Journal:

Article Title: The p300/CBP acetyltransferases function as transcriptional coactivators of ?-catenin in vertebrates

doi: 10.1093/emboj/19.8.1839

Figure Lengend Snippet: Fig. 1. β-catenin utilizes p300 as coactivator at the siamois promoter. (A) Schematic representation of β-catenin and p300. In β-catenin (hatched bar) the Armadillo repeats (gray boxes) and interaction domains for various binding partners are marked. The GSK-3β target sequence (GSK) and the transactivating elements (N-TAs, C-TAs) are also shown. Autonomous TAs: dark gray; auxiliary TAs: light gray. In β-catS33A serine/threonine residues at positions 33, 37, 41 and 45 in the GSK-3β target sequence were substituted with alanines. In p300 the cysteine/histidine-rich elements CH-1, CH-2, and CH-3, the Bromo domain (gray boxes), the HAT domain (black bar) and regions interacting with nuclear hormone receptors (NHR), other transcription factors and E1A are denoted. (B) 293 cells were transfected with luciferase reporter plasmids harboring either the WT siamois promoter or a deletion mutant lacking the TCF binding sites (1.0 µg), the lacZ expression plasmid pCMVβ (Promega) (0.5 µg), a construct for expression of β-catS33A (0.5 µg) and increasing amounts of expression vector for p300 (0.5, 1.0, 2.0 µg) as indicated. Luciferase activities were determined 40 h post transfection, normalized against β-galactosidase values and expressed as relative activity compared with cells transfected with the WT siamois reporter alone (rel. activity: 1). (C) 293 cells were transfected with the WT siamois reporter, pCMVβ and expression vectors for β-catS33A (0.5 µg), p300 (2.0 µg) and WT E1A or mutant E1A (0.2 µg) as indicated. Reporter activity without β-catenin and p300: 1. A diagram of 12S-E1A is depicted. Conserved regions 1 and 2 (CR1, CR2), domains involved in binding Rb-family members, p300/CBP and the CtBP transcriptional repressor, and the location of the mRb and mCBP mutations are indicated. (B, C) Average values from at least three independent experiments are shown with standard deviations. (D) SW480 cells were transfected with expression vectors (2.5 µg each) for the E1A proteins as indicated. Whole cell lysates were prepared 40 h after transfection and E1A expression levels were analyzed by Western blotting with a monoclonal antibody. Mw: molecular weight markers.

Article Snippet: In p300 the cysteine/histidine-rich elements CH-1, CH-2, and CH-3, the Bromo domain (gray boxes), the HAT domain (black bar) and regions interacting with nuclear hormone receptors (NHR), other transcription factors and E1A are denoted. ( B ) 293 cells were transfected with luciferase reporter plasmids harboring either the WT siamois promoter or a deletion mutant lacking the TCF binding sites (1.0 μg), the lacZ expression plasmid pCMVβ (Promega) (0.5 μg), a construct for expression of β-catS33A (0.5 μg) and increasing amounts of expression vector for p300 (0.5, 1.0, 2.0 μg) as indicated.

Techniques: Binding Assay, Sequencing, Transfection, Luciferase, Mutagenesis, Expressing, Plasmid Preparation, Construct, Activity Assay, Western Blot, Molecular Weight

Journal:

Article Title: The p300/CBP acetyltransferases function as transcriptional coactivators of ?-catenin in vertebrates

doi: 10.1093/emboj/19.8.1839

Figure Lengend Snippet: Fig. 2. Promoter-specific differences in transcriptional activation mediated by β-catenin and p300. (A) 293 cells were transfected with the TOPFLASH or FOPFLASH reporter plasmids (1.0 µg), pCMVβ (0.5 µg), a construct for expression of β-catS33A (0.5 µg) and increasing amounts of expression vector for p300 (0.5, 1.0, 2.0 µg) as indicated. Luciferase activities were determined as before. (B) Same experiment as in (A) but with a reporter construct harboring the cyclin D1 promoter. Reporter activity in (A) and (B) without β-catenin and p300: 1.

Article Snippet: In p300 the cysteine/histidine-rich elements CH-1, CH-2, and CH-3, the Bromo domain (gray boxes), the HAT domain (black bar) and regions interacting with nuclear hormone receptors (NHR), other transcription factors and E1A are denoted. ( B ) 293 cells were transfected with luciferase reporter plasmids harboring either the WT siamois promoter or a deletion mutant lacking the TCF binding sites (1.0 μg), the lacZ expression plasmid pCMVβ (Promega) (0.5 μg), a construct for expression of β-catS33A (0.5 μg) and increasing amounts of expression vector for p300 (0.5, 1.0, 2.0 μg) as indicated.

Techniques: Activation Assay, Transfection, Construct, Expressing, Plasmid Preparation, Luciferase, Activity Assay

Journal:

Article Title: The p300/CBP acetyltransferases function as transcriptional coactivators of ?-catenin in vertebrates

doi: 10.1093/emboj/19.8.1839

Figure Lengend Snippet: Fig. 3. (A) Endogenous β-catenin in SW480 cells utilizes p300 as coactivator. SW480 cells were transfected with pCMVβ (0.5 µg), the TOPFLASH/FOPFLASH (1.5 µg) or siamois promoter constructs (2.5 µg) and expression vectors for p300 (2.0 µg) or dominant negative ΔN-TCF4 (2.0 µg) as indicated. To interfere with p300-mediated transcriptional activation WT E1A, E1AmRb, E1AmCBP or E1AΔCR1 were cotransfected (0.2 µg each). Luciferase activities were determined as before. Activity of the TOPFLASH or WT siamois reporter without exogenous p300, ΔN-TCF4 or E1A was set as 100% (n.d.: not done). (B) Functional similarity of p300 and CBP. SW480 cells were transfected with pCMVβ (0.5 µg), the TOPFLASH/FOPFLASH reporter constructs (1.5 µg) and expression vectors for p300 (4.0 µg) or CBP (1.0, 2.0 and 4.0 µg) as indicated. Luciferase activities, determined as before, are expressed as relative activities compared with cells transfected with FOPFLASH alone. (C) HAT activity of p300 is not required for its function as coactivator of β-catenin. 293 cells were transfected with the siamois reporter (0.5 µg), pCMVβ (0.1 µg), and expression vectors for β-catS33A (0.5 µg), WT p300, or HAT-defective p300mut (0.5 and 2.5 µg) as indicated. Control transfectants received 2.5 µg of empty expression vector or p300 plasmids. Luciferase activities were determined as before. Reporter activity without β-catenin and p300: 1.

Article Snippet: In p300 the cysteine/histidine-rich elements CH-1, CH-2, and CH-3, the Bromo domain (gray boxes), the HAT domain (black bar) and regions interacting with nuclear hormone receptors (NHR), other transcription factors and E1A are denoted. ( B ) 293 cells were transfected with luciferase reporter plasmids harboring either the WT siamois promoter or a deletion mutant lacking the TCF binding sites (1.0 μg), the lacZ expression plasmid pCMVβ (Promega) (0.5 μg), a construct for expression of β-catS33A (0.5 μg) and increasing amounts of expression vector for p300 (0.5, 1.0, 2.0 μg) as indicated.

Techniques: Transfection, Construct, Expressing, Dominant Negative Mutation, Activation Assay, Luciferase, Activity Assay, Functional Assay, Control, Plasmid Preparation

Journal:

Article Title: The p300/CBP acetyltransferases function as transcriptional coactivators of ?-catenin in vertebrates

doi: 10.1093/emboj/19.8.1839

Figure Lengend Snippet: Fig. 5. Interactions between p300 and β-catenin in vivo. (A) Co-immunoprecipitation of p300 and β-catenin. 293 cells were transfected with expression plasmids for HA-tagged p300 and myc-tagged β-catS33A as indicated. Cell extracts were prepared 40 h after transfection and used for immunoprecipitations with anti-HA or anti-myc antibodies. Immunoprecipitated material and a fraction of each lysate was resolved by SDS–PAGE and analyzed by Western blotting with antibodies as shown. IP: immunoprecipitation; IB: immunoblot. (B) Mammalian two-hybrid analyses of interactions between β-catenin and p300. NIH 3T3 cells were transfected with pCMVβ (0.05 µg), the Gal4-dependent pG5E1bLuc reporter construct (0.1 µg) and empty control vectors or expression vectors for Gal4–p300 (1737–1891) (0.5 µg), LEFΔN–VP16 (0.5 µg) and β-catenin–VP16 fusions (0.5 µg) as indicated. Luciferase activities, determined as before, are expressed as relative activity compared with cells transfected with the pG5E1bLuc reporter and empty expression vector (rel. activity: 1). The diagram on the right shows the structure of the β-catenin–VP16 and the LEFΔN–VP16 prey fusions, the Gal4–p300 (1737–1891) bait and the pG5E1bLuc reporter. In β-catenin, the GSK recognition sequence, N-terminal and C-terminal transactivating elements (N-TAs, C-TAs) and the p300 interaction domain (black bar) are marked. LEFΔN–VP16 contains the HMG-box (hatched box) but lacks the β-catenin binding site. The Gal4–p300 fusion harbors the C-terminal portion of the CH-3 domain (dark gray box). VP16: VP16 transactivation domain.

Article Snippet: In p300 the cysteine/histidine-rich elements CH-1, CH-2, and CH-3, the Bromo domain (gray boxes), the HAT domain (black bar) and regions interacting with nuclear hormone receptors (NHR), other transcription factors and E1A are denoted. ( B ) 293 cells were transfected with luciferase reporter plasmids harboring either the WT siamois promoter or a deletion mutant lacking the TCF binding sites (1.0 μg), the lacZ expression plasmid pCMVβ (Promega) (0.5 μg), a construct for expression of β-catS33A (0.5 μg) and increasing amounts of expression vector for p300 (0.5, 1.0, 2.0 μg) as indicated.

Techniques: In Vivo, Immunoprecipitation, Transfection, Expressing, SDS Page, Western Blot, Construct, Control, Luciferase, Activity Assay, Plasmid Preparation, Sequencing, Binding Assay

Journal: bioRxiv

Article Title: Cell Type- and Tissue-specific Enhancers in Craniofacial Development

doi: 10.1101/2023.06.26.546603

Figure Lengend Snippet: a. in vivo activity pattern of select craniofacial enhancers (hs1431, hs746, hs521) at e11.5, visualized by lacZ -reporter assays (top). In separate experiments, the same enhancers were coupled to an mCherry -fluorescent reporter gene and examined by scRNA-seq of craniofacial tissues of resulting embryos. UMAPs show enhancer-driven mCherry expression (see for reference). b. Location of enhancers hs1431, hs746 and hs521 in their respective genomic context (red vertical lines), along with protein-coding genes within the genomic regions and local conservation profile (PhyloP). c. Average expression of genes (Seurat) in the vicinity of the respective enhancers, and proportion (percent) of cells expressing the genes in specific cell types. Enhancer-driven mCherry signal is plotted in the center in lieu of the approximate enhancer location in its endogenous genomic context. Bottom panels show expression of Snai2 , Msx1, and Gbx2 as likely candidate target genes for each of the enhancers hs1431, hs746 and hs521 across UMAPs. IsO: Isthmic Organizer Cells.

Article Snippet: The selected genomic region was PCR amplified from human or mouse genomic DNA where applicable; the PCR amplicon was cloned into a lacZ -reporter vector (Addgene #139098) or mCherry reporter vector (available upon request) using Gibson assembly (New England Biolabs) .

Techniques: In Vivo, Activity Assay, Expressing