Epigenetic regulation of ELF3 transcriptional activation in human chondrocytes in vivo and in vitro

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.

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CDK13 upregulation-induced formation of the positive feedback loop among circCDK13, miR-212-5p/miR-449a and E2F5 contributes to prostate carcinogenesis

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-020-01814-5 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Jin-Chun Qi ◽

Zhan Yang ◽

Tao Lin ◽

Long Ma ◽

Ya-Xuan Wang ◽

...

Keyword(s):

Cell Proliferation ◽

Transcriptional Activation ◽

Positive Feedback ◽

Feedback Loop ◽

Biological Effects ◽

Therapeutic Benefit ◽

Loss Of Function ◽

Positive Feedback Loop

Abstract Background Both E2F transcription factor and cyclin-dependent kinases (CDKs), which increase or decrease E2F activity by phosphorylating E2F or its partner, are involved in the control of cell proliferation, and some circRNAs and miRNAs regulate the expression of E2F and CDKs. However, little is known about whether dysregulation among E2Fs, CDKs, circRNAs and miRNAs occurs in human PCa. Methods The expression levels of CDK13 in PCa tissues and different cell lines were determined by quantitative real-time PCR and Western blot analysis. In vitro and in vivo assays were preformed to explore the biological effects of CDK13 in PCa cells. Co-immunoprecipitation anlysis coupled with mass spectrometry was used to identify E2F5 interaction with CDK13. A CRISPR-Cas9 complex was used to activate endogenous CDK13 and circCDK13 expression. Furthermore, the mechanism of circCDK13 was investigated by using loss-of-function and gain-of-function assays in vitro and in vivo. Results Here we show that CDK13 is significantly upregulated in human PCa tissues. CDK13 depletion and overexpression in PCa cells decrease and increase, respectively, cell proliferation, and the pro-proliferation effect of CDK13 is strengthened by its interaction with E2F5. Mechanistically, transcriptional activation of endogenous CDK13, but not the forced expression of CDK13 by its expression vector, remarkably promotes E2F5 protein expression by facilitating circCDK13 formation. Further, the upregulation of E2F5 enhances CDK13 transcription and promotes circCDK13 biogenesis, which in turn sponges miR-212-5p/449a and thus relieves their repression of the E2F5 expression, subsequently leading to the upregulation of E2F5 expression and PCa cell proliferation. Conclusions These findings suggest that CDK13 upregulation-induced formation of the positive feedback loop among circCDK13, miR-212-5p/miR-449a and E2F5 is responsible for PCa development. Targeting this newly identified regulatory axis may provide therapeutic benefit against PCa progression and drug resistance.

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Reversal of senescence-associated beta-galactosidase expression during in vitro three-dimensional tissue-engineering of human chondrocytes in a polymer scaffold

Scientific Reports ◽

10.1038/s41598-021-93607-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Shojiro Katoh ◽

Atsuki Fujimaru ◽

Masaru Iwasaki ◽

Hiroshi Yoshioka ◽

Rajappa Senthilkumar ◽

...

Keyword(s):

Regenerative Medicine ◽

Replicative Senescence ◽

Cultured Cells ◽

Three Dimensional ◽

Human Chondrocytes ◽

Cartilage Tissue ◽

Optimal Method ◽

Beta Galactosidase

AbstractRegenerative medicine applications require cells that are not inflicted with senescence after in vitro culture for an optimal in vivo outcome. Methods to overcome replicative senescence include genomic modifications which have their own disadvantages. We have evaluated a three-dimensional (3D) thermo-reversible gelation polymer (TGP) matrix environment for its capabilities to reverse cellular senescence. The expression of senescence-associated beta-galactosidase (SA-βgal) by human chondrocytes from osteoarthritis-affected cartilage tissue, grown in a conventional two-dimensional (2D) monolayer culture versus in 3D-TGP were compared. In 2D, the cells de-differentiated into fibroblasts, expressed higher SA-βgal and started degenerating at 25 days. SA-βgal levels decreased when the chondrocytes were transferred from the 2D to the 3D-TGP culture, with cells exhibiting a tissue-like growth until 42–45 days. Other senescence associated markers such as p16INK4a and p21 were also expressed only in 2D cultured cells but not in 3D-TGP tissue engineered cartilage. This is a first-of-its-kind report of a chemically synthesized and reproducible in vitro environment yielding an advantageous reversal of aging of human chondrocytes without any genomic modifications. The method is worth consideration as an optimal method for growing cells for regenerative medicine applications.

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NR5A2 transcriptional activation by BRD4 promotes pancreatic cancer progression by upregulating GDF15

Cell Death Discovery ◽

10.1038/s41420-021-00462-8 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Feng Guo ◽

Yingke Zhou ◽

Hui Guo ◽

Dianyun Ren ◽

Xin Jin ◽

...

Keyword(s):

Pancreatic Cancer ◽

Cancer Cells ◽

Cancer Progression ◽

Transcriptional Activation ◽

Ductal Adenocarcinoma ◽

Pancreatic Cancer Cells ◽

Gene Sets ◽

And Migration

AbstractNR5A2 is a transcription factor regulating the expression of various oncogenes. However, the role of NR5A2 and the specific regulatory mechanism of NR5A2 in pancreatic ductal adenocarcinoma (PDAC) are not thoroughly studied. In our study, Western blotting, real-time PCR, and immunohistochemistry were conducted to assess the expression levels of different molecules. Wound-healing, MTS, colony formation, and transwell assays were employed to evaluate the malignant potential of pancreatic cancer cells. We demonstrated that NR5A2 acted as a negative prognostic biomarker in PDAC. NR5A2 silencing inhibited the proliferation and migration abilities of pancreatic cancer cells in vitro and in vivo. While NR5A2 overexpression markedly promoted both events in vitro. We further identified that NR5A2 was transcriptionally upregulated by BRD4 in pancreatic cancer cells and this was confirmed by Chromatin immunoprecipitation (ChIP) and ChIP-qPCR. Besides, transcriptome RNA sequencing (RNA-Seq) was performed to explore the cancer-promoting effects of NR5A2, we found that GDF15 is a component of multiple down-regulated tumor-promoting gene sets after NR5A2 was silenced. Next, we showed that NR5A2 enhanced the malignancy of pancreatic cancer cells by inducing the transcription of GDF15. Collectively, our findings suggest that NR5A2 expression is induced by BRD4. In turn, NR5A2 activates the transcription of GDF15, promoting pancreatic cancer progression. Therefore, NR5A2 and GDF15 could be promising therapeutic targets in pancreatic cancer.

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Expression of laminin γ 1 cultured hepatocytes involves repeated CTC and GC elements in the LAMC1 promoter

Biochemical Journal ◽

10.1042/bj3130745 ◽

1996 ◽

Vol 313 (3) ◽

pp. 745-752 ◽

Cited By ~ 5

Author(s):

Françoise LEVAVASSEUR ◽

Jocelyne LIÉTARD ◽

Kohei OGAWA ◽

Nathalie THÉRET ◽

Peter D. BURBELO ◽

...

Keyword(s):

Transcriptional Activation ◽

Hepatoma Cell ◽

Primary Cultures ◽

Regulatory Elements ◽

Hepatoma Cells ◽

Basement Membranes ◽

Major Protein ◽

Cultured Hepatocytes

Laminin γ1 chain is present in all basement membranes and is expressed at high levels in various diseases, such as hepatic fibrosis. We have identified cis- and trans-acting elements involved in the regulation of this gene in normal rat liver, as well as in hepatocyte primary cultures and hepatoma cell lines. Northern-blot analyses showed that laminin γ1 mRNA was barely detectable in freshly isolated hepatocytes and expressed at high levels in hepatocyte primary cultures, as early as 4 h after liver dissociation. Actinomycin D and cycloheximide treatment in vivo and in vitro indicated that laminin γ1 overexpression in cultured hepatocytes was under the control of transcriptional mechanisms. Transfection of deletion mutants of the 5´ flanking region of murine LAMC1 gene in hepatoma cells that constitutively express laminin γ1 indicated that regulatory elements were located between -594 bp and -94 bp. This segment included GC- and CTC-containing motifs. Gel-shift analyses showed that two complexes were resolved with different affinity for the CTC sequence depending on the location of the GC box. The pattern of complex formation with nuclear factors from freshly isolated and cultured hepatocytes was different from that obtained with total liver and similar to that with hepatoma cells. Southwestern analysis indicated that several polypeptides bound the CTC-rich sequence. Affinity chromatography demonstrated that a Mr 60000 polypeptide was a major protein binding to the CTC motif. This polypeptide is probably involved in the transcriptional activation of various proto-oncogenes and extracellular matrix genes that are expressed at high levels in both hepatoma cells and early hepatocyte cultures.

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Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

Molecular and Cellular Biology ◽

10.1128/mcb.00842-15 ◽

2015 ◽

Vol 36 (6) ◽

pp. 913-922 ◽

Cited By ~ 17

Author(s):

Nallani Vijay Kumar ◽

Jianbo Yang ◽

Jitesh K. Pillai ◽

Swati Rawat ◽

Carlos Solano ◽

...

Keyword(s):

Transcription Factor ◽

Transcriptional Activation ◽

Transcriptional Response ◽

Transcriptional Regulator ◽

Yeast Protein ◽

Content Type ◽

Arsenic Tolerance ◽

Arsenic Sensing

The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeastSaccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)]in vitroandin vivoand that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation.

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Apoptosis in metanephric development.

The Journal of Cell Biology ◽

10.1083/jcb.119.5.1327 ◽

1992 ◽

Vol 119 (5) ◽

pp. 1327-1333 ◽

Cited By ~ 200

Author(s):

C Koseki ◽

D Herzlinger ◽

Q al-Awqati

Keyword(s):

Spinal Cord ◽

Protein Kinase ◽

Transcriptional Activation ◽

Phorbol Esters ◽

Dna Degradation ◽

Morphological Evidence ◽

Metanephric Mesenchyme ◽

Embryonic Spinal Cord

During metanephric development, non-polarized mesenchymal cells are induced to form the epithelial structures of the nephron following interaction with extracellular matrix proteins and factors produced by the inducing tissue, ureteric bud. This induction can occur in a transfilter organ culture system where it can also be produced by heterologous cells such as the embryonic spinal cord. We found that when embryonic mesenchyme was induced in vitro and in vivo, many of the cells surrounding the new epithelium showed morphological evidence of programmed cell death (apoptosis) such as condensed nuclei, fragmented cytoplasm, and cell shrinking. A biochemical correlate of apoptosis is the transcriptional activation of a calcium-sensitive endonuclease. Indeed, DNA isolated from uninduced mesenchyme showed progressive degradation, a process that was prevented by treatment with actinomycin-D or cycloheximide and by buffering intracellular calcium. These results demonstrate that the metanephric mesenchyme is programmed for apoptosis. Incubation of mesenchyme with a heterologous inducer, embryonic spinal cord prevented this DNA degradation. To investigate the mechanism by which inducers prevented apoptosis we tested the effects of protein kinase C modulators on this process. Phorbol esters mimicked the effects of the inducer and staurosporine, an inhibitor of this protein kinase, prevented the effect of the inducer. EGF also prevented DNA degradation but did not lead to differentiation. These results demonstrate that conversion of mesenchyme to epithelial requires at least two steps, rescue of the mesenchyme from apoptosis and induction of differentiation.

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Targeting of p38 Mitogen-Activated Protein Kinases to MEF2 Transcription Factors

Molecular and Cellular Biology ◽

10.1128/mcb.19.6.4028 ◽

1999 ◽

Vol 19 (6) ◽

pp. 4028-4038 ◽

Cited By ~ 172

Author(s):

Shen-Hsi Yang ◽

Alex Galanis ◽

Andrew D. Sharrocks

Keyword(s):

Transcription Factors ◽

Transcriptional Activation ◽

Cellular Response ◽

Map Kinases ◽

Biological Response ◽

Extracellular Signals ◽

Extracellular Signal ◽

Mitogen Activated Protein

ABSTRACT Mitogen-activated protein (MAP) kinase-mediated signalling to the nucleus is an important event in the conversion of extracellular signals into a cellular response. However, the existence of multiple MAP kinases which phosphorylate similar phosphoacceptor motifs poses a problem in maintaining substrate specificity and hence the correct biological response. Both the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) subfamilies of MAP kinases use a second specificity determinant and require docking to their transcription factor substrates to achieve maximal substrate activation. In this study, we demonstrate that among the different MAP kinases, the MADS-box transcription factors MEF2A and MEF2C are preferentially phosphorylated and activated by the p38 subfamily members p38α and p38β2. The efficiency of phosphorylation in vitro and transcriptional activation in vivo of MEF2A and MEF2C by these p38 subtypes requires the presence of a kinase docking domain (D-domain). Furthermore, the D-domain from MEF2A is sufficient to confer p38 responsiveness on different transcription factors, and reciprocal effects are observed upon the introduction of alternative D-domains into MEF2A. These results therefore contribute to our understanding of signalling to MEF2 transcription factors and demonstrate that the requirement for substrate binding by MAP kinases is an important facet of three different subclasses of MAP kinases (ERK, JNK, and p38).

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Extracellular Signal-Regulated Kinase Binds to TFII-I and Regulates Its Activation of the c-fosPromoter

Molecular and Cellular Biology ◽

10.1128/mcb.20.4.1140-1148.2000 ◽

2000 ◽

Vol 20 (4) ◽

pp. 1140-1148 ◽

Cited By ~ 37

Author(s):

Dae-Won Kim ◽

Brent H. Cochran

Keyword(s):

Map Kinase ◽

Transcriptional Activation ◽

Dominant Negative ◽

Binding Motif ◽

Dependent Manner ◽

Consensus Map ◽

Interaction Domain ◽

Extracellular Signal

ABSTRACT We have previously shown that TFII-I enhances transcriptional activation of the c-fos promoter through interactions with upstream elements in a signal-dependent manner. Here we demonstrate that activated Ras and RhoA synergize with TFII-I for c-fospromoter activation, whereas dominant-negative Ras and RhoA inhibit these effects of TFII-I. The Mek1 inhibitor, PD98059 abrogates the enhancement of the c-fos promoter by TFII-I, indicating that TFII-I function is dependent on an active mitogen-activated protein (MAP) kinase pathway. Analysis of the TFII-I protein sequence revealed that TFII-I contains a consensus MAP kinase interaction domain (D box). Consistent with this, we have found that TFII-I forms an in vivo complex with extracellular signal-related kinase (ERK). Point mutations within the consensus MAP kinase binding motif of TFII-I inhibit its ability to bind ERK and its ability to enhance the c-fos promoter. Therefore, the D box of TFII-I is required for its activity on the c-fos promoter. Moreover, the interaction between TFII-I and ERK can be regulated. Serum stimulation enhances complex formation between TFII-I and ERK, and dominant-negative Ras abrogates this interaction. In addition, TFII-I can be phosphorylated in vitro by ERK and mutation of consensus MAP kinase substrate sites at serines 627 and 633 impairs the phosphorylation of TFII-I by ERK and its activity on the c-fos promoter. These results suggest that ERK regulates the activity of TFII-I by direct phosphorylation.

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p53 upregulated by HIF-1α promotes hypoxia-induced G2/M arrest and renal fibrosis in vitro and in vivo

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjy042 ◽

2018 ◽

Vol 11 (5) ◽

pp. 371-382 ◽

Cited By ~ 15

Author(s):

Limin Liu ◽

Peng Zhang ◽

Ming Bai ◽

Lijie He ◽

Lei Zhang ◽

...

Keyword(s):

Cell Cycle ◽

Transcriptional Activation ◽

Renal Fibrosis ◽

Intraperitoneal Administration ◽

Luciferase Reporter ◽

Loss Of Function ◽

P53 Expression ◽

Tubular Cells

Abstract Hypoxia plays an important role in the genesis and progression of renal fibrosis. The underlying mechanisms, however, have not been sufficiently elucidated. We examined the role of p53 in hypoxia-induced renal fibrosis in cell culture (human and rat renal tubular epithelial cells) and a mouse unilateral ureteral obstruction (UUO) model. Cell cycle of tubular cells was determined by flow cytometry, and the expression of profibrogenic factors was determined by RT-PCR, immunohistochemistry, and western blotting. Chromatin immunoprecipitation and luciferase reporter experiments were performed to explore the effect of HIF-1α on p53 expression. We showed that, in hypoxic tubular cells, p53 upregulation suppressed the expression of CDK1 and cyclins B1 and D1, leading to cell cycle (G2/M) arrest (or delay) and higher expression of TGF-β, CTGF, collagens, and fibronectin. p53 suppression by siRNA or by a specific p53 inhibitor (PIF-α) triggered opposite effects preventing the G2/M arrest and profibrotic changes. In vivo experiments in the UUO model revealed similar antifibrotic results following intraperitoneal administration of PIF-α (2.2 mg/kg). Using gain-of-function, loss-of-function, and luciferase assays, we further identified an HRE3 region on the p53 promoter as the HIF-1α-binding site. The HIF-1α–HRE3 binding resulted in a sharp transcriptional activation of p53. Collectively, we show the presence of a hypoxia-activated, p53-responsive profibrogenic pathway in the kidney. During hypoxia, p53 upregulation induced by HIF-1α suppresses cell cycle progression, leading to the accumulation of G2/M cells, and activates profibrotic TGF-β and CTGF-mediated signaling pathways, causing extracellular matrix production and renal fibrosis.

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