scholarly journals Rhythmic SAF-A Binding Underlies Circadian Transcription of the Bmal1 Gene

2008 ◽  
Vol 28 (10) ◽  
pp. 3477-3488 ◽  
Author(s):  
Yoshiaki Onishi ◽  
Syuji Hanai ◽  
Tomoya Ohno ◽  
Yasuhiro Hara ◽  
Norio Ishida
Keyword(s):  
Chromatin Structure ◽  
Matrix Protein ◽  
Open Chromatin ◽  
Nuclear Matrix Protein ◽  
Actual Mechanism ◽  
Gene Assay ◽  
Flanking Region ◽  
Circadian Transcription

ABSTRACT Although Bmal1 is a key component of the mammalian clock system, little is understood about the actual mechanism of circadian Bmal1 gene transcription, particularly at the chromatin level. Here we discovered a unique chromatin structure within the Bmal1 promoter. The RORE region, which is a critical cis element for the circadian regulation of the Bmal1 gene, is comprised of GC-rich open chromatin. The 3′-flanking region of the promoter inhibited rhythmic transcription in the reporter gene assay in vitro even in the presence of RORα and REV-ERBα. We also found that the nuclear matrix protein SAF-A binds to the 3′-flanking region with circadian timing, which was correlated with Bmal1 expression by footprinting in vivo. These results suggest that the unique chromatin structure containing SAF-A is required for the circadian transcriptional regulation of the Bmal1 gene in cells.

scholarly journals The amino-terminal region of the retinoblastoma gene product binds a novel nuclear matrix protein that co-localizes to centers for RNA processing.

1994 ◽  
Vol 127 (3) ◽  
pp. 609-622 ◽  
Author(s):  
T Durfee ◽  
M A Mancini ◽  
D Jones ◽  
S J Elledge ◽  
W H Lee
Keyword(s):  
Nuclear Matrix ◽  
Rna Processing ◽  
Matrix Protein ◽  
Terminal Region ◽  
Cellular Proteins ◽  
Amino Terminus ◽  
Nuclear Matrix Protein ◽  
Amino Terminal

The tumor suppressing capacity of the retinoblastoma protein (p110RB) is dependent on interactions made with cellular proteins through its carboxy-terminal domains. How the p110RB amino-terminal region contributes to this activity is unclear, though evidence now indicates it is important for both growth suppression and regulation of the full-length protein. We have used the yeast two-hybrid system to screen for cellular proteins which bind to the first 300 amino acids of p110RB. The only gene isolated from this screen encodes a novel 84-kD nuclear matrix protein that localizes to subnuclear regions associated with RNA processing. This protein, p84, requires a structurally defined domain in the amino terminus of p110RB for binding. Furthermore, both in vivo and in vitro experiments demonstrate that p84 binds preferentially to the functionally active, hypophosphorylated form of p110RB. Thus, the amino terminus of p110RB may function in part to facilitate the binding of growth promoting factors at subnuclear regions actively involved in RNA metabolism.

scholarly journals SRm160 Splicing Coactivator Promotes Transcript 3′-End Cleavage

2002 ◽  
Vol 22 (1) ◽  
pp. 148-160 ◽  
Author(s):  
Susan McCracken ◽  
Mark Lambermon ◽  
Benjamin J. Blencowe
Keyword(s):  
Gene Expression ◽  
Matrix Protein ◽  
Mrna Processing ◽  
Nuclear Matrix Protein ◽  
Mrna Transcripts ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
Cytoplasmic Accumulation

ABSTRACT Individual steps in the processing of pre-mRNA, including 5′-end cap formation, splicing, and 3′-end processing (cleavage and polyadenylation) are highly integrated and can influence one another. In addition, prior splicing can influence downstream steps in gene expression, including export of mRNA from the nucleus. However, the factors and mechanisms coordinating these steps in the maturation of pre-mRNA transcripts are not well understood. In the present study we demonstrate that SRm160 (for serine/arginine repeat-related nuclear matrix protein of 160 kDa), a coactivator of constitutive and exon enhancer-dependent splicing, participates in 3′-end formation. Increased levels of SRm160 promoted the 3′-end cleavage of transcripts both in vivo and in vitro. Remarkably, at high levels in vivo SRm160 activated the 3′-end cleavage and cytoplasmic accumulation of unspliced pre-mRNAs, thereby uncoupling the requirement for splicing to promote the 3′-end formation and nuclear release of these transcripts. Consistent with a role in 3′-end formation coupled to splicing, SRm160 was found to associate specifically with the cleavage polyadenylation specificity factor and to stimulate the 3′-end cleavage of splicing-active pre-mRNAs more efficiently than that of splicing-inactive pre-mRNAs in vitro. The results provide evidence for a role for SRm160 in mRNA 3′-end formation and suggest that the level of this splicing coactivator is important for the proper coordination of pre-mRNA processing events.

Induction of apoptosis in breast cancer cells in response to vitamin D and antiestrogens

1994 ◽  
Vol 72 (11-12) ◽  
pp. 537-545 ◽  
Author(s):  
JoEllen Welsh
Keyword(s):  
Breast Cancer ◽  
Vitamin D ◽  
Nuclear Matrix ◽  
Matrix Protein ◽  
Combined Treatment ◽  
Cell Number ◽  
Nuclear Matrix Protein ◽  
Mcf 7

1,25-Dihydroxycholecalciferol D3 (1,25(OH)2D3), the active metabolite of vitamin D, is a potent inhibitor of breast cancer cell growth both in vivo and in vitro. We have shown that MCF-7 cells treated with 100 nM 1,25(OH)2D3 exhibit characteristic apoptotic morphology (pyknotic nuclei, chromatin and cytoplasmic condensation, nuclear matrix protein reorganization) within 48 h. In the experiments reported here, we examined the interactions between 1,25(OH)2D3 and the antiestrogen 4-hydroxytamoxifen (TAM), which also induces apoptosis in MCF-7 cells. Our data suggest that TAM significantly potentiates the reduction in cell number induced by 1,25(OH)2D3 alone. Combined treatment with 1,25(OH)2D3 and TAM enhances the degree of apoptosis assessed using morphological markers that identify chromatin and nuclear matrix protein condensation. We have selected a subclone of MCF-7 cells resistant to 1,25(OH)2D3 (MCF-7D3Res). These cells express the vitamin D receptor and exhibit doubling times comparable to the parental MCF-7 cells, even when grown in 100 mM 1,25(OH)2D3. Treatment of both parental and resistant MCF-7 cells with TAM induces apoptosis and clusterin. These data emphasize that apoptosis can be induced in MCF-7 cells either by activation of vitamin-D-mediated signalling or disruption of estrogen-dependent signalling.Key words: apoptosis, breast, vitamin D, antiestrogen, gene expression, clusterin.

scholarly journals Impact of RARα and miR-138 on retinoblastoma etoposide resistance

Tumor Biology ◽  
2021 ◽  
Vol 43 (1) ◽  
pp. 11-26
Author(s):  
Maike Busch ◽  
Natalia Miroschnikov ◽  
Jaroslaw Thomas Dankert ◽  
Marc Wiesehöfer ◽  
Klaus Metz ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Lines ◽  
Cancer Progression ◽  
Treated Patient ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
The Impact

BACKGROUND: Retinoblastoma (RB) is the most common childhood eye cancer. Chemotherapeutic drugs such as etoposide used in RB treatment often cause massive side effects and acquired drug resistances. Dysregulated genes and miRNAs have a large impact on cancer progression and development of chemotherapy resistances. OBJECTIVE: This study was designed to investigate the involvement of retinoic acid receptor alpha (RARα) in RB progression and chemoresistance as well as the impact of miR-138, a potential RARα regulating miRNA. METHODS: RARα and miR-138 expression in etoposide resistant RB cell lines and chemotherapy treated patient tumors compared to non-treated tumors was revealed by Real-Time PCR. Overexpression approaches were performed to analyze the effects of RARα on RB cell viability, apoptosis, proliferation and tumorigenesis. Besides, we addressed the effect of miR-138 overexpression on RB cell chemotherapy resistance. RESULTS: A binding between miR-138 and RARα was shown by dual luciferase reporter gene assay. The study presented revealed that RARα is downregulated in etoposide resistant RB cells, while miR-138 is endogenously upregulated. Opposing RARα and miR-138 expression levels were detectable in chemotherapy pre-treated compared to non-treated RB tumor specimen. Overexpression of RARα increases apoptosis levels and reduces tumor cell growth of aggressive etoposide resistant RB cells in vitro and in vivo. Overexpression of miR-138 in chemo-sensitive RB cell lines partly enhances cell viability after etoposide treatment. CONCLUSIONS: Our findings show that RARα acts as a tumor suppressor in retinoblastoma and is downregulated upon etoposide resistance in RB cells. Thus, RARα may contribute to the development and progression of RB chemo-resistance.

scholarly journals Low-Intensity Pulsed Ultrasound Promotes Autophagy-Mediated Migration of Mesenchymal Stem Cells and Cartilage Repair

2021 ◽  
Vol 30 ◽  
pp. 096368972098614
Author(s):  
Peng Xia ◽  
Xinwei Wang ◽  
Qi Wang ◽  
Xiaoju Wang ◽  
Qiang Lin ◽  
...  
Keyword(s):  
Cartilage Repair ◽  
Matrix Protein ◽  
Extracellular Matrix Protein ◽  
Histopathological Analysis ◽  
Pulsed Ultrasound ◽  
Low Intensity Pulsed Ultrasound ◽  
Low Intensity ◽  
Autophagy Inhibitor

Mesenchymal stem cell (MSC) migration is promoted by low-intensity pulsed ultrasound (LIPUS), but its mechanism is unclear. Since autophagy is known to regulate cell migration, our study aimed to investigate if LIPUS promotes the migration of MSCs via autophagy regulation. We also aimed to investigate the effects of intra-articular injection of MSCs following LIPUS stimulation on osteoarthritis (OA) cartilage. For the in vitro study, rat bone marrow-derived MSCs were treated with an autophagy inhibitor or agonist, and then they were stimulated by LIPUS. Migration of MSCs was detected by transwell migration assays, and stromal cell-derived factor-1 (SDF-1) and C-X-C chemokine receptor type 4 (CXCR4) protein levels were quantified. For the in vivo study, a rat knee OA model was generated and treated with LIPUS after an intra-articular injection of MSCs with autophagy inhibitor added. The cartilage repair was assessed by histopathological analysis and extracellular matrix protein expression. The in vitro results suggest that LIPUS increased the expression of SDF-1 and CXCR4, and it promoted MSC migration. These effects were inhibited and enhanced by autophagy inhibitor and agonist, respectively. The in vivo results demonstrate that LIPUS significantly enhanced the cartilage repair effects of MSCs on OA, but these effects were blocked by autophagy inhibitor. Our results suggest that the migration of MSCs was enhanced by LIPUS through the activation autophagy, and LIPUS improved the protective effect of MSCs on OA cartilage via autophagy regulation.

Contrasting migratory response of astrocytoma cells to tenascin mediated by different integrins

1996 ◽  
Vol 109 (8) ◽  
pp. 2161-2168 ◽  
Author(s):  
A. Giese ◽  
M.A. Loo ◽  
S.A. Norman ◽  
S. Treasurywala ◽  
M.E. Berens
Keyword(s):  
Cell Adhesion ◽  
Matrix Protein ◽  
Glioma Cells ◽  
Extracellular Matrix Protein ◽  
Integrin Receptors ◽  
Migration Assay ◽  
Dose Dependent Fashion ◽  
Beta 1 Integrin

Tenascin, an extracellular matrix protein, is expressed in human gliomas in vitro and in vivo. The distribution of tenascin at the invasive edge of these tumors, even surrounding solitary invading cells, suggests a role for this protein as a regulator of glioma cell migration. We tested whether purified tenascin, passively deposited on surfaces, influenced the adhesion or migration of a human gliomaderived cell line, SF-767. Adhesion of glioma cells to tenascin increased in a dose-dependent fashion up to a coating concentration of 10 micrograms/ml. Higher coating concentrations resulted in progressively fewer cells attaching. Cell adhesion could be blocked to basal levels using anti-beta 1 integrin antibodies. In contrast, when anti-alpha v antibodies were added to the medium of cells on tenascin, cell adhesion was enhanced slightly. Using a microliter scale migration assay, we found that cell motility on tenascin was dose dependently stimulated at coating concentrations of 1 and 3 micrograms/ml, but migration was inhibited below levels of non-specific motility when tested at coating concentrations of 30 and 100 micrograms/ml. Migration on permissive concentrations of tenascin could be reversibly inhibited with anti-beta 1, while treatment with anti-alpha v antibodies increased migration rates. We conclude that SF-767 glioma cells express two separate integrin receptors that mediate contrasting adhesive and migratory responses to tenascin.

Evaluation of Estrogenic Activity of Wastewater: Comparison Among In Vitro ERα Reporter Gene Assay, In Vivo Vitellogenin Induction, and Chemical Analysis

2015 ◽  
Vol 49 (10) ◽  
pp. 6319-6326 ◽  
Author(s):  
Masaru Ihara ◽  
Tomokazu Kitamura ◽  
Vimal Kumar ◽  
Chang-Beom Park ◽  
Mariko O. Ihara ◽  
...  
Keyword(s):  
Chemical Analysis ◽  
Reporter Gene ◽  
Estrogenic Activity ◽  
Reporter Gene Assay ◽  
Gene Assay

scholarly journals Stable Transfection of the BovineNRAMP1 Gene into Murine RAW264.7 Cells: Effect onBrucella abortus Survival

2001 ◽  
Vol 69 (5) ◽  
pp. 3110-3119 ◽  
Author(s):  
Robert Barthel ◽  
Jianwei Feng ◽  
Jorge A. Piedrahita ◽  
David N. McMurray ◽  
Joe W. Templeton ◽  
...  
Keyword(s):  
Cell Lines ◽  
In Vitro Model ◽  
Regulatory Control ◽  
Natural Resistance ◽  
Ifn Γ ◽  
Flanking Region ◽  
Vitro Model ◽  
Raw264.7 Macrophage

ABSTRACT Genetically based natural resistance to brucellosis in cattle provides for novel strategies to control zoonotic diseases. BovineNRAMP1, the homologue of a murine gene (Bcg), has been identified as a major candidate for controlling the in vivo resistant phenotype. We developed an in vitro model for expression of resistance- and susceptibility-associated alleles of bovine NRAMP1 as stable transgenes under the regulatory control of the bovineNRAMP1 promoter in the murine RAW264.7 macrophage cell line (Bcg s ) to analyze the regulation of the NRAMP1 gene and its role in macrophage function. We demonstrated that the 5′-flanking region of bovineNRAMP1, despite the lack of TATA and CAAT boxes, has a functional promoter capable of driving the expression of a transgene in murine macrophages. A polymorphism within a microsatellite in the 3′ untranslated region critically affects the expression of bovineNRAMP1 and the control of in vitro replication ofBrucella abortus but not Salmonella enterica serovar Dublin. We did not observe any differences in the production of NO by resting or gamma interferon (IFN-γ)- and IFN-γ–lipopolysaccharide (LPS)-treated transfected cell lines, yet the resistant transfected cell lines produced significantly less NO than other cell lines, following stimulation with LPS at 24 and 48 h.

scholarly journals Early Embryonic Lethality of Mice Lacking the Essential Protein SNEV

2007 ◽  
Vol 27 (8) ◽  
pp. 3123-3130 ◽  
Author(s):  
Klaus Fortschegger ◽  
Bettina Wagner ◽  
Regina Voglauer ◽  
Hermann Katinger ◽  
Maria Sibilia ◽  
...  
Keyword(s):  
Matrix Protein ◽  
Replicative Senescence ◽  
Inner Cell Mass ◽  
Umbilical Vein ◽  
Cell Mass ◽  
Preimplantation Embryos ◽  
Proliferative Potential ◽  
Mouse Development

ABSTRACT SNEV (Prp19, Pso4, NMP200) is a nuclear matrix protein known to be involved in pre-mRNA splicing, ubiquitylation, and DNA repair. In human umbilical vein endothelial cells, SNEV overexpression delayed the onset of replicative senescence. Here we analyzed the function of the mouse SNEV gene in vivo by employing homologous recombination in mice and conclude that SNEV is indispensable for early mouse development. Mutant preimplantation embryos initiated blastocyst formation but died shortly thereafter. Outgrowth of SNEV-null blastocysts showed a lack of proliferation of cells of the inner cell mass, which subsequently underwent cell death. While SNEV-heterozygous mice showed no overt phenotype, heterozygous mouse embryonic fibroblast cell lines with reduced SNEV levels displayed a decreased proliferative potential in vitro. Our experiments demonstrate that the SNEV protein is essential, functionally nonredundant, and indispensable for mouse development.

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