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

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.

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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 influence 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 has a high intrinsic affinity for RNAP II and with p21 being the stronger template (Fig. 1A,B). Al- rapidly assembles PICs in vitro, whereas the opposite is though the Fas/APO1 gene has been reported to have observed for Fas/APO1. Although Fas/APO1 has a low multiple start sites within a relatively close region of affinity for RNAP II, once a PIC is formed, this core ;200 base pairs (bp), only one major initiation site was promoter directs very efficient reinitiation of transcrip- used under in vitro transcription conditions. This mapped tion, in marked contrast to p21, which reinitiates poorly. to ;80 bp upstream of the ATG start codon and within Consequently, affinity for RNAP II does not necessarily 4 bp of previously reported start site predictions and dictate prolonged gene expression through high reinitia- RACE analysis (Behrmann et al. 1994; Cheng et al. 1995). tion frequency, and these two important aspects of To define the relative promoter strength and affinity of transcription are independently regulated. Upon muta- the transcriptional machinery, the p21 and Fas/APO1 genesis of the p21 and Fas/APO1 core promoters, we DNA templates were transcribed after PIC formation defined the TATA box in p21 and a downstream element in the presence of the detergent sarkosyl, which limits in Fas as key regulators of promoter-specific RNAP II transcription to a single round by inhibiting RNAP II activity. Additionally, we identified the bifunctional reinitiation (diagrammed in Fig. 1C; Cai and Luse 1987; transcription factor NF-Y (nuclear transcription factor Hawley and Roeder 1987; Kadonaga 1990). By performing

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Figure 1. Functional characterization of the p21 and Fas/APO1 core promoters. (A) Schematic diagram of p21 and Fas/APO1 promoters, including their respective p53-binding sites. (B) In vitro transcription of p21 or Fas/APO1 plasmids was performed and analyzed by primer extension. (C) Schematic diagram of single- and multiple-round transcription in the presence of sarkosyl. Addition of sarkosyl to form PICs prevents new PIC formation. Templates without sarkosyl undergo multiple rounds of transcription. (D) Rates of PIC formation were analyzed by adding sarkosyl just before (time 0), or various times after, combining DNA templates with HeLa nuclear extracts (HNEs). Transcription was initiated by adding NTPs. (E) Quantification and graphical representation of D.(F) Rounds of transcription were analyzed by allowing PICs to form for 1 h before initiating transcription (time 0). The initiated reactions then underwent multiple rounds of transcription before sarkosyl addition at intervals from 15 sec to 30 min. The first lane shows that sarkosyl addition to DNA templates before exposure to HeLa extract prevents formation of functional PICs. (G) Rounds of transcription were also measured using DNA templates immobilized on magnetic beads. Extract and immobilized DNA were mixed for 1 h to allow PIC formation similar to C. For single-round transcription, the DNA template was washed with buffer followed by addition of NTPs to allow elongation, whereas this wash step was not included for multiple rounds of transcription. (H) Rounds of transcription were quantified, and the signal at each time point was compared with time 0 to calculate the rounds of reinitiation. kinetic analyses of transcription in the presence and Next, we analyzed the RNAP II reinitiation frequency absence of sarkosyl, we could measure two important by measuring the approximate rounds of transcription parameters: the rate of PIC formation, and the number directed from each promoter in vitro. PIC formation was of rounds of reinitiation directed by each promoter. To allowed to occur on each template for 1 h, followed by the analyze the rate of PIC formation, PICs were allowed to addition of NTPs to initiate RNA synthesis. Once tran- assemble on each promoter between 0 and 2 h, with scription was initiated, we conducted a time course of further PIC formation or RNAP II reinitiation blocked by sarkosyl addition from 0 to 30 min to stop further the addition of sarkosyl to 0.04%. Immediately afterward, reinitiation at specific time intervals (Fig. 1F). These transcription was initiated by addition of nucleotide results were quantified using a PhosphorImager by com- triphosphates (NTPs) (Fig. 1D,E). This revealed that the paring the transcriptional output at each time point with p21 promoter was activated as early as 5 min after that of a single round at time 0. Unexpectedly, this addition of NTPs, reaching a plateau at ;1 h. In contrast, analysis revealed that, although the p21 promoter can the Fas/APO1 promoter was much less efficient than assemble a PIC far more rapidly than Fas/APO1 (Fig. 1E), p21 in assembling PICs, with negligible transcription it is much less efficient in directing RNAP II reinitiation detected under these single-round conditions even after (four rounds) than Fas/APO1 (21 rounds). Previous in 2 h (Fig. 1D,E). We also observe similar kinetics on the vitro assays using sarkosyl on more classical promoters APAF-1 promoter (Supplemental Fig. 2). These results, on have reported a maximal two to six rounds of transcrip- naked DNA templates, were surprisingly consistent with tion using mammalian or Drosophila protein extracts the chromatin immunoprecipitation (ChIP) analysis in (Hawley and Roeder 1987; Kadonaga 1990). To confirm unstressed cells, showing dramatic differences in levels of our results, we tested whether differences in the general RNAP II binding to the endogenous p21 and Fas/APO1 sensitivity of sarkosyl toward transcription of p21 and promoters (Espinosa et al. 2003). This suggests that Fas/APO1 existed, but found none (Supplemental Fig. variations in RNAP II-binding affinities are directed by 1). As a more rigorous verification of the large differ- intrinsic features of p53 target promoters encoded in their ence in reinitiation that we observed between p21 and DNA sequence, since neither chromatin nor p53 was Fas/APO1, we developed an assay to estimate rounds of required for this level of basic regulation, although both transcription without using sarkosyl. First, the p21 or obviously modulate transcriptional activity at more com- Fas/APO1 templates were immobilized on magnetic plex stages. beads and incubated with HeLa extracts to form a PIC

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Morachis et al. on each promoter. Then, instead of adding sarkosyl to RNAP II binding to p53 target genes in unstressed cells eliminate reinitiation, unbound RNAP II and other pro- and the kinetics of gene expression upon stress induction teins were washed away from each template with buffer are strongly influenced by intrinsic features of structur- before starting transcription by the addition of NTPs (Fig. ally diverse core promoters at the level of DNA. 1G). By this means, only engaged RNAP II complexes at the time of washing were capable of initiating single- Mapping critical elements within the p21 round transcription, whereas templates left with excess and Fas/APO1 core promoters unbound RNAP II (unwashed) could reinitiate multiple To extend our observation that the p21 and Fas/APO1 rounds of RNA synthesis. The results of this experiment core promoters were each sufficient to direct dissimilar were quantified as before and closely matched that RNAP II-binding kinetics (Fig. 2), we mapped the func- obtained under sarkosyl conditions, with p21 undergoing tional elements within each promoter by scanning mu- seven rounds of transcription and Fas/APO1 undergoing tagenesis of progressive 10-bp blocks to the transverse 18 rounds (Fig. 1H). bases using a PCR-based strategy (Invitrogen) (Fig. 3A,B). The preceding experiments were performed using full- All mutations were generated in the context of the full- length, natural p21 and Fas/APO1 recombinant pro- length promoters and were assessed by in vitro transcrip- moters (Fig. 1A). We now asked whether the pronounced tion using HeLa extracts. An examination of the p21 differences in RNAP II behavior observed between the promoter set revealed that the TATA box was the most two templates were regulated by DNA sequences within critical sequence in the core region, since mutation of or outside of their respective core promoters. To this end, this element decreased transcription to a negligible ex- we generated templates containing core promoter se- tent (Fig. 3C, Scan C). Mutation of other sequences had quences from À149 to +42 of p21 and from À50 to +78 far less deleterious effects, except to change the major of Fas/APO1 (Fig. 2A). Both the full-length and core transcriptional start site, such as Scan D (adjacent to the promoters of each gene generated approximately equal TATA box) and Scan F (impairs the Initiator), or create amounts of RNA synthesis (Fig. 2B), consistent with the additional initiation sites like Scan B (disrupts an Sp1 site general observation that in vitro transcription of DNA flanking the TATA box) and Scan E (disrupts sequence templates mainly reflects their core promoter activity. A just upstream of the start site). time course of sarkosyl addition during PIC formation A similar analysis of the Fas/APO1 promoter set re- followed by NTP addition, similar to that shown in Figure vealed little change in transcriptional efficiency of tem- 1D, demonstrated that the core promoters of p21 and plates containing scanning mutations of sequences from Fas/APO1 direct PIC formation and reinitiation effi- À52 to the Initiator region (Scans A–E) (Fig. 3B). Interest- ciency in an identical manner as that observed with the ingly, mutation of sequences just 39 of the Initiator from full-length promoters (Fig. 2C). The activities were quan- +7 (Scans F–G) completely abolished transcription, tified, and a graphical representation of the results is whereas adjacent 39 sequences (Scans H–I) had no effect. presented (Supplemental Fig. 5). Taken together, these To further define this essential region, transversion results support the notion that the differences in levels of mutations in consecutive blocks of 5 bp were created within Scans F–G. After transcriptional analysis of these mutated templates, the essential sequences for Fas/APO1 expression in vitro were localized to a 15-bp element (Scans F.2, G.1, and G.2) residing between +12 to +26 (Fig. 3E). The reactions were quantified, and a graphical representation of the results is shown (Supplemental Fig. 6). To verify that this region was also responsible for basal transcription in vivo, we created a luciferase reporter system using the core promoters of p21 and Fas/APO1 and compared the wild-type activity with the mutated regions important for transcription (Fig. 3F). Similar to previous studies, the TATA-box mutation (p21 C) drasti- cally reduced luciferase activity from the p21 promoter. The core promoter mutations within the Fas Scan F and Scan G mutations also decreased the basal activity of Fas/APO1 to varying degrees. Thus, our mutagenesis studies identified the most critical sequences for p21 Figure 2. Analysis of PIC assembly kinetics on full-length or and Fas/APO1 intrinsic core promoter function as the core p21 and Fas/APO1 promoters. (A) Diagram of the full- p21 TATA box and a Fas/APO1 downstream element. length promoters containing p53 response elements or core promoters. (B) In vitro transcription reactions using HeLa Analysis of the TATA box and Fas/APO1 downstream extracts to compare promoter strength between full-length and elements using chimeric p21 and Fas/APO1 promoters core promoter templates. (C) Rates of PIC formation on p21 and Fas/APO1 full-length or core promoters were measured by in Next, we examined whether the distinct characteristics vitro transcription under conditions similar to Figure 1D. of RNAP II binding and reinitiation observed on the p21

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Figure 3. Functional core promoter elements mapped by scanning mutagenesis. (A,B) Diagram of p21 (A) and Fas/APO1 (B) promoter sequences indicating the location of scanning mutations. Progressive 10-bp transversion mutations were generated in the context of the full-length promoters. (C) In vitro transcription reactions of p21 scanning mutations. The two left lanes labeled ‘‘G’’ and ‘‘A’’ contain DNA sequencing reactions used to map the transcription start site(s). (D) Same as C but using Fas/APO1 scanning mutations. (E) Transcriptional analysis of the scanning mutations ‘‘F’’ and ‘‘G’’ of Fas/APO1 that were further defined by creating four 5-bp block mutations between Scans F and G to create Fas scan mutants F.1, F.2, G.1, and G.2. (F) Luciferase expression analysis in HCT116 cells using transiently transfected p21 or Fas/APO-1 core promoters within a pGL3 luciferase plasmid.

and Fas/APO1 core promoters were regulated by the p21 complex can act relatively independently in these in vitro TATA box or the Fas/APO1 downstream element. To assays (Fig. 3A). address this, a chimeric promoter called Fas-TATA was We further analyzed the function of the Fas down- constructed in which the p21 TATA sequence (ATAT stream element (Scans F–G) by placing that sequence CAG) was inserted into the Fas/APO1 promoter to re- (+7to+26 bp) in the p21 promoter at the corresponding place the region from À29 to À23 (GAGGCCA) and location from +7to+26 (Fig. 4E; represented graphically generate the sequence TATATCAGG beginning at À30 in Supplemental Fig. 7). We discovered that the Fas (Fig. 4A). A functional analysis of the Fas-TATA promoter downstream element repressed p21 transcription, and template revealed that it was much more efficiently this was mediated specifically by the Scan F sequence transcribed in vitro than templates containing the wild- +7to+16 bp (Fig. 4E, cf. lanes 2 and 3). When either the type Fas/APO1 (Fas-wt) promoter (Fig. 4B). Moreover, Scan F or G downstream element was inserted in p21 a time course of sarkosyl addition under single-round promoters lacking a functional TATA box, only negligible transcription conditions, analogous to the experiment transcription was obtained. This indicates that, in the shown in Figure 1D, showed that the Fas-TATA pro- context of the p21 promoter, unlike the Fas/APO1 promoter was capable of forming detectable functional PICs moter, the Fas downstream element cannot synergize much faster, like p21 but unlike Fas/APO1 wild type, and with the TATA box or functionally replace it (Fig. 4E, could still direct multiple rounds of reinitiation (Fig. 4C; lanes 6–8). represented graphically in Supplemental Fig. 7). Thus, the chimeric promoter has the dual capacity of high- The Fas/APO1 downstream core promoter element affinity PIC formation, conferred by the TATA box, and specifically interacts with NF-Y efficient rates of reinitiation, like Fas/APO1. Interest- ingly, mutation of the Fas downstream element (Scan F) We next wished to identify which proteins associated within the Fas-TATA hybrid template (‘‘F-TATA’’) re- with the critical core promoter elements of p21 and sulted in a severe reduction of transcription (Fig. 4D). Fas/APO1. A DNase protection analysis demonstrated This indicates that the TATA box can synergize with that both native TFIID and recombinant TATA-binding the Fas downstream element within the context of the protein (TBP) could interact with the p21 TATA box; Fas/APO1 full-length promoter to greatly stimulate ex- however, no binding was observed on Fas/APO1. This sub- pression, but that the downstream element is an essential stantiates the notion that significant differences in bind- feature that cannot be functionally replaced by the TATA ing affinities for general transcriptional factors exist be- box. No sequences homologous to the Fas downstream tween these diverse core promoters, which may result in element have been found within the p21 core promoter, the assembly of compositionally distinct PICs (Supple- and none of our scanning p21 mutations significantly mental Fig. 3). To determine whether the DPE that is es- impaired TATA function, suggesting that the TFIID sential for Fas/APO1 transcription specifically interacts

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a magnet to remove unbound material. We also incorpo- rated a ‘‘preclearing’’ step in which HeLa extracts plus dI–dC were initially incubated with the immobilized, mutated Fas downstream element (which abolishes specific protein binding as determined by EMSA) to further minimize contaminating proteins in our subsequent analysis. Proteins that ‘‘flowed through’’ the precleared chromatographic steps were enriched for factors that specifically interact with the Fas downstream element rather than those that spuriously bind to mutated sequences. The immobilized templates were then incubated with HeLa extract containing the potential Fas downstream element-interacting protein(s), washed, and step-eluted with 0.1, 0.25, 0.5, and 1 M NaCl buffer (diagrammed in Fig. 5D). The eluates were analyzed by EMSA, which showed that the downstream element- binding activity eluted mainly between 0.1 and 0.25 M NaCl (Fig. 5E). Figure 4. Analysis of critical core promoter elements using These fractions, eluted from immobilized wild-type chimeric templates. (A) Diagram of the Fas/APO1 promoter and mutated downstream element oligos, were compared sequence indicating the location in which the p21 ATATCAG by electrophoresis on SDS-PAGE. Analysis of silver- sequence was inserted to replace À23 to À29 and create the Fas- TATA promoter. (B) In vitro transcription to examine the stained proteins revealed a specific band between 49 activity of Fas-TATA compared with the Fas/APO1 promoter. and 64 kDa in the 0.1 and 0.25 M NaCl elutions that (C) In vitro transcription analyzing the rate of PIC formation was not bound to the mutated template (Supplemental of p21, Fas/APO1, and Fas-TATA. HeLa nuclear extract was Fig. 3A). This protein band (along with the corresponding allowed to bind to template for 0–2 h before addition of sarkosyl, area of the mutant lane in Supplemental Fig. 3A) was similar to Figure 2D. To generate multiple rounds of transcrip- excised from the gel, digested into peptides, and analyzed tion, sarkosyl was not added to the last lane (2 h*). (D) Transcrip- by MALDI-TOF mass spectrometry. One of the top tional analysis of Fas-TATA compared with F-TATA (Fas-TATA candidates from the mass spectrometry analysis was with the Scan F mutation). (E) Analysis of the Fas downstream NF-Y subunit C, which was not detected in the corre- element within the p21 wild-type or mutated full-length sponding mutant oligo sample. Western blot analysis promoter. against the NF-Y subunit C confirmed the mass spectrometry results (Supplemental Fig. 3B). NF-Yis a trimeric with proteins present in our HeLa transcription extracts, complex that is comprised of subunits A, B, and C we performed an electrophoretic mobility shift assay (Mantovani 1999). To verify that the trimeric complex (EMSA) using oligonucleotides that span À1to+35 bp, actually binds to the downstream Fas promoter element, which includes the sequences in Scans F–G (Fig. 5A). We we performed a supershift EMSA assay using antibodies observed specific protein complex formation using wild- against two of the three NF-Y subunits (anti-NF-YA and type oligos that was not generated with oligos containing anti-NF-YC), using anti-YY1 and anti-Bmi as controls. mutations in the 20 bp corresponding to elements F–G The specific band observed previously by EMSA was (Fig. 5B). Although a different shift was observed using clearly supershifted using either anti-NF-YA or anti-NF- mutated oligos, this is likely to be a distinct protein, since YC antibody, whereas no supershift was observed with mutations were generated across the functional se- the control antibodies (Fig. 5F). The reactions in lanes quences (middle 20 bp out of 36), and even a 10-fold 8 and 9 of Figure 5F are similar to lanes 5 and 7, re- excess of mutant oligo failed to compete for binding of the spectively, except that HeLa extract was incubated with protein to the wild-type oligo (Fig. 5C). the oligo probe before antibody addition. We next identified and characterized the protein(s) binding to this DNA region by oligonucleotide affinity chro- NF-Y binds to the Fas/APO1 promoter in vivo matography, similar to what has been used previously to and activates Fas/APO1, but not p21, transcription purify a corepressor of p53, SnoN, on the a-fetoprotein gene (Wilkinson et al. 2005). In this approach, DNA NF-Y is a conserved transcription factor that binds with sequences comprising the Fas activator element (Scans high specificity to CCAAT (or reverse: ATTGG) motifs in F–G) that are capable of protein binding by EMSA were the promoter regions in a variety of genes. The C subunit immobilized as multiple copies on streptavidin-coated forms a tight dimer with the B subunit, a prerequisite for magnetic beads. The multimers were produced using association with subunit A. The resulting trimer binds to a self-primer PCR method (Hemat and McEntee 1994; DNA with high specificity and affinity and is responsible Yaneva and Tempst 2006). Protein–DNA-binding reac- for transcriptional regulation of numerous promoters tions were conducted in the presence of the synthetic (McNabb et al. 1995; Sinha et al. 1995; Caretti et al. polymer dI–dC to reduce nonspecific interactions, and 2003). NF-Y became an obvious candidate after its iden- specific protein–DNA complexes were captured with tification from the chromatographic elutions because the

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Figure 5. The NF-Y complex can bind to the critical Fas downstream element. (A) Sequences of the wild-type or mutant probes used for EMSA. (B) EMSA analysis of Fas downstream element (Fas ‘‘F–G’’)-binding protein(s) from HeLa nuclear extracts. (C) EMSA competition assay using cold wild-type or mutated competitor F–G oligonucleotides. (First lane) Wild-type Fas ‘‘F–G’’ oligos were shifted with 4 mg of HNE in the absence or presence of cold wild-type or mutated competitor DNA. (D) Diagram indicating the steps to capture and characterize the complex binding the Fas downstream element. The DNA affinity pull-down was performed using immobilized multimers of the DNA sequence used for EMSA. (E) Proteins that bound to the wild-type Fas downstream element or the mutated sequence were step-eluted with buffer containing 0.1 M, 0.25 M, 0.5 M, or 1 M NaCl and tested for binding activity by EMSA. (F) Supershift analysis to test the specificity of NF-Y binding. In lanes 1–7, EMSA reactions were incubated for 30 min followed by addition of the specified antibody and incubated for 15 min. For lanes 8 and 9, antibodies were incubated with HNE for 10 min before mixing with DNA probe.

DNA sequence within the critical promoter region for the Fas downstream core promoter element by EMSA and Fas/APO1 transcription contains ‘‘...GGGTTGGTGG...’’ recruitment to immobilized Fas/APO1 templates (Fig. 5), and is very similar, although not identical, to the reverse and thereby confirm the biological relevance of this ATTGG NF-Y consensus sequence. To examine the protein–DNA interaction. We next analyzed whether physiological relevance of NF-Y to Fas/APO1 gene regu- NF-Y influenced Fas/APO1 transcription in vivo. To this lation, we used the human breast cancer MCF7 cell line, end, the three subunits of NF-Y were each overexpressed since previous studies have shown that Fas/APO1 plays to similar levels in MCF7 cells using CMV-driven cDNA a role in apoptosis in these cells (Ruiz-Ruiz et al. 2003; constructs (Fig. 6C), and mRNA levels of the endogenous Hernandez-Vargas et al. 2006). Upon treatment of MCF7 Fas/APO1 gene were subsequently measured by quanti- cells with 5-fluorouracil (5-FU) for 14 h to induce DNA tative PCR (qPCR). We found that overexpression of damage, Fas/APO1 mRNA synthesis was highly elevated the NF-Y trimeric complex activated Fas/APO1 mRNA (Fig. 6A). A ChIP analysis was performed in these cells, in nearly twofold, whereas transcription of endogenous p21 the presence or absence of 5-FU, to determine whether and a control gene, SDHA, remained unchanged (Fig. 6D). NF-Y was associated with the Fas/APO1 promoter in This suggests that NF-Y positively regulates Fas/APO1 vivo. For this purpose, we used an antibody to the NF-Y expression and can function selectively on p53 target subunit A and mapped protein binding with primers near genes, consistent with our biochemical studies. These the transcriptional start site of Fas/APO1 and a control results also support the notion that the specialized roles region far downstream. The p21 and CCNB1 promoters of NF-Y and other components of the basal transcription were used as negative and positive controls, respectively, machinery in regulating the p53 response are directed by for NF-YA interaction. These studies show that NF-YA is diverse core promoter structures within p53 target genes. bound near the Fas/APO1 initiator region (Inr) in unstressed MCF7 cells, and this interaction increases upon Discussion 5-FU stress induction. In contrast, negligible NF-YA binding was observed on the p21 promoter in treated In unstressed cells, certain p53 target promoters, like and untreated MCF7 cells. A strong ChIP signal to NF-YA p21, are ‘‘preloaded’’ with paused RNAP II, whereas was also generated on the known target promoter CCNB1 proapoptotic promoters, among others, have negligible (Fig. 6B). These ChIP results are consistent with our RNAP II association. Such striking variation in levels of biochemical data showing specific binding of NF-Y to promoter-bound RNAP II may have direct bearing on the

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Figure 6. NF-Y binds to and regulates the FAS/APO-1 promoter in MCF7 cells. (A) mRNA analysis of Fas/APO1 activation upon 5-FU (50 ng/mL) treatment for 14 h. (B) ChIPs of the FAS/APO-1, p21, and CCNB1 genes were assayed for the presence of NF-Y in MCF7 cells. (C) Western blot analysis of NF-Y subunits overexpressed in MCF7 cells. CMV-driven NF-YA, NF-YB, and NF-YC plasmids were used for transfection and assayed with subunit-specific antibodies. (D) mRNA from MCF7 cells overexpressing NF-Y were analyzed by qPCR.

differential activation kinetics observed after stress in- core promoters may be evolutionarily beneficial to pro- duction of p53-responsive genes (Espinosa et al. 2003). tect cells against unwarranted cell proliferation after The existence of such regulatory mechanisms acting damage or apoptosis when damage can be repaired. For before DNA damage to establish a default programmatic Fas/APO1, and potentially other proapoptotic genes, transcriptional response to stress is very intriguing. Our having a fail-safe mechanism embedded in the promoter data suggest that the intrinsic properties of diverse p53 that prevents rapid transcription may be required to act as core promoters play a key role in regulating RNAP II a buffer until critical signaling thresholds are surpassed. affinity and dynamics to coordinate appropriate responses Once this point is reached, apoptotic genes can be ac- to different stress conditions. We find an unexpected level tively transcribed and undergo multiple rounds of reini- of transcriptional regulation governing RNAP II dynam- tiation to guarantee sufficient mRNA synthesis to drive ics that is encoded within the DNA sequence of diverse cell suicide. Our data reveal that architecturally distinct core promoters that drives expression of p53-responsive core promoters, independent of chromatin or p53 binding, genes (Fig. 7). The TATA box within the p21 promoter has contain the information to impart these properties. a critical role in recruiting the transcriptional machinery by promoting rapid formation of a functional PIC that is Role of the TATA box in p21 promoter regulation poised for initiation. However, the p21 core promoter is The TATA box was the first core promoter element to be intrinsically inefficient for reinitiation, which may be identified and was originally believed to be required for enhanced by signal-dependent components acting at all promoters. However, recent studies indicate that as other levels of regulation to facilitate PIC reformation few as 10% of all core promoters actually have a TATA and prolonged RNA synthesis. In contrast to p21, the Fas/APO1 promoter does not contain a TATA box or other well-characterized core motifs, and the rate of PIC formation is very slow. A Fas downstream element that binds to NF-Y is essential for core promoter activity in vitro, and may nucleate PIC assembly by direct interaction with the general transcription machinery. Surpris- ingly, once transcription is engaged, the Fas/APO1 promoter is capable of efficient RNAP II reinitiation events. Published reports have demonstrated that initiation and reinitiation can be experimentally uncoupled and, in one example, reinitiation is faster than initiation (Jiang and Gralla 1993), which we also observe with Fas/APO1. Transcriptional activation of genes responsible for important cellular events such as cell cycle arrest and apoptosis must be highly regulated. For example, it is advantageous for the cell to have a poised RNAP II on the p21 promoter to ensure its rapid activation upon stress Figure 7. Schematic model of p21 and Fas/APO1 PIC formation and reinitiation kinetics. The p21 core promoter supports signaling in order to quickly halt cell cycle progression. In efficient PIC assembly through the TATA box for rapid tran- contrast, it is equally advantageous to program proapop- scriptional activation, but only poor reinitiation capability. In totic genes, like Fas/APO1, for slow transcriptional contrast to p21, the Fas/APO1 core promoter has low affinity for initiation followed by rapid reinitiation as a safeguard PIC recruitment but supports multiple reinitiation events. The against inappropriately timed cell death. Thus, a genetic downstream NF-Y-binding element is required for core pro- system that is ‘‘hard-wired’’ within the DNA sequence of moter activity and may facilitate nucleation of the PIC.

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Diverse p53 core promoter regulation sequence (Suzuki et al. 2001; Yang et al. 2007). For TATA- NF-Y can associate with TFIID, TBP, and RNAP II, it may containing genes, this motif has been shown to be facilitate nucleation of PIC components and/or help the important in transcriptional activity and PIC formation, core promoter to retain a partial PIC ‘‘scaffold’’ to en- with TFIID recruited before other general PIC compo- hance reinitiation events. Another possibility is that nents (Mathis and Chambon 1981; O’Shea-Greenfield and NF-Y bends the DNA sequence in such a way as to in- Smale 1992; Ranish et al. 1999). The core promoter of p21 crease transcriptional output from the core promoter. In contains a nearly perfect TATA sequence at about À30 bp this regard, the observation from chimeric promoters that that is capable of interacting with either TBP or the TFIID the NF-Y-binding motif (Fas downstream element) works holocomplex (Supplemental Fig. 3). We show that the positively with the TATA box in the Fas/APO1 promoter TATA box is not only critical for p21 transcription, but it but negatively when it is placed in the p21 promoter enables the p21 promoter to efficiently assemble a poised suggests that NF-Y has a unique activity and must func- PIC before transcriptional initiation. This intrinsic fea- tion in particular promoter contexts, underscoring its ture presumably facilitates the rapid stress-induced p21 importance. expression kinetics observed in vivo. Interestingly, ChIP As a gatekeeper of cell growth and division, p53 must analyses of the p21 promoter in wild-type and p53-null orchestrate the activities of numerous factors, such as cells show greatly reduced PIC formation in cells lacking NF-Y, that regulate transcription to ensure orderly cell p53 (Espinosa et al. 2003). In this case, p53 may function cycle progression. Several studies have implicated a re- to relieve chromatin compaction through recruitment of lationship between p53 and NF-Y in which p53 interacts cofactors, such as p300, to generate greater nucleosome directly with NF-Y to repress various cell cycle genes accessibility for PIC formation before stress. In addition, (Imbriano et al. 2005). Interestingly, NF-Y knockdown by specific post-translational modifications of p53 may also siRNA causes apoptosis while activating many p53 target facilitate increased PIC interaction with the p21 pro- genes. We analyzed RNA levels in NF-Y knockdown cells moter. In this regard, acetylation of Lys 373 and Lys 382 and observed activation of Fas/APO1, p21, and PUMA on p53 has been shown to directly promote TFIID binding genes, possibly by indirect induction (Supplemental Fig. to the p21 core promoter (Li et al. 2007). 8). This is consistent with previous studies, although Fas/APO1 expression was not measured (Benatti et al. 2008). Activation of p53 target genes is not entirely Regulation of the FasAPO1 core promoter downstream surprising, since Benatti et al. (2008) demonstrated that element by NF-Y NF-Y depletion from HCT116 cells resulted in down- One explanation for the ability of the Fas/APO1 promoter regulation of 478 genes and up-regulation of 803 genes, to undergo multiple rounds of transcription in vitro is indicating that NF-Y knockdown affects multiple genes through a particular multifunctional protein complex. in a global manner. We hypothesize that NF-Y is a critical, Using immobilized DNA affinity assays, we captured functionally diverse transcription factor and, upon its and identified NF-Y as specifically binding to the Fas cellular depletion, a crisis ensues that results in apopto- DPE. The physiological relevance was confirmed by dem- sis. The multifunctional nature of NF-Y on its distinct onstrating that NF-Y interacts with the endogenous target genes may be conferred by phosphorylation of Fas/APO1 promoter and that overexpression of the NF-Y NF-YA by CDK2 (Chae et al. 2004). Interestingly, p53- complex can stimulate basal Fas/APO1 transcription in dependent activation of p21, an inhibitor of CDK2, may vivo. Interestingly, NF-Y is known to activate or repress create a regulatory loop that ultimately affects NF-Y numerous promoters and associate with general tran- phosphorylation and activity (Yun et al. 2003). With scription factors such as RNAP II, TFIID, TBP, and PC4, regard to Fas/APO1, we speculate that stress-dependent as well as cofactors such as histone acetyl transferases gene activation requires a positive interaction between (HATs) and histone deacetylases (HDACs) (Bellorini et al. promoter-bound p53 and NF-Y, and the involvement of 1997; Currie 1998; Caretti et al. 2003; Peng and Jahroudi post-translational modifications as well as specific inter- 2003; Kabe et al. 2005). Previous studies have shown that actions with Mediator, TFIID-associated factors (TAFs), certain promoters have faster reinitiation rates by using or chromatin-modifying enzymes to generate a fully reg- a ‘‘scaffold,’’ consisting of TFIID, TFIIA, TFIIH, TFIIE, and ulated transcriptional response (An et al. 2004; Donner Mediator, which remains stably bound to the promoter et al. 2007; Sullivan and Lu 2007). after the first round of transcription (Zawel et al. 1995; Yudkovsky et al. 2000). This scaffold may facilitate Core promoter diversity RNAP II reinitiation by avoiding the requirement to reassemble a full PIC after each round of transcription. Our results provide insight into how TATA-less pro- Our in vitro observations of slow initiation followed moters are transcribed. Previous studies demonstrated by efficient, multiple rounds of transcription from the that TATA-less promoters rely on other core elements Fas/APO1, but not p21, promoter are consistent with the that interact directly with various TAFs (Martinez et al. existence of a scaffold. However, scaffold retention on 1994; Burke and Kadonaga 1996; Smale and Kadonaga Fas/APO1 may be achieved by an unconventional mech- 2003). Here we show that Fas/APO1 can use an alterna- anism, since this promoter does not contain typical core tive promoter element that associates with NF-Y and elements, but instead uses a near-perfect NF-Y-binding potentially facilitates PIC assembly. In addition, our anal- site (an inverted CCAAT box centered at +20). Because ysis of chimeric promoters demonstrates the importance

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Morachis et al. of the relative context of core elements. An inserted genes identically. If this were true, apoptosis would likely TATA box within Fas/APO1 increases transcription but override the cell cycle arrest program without allowing cannot functionally replace the Fas downstream element. the cell to recover from stress or DNA damage. Further However, the Fas downstream element placed within the investigation into the default mechanisms used by struc- p21 promoter negatively affects transcription and does turally diverse p53 target genes may provide insight into not rescue a mutated TATA box. It is clear that the how the programmatic response to stress is regulated and interplay between distinct core promoter elements con- how it can be manipulated for targeted therapies. fers another level of promoter regulation and can result in diverse transcriptional outputs. Recent studies have shed Materials and methods light on how these core regulatory circuits control RNAP II activity. One seminal report demonstrated cross-talk In vitro transcription assays between the TATA box and DPE in which factors bound Nuclear protein extracts from HeLa cells were prepared as specifically to one element had an inhibitory effect on described (Dignam et al. 1983). Transcription reactions included transcription initiated at the other (Hsu et al. 2008). 10 mL(;5–6 mg/mL) of HeLa nuclear extract (HNE), 15 mLof Furthermore, TFIID has been shown to regulate pro- HeLa Dialysis Buffer (HDB) (20 mM Hepes at pH 7.9, 50 mM KCl, moters through two distinct motifs—the DPE and 1 mM DTT, 0.2 mM EDTA, 10% glycerol), and 25 mLof DCE—in a phosphorylation-dependent manner driven transcription mix (0.4 mg/mL BSA, 20 mM HEPES at pH 7.9, by CK2 (Lewis et al. 2005). This is significant because it 70 mM KCl, 3 mM DTT, 1.2 mM NTPs, 1–3 mM MgCl2, 0.5 mL demonstrates that extracellular signals can impact gene of RNase inhibitor per reaction), and 500 ng of DNA templates. For the PIC kinetics analyses, NTPs were omitted from the activity by determining which core element a given transcription mix, and the PIC was allowed to form for 0 min to transcription factor will function through. 2 h at 25°C before adding 2 mL of NTP mix to start the reaction The diversity of core promoter structures within p53- (final volumes were adjusted with HDB). Sarkosyl (Sigma- regulated genes implies that each gene may be uniquely Aldrich) was introduced in each reaction by adding 2 mLof regulated. However, the fact that most apoptotic target a 1% stock to 0.04% final concentration unless otherwise noted. genes have much lower levels of poised RNAP II com- Transcription reactions were incubated in a 30°C water bath or pared with cell cycle arrest genes suggests that there are at room temperature. Reactions were stopped and processed some general similarities. A comparison of core pro- using reagents from Zymo Research (RNA Clean-up Kit-5) by moters from several p53 target genes reveals that most adding 200 mL of RNA-binding buffer, applying the mixture to cell cycle arrest genes have focused promoters (single start columns, washing two times with wash buffer, and then eluting with 8 mL of RNase-free water. Primer extension was performed sites and typical core elements), whereas most apoptotic by adding 3 mL of primer annealing mix (10 mM Tris, 1 mM genes including Fas/APO1, PUMA, and APAF-1 appear to EDTA, 1.25 M KCl) to each reaction and heating for 2–3 min at resemble dispersed promoters (several start sites over 50– 75°C in heating blocks. Reactions were removed from the heat 100 nucleotides and few typical core elements) (Juven- blocks and allowed to cool slowly to ;37°C. This was followed Gershon and Kadonaga 2009; JM Morachis, E Scheeff, and by addition of 23 mL of reverse transcription mix (20 mM Tris-

G Manning, unpubl.). We speculate that differences in HCl at pH 8, 10 mM MgCl2, 0.1 mg/mL Actinomycin D, 5 mM core promoter structures exist to establish a default DTT, 0.33 mM dNTP) and 0.5 mL of M-MLV Reverse Transcrip- transcriptional state, with respect to PIC formation and tase (Promega) per reaction, and incubation for 1 h at 37°C. Final dynamics, which insures an appropriate p53 program- reactions were precipitated and washed with ethanol and placed matic response to diverse forms of stress. in a speedvac for 5 min. DNA pellets were each resuspended in 10 mL of formamide with 1 mM EDTA/0.1 mM NaOH (2:1) and Thus, two critical parameters of p53-dependent gene heated for 2–3 min to 95°C followed by snap-cooling on ice. activation—the kinetics of induction and duration of Samples were electrophoresed through 8% polyacrylamide/TBE expression through frequent reinitiation—are intrinsic, gels (SequaGel-8, National Diagnostics). For transcription re- DNA-encoded features of diverse core promoters that actions using immobilized DNA templates, plasmids containing may be fundamental to anticipatory programming of p53 the p21 and Fas full promoters were first linearized by restriction response genes. The default mode, as seen at the p21 enzyme cleavage with NotI, followed by cleavage with a second promoter, is to rapidly form a PIC but undergo few rounds restriction enzyme, EcoRI, to generate sticky ends that were of reinitiation, whereas that of Fas/APO1 is the opposite. filled in by Klenow DNAP with biotinylated dATP and dUTP. Of course, sustained p21 expression requiring multiple After removal of excess nucleotides, the biotinylated fragments rounds of RNAP II reinitiation and reduced Fas/APO1 were incubated with streptavidin-coated magnetic beads (Dynal, Invitrogen) and purified from unbiotinylated DNA using a mag- expression by infrequent reinitiation can be achieved by net. The immobilized p21 or Fas/APO1 DNA templates (250 overriding the default programming through sophisti- fmol) were each incubated with HNE for in vitro transcription as cated epigenetic processes that are tailored to specific described. stress environments. Considering the advantage of pre- serving flexibility to fine-tune cell fate decisions, having Plasmids and mutagenesis a default, genetic program embedded in core promoter The p21 (À2.4 kb to +42 bp) and Fas/APO1 (À1kbto+700 bp) DNA would safeguard against misregulation, particularly promoters were each cloned into pBSKII plasmids using EcoRI of apoptotic genes. This may reflect an evolutionary need and XbaI and named p21DPE-A and FasMut2, respectively. For to balance cell growth while limiting the ability to self- the ‘‘core’’ p21 and Fas promoters, PCR was used to generate the destruct. It is difficult to envision a cellular system that sequence of À149 to 42 bp for p21 and À50 to 78 bp for Fas and would evolve to activate cell cycle arrest and apoptotic then subcloned back into pBSKII. All mutational analyses were

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Diverse p53 core promoter regulation performed by progressively generating 10-bp transversion muta- p21-pGL3, or Fas-pGL3 core promoter constructs and 200 ng of tions in the context of the full-length promoters using the renilla (pRL-TK) were used per well for transfection with Fugene GeneTailor site-directed mutagenesis system following the HD (Roche). After transfection, cells were maintained in 500 mL manufacturer’s instructions (Invitrogen). Fas-TATA was created of DMEM growth media overnight. Cells were then lysed in 100 using Fas/APO1 promoter sequence and inserting the p21 mLof13 passive lysis buffer (PLB buffer, Promega) for 15 min at ATATCAG sequence to replace À23 to À29 and create the Fas- room temperature. For dual luciferase assays, 20 mL of lysate per TATA promoter. F-TATA was created using Fas-TATA and well were aliquoted into a 96-well plate and analyzed using mutating the Scan F region as in Scan F of Figure 3B. p21 wild- firefly luciferase (50 mL of LAR II) and renilla (50 mL of stop and type (TATA) or p21 Scan C (mutated TATA) promoters were glow buffer). All analyses were performed in duplicate, with each mutated by inserting the F, G, or F + G elements of Fas/APO1 at experiment performed at least twice. the same position with respect to the transcription start site For overexpression of NF-Y, MCF7 cells were plated in 12-well (from +7to+26 bp). All of the templates used for in vitro plates and grown to 80% confluency for transfection. We used transcription contained a 27-bp sequence from the luciferase NF-YA(SC112917), NF-YB(SC116285), and NF-YC(SC112622) gene (59-GCGTCTTCCATTTTACCAACAGTACCG-39)thatwas human cDNA clones in pCMV6-XL5 plasmids (Origene) and used for primer extension. For the luciferase reporter assays, the pCMV-GPF as a control. Fugene HD (Roche) transfection reagent core promoters of p21 (À134 to +42 bp) and Fas (À130 to +46 bp) was used at an 8:2 DNA:reagent ratio, transfecting 20 mLof were generated by PCR, and each was subcloned into pGL3 re- transfection mix and 800 ng of total DNA. Cells were collected porter plasmids. 24 h after transfection and processed for mRNA purification (Qiagen) or lysed for Western blot analysis. EMSA Protein–DNA-binding reactions included 2 mg of HNE, 250 ng of Real-time RT-PCR reactions dI–dC, and 5 mL of premix (1 mLof53 shift buffer [100 mM Cells transfected with NF-Y expression plasmids were collected HEPES, 350 mM KCl, 25 mM MgCl2, 15 mM DTT]), 0.5 mLof 24 h later, and total RNA was prepared with the Qiagen 4 mg/mL BSA, and 1.5 mL 15% Ficoll adjusted to 10-mL final RNAeasy Kit. RT reactions were performed with SuperScriptase volume with HDB. The premix was incubated for 15 min at 4°C III (Invitrogen) using the random hexamer protocol following the 32 followed by the addition of 1 mL (15 fmol/mL) of P-labeled manufacturer’s instructions. Primers for FAS/APO-1, p21, and oligonucleotide. Binding reactions were performed for 30 min at SDHA (a control housekeeping gene) were used to analyze gene room temperature. For antibody ‘‘supershift’’ assays, 2 mg of anti- expression using SYBR Green, and values were normalized to NF-YA (C-18), anti-NF-YC (H-120), anti-YY1 (H-414), and anti- b-actin. Bmi-1 (C-20) from Santa Cruz Biotechnologies were incubated with binding reactions for 15 min before gel loading. Samples were then electrophoresed through 4% PAGE (39:1 acrylamide/ ChIP assays bis) in TBE and scanned using a Fuji FLA-5100 PhosphorImager. MCF7 cultures were grown to 50%–60% confluency and were treated with 50 ng/mL 5-FU (Sigma-Aldrich) for 14 h. After wash- DNA affinity protein chromatography ing with PBS, cells were cross-linked with a 1% formaldehyde/ PBS solution for 15 min at room temperature. Cross-linking was 59-Biotin-labeled DNA primers (Supplemental Table 1) were used stopped by addition of glycine to 125 mM final concentration. in an efficient PCR method (Hemat and McEntee 1994) to Cells were washed twice with cold PBS and harvested in RIPA generate ;500-bp multimers of the wild-type and mutated buffer (150 mM NaCl, 1% Igepal CA-630, 0.5% deoxycholate, DNA sequences used in the EMSA reactions. The stock bead 0.1% SDS, 50 mM Tris-HCl at pH 8, 5 mM EDTA, 20 mM NaF, reaction contained 5 mg of DNA and 2 mg of Streptavidin-coated 0.2 mM sodium orthovanadate, 5 mM trichostatin A, 5 mM magnetic beads (Dynal, Invitrogen) in 400-mL final volume. For sodium butyrate, protease inhibitors). Samples were sonicated the recruitment assay, HeLa nuclear extracts were first pre- to generate DNA fragments <1000 bp. For immunoprecipitation, cleared by mixing 100 mL(;600 mg) of HNE with 100 mLof 1 mg of protein extract was precleared for 1 h with 40 mL of 50% ‘‘premix’’ (similar to EMSA buffer) and passing the reaction A/G protein–Sepharose slurry before addition of indicated anti- through 30 mL(;450 ng) of immobilized mutated DNA three bodies. The antibodies used were anti-NF-YA (H-209) or rabbit times. The precleared extract was then separated from the beads, IgG (Santa Cruz Biotechnologies). Each antibody (2 mg) was added mixed with immobilized wild-type or mutated DNA, and al- to the samples and incubated overnight at 4°C in the presence lowed to bind for 30 min at room temperature. Reactions were of 40 mL of protein G beads preblocked with 1 mg/mL BSA and washed three times with HDB containing 0.1 mg/mL of single- 0.3 mg/mL salmon sperm DNA. Beads were washed once with and double-stranded Escherichia coli DNA. Bound proteins were RIPA buffer, three times with ChIP wash buffer (100 mM Tris- then eluted with 20 mL of HDB containing 100 mM, 250 mM, HCl at pH 8.5, 500 mM LiCL, 1% [v/v] NP-40, 1% [w/v] 500 mM, or 1 M NaCl. These fractions were analyzed for protein deoxycholic acid), once again with RIPA buffer, and twice with composition by SDS-PAGE (Western blot, silver staining, and 13 TE. Immunocomplexes were eluted for 10 min at 65°C with mass spectrometry) and for specific DNA binding by EMSA. 1% SDS, and cross-linking was reversed by adjusting to 200 mM NaCl and incubating 5 h at 65°C. DNA was purified, and Mass spectrometry a fraction was used as template in real-time PCR reactions. A detailed description of the mass spectrometry used is included in the Supplemental Material. Western blot analysis Proteins were electrophoresed through 10% SDS-PAGE and Transfection for dual luciferase assay and NF-Y transferred onto nitrocellulose membranes (Amersham Biosci- overexpression ences) before being probed with the following antibodies: anti- HCT116 cells were plated in a 12-well plate and grown at 70% NF-YA (H-209), anti-NF-YB (FL-207), anti-NF-YC (H-120), anti- confluency for transfection. For luciferase assays, 1.8 mg of pGL3, RNAP II (H224) (Santa Cruz Biotechnologies).

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Acknowledgments Espinosa JM, Verdun RE, Emerson BM. 2003. p53 functions through stress- and promoter-specific recruitment of tran- We are grateful to Wolfgang Fischer and Jessica Read of the Salk scription initiation components before and after DNA dam- Institute Mass Spectrometry Core Facility for their identifica- age. Mol Cell 12: 1015–1027. tion of NF-Y. We thank Michael Witcher and other members of Gomes NP, Bjerke G, Llorente B, Szostek SA, Emerson BM, the Emerson laboratory for reviewing the manuscript and pro- Espinosa JM. 2006. Gene-specific requirement for P-TEFb viding significant technical advice and reagents, Cheng-Ming activity and RNA polymerase II phosphorylation within the Chiang and Shwu-Yuan (Sue) Wu for their generous gifts of p53 transcriptional program. Genes & Dev 20: 601–612. purified TBP and TFIID, and Eric Scheeff and Gerard Manning for Hawley DK, Roeder RG. 1987. Functional steps in transcription a bioinformatics analysis of core promoters. 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Diverse p53 core promoter regulation

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GENES & DEVELOPMENT 147 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

José M. Morachis, Christopher M. Murawsky and Beverly M. Emerson

Genes Dev. 2010, 24: originally published online December 29, 2009 Access the most recent version at doi:10.1101/gad.1856710

Supplemental http://genesdev.cshlp.org/content/suppl/2009/12/22/gad.1856710.DC1 Material

Related Content Differential regulation of p53 target genes: it's (core promoter) elementary Nathan P. Gomes and Joaquín M. Espinosa Genes Dev. January , 2010 24: 111-114

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