TBP2 is a TATA-binding basal transcription factor. (A) Gel-shift assay with control (Ctrl), TBP, and TBP2 in vitro translation lysates (2 and 6 μl, compare with C), with or without the addition of recombinant TFIIA (25 ng). F, free probe, Adenovirus Major Late promoter TATA box. (B) In vitro transcription in Xenopus egg extract with recombinant TBP or TBP2. Eggs (matured oocytes) are naturally devoid of TBP, so that TBP stimulates transcription >50-fold in egg extracts (23). H2B, correctly initiated transcript from the H2B promoter as detected by primer extension. IC, internal control for primer extension. (C) In vitro translation of TBP and TBP2 using reticulocyte lysates and [³⁵S]methionine, showing that TBP and TBP2 were used in approximately equal amounts in the gel-shift experiment (A).

TBP2 is required for gastrulation and embryonic transcription. (A) Effects of TBP2 knockdown on development. (Top) Control embryos at gastrula and larval stages (stages 13 and 25, respectively). (Middle) Typical examples of embryos injected with 1 ng of TBP2-AS167 oligonucleotide at the same stages. (Bottom) Embryos injected with 1 ng of TBP2-AS167 and 0.05–0.2 ng of synthetic antisense-resistant TBP2 mRNA. (B) Expression analysis of TBP2 knockdown embryos by qRT-PCR. mRNA levels of TBP2, EF1α, Rb, XK81A1, and Goosecoid were determined in control embryos (Ctrl) and embryos injected with TBP2-AS167 oligonucleotide (1, 2, or 3 ng). Control embryos had reached stage 12.5 when samples were taken for RNA isolation.

Characterization of TBP2. (A) Quantitation of TBP and TBP2 mRNA levels in oocytes and embryos by qRT-PCR, expressed as molecules per oocyte or embryo. (B) Western blot analysis of TBP and TBP2 expression (antibody 58C9). TBP2 protein is abundant in oocytes where it localizes to the nucleus, as does the TFIIB transcription factor. TBP is more abundant during embryogenesis, but low levels of TBP2 protein persist during embryogenesis (see C). Oo, stage VI oocyte, numbers refer to Nieuwkoop–Faber stages of development; Cyt, oocyte cytoplasm; nucl, oocyte nucleus (GV). (C) Long exposure of a Western blot reveals low levels of TBP2 persisting during embryogenesis (antibody 58C9). (D) Immunoprecipitation experiments using reticulocyte lysates programmed with TBP or TBP2 mRNA in the presence of [³⁵S]methionine (first two lanes). Input, 20% of the amount of mixed TBP and TBP2 lysate used in immunoprecipitations. Beads, no-antibody control (nonspecific interaction with beads). 58C9 precipitates both TBP and TBP2, and SL27 and SL30 selectively precipitate TBP, whereas 1G6 and 4H7 preferentially precipitate TBP2. (E) Western blot analysis using oocyte GV extracts. 58C9, 1G6, and 4H7 all bind to TBP2.

Recruitment of TBP and TBP2 as assayed by ChIP. (A) TBP2 is recruited to promoters injected into oocyte nuclei. ChIP assay with two TBP antibodies (SL27, SL30) and two TBP2 antibodies (1G6, 4H7) on episomal chromatin isolated from GV-injected oocytes. The no-antibody control defines the nonspecific binding of chromatin to the protein G beads. Enrichment is the signal relative to background (no-antibody control). (B) Recruitment to endogenous promoters during embryogenesis. A switch in recruitment of TBP and TBP2 is observed at the EF1α promoter during development (stages 12, 24–28, 38–42, and 46).

TBP2 can partly substitute for TBP function. (A) TBP2 overexpression partially rescues the TBP knockdown phenotype. Control embryos, TBP-AS injected embryos, embryos injected with both TBP-AS and TBP2 mRNA, and TBP2 overexpression embryos are shown at the time when control embryos reached stage 30. (B) Coinjection of TBP-AS and TBP2 mRNA replaces TBP with similar levels of TBP2. Western blotting of injected stage 30 embryos by using 58C9, a monoclonal antibody that recognizes the core domain of TBP and TBP2. Similar results were obtained when extracts from gastrula embryos (stage 12) were used. Antibodies against TFIIB and α-tubulin were used as loading control. (C) Northern blotting analysis using probes to determine expression of MyoDb, EF1α, Gsc, and XK81A1. The RNA used for this analysis was isolated from stage 12 embryos, except for the analysis of XK81A1 expression, in which case stage 18 embryos were used. Methylene blue-stained 18S rRNA was used as loading control.