1. Volume 5, Issue 4, Pages 683-693 (April 2000)
Involvement of the TRAP220 Component of the TRAP/SMCC Coactivator Complex in Embryonic Development and Thyroid Hormone Action 
Mitsuhiro Ito, Chao-Xing Yuan, Hirotaka J Okano, Robert B Darnell, Robert G Roeder 
Molecular Cell 
Volume 5, Issue 4, Pages (April 2000)
DOI: /S (00)

2. Figure 1 Disruption of the Trap220 Gene
(A) The targeting vector (middle) was designed to replace the SacII-SacI fragment of the mouse Trap220 gene (“Wild allele”) with lacZ and PGK-neo cassettes. The predicted mutant allele resulting from homologous recombination is shown as “Mutant allele.” Cross-hatched boxes indicate exons without replacement. Restriction sites indicated are PstI (P), SacI (S), SacII (SII), and SmaI (Sm).
(B) Southern blot analysis of offspring obtained by a heterozygous cross. High molecular weight DNAs from yolk sacs of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) F2 embryos were digested with PstI and SacI. Each digest was hybridized with 5′ external probe A, 3′ external probe B, or lacZ. The positions of the bands corresponding to the wild allele (9.8 kb and 8.7 kb) and the mutant allele (8.1 kb and 11.4 kb) are indicated.
(C) Northern blot analysis of TRAP220 mRNA and actin mRNA in E10.5 embryos of the indicated genotypes.
(D) Western blot analysis of TRAP220 protein in E10.5 embryos of the indicated genotypes.
(E) Expression of TRAP220 protein during embryogenesis as visualized by whole-mount lacZ staining.
Molecular Cell 2000 5, DOI: ( /S (00) )

3. Figure 2 Histopathology of Trap220−/− Embryos
Sagittal sections of Trap220−/− embryos (A, C, E, G, and I) and Trap220+/+ littermates (B, D, F, H, and J) are presented.
(A and B) Overview of E9.5 embryos. Note the underdeveloped telencephalon (arrows) in the Trap220−/− embryo (A) compared to the wild type (B).
(C and D) Overview of E10.5 embryos. Note the significantly smaller size of the Trap220−/− embryo (C) relative to the Trap220+/+ embryo (D) and marked retardation of the heart development in the Trap220−/− embryo (arrows).
(E and F) Forebrain of E9.5 embryos. Note the poor brain development in Trap220−/− embryos (E) relative to that of the wild-type littermate (F).
(G and H) Immunohistochemical staining with anti-MAP-2 monoclonal antibody. Note that some cells of the superficial postmitotic telencephalic layer in E10.5 Trap220−/− embryos are differentiated into neurons (arrows) (G), although their number is much lower than observed in the Trap220+/+ embryos (H).
(I and J) TUNEL staining of a serial section of the cortical plate. Note the apoptotic cells (arrows) in the Trap220−/− embryos (I) compared to the wild-type control (J). Large arrows indicate the whole layer of the embryonic cortex. V, ventricle.
Scale bars: (A–D), 625 μm; (E and F), 20 μm; (G and H), 80 μm; (I and J), 40 μm.
Molecular Cell 2000 5, DOI: ( /S (00) )

4. Figure 3 Heart Malformation in Trap220−/− Embryos
Sagittal sections of Trap220−/− embryos (A, C, and E) and Trap220+/+ littermates (B, D, and F) are shown.
(A and B) E8.5 embryos.
(C and D) E9.5 embryos. Note the smaller size of the heart and poor development of the trabecular layer in the Trap220−/− embryo (arrow).
(E and F) E10.5 embryos. The thickness of the subepicardial layer is markedly reduced (arrowhead) and the formation of the trabecular layer is severely impaired (arrows) in the Trap220−/− embryo.
Scale bar, 80 μm.
Molecular Cell 2000 5, DOI: ( /S (00) )

5. Figure 4
Defective Cell Cycle Regulation in Trap220−/− Mouse Embryonic Fibroblasts and DNA Fragmentation of Trap220−/− Embryos
(A) Trap220−/− MEFs grow slower than Trap220+/+ and Trap220+/− MEFs. Each value represents the mean ± SD of a representative experiment performed in duplicate.
(B) Entrance into S phase is impaired in Trap220−/− MEFs. Cells (5 × 104) were cultured in complete medium in the presence of [methyl-3H]thymidine (1 μCi/ml, 25 mCi/mmol) for 24 hr, and the incorporated radioactivity was counted (means ± SD of a representative experiment performed in triplicate.)
(C) DNA fragmentation in Trap220−/− embryonic genomic DNA. Genomic DNAs (100 ng) extracted from E11.5 embryos of each genotype were end-labeled with [32P]dCTP, separated with a 2% agarose gel, and visualized by autoradiography.
Molecular Cell 2000 5, DOI: ( /S (00) )

6. Figure 5
Defective TR-Driven Transcriptional Activation in Trap220−/− MEFs
(A) Gene dose-dependent decrease of TR-driven transcriptional activation in Trap220+/− and Trap220−/− MEFs. The MEFs of each genotype were transfected with 30 ng human TRα, 170 ng TRE5-luciferase reporter, 10 ng control luciferase pRL-SV40, and indicated amounts of the human TRAP220 construct. Cells were cultured in the absence or presence of the ligand, T3 (10−7 M), and dual luciferase activities were measured 48 hr after transfection. Values (means ± SD of a representative experiment performed in duplicate) are plotted as a fold increase against the value of Trap220+/+ MEFs without a ligand.
(B, C, and D) Gal4-RARα- and Gal4-RXRα-driven transcription is not affected in Trap220−/− MEFs. The MEFs of each genotype were transfected with 30 ng human full-length RARα (B) or RARα without AF1 domain (C) or AF2 ligand-binding domain of RXRα (D) fused to Gal4, 100 ng Gal-luciferase reporter, and 10 ng control luciferase pRL-SV40 and were cultured in the absence or presence of the ligand, all-trans retinoic acid (ATRA) or 9-cis retinoic acid (9-cis RA) (10−6 M). Dual luciferase activities were measured likewise.
(E) Gal4-VP16- and p53-driven transcription is not affected in Trap220−/− MEFs. The MEFs of each genotype were transfected with either Gal-luciferase or MDM2-luciferase reporter and with or without activator constructs. Values (means ± SD of a representative experiment performed in duplicate) are plotted as a fold increase against the value of Trap220+/+ MEFs without Gal4-VP16.
Molecular Cell 2000 5, DOI: ( /S (00) )

7. Figure 6
Growth Retardation and Pituitary Hypothyroidism in Trap220+/− F1 Mice
(A) Growth curve of the males. Trap220+/− F1 mice grow significantly slower than the wild-type F1 mice (129SvJ and C57BL/6J hybrids). Values are means ± SEM of at least 11 mice each.
(B) Serum levels of free T3 and free T4 were measured in five 12-week-old male mice of each genotype. Values are means ± SD.
(C) Northern blot of pooled pituitary total RNAs of the same mice. A significant decrease in the TSHβ signal without significant change in TSHα and GH is shown in Trap220+/− F1 mice. The results were reproducible in four independent experiments.
Molecular Cell 2000 5, DOI: ( /S (00) )

8. Figure 7
Broadly Impaired Transcription and Posttranscriptional Regulation in the Trap220+/− Testis
(A and B) Relative signal intensities of RNA blot analyses (five pure 129SvJ mice each) in testis (A) and in brain (B) were quantified with an image analyzer. The results are expressed as a relative change ± SD of the mean expression level of each mRNA in wild-type mice. The levels of ribosomal RNAs were exactly the same (data not shown).
Molecular Cell 2000 5, DOI: ( /S (00) )