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1.
Fig. 5.

Fig. 5. From: GRIP1-associated SET-domain methyltransferase in glucocorticoid receptor target gene expression.

Depletion of endogenous Suv4-20h1.1 potentiates hormonal induction of a subset of GR target genes. U2OS-GR cells were nucleofected with scrambled (siC, 1 μg per 2 × 106 cells) or Suv4-20h1.1-specific (siSuv; 1 or 2 μg per 2 × 106 cells) siRNA. Twenty-four hours later, cells were treated with 10 nM Dex for 2 h, as indicated, and the expression of Suv4-20h1.1 (A) and IGFBP1, GILZ, and I6PK (B) was analyzed as in Fig. 4C. In A, relative amount of Suv4-20h1.1 mRNA in siC-transfected cells is set as 1. In B, mRNA level of each gene in untreated cells transfected with siCon is set as 1.

Yurii Chinenov, et al. Proc Natl Acad Sci U S A. 2008 December 23;105(51):20185-20190.
2.
Fig. 2.

Fig. 2. From: GRIP1-associated SET-domain methyltransferase in glucocorticoid receptor target gene expression.

Suv4-20h1 expression in mammalian cells. (A) Suv4-20h1 is a ubiquitous protein. Extracts from indicated cell lines were separated by SDS/PAGE and probed with antibodies to Suv4-20h1. (B) Suv4-20h1 resides in the nucleus. YFP or YFP-Suv4-20h1 were transfected into U2OS-GR cells and images were acquired 18 h later under fluorescent microscope with the FITC filter set. (C) Suv4-20h1 tightly associates with chromatin. U2OS-GR cells were transfected with FLAG-Suv4-20h1 (or “vector” control) and either boiled in 2× SDS sample buffer (total cell lysate) or extracted in a lysis buffer containing 150 or 400 mM NaCl, as indicated, before boiling in 2× SDS. Proteins were separated by SDS/PAGE and probed with anti-FLAG antibodies.

Yurii Chinenov, et al. Proc Natl Acad Sci U S A. 2008 December 23;105(51):20185-20190.
3.
Fig. 6.

Fig. 6. From: GRIP1-associated SET-domain methyltransferase in glucocorticoid receptor target gene expression.

GRIP1 binding and catalytic activities are required for Suv4-20h1-mediated regulation of GR target genes. (A) Suv4-20h1 long and short isoforms, the mutant proteins deficient in GRIP1 binding (N409) or catalysis (H264I), and the SET domain core with 4 conserved motifs (I-IV) are shown on the Left. We replaced a conserved histidine in motif III (H264, Right), involved in AdoMet binding with isoleucine. (B) U2OS-GR cells were transfected with an empty vector, wt Suv4-20h1, or its N409 or H264I mutants, as indicated. Twenty hours later, cells were treated with Dex for 2 h as shown, and the expression of indicated GR target genes were assessed as in Fig. 4C. Ectopic expression of Suv4-20h1.1 derivatives was assessed by anti-Flag immunoblotting (Inset).

Yurii Chinenov, et al. Proc Natl Acad Sci U S A. 2008 December 23;105(51):20185-20190.
4.
Fig. 3.

Fig. 3. From: GRIP1-associated SET-domain methyltransferase in glucocorticoid receptor target gene expression.

Suv4-20h1.1 interacts with GRIP1 in mammalian cells. (A) A schematic of the mammalian 2-hybrid system with the diagrammed GRIP1 and Suv4-20h1 derivatives. Blue circles and green rectangles indicate Gal4 DBD and VP16 AD, respectively. (B) GRIP1 RD mediates the interaction with Suv4-20h1.1. U2OS-GR cells were cotransfected with Gal4 DBD alone or fused to NID or 3RD (Gal, Gal-NID and Gal-3RD), VP16 or VP16 418C, as indicated, a UAS-driven luciferase reporter and β-actin LacZ. Luciferase activity was assayed 16 h later, normalized to β-Gal activity and expressed as relative luminescence units (RLU). (C) The C-terminal 91aa of Suv4-20h1.1 are necessary but not sufficient for binding 3RD or full-length GRIP1 in U2OS cells. Indicated GRIP1 and Suv4-20h1 fragments were cotransfected into U2OS cells and luciferase activity was determined as in B. (D) Full-length Suv4-20h1.1 interacts with GRIP1 in U2OS cells. The assay was performed as in B.

Yurii Chinenov, et al. Proc Natl Acad Sci U S A. 2008 December 23;105(51):20185-20190.
5.
Fig. 1.

Fig. 1. From: GRIP1-associated SET-domain methyltransferase in glucocorticoid receptor target gene expression.

GRIP1 and Suv4-20h1 interact in yeast and in vitro. (A) GRIP1 and Suv4-20h1 derivatives used in binding assays. Suv4-20h1 SET domain and the original yeast 2-hybrid isolate 795C are diagrammed. GRIP1 functional domains including NR boxes 1, 2, and 3, the repression domain (RD) and activation domains (AD) 1 and 2 are indicated. GRIP1 deletion mutants included the NID-RD bait, NID, 2RD, 3RD, RD and 3RD-mt (where asterisk denotes the NR box-3 LL → AA substitution). (B) The C-terminal fragment of Suv4-20h1.1 interacts with GRIP1 NID-RD in yeast. LexOP-driven LacZ reporter and Suv4–20 795C fused to LexA DBD were cotransformed into yeast with B42AD alone, fused to GRIP1 NID or NID-RD (as indicated) and replica-plated onto glucose (Upper) or galactose/raffinose (Lower) in the presence of X-Gal. (C) Suv4-20h1 795C interacts with GRIP1 in vitro. 35S-labeled GRIP1 derivatives (shown above the autoradiogram) were produced in vitro (“inputs”) and tested for their ability to interact with recombinant GST or GST-795C, as indicated. Equal amounts of GST or GST-795C in binding reactions were assessed by Coomassie blue staining (Lower).

Yurii Chinenov, et al. Proc Natl Acad Sci U S A. 2008 December 23;105(51):20185-20190.
6.
Fig. 4.

Fig. 4. From: GRIP1-associated SET-domain methyltransferase in glucocorticoid receptor target gene expression.

Overexpression of Suv4-20h1.1 antagonizes GR-dependent activation. (A) Suv4-20h1.1 abolishes glucocorticoid activation of the chromatinized GR-responsive reporter. UL3 cells containing an integrated MMTV-LTR-Luc reporter were transfected with increasing amounts of pCDNA3-Suv4-20h1.1 (balanced by “empty” pCDNA3) along with the β-actin LacZ normalization vector and induced with Dex for 18 h. Luciferase activity was assessed as in Fig. 2B. (B) GRIP1 and GR are abundantly expressed in U2OS-GR (U) and A549 (A) cells. Protein extracts were separated by SDS/PAGE and probed with antibodies to GRIP1, GR, or ERK as a loading control. (C) Suv4-20h1.1 overexpression antagonizes hormonal induction of endogenous GR targets in U2OS-GR cells. U2OS-GR cells were transfected with pCDNA3 vector or pCDNA3-Suv4-20h1.1 (400 ng per 3 × 105 cells) and 18 h later, treated with vehicle or 100 nM Dex for 2 h. Total RNA was isolated, reverse-transcribed and mRNA levels of indicated genes were measured by qPCR, using β-actin as normalization control. mRNA level of each gene in untreated cells transfected with pCDNA3 was set as 1. Error bars represent standard deviations of duplicated experiments calculated as in Applied Biosystems User Bulletin no. 2 (http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_040980.pdf). (D) Glucocorticoid induction of endogenous GR targets in A549 cells is diminished by Suv4-20h1.1 overexpression. Transfections were performed as in C, using 2 μg of DNA per 3 × 105 cells. qPCR data (collected as in C) were expressed as a fold induction of each mRNA in Dex-treated over untreated cells. (E) Suv4-20h1.1 inhibits GRIP1-mediated activation in heterologous context in GRIP1 RD-dependent manner. Gal4DBD alone (Gal) or fused to GRIP1 or ΔRD were transfected into U2OS-GR cells along with UAS-Luc reporter, β-actin LacZ (40 ng per 2 × 104 cells each) and pCDNA3 vector or pCDNA3-Suv4-20h1.1 (20 ng per 2 × 104 cells). Luciferase activity was assayed as in Fig. 3A.

Yurii Chinenov, et al. Proc Natl Acad Sci U S A. 2008 December 23;105(51):20185-20190.

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