tsai ◽

Edgar M Gutierrez ◽

...

Keyword(s):

Transcriptional Regulation ◽

Cell Lines ◽

Promoter Region ◽

Core Promoter ◽

Dnase I ◽

Fc Receptor ◽

Minimal Promoter ◽

Luciferase Reporter ◽

Core Promoter Region

The regulatory elements that control the transcriptional regulation of the intestinal Fc receptor ( FcRn) have not been elucidated. The objective of this study was to characterize the core promoter region of the rat FcRn gene. Chimeric clones that contained various regions of the promoter located upstream of the luciferase reporter were transiently transfected into either IEC-6 or Caco-2 cell lines and nuclear extracts were used to perform DNase I footprint and DNA binding assays (EMSA). Transfection of chimeric upstream nested deletions-luciferase reporter clones into either of these cell lines supported robust reporter activity and identified the location of the minimal promoter at −157/+135. DNase I footprint analysis revealed two complexes located within the gene's core promoter region, and site-directed mutagenesis identified two regions that were critical to maintain basal expression. EMSA identified the presence of five Sp elements within the immediate promoter region that are capable of binding members of the Sp family of proteins. Among the five Sp elements, one element appears to not bind Sp1, Sp2, or Sp3 while influencing the interaction of Sp proteins with an adjacent Sp site. Overexpression of either Sp1 or Sp3 augments activity of the minimal promoter in Sp-deficient Drosophila SL2 cells. In summary, we report on the characterization of the rat FcRn minimal promoter, including the characterization of five Sp elements within this region that interact with members of the Sp family of transcriptional factors and drive promoter activity in intestinal cell lines.

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PRPF6 Induces the Alternative Splicing of lncRNA SNHG16 To Promote Progression and Paclitaxel Resistance of Ovarian Cancer via Regulating GATA3 Transcription

10.21203/rs.3.rs-1045802/v1 ◽

2021 ◽

Author(s):

Han Wang ◽

Yingying Zhou ◽

Siyang Zhang ◽

Ya Qi ◽

Min Wang

Keyword(s):

Ovarian Cancer ◽

Alternative Splicing ◽

Epithelial To Mesenchymal Transition ◽

Binding Protein ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Mesenchymal Transition ◽

Gene Assay

Abstract Background Small nucleolar RNA host gene 16 (SNHG16) and pre-mRNA processing factor 6(PRPF6) play vital roles in regulatory mechanisms of multiple cancers, but the mechanisms in ovarian cancer (OC) remains poorly understood. Methods The expression of SNHG16 transcripts-SNHG16-L/S in OC tissues were analyzed by real-time PCR (RT-PCR). The expression of PRPF6 in OC tissues were detected by Immunohistochemistry (IHC). Tumorigenesis, epithelial-to-mesenchymal transition (EMT) and PTX-resistance were detected by western blot, transwell, CCK-8 assays, colony formation assays and flow cytometry analyses. Molecular interactions were examined by dual-luciferase reporter gene assay, RNA immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP). Results The results indicated the expression of SNHG16-L/S was opposite in chemo-resistance and chemo-sensitivity tissues of OC. And SNHG16-L/S had different effects on the progression and PTX-resistance of OC cells. SNHG16-L inhibited GATA binding protein 3 (GATA3) transcription through CCAAT/enhancer-binding protein b (CEBPB) to further promote tumorigenesis, EMT and PTX-resistance of OC. Moreover, PRPF6 was upregulated in chemo-resistance tissues of OC. PRPF6 promoted tumorigenesis and PTX-resistance in vitro and in vivo. Mechanistically, PRPF6 induced the alternative splicing of SNHG16 to downregulate SNHG16-L, which further mediated progression and PTX-resistance through upregulating GATA3 in OC. Conclusions Totally, the results demonstrated that PRPF6 promoted progression and PTX-resistance in OC through SNHG16-L/CEBPB/GATA3 axis. Thus, PRPF6 may become a valuable target for OC therapy.

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Interplay between cancer cells and M2 macrophages is necessary for miR-550a-3-5p down-regulation-mediated HPV-positive OSCC progression

10.21203/rs.3.rs-23980/v1 ◽

2020 ◽

Author(s):

Mingxin Cao ◽

Weilong Zhang ◽

Xianghua Yu ◽

Jiashun Wu ◽

Xinwei Qiao ◽

...

Keyword(s):

Cancer Cells ◽

Critical Role ◽

Differentially Expressed ◽

Luciferase Reporter ◽

Vehicle Group ◽

M2 Macrophages ◽

Tumor Associated Macrophages ◽

Down Regulation ◽

Macrophages Polarization ◽

Gene Assay

Abstract Background: Human papillomavirus (HPV)-positive oral squamous cell carcinoma (OSCC) is increasing worldwide with typically higher grade and stage. Studies suggested that microRNAs (miRNAs) play a critical role in cancer; However, their role in HPV-positive OSCC progression remains unclear.Methods: miRNA microarray was performed to identify differentially expressed miRNAs. qRT-PCR and FISH were performed to determine the relative expression of miR-550a-3-5p. CCK-8, Flow cytometry, Wound healing, Cell invasion assays and xenograft experiments were conducted to analyze the biological roles of miR-550a-3-5p. Tumor-associated macrophages (TAMs) generation, co-culturing of cancer cells with TAMs, Western blot, Dual-luciferase reporter gene assay, Immunohistochemistry and animal studies were performed to explore the mechanisms underlying the functions of miR-550a-3-5p. Results: In this study, we identified 19 miRNAs differentially expressed in HPV-positive OSCC specimens. One of these, miR-550a-3-5p, was down-regulated in HPV-positive OSCC. This down-regulation correlated with higher tumor size and nodal metastasis. Biofunctional investigations revealed that miR-550a-3-5p inhibited tumor growth and progression in nude mice models without altering the in vitro migration, invasion and EMT of HPV-positive OSCC cells. After co-culturing cancer cells with tumor-associated macrophages (TAMs), we found that the effects of miR-550a-3-5p on suppressing migration, invasion and EMT of HPV-positive OSCC cells were dependent on decreasing M2 macrophages polarization. Moreover, we identified that miR-550a-3-5p, down-regulated by E6 oncoprotein, inhibited M2 macrophages polarization by YAP/CCL2 signaling, which in turn abrogating EMT program in HPV-positive OSCC cells. Using YAP inhibitor, verteporfin (VP) in a HPV-positive OSCC model of transgenic mice also showed that tumors were less progressive when compared to those in Vehicle group. In both xenografts and clinical HPV-positive OSCC samples, miR-550a-3-5p levels were inversely associated with YAP, CCL2 expressions and the number of M2 macrophages.Conclusions: E6/miR-550a-3-5p/YAP/CCL2 signaling induces M2 macrophages polarization to enhance EMT and progression, revealing a novel crosstalk between cancer cells and immune cells in HPV-positive OSCC microenvironment.

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COX2 Enhances Neovascularization of Inflammatory Tenocytes Through the HIF-1α/VEGFA/PDGFB Pathway

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.670406 ◽

2021 ◽

Vol 9 ◽

Author(s):

Bin Deng ◽

Pu Xu ◽

Bingyu Zhang ◽

Qing Luo ◽

Guanbin Song

Keyword(s):

Growth Factor ◽

Trichostatin A ◽

Critical Role ◽

Tendon Injuries ◽

Luciferase Reporter ◽

Inflammatory Factor ◽

Vascular Endothelial ◽

Expression Levels ◽

Transcriptional Targets ◽

Gene Assay

Tendon injuries are among the most challenging in orthopedics. During the early tendon repair, new blood vessel formation is necessary. However, excessive angiogenesis also exacerbates scar formation, leading to pain and dysfunction. A significantly worse outcome was associated with higher expression levels of hypoxia-inducible factor-1 alpha (HIF-1α), and its transcriptional targets vascular endothelial growth factor A (VEGFA) and platelet-derived growth factor B (PDGFB), but the underlying molecular mechanisms remain unclear. In this study, lipopolysaccharide (LPS) was used to induce an inflammatory response in tenocytes. LPS increased the tenocytes’ inflammatory factor COX2 expression and activated the HIF-1α/VEGFA/PDGFB pathway. Moreover, the conditioned medium from the tenocytes boosted rat aortic vascular endothelial cell (RAOEC) angiogenesis. Furthermore, Trichostatin A (TSA), an inhibitor of histone deacetylase, was used to treat inflammatory tenocytes. The expression levels of HIF-1α and its transcriptional targets VEGFA and PDGFB decreased, resulting in RAOEC angiogenesis inhibition. Finally, the dual-luciferase reporter gene assay and chromatin immunoprecipitation (ChIP) assay proved that the HIF-1α/PDGFB pathway played a more critical role in tenocyte angiogenesis than the HIF-1α/VEGFA pathway. TSA could alleviate angiogenesis mainly through epigenetic regulation of the HIF-1α/PDGFB pathway. Taken together, TSA might be a promising anti-angiogenesis drug for abnormal angiogenesis, which is induced by tendon injuries.

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YY1 Activates EMI2 and Promotes the Progression of Cholangiocarcinoma Through the PI3K/Akt Signaling Axis

10.21203/rs.3.rs-792593/v1 ◽

2021 ◽

Author(s):

Shuai zhou ◽

Kang Lin Qu ◽

JinAng Li ◽

Shilei Chen ◽

Yi Gang Zhang ◽

...

Keyword(s):

Transcription Factors ◽

Bile Duct ◽

Signaling Pathway ◽

Akt Signaling ◽

Reporter Gene Assay ◽

Luciferase Reporter ◽

Akt Signaling Pathway ◽

Luciferase Reporter Gene ◽

Luciferase Reporter Gene Assay ◽

Gene Assay

Abstract Background: Cholangiocarcinoma (CCA) is one of the deadliest cancers of the digestive tract. The prognosis of CCA is poor and the 5-year survival rate is low. Bioinformatic analysis showed that early mitotic inhibitor 2 (EMI2) was overexpressed in CCA but the underlying mechanism is not known.Methods: The data on bile duct carcinoma from TCGA and GEO databases were used to detect the expression of EMI2. The transcription factors of EMI2 were predicted using JASPAR and PROMO databases. Among the predicted transcription factors, YY1 has been rarely reported in cholangiocarcinoma, and was verified using the luciferase reporter gene assay. RT-PCR was performed to predict the downstream pathway of EMI2, and PI3K/Akt was suspected to be associated with it. Subsequently, in vivo and in vitro experiments were conducted to verify the effects of silencing and overexpressing EMI2 and YY1 on the proliferation, invasion, and metastasis of the bile duct cancer cells.Results: EMI2 was highly expressed in CCA. Silencing EMI2 inhibited the proliferation, invasion, and migration of CCA cells, arrested cell cycle in the G1 phase, and inhibition of apoptosis. The luciferase reporter gene assay showed that YY1 bound to the promoter region of EMI2, and after silencing YY1, the expression of EMI2 decreased and the progression of CCA was inhibited. Moreover, key proteins in the PI3K/Akt signaling pathway decreased after silencing EMI2.Conclusion: EMI2 may be one of the direct targets of YY1 and promotes the progression of CCA through the PI3K/Akt signaling pathway.

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A novel peptide RIFV suppresses human adipocyte differentiation through the inhibition of C/EBP-β expression

Nutrition & Metabolism ◽

10.1186/s12986-019-0414-z ◽

2019 ◽

Vol 16 (1) ◽

Author(s):

Wen Zhang ◽

Dan Shen ◽

Yun Li ◽

Hong Zhong ◽

Xing Wang ◽

...

Keyword(s):

Adipocyte Differentiation ◽

Inhibitory Effect ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Epidemic Disease ◽

Global Epidemic ◽

Protein Levels ◽

Therapeutic Drugs ◽

Peptide Database ◽

Gene Assay

Abstract Background Obesity is a global epidemic disease that increases the risk of metabolic syndrome. However, therapeutic drugs for obesity are still scarce. In recent years, peptides have been identified as new biological regulators. RIFV (R-I-F-V-P-I-K-G-R-P-A-P), a novel active peptide from our peptide database. Methods We performed oil red O staining and triglyceride measurement to analyze the influence of RIFV on white preadipocytes differentiation. Then the effects of RIFV on cell proliferation, apoptosis and cell cycle were determined by using CCK-8 assay and flow cytometry. The mRNA and protein levels of adipogenesis-related genes were respectively detected by qRT-PCR and western blot. Rescue experiment was conducted to confirm whether RIFV could regulate adipocytes differentiation via targeting C/EBP-β. Finally, the luciferase reporter gene assay was performed to verify the regulation of RIFV on C/EBP-β gene. Results RIFV was revealed to inhibit the differentiation of human white adipocytes without affecting their proliferation. Additionally, RIFV could also suppress the differentiation of mouse primary white preadipocytes isolated from inguinal fat tissues. Furthermore, RIFV may have an inhibitory effect on adipogenesis by inhibiting the regulation of the adipogenic gene C/EBP-β. Conclusions Our results indicated that RIFV may be a novel essential regulator of adipocyte differentiation and represents a therapeutic strategy for obesity and related complications.

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Cellular Retinol-Binding Protein Type I (CRBP-I) Regulates Adipogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.00014-10 ◽

2010 ◽

Vol 30 (14) ◽

pp. 3412-3420 ◽

Cited By ~ 33

Author(s):

C. F. Zizola ◽

S. K. Frey ◽

S. Jitngarmkusol ◽

B. Kadereit ◽

N. Yan ◽

...

Keyword(s):

Transcription Factors ◽

High Fat Diet ◽

Adipocyte Differentiation ◽

Binding Protein ◽

Retinol Binding Protein ◽

Type I ◽

High Fat ◽

Embryonic Fibroblasts ◽

Glucose Tolerant ◽

Cellular Retinol Binding Protein

ABSTRACT Adipogenesis is governed by a well-documented cascade of transcription factors. However, less is known about non-transcription factors that govern early stages of adipogenesis. Here we show that cellular retinol-binding protein type I (CRBP-I), a small cytosolic binding protein for retinol and retinaldehyde, is specifically restricted to preadipocytes in white adipose tissue. The absence of CRBP-I in mice (CRBP-I-KO mice) leads to increased adiposity. Despite increased adiposity, CRBP-I-KO mice remain more glucose tolerant and insulin sensitive during high-fat-diet feeding. 3T3-L1 cells deficient in CRBP-I or mouse embryonic fibroblasts derived from CRBP-I-KO mice had increased adipocyte differentiation and triglyceride (TG) accumulation. This was due to increased expression and activity of PPARγ, while other transcription factor pathways in early and late differentiation remained unchanged. Conversely, the overexpression of CRBP-I in 3T3-L1 cells results in decreased TG accumulation. In conclusion, CRBP-I is a cytosolic protein specifically expressed in preadipocytes that regulates adipocyte differentiation in part by affecting PPARγ activity.

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