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Regulation of the P53 Transcriptional Response by Structurally Diverse Core Promoters

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Regulation of the P53 Transcriptional Response by Structurally Diverse Core Promoters Downloaded from genesdev.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press Regulation of the p53 transcriptional response by structurally diverse core promoters Jose´ M. Morachis, Christopher M. Murawsky, and Beverly M. Emerson1 Regulatory Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA p53 target promoters are structurally diverse and display pronounced differences in RNA polymerase II (RNAP II) occupancy even in unstressed cells, with higher levels observed on cell cycle arrest genes (p21) compared with apoptotic genes (Fas/APO1). This occupancy correlates well with their ability to undergo rapid or delayed stress induction. To understand the basis for such distinct temporal assembly of transcription complexes, we examined the role of core promoter structures in this process. We find that the p21 core promoter directs rapid, TATA box- dependent assembly of RNAP II preinitiation complexes (PICs), but permits few rounds of RNAP II reinitiation. In contrast, PIC formation at the Fas/APO1 core promoter is very inefficient but supports multiple rounds of transcription. We define a downstream element within the Fas/APO1 core promoter that is essential for its activation, and identify nuclear transcription factor Y (NF-Y) as its binding partner. NF-Y acts as a bifunctional transcription factor that regulates basal expression of Fas/APO1 in vivo. Thus, two critical parameters of the stress-induced p53 transcriptional response are the kinetics of gene induction and duration of expression through frequent reinitiation. These features are intrinsic, DNA-encoded properties of diverse core promoters that may be fundamental to anticipatory programming of p53 response genes upon stress. [Keywords: p53; RNA polymerase II; transcription; core promoters; p21; Fas/APO1; NF-Y] Supplemental material is available at http://www.genesdev.org. Received August 24, 2009; revised version accepted November 30, 2009. The ability of cells to undergo cell cycle arrest or ap- control being expressed early and proapoptotic genes optosis after acquiring malignant alterations is of funda- being expressed later (Zhao et al. 2000). mental importance to our normal surveillance mecha- A critical issue that remains to be elucidated is how p53 nisms that are designed to prevent tumor progression. chooses which of its multiple target genes to activate or The tumor suppressor protein p53 is a critical component repress in response to a given stress. In this regard, an of this anti-tumor response by regulating diverse gene important source of p53 functional diversity that could pathways that control cell cycle arrest, angiogenesis, contribute to selective gene regulation and cell fate DNA repair, senescence, and apoptosis (Murray-Zmijewski choice resides within the core promoters of p53 target et al. 2008; Vousden 2009; Vousden and Prives 2009). genes. The core promoter is defined as the DNA sequence Induction of cell cycle arrest (at G1–S) by p53 results from required to direct accurate transcriptional initiation by transcriptional activation of CDKN1A (p21), leading to the RNA polymerase II (RNAP II) complex. It contains inhibition of cyclin-dependent kinase (CDK)–cyclin com- the region around the initiation site and usually one or plexes and proliferating nuclear antigen (PCNA) (el-Deiry more conserved sequence motifs such as the TATA box, et al. 1993; Abbas and Dutta 2009). The molecular events initiator (Inr), TFIIB recognition element (BRE), down- that promote p53-dependent apoptosis are more complex stream promoter element (DPE), and downstream core el- and occur through activation of critical genes involved in ement (DCE) that impose different requirements for tran- the mitochondrial apoptotic pathway (PUMA) and the scription initiation (Heintzman and Ren 2007; Sandelin death receptor pathway (Fas/APO1). The kinetics of ex- et al. 2007; Juven-Gershon and Kadonaga 2009). The pression after p53 induction varies considerably among series of regulatory events that direct the activity of p53 different target genes, with those involved in cell cycle target promoters must ultimately relay through the basal RNAP II machinery. Thus, it is important to understand not only the relationship of p53 to the RNAP II complex, 1Corresponding author. but also how architectural diversity among its promoters E-MAIL [email protected]; FAX (858) 535-8194. Article published online ahead of print. Article and publication date are affects this relationship and contributes to the overall online at http://www.genesdev.org/cgi/doi/10.1101/gad.1856710. stress-induced transcriptional program. GENES & DEVELOPMENT 24:135–147 Ó 2010 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/10; www.genesdev.org 135 Downloaded from genesdev.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press Morachis et al. Previous studies have shown that different levels of Y) as specifically interacting with the Fas downstream RNAP II transcription preinitiation complexes (PICs) are element and regulating basal expression of endogenous assembled on endogenous p53 target promoters even Fas/APO1 in vivo. before stress induction (Espinosa et al. 2003). These levels These studies support the notion that diverse core correlate with the timing of transcription activation promoters among p53 target genes play a fundamental during the stress response. The pro-cell cycle arrest gene role in stress-induced cell fate decisions by regulating the p21 contains high levels of RNAP II and other initiation kinetics of gene activation and the duration of expression components in unstressed cells and is rapidly induced by through RNAP II reinitiation. Interestingly, these intrin- DNA-damaging agents. This is achieved by conversion of sic features of distinct core promoter structures are ‘‘hard- RNAP II to an elongating form through recruitment of wired’’ into their DNA without the need for chromatin, elongation factors to distinct regions of the p21 gene p53, or coactivators, which act at other levels of control. (Espinosa et al. 2003). In contrast, proapoptotic genes like Thus, the programming of p53 response genes in un- Fas/APO1, PUMA, and APAF-1 contain very low levels of stressed cells to be ‘‘poised’’ by engaged RNAP II for rapid RNAP II and display delayed induction kinetics relative activation or ‘‘unpoised’’ for delayed, but sustained ex- to p21. These genes are more likely to be controlled at the pression is, in part, genetically determined to antici- level of initiation. Interestingly, high levels of promoter- pate how and when these genes will need to function in bound RNAP II complexes do not correlate with the the stress response. This default, intrinsic programming duration of gene expression during the damage response, can be modulated by epigenetic events and specific p53 since mRNA synthesis from proapoptotic genes can equal and coactivator complexes that tailor gene activity and or exceed that of p21. Prolonged transcription after cell fate choices in a tissue- and stress-specific manner damage may depend, in part, on the efficiency of RNAP (Espinosa 2008). Thus, core promoter architecture pro- II reinitiation from specific promoters. In addition, p53 is vides another important mechanistic level by which p53 required to assemble the RNAP II complex on the coordinates a physiologically appropriate response to di- endogenous p21 promoter before stress, and to differen- verse stress conditions. tially recruit and retain specific initiation and elongation factors after distinct types of DNA damage in vivo (Espinosa et al. 2003; Gomes et al. 2006). However, since Results p53 has been shown to interact with both p21 and Intrinsic features of diverse p53 core promoters proapoptotic genes before stress (Szak et al. 2001; Kaeser regulate differences in RNAP II-binding affinity and Iggo 2002; Espinosa et al. 2003), the variation in and reinitiation kinetics promoter structure among these genes is likely to in- fluence the composition and rate of assembly of pro- To understand the function of diverse core promoter moter-specific RNAP II transcription complexes. This, in structures that exist among p53 target genes, we exam- turn, affects whether activation of specific genes occurs ined which features may influence the differences in with early or delayed kinetics, and how long expression is apparent RNAP II-binding affinities and expression rates sustained during the stress response. observed among p53 target genes using an in vitro We investigated the role of core promoter architecture transcription system. Initially, we assumed that p53 in- in directing the transcriptional kinetics of two function- teraction with chromatin-assembled recombinant genes ally and structurally diverse p53 target genes, p21 and would be required to recapitulate these aspects of reg- Fas/APO1, using biochemical and cell-based approaches. ulation. We began by simply measuring the relative Surprisingly, we find that differences in RNAP II affinity amounts of transcription obtained from cloned natural and reinitiation kinetics observed between the endoge- p21 and Fas/APO1 promoter–reporter plasmids as naked nous p21 and Fas/APO1 genes can be recapitulated in DNA in vitro using protein extracts from unstressed vitro using DNA templates in a manner dependent on HeLa cells. These promoters have very dissimilar struc- their respective core promoters. We find that the p21 core tures, but each directed efficient RNA synthesis in vitro, promoter
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