p53 Dynamically Directs TFIID Assembly on Target Gene Promoters

Abstract

p53 is a central regulator that turns on vast gene networks to maintain cellular integrity in the presence of various stimuli. p53 activates transcription initiation in part by aiding recruitment of TFIID to the promoter. However, the precise means by which p53 dynamically interacts with TFIID to facilitate assembly on target gene promoters remains elusive. To address this key issue, we have undertaken an integrated approach involving single-molecule fluorescence microscopy, single-particle cryo-electron microscopy, and biochemistry. Our real-time single-molecule imaging data demonstrate that TFIID alone binds poorly to native p53 target promoters. p53 unlocks TFIID's ability to bind DNA by stabilizing TFIID contacts with both the core promoter and a region within p53's response element. Analysis of single-molecule dissociation kinetics reveals that TFIID interacts with promoters via transient and prolonged DNA binding modes that are each regulated by p53. Importantly, our structural work reveals that TFIID's conversion to a rearranged DNA binding conformation is enhanced in the presence of DNA and p53. Notably, TFIID's interaction with DNA induces p53 to rapidly dissociate, which likely leads to additional rounds of p53-mediated recruitment of other basal factors. Collectively, these findings indicate that p53 dynamically escorts and loads TFIID onto its target promoters. KEYWORDS p53, TFIID, transcription, structure, single-molecule microscopy M ore than 50% of cancer patients harbor p53 mutations, highlighting the essential role of this protein in tumor suppression (1). To properly maintain genomic stability, p53 induces vast gene networks involved in diverse cellular pathways, including the cell cycle arrest, apoptosis, and DNA repair pathways (2). In response to various stress stimuli, p53 acts as a transcriptional activator that specifically binds consensus response elements (REs; two 10-base-pair half-sites [RRRCWWGYYY]) within its target genes to directly stimulate gene expression (2). p53 utilizes its ability to nonspecifically bind and slide along the DNA to expedite the search for target REs (3). Upon recognition of its response genes, p53 facilitates transcription at least in part by targeting the TFIID-mediated transcription machinery to the promoter (4-7). TFIID is composed of TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). TFIID recognizes and binds multiple core promoter elements (e.g., the TATA box, the initiator, and downstream core promoter elements [DPE]) surrounding the transcription start site (TSS) (8). Once bound, TFIID serves as a central scaffold for six basal factors, including RNA polymerase II, to form a preinitiation complex (PIC) directing transcription initiation. Importantly, without the assistance of activators such as p53, TFIID's core promoter recognition is weak and rate limiting for transcription initiation due to inefficient PIC assembly (9-16). As TFIID is responsible for transcription of at least ϳ90% of proteincoding genes (17), unraveling how p...

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“…Notably, PBAF's residence time on chromatin is highly similar to activators (Sox2 and p53, ~15 seconds Coleman et al 2017)) and the Polycomb repressor (Cbx7, ~7 seconds (Zhen et al 2016)) as measured using single molecule tracking. This further indicates that a variety of transcription factors dynamically access their chromatin targets on timescales of seconds.…”

A new era of studying p53-mediated transcription activation

“…Our cryo-EM studies further suggest that p53 aids in TFIID's transition to the rearranged DNA-binding conformation. 7 More recently, armed with significant advancement of cryo-EM, we have captured the first 3D structure of wild-type p53 bound to human Pol II 13 (Fig. 1).…”

“…It is established that p53 directly binds and recruits several components of the transcription initiation machinery (i.e., Pol II, TFIIB, TFIID, TFIIH, Mediator) to promote PIC assembly on the promoter. [6][7][8][9][10][11][12] Researchers have long questioned how p53 can simultaneously recruit multiple factors during PIC assembly. It is also unclear which oligomeric form of p53 is actually responsible for navigating these multivalent interactions.…”

“…6,7 p53 was shown to bind several PIC components (i.e., TFIIB, TFIID, TFIIH, Mediator, Pol II) and subsequently aid their recruitment to the core promoter. [6][7][8][9][10][11][12][13] Thus, the interactions of p53 with multiple components of the PIC are crucial for transcription initiation. Researchers have studied stimulation of eukaryotic transcription by activators (e.g., p53) for the last 30 years.…”

Transcriptional activation is weakened when Taf1p N-terminal domain 1 is substituted with its <i>Drosophila</i> counterpart in yeast TFIID

“…This factor can directly recognize a variety of core promoter structures such as the TATA box, initiator (Inr), motif ten element (MTE), downstream promoter element (DPE) or downstream core element (DCE) to initiate accurate transcription from transcriptional start sites (TSSs) (Thomas and Chiang, 2006;Kadonaga, 2012;Lenhard et al, 2012;Nogales et al, 2017). Furthermore, when TFIID interacts with transcriptional activators on the promoter DNA, its conformation may be altered to recruit other GTFs (i.e., TFIIA, B, E, F and H) as well as RNA polymerase II to the core promoter region, culminating in assembly of the preinitiation complex (PIC) (Johnson and Carey, 2003;Liu et al, 2009;Coleman et al, 2017).…”