TBP Dynamics in Living Human Cells: Constitutive Association of TBP with Mitotic Chromosomes

Abstract

The recruitment of TATA binding protein (TBP) to gene promoters is a critical rate-limiting step in transcriptional regulation for all three eukaryotic RNA polymerases. However, little is known regarding the dynamics of TBP in live mammalian cells. In this report, we examined the distribution and dynamic behavior of green fluorescence protein (GFP)-tagged TBP in live HeLa cells using fluorescence recovery after photobleaching (FRAP) analyses. We observed that GFP-TBP associates with condensed chromosomes throughout mitosis without any FRAP. These results suggest that TBP stably associates with the condensed chromosomes during mitosis. In addition, endogenous TBP and TBP-associated factors (TAFs), specific for RNA polymerase II and III transcription, cofractionated with mitotic chromatin, suggesting that TBP is retained as a TBP-TAF complex on transcriptionally silent chromatin throughout mitosis. In interphase cells, GFP-TBP distributes throughout the nucleoplasm and shows a FRAP that is 100-fold slower than the general transcription factor GFP-TFIIB. This difference supports the idea that TBP and, most likely, TBP-TAF complexes, remain promoter-bound for multiple rounds of transcription. Altogether, our observations demonstrate that there are cell cycle specific characteristics in the dynamic behavior of TBP. We propose a novel model in which the association of TBP-TAF complexes with chromatin during mitosis marks genes for rapid transcriptional activation as cells emerge from mitosis. INTRODUCTIONIn eukaryotic cells, the three RNA polymerases I, II, and III are dedicated to the transcription of distinct classes of genes. Distinct promoter architectures and the assembly of polymerase-specific initiation complexes at gene promoters are keys that dictate the recruitment of the particular class of polymerases. TATA binding protein (TBP) interacts with a variety of TBP-associated factors (TAFs) to form the selectivity factor-1 (SL1), transcription factor TFIID, and TFIIIB complexes that are important for specifying RNA polymerase I, II, and III transcription, respectively (Hernandez, 1993). TBP-TAF complexes are critical players in determining levels of transcription initiation. Thus, the formation of specific TBP-TAF complexes potentially regulates transcription of specific genes under different growth conditions. Increasing the recruitment of these complexes to gene promoters by regulatory proteins is one mechanism for transcriptional activation (Albright and Tjian, 2000;Hampsey and Reinberg, 1999;Hernandez, 1993;Lee and Young, 1998). Once recruited to a promoter, TBP-TAF complexes can perform additional functions that are important for transcriptional regulation, including recruitment of additional members of the general transcriptional machinery to the promoter, induction of conformational changes in DNA topology, and recruitment of coactivator or corepressor proteins that influence gene transcription (reviewed in Tansey and Herr, 1997).The various TBP-TAF complexes have been purified and characterized extensiv...

Coactivators and general transcription factors have two distinct dynamic populations dependent on transcription

“…First, we chose RPB1 (the largest subunit of Pol II) known to have stable interactions with chromatin (Kimura et al , 2002; Hieda et al , 2005; Boireau et al , 2007; Darzacq et al , 2007; Fromaget & Cook, 2007) and also highly dynamic nuclear factors, like the TFIID subunit TAF5 and the GTF TFIIB (Chen et al , 2002; Sprouse et al , 2008; de Graaf et al , 2010; Ihalainen et al , 2012). In addition, eGFP was used as a control for a freely diffusing protein that should exhibit no specific interactions with the nuclear environment.…”

Two-step Mechanism for Modifier of Transcription 1 (Mot1) Enzyme-catalyzed Displacement of TATA-binding Protein (TBP) from DNA

“…In yeast, TBP is highly mobile, and this mobility is Mot1-dependent (27). A significant proportion of the TBP pool is highly dynamic in human cells as well (28,29). This contrasts with the lifetime of the isolated TBP-DNA complex in vitro, which is much longer (30 -32).…”

Transcriptional Coactivator PC4, a Chromatin-Associated Protein, Induces Chromatin Condensation

“…Preferential interactions of PC4 with different core histones. The stable association of PC4 to the chromatin could occur through its nonspecific DNA binding ability (34), interaction with bookmarked general transcription factors (12,13,50) or other nonhistone chromatin-associated proteins (33,37), or direct interactions with the core histones. Direct interactions of several nonhistone CAPs with the core histones have been shown to contribute to their association with the chromatin (43,54).…”

“…Several TAFs associated with the mitotic chromatin get phosphorylated and consequently cannot modulate activator-dependent transcription, which is restored upon dephosphorylation (50). Apart from transcription factor IID (TFIID), some amount of TFIIB also remains associated with the previously active promoters during mitosis, whereas RNA polymerase II (Pol-II) and NC2 (which can function both as an activator and a repressor) are displaced (12,13). In general, association of transcription factors with the chromosome and/or chromatin is found to be a highly dynamic process, which depends upon the stages of cell cycle.…”