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• Journal List
• Genome Res
• 5.29(1); 2019 Jan
• PMC6314159

Genome Res. 2019 Jan; 29(one): 107–115.

Defining TP53 pioneering capabilities with competitive nucleosome binding assays

Xinyang Yu

¹New York State Middle of Excellence in Bioinformatics and Life Sciences and Department of Biochemistry, State University of New York at Buffalo, Buffalo, New York 14203, United states;

Michael J. Cadet

¹New York State Middle of Excellence in Bioinformatics and Life Sciences and Section of Biochemistry, State University of New York at Buffalo, Buffalo, New York 14203, USA;

ⁱⁱDepartment of Biomedical Informatics, State University of New York at Buffalo, Buffalo, New York 14203, USA

Received 2017 Dec 29; Accepted 2018 Nov 1.

Supplementary Materials

Supplemental Material

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Abstract

Accurate factor expression requires the targeting of transcription factors (TFs) to regulatory sequences often occluded within nucleosomes. The ability to target a TF bounden site (TFBS) within a nucleosome has been the defining feature for a special form of TFs known every bit pioneer factors. Recent studies advise TP53 functions as a pioneer gene that can target its TFBS within nucleosomes, but information technology remains unclear how TP53 binds to nucleosomal DNA. To comprehensively examine TP53 nucleosome binding, we competitively bound TP53 to multiple in vitro–formed nucleosomes containing a high- or depression-affinity TP53 TFBS located at differing translational and rotational positions within the nucleosome. Stable TP53–nucleosome complexes were isolated and quantified using next-generation sequencing. Our results demonstrate TP53 binding is limited to nucleosome edges with pregnant binding inhibition occurring within 50 bp of the nucleosome dyad. Binding site affinity only affects TP53 binding for TFBSs located at the same nucleosomal positions; otherwise, nucleosome position takes precedence. Furthermore, TP53 has stiff nonspecific nucleosome bounden facilitating its interaction with chromatin. Our in vitro findings were confirmed by examining TP53-induced bounden in a cell line model, showing induced binding at nucleosome edges flanked by a nucleosome-free region. Overall, our results suggest that the pioneering capabilities of TP53 are driven by nonspecific nucleosome binding with specific binding at nucleosome edges.

Appropriate gene expression is vital for the success of countless cellular processes, including growth, evolution, and metabolism. Amongst the factors known to regulate gene expression is a highly characterized subset of proteins known as transcription factors (TFs). TFs regulate genes by binding to specific Deoxyribonucleic acid sequences in the genomic vicinity of each regulated gene and inducing a alter in expression. TFs recognize degenerate sites as TF binding sites (TFBSs) that announced thousands of times across a given eukaryotic genome. In vivo, most TFBSs are never targeted, likely due to the fact that they are inaccessible due to binding by nucleosomes.

Nucleosomes are the chief unit of measurement of chromatin structure equanimous of a 147-bp department of Dna wrapped around a histone poly peptide core with neighboring nucleosomes separated by accessible linker Deoxyribonucleic acid (Kornberg and Lorch 1999; Jiang and Pugh 2009). In theory, the steric hindrance of nucleosome–Dna interactions could inhibit TFs binding to all nucleosome-jump DNA. In reality, however, nucleosome inhibition of TFs binding is variable both across a genome and even within nucleosomes (Buck and Lieb 2006). Equally Dna duplex molecule bends and twists around the histone octamer, 1 side of it directly contacts the histone surface and gets buried inside, while the other side is exposed to the solvent and is accessible, with a curtained/exposed periodicity of 5 to vi bp. This well-organized architecture maintains nucleosome stability via electrostatic interactions and hydrogen bonds between histone protein side bondage and phosphate groups in the DNA backbone. As a result, this highly distorted and partially overlaid nucleosomal Deoxyribonucleic acid cannot exist accessed readily by TFs (Vermaak et al. 2003; Luger et al. 2012).

The location of TFBSs within a nucleosome tin significantly affect TF–nucleosome binding. TFBSs tin can take various positions within a nucleosome core (known equally translational setting), from near the edge to the center of the nucleosome. For the glucocorticoid receptor, a TFBS located near the edge is bound fourfold better compared with an identical TFBS positioned twenty bp from the dyad (Li and Wrange 1993). Other TFs are inhibited by the translational settings by differing amounts from two- to 100-fold (Vettese-Dadey et al. 1994; Blomquist et al. 1996; Angelov et al. 2004). These divergences in nucleosome inhibition of TF binding are likely driven past differences in how TFs recognize their bounden sites on nucleosomes. The orientation of a TFBS on a nucleosome (known as "rotational setting") besides influences how nucleosomes inhibit TF binding. TFBSs located along a nucleosome surface can face either in or outward due to the twisting of Deoxyribonucleic acid'southward helical structure. For FOXA, a TFBS located almost the nucleosome dyad at a specific rotational setting is significantly bound in in vitro binding assays, while a rotational shifted TFBS located 5 bp away is not bound (Sekiya et al. 2009).

FOXA represents a special course of TFs known as pioneer factors. Pioneer factors were first described in 2002, as regulatory proteins capable of targeting Deoxyribonucleic acid sequences even within compacted, closed chromatin, while other TFs cannot (Cirillo et al. 2002). Amid all known TFs, simply a few have been characterized as pioneer TFs (Iwafuchi-Doi and Zaret 2014). Contempo studies suggest that TP53 functions every bit a pioneer factor at some of its binding sites (Sammons et al. 2015). Sammons et al. (2015) showed that activated TP53 spring to 4416 new bounden sites, 44% of which reside within inactive (H3K4me1- and H3K4me3-) and inaccessible chromatin (Sammons et al. 2015). At the CDKN1A gene, TP53 binds at a higher affinity to its bounden site within chromatin compared with naked DNA (Espinosa and Emerson 2001). Furthermore, throughout the human genome TP53 TFBSs (p53BS) occur in regions with strong nucleosome positioning sequences (Lidor Nili et al. 2010). These findings advise that TP53 functions as a pioneer factor that tin target its binding sites within nucleosomes.

TP53 is a Dna binding TF that acts as a tumor suppressor and has its DNA-bounden domain being frequently mutated during cancer (Rivlin et al. 2011). TP53 integrates multiple stress-induced signals and acts as a transcriptional regulator for a wide variety of genes involved in DNA repair, cell-cycle abort, and apoptosis. TP53'due south ability to regulate these transcriptional responses requires information technology to specifically target and demark its bounden sites throughout the genome. TP53's recognition of naked Dna has been extensively studied and TP53 binds to Deoxyribonucleic acid as a human-tetramer recognizing two decamers of RRRCWWGYYY (el-Deiry et al. 1992), each decamer is chosen a "one-half site" with underlined iv nucleotides (nt) CWWG as the cadre, ordinarily i full site consists of ii one-half sites. TP53 bounden to nucleosomes has been previously tested with conflicting findings. Laptenko et al. (2011) showed that a p53BS located about the dyad had undetectable binding, while sites near the nucleosome edge are bound. Sahu et al. (2010) showed that TP53 can bind to sites near the nucleosome dyad when in the advisable rotational position. Therefore, to address these conflicting results, we adult a quantitative and competitive binding assay allowing the direct comparison of multiple nucleosome sequences in a single binding assay.

Results

Nucleosome design and formation

To determine the specificity of TP53 to nucleosomal DNA, we developed a competitive nucleosome binding analysis (Fig. oneA). Starting with the Widom 601 nucleosome positioning sequence, 14 templates were designed and compared to nonspecific binding to two control sequences (Fig. aneB). With increasing distance to the dyad axis, 3 translational settings were tested: dyad (superhelix location [SHL] 0, 0.5), intermediate (SHL iv, 4.five), and border (SHL 6.5, 7) (Fig. 1B). At each translational position, two rotational settings were tested by shifting the p53BS 5 bp to the right. p53BS accessibility was adamant past modeling the TFBS position onto the nucleosome crystal structure formed from the Widom 601 sequence (Makde et al. 2010). In addition, a site in the linker region (SHL 8), which is outside the nucleosome core, was examined besides. We selected two p53BSs: The outset is from its in vivo target CDKN1A promoter with relative low analogousness; the other 1 is a modified sequence with high TP53 binding affinity. The two p53BSs were then separately added to Widom 601 Deoxyribonucleic acid by replacing the base pairs at the selected locations. Consequently, we obtained xiv different nucleosomal templates having p53BS with increasing distance to the nucleosome dyad and existence placed in either an exposed or concealed orientation (Fig. 1C). Nucleosomes were and so assembled using table salt slope dialysis on all nucleosome sequences simultaneously (Supplemental Fig. S1).

Blueprint of nucleosome templates with p53BS. (A) A 217-bp dsDNA library was designed containing p53BS in various nucleosomal positions. Nucleosomes were formed and purified, generating a nucleosome library that was incubated with increasing amounts of TP53 protein. TP53–nucleosome complexes were separated by EMSA, and the bound and unbound Deoxyribonucleic acid was recovered, quantified by qPCR, and sequenced. (B) A twenty-bp-long p53BS (low and high analogousness) was placed at different positions 0, 5, 41, 46, 66, 71, and 81 bp away from the dyad. The superhelix location (SHL) is designated for each nucleosome sequence. (C) Within the nucleosome, three different positions (dyad, intermediate, and edge) were chosen with increasing distance to nucleosome dyad.

Since we are modifying the Widom 601 nucleosome positioning sequence past inserting a p53BS, we validated nucleosome formation efficiency for each sequence compared with the unmodified Widom 601 sequence. After nucleosome assembly and gel shift analysis, the nucleosomal Dna and free Deoxyribonucleic acid were gel-extracted and sequenced. By comparing the number of reads for each template sequence in the nucleosome band with the number of reads for the 601 control sequence, we tin can determine the relative nucleosome formation efficiency. Nucleosome formation efficiency for each template was extremely consistent across the replicates with only modest differences among nucleosome templates, <20% (Supplemental Fig. S1B).

TP53 is occluded from binding nucleosome core region

To go a comprehensive idea of how TP53 binds to nucleosomes, we combined the traditional TF–nucleosome binding assay with high-throughput sequencing, so that we tin analyze multiple positions on the nucleosome simultaneously with one bounden reaction. Nosotros added TP53 protein to 0.16 pmol purified nucleosome with increasing amount of TP53 (0–ii pmol, 0–286 nM). After a brusk incubation, the binding reactions were separated on a native polyacrylamide gel to detect the TP53–nucleosome complex (Fig. iiA). The offset lane contained just nucleosomes and was used to measure background and input levels for each experimental replicate. As the concentration of TP53 increased, the offset supershift band intensity increased and higher-order bands appeared. Information technology is also noteworthy that every bit the TP53 amount increased, the intensity of nucleosome-only band significantly decreased. To decide the makeup of the shifted complex, we performed a modified western blot from the EMSA gel (see Supplemental Methods). This consequence confirmed a stable ternary complex equanimous of TP53 and nucleosome (Supplemental Fig. S2).

TP53 is occluded from binding the nucleosome core region. (A) Nucleosomes containing 16 dissimilar sequences were bound to increasing amounts of TP53 protein and separated by native PAGE. Lanes contain the following: (1) 0.sixteen pmol nucleosomes; (two–v) 0.xvi pmol of nucleosomes with 36, 71, 142, or 286 nM of TP53 (0.25, 0.5, 1, or 2 pmol). Nucleosome and the major supershift bands are indicated (Ss-1, Ss-ii, Ss-3). (B–D) Relative supershift for each nucleosome is adamant by counting the frequency of each sequence within the supershift and comparison it to nonspecific binding to the 601 sequence. This value is so normalized to the input ratios of nucleosomes (see Equation 1). Mistake confined, SEM; P-values are shown comparing each nucleosome sequence at a specific TP53 concentration to background levels in the input lane. (B) First supershift ring (Ss-1). (C) Second supershift band (Ss-2). (D) 3rd supershift band (Ss-three). (E) Nucleosome sequences that are lost from the nucleosome ring were quantified relative to the input nucleosomes. Strongly spring nucleosomes will generate negative relative shifts.

To determine which nucleosome TP53 preferentially binds, all shifted bands were gel extracted, DNA purified, quantified by qPCR, and sequenced. The sequencing results were and then analyzed and mapped dorsum to the original 16 nucleosome sequences. The resulting information fix was then analyzed past multiple methods, all producing the aforementioned conclusions. In the first method, raw sequence counts were analyzed by comparison each template sequence to the control nonspecific sequence in the same binding experiment (see Equation 1). By comparing each nucleosome sequence to the nonspecific control sequences from the same lane, loading, PCR amplification, and next-generation sequencing (NGS) are all internally controlled. This approach is performed on each shifted band independently (Fig. 2A–D). Statistical significance for each template sequence was so determined by comparing to background measurements from the TP53-null lane. This analysis demonstrates that nucleosome sequences containing a p53BS located outside of the nucleosome are spring outset at the everyman concentrations. Sites located near the nucleosome edge are also spring at low concentrations, while the nucleosomes containing a p53BS located near the dyad (±50 bp) are not specifically spring compared with the command nucleosomes. At higher concentrations of TP53, 142 and 286 nM, binding is no longer significantly different than nonspecific bounden to the control nucleosomes.

To confirm these results from the shifted bands, we also examined the nonshifted nucleosome fragments (Fig. 2E). In this analysis, we examined the loss of DNA fragments relative to the starting amount (TP53-zilch nucleosome band). Nucleosomes that are jump strongly past TP53 will exist shifted out of the nucleosome band and generate a negative relative shift. The advantage of this approach is that information technology allows the decision of binding regardless of TP53/nucleosome complex structure and location of the supershift. Previous studies have shown that TP53 oligomerizes into larger complexes (Stenger et al. 1992; Lee et al. 1994; Chène 2001; Kearns et al. 2016). These results confirm specific binding at nucleosome edges and linker. In this analysis, we practice not meet a drib-off in binding at higher TP53 concentrations relative to the command because one time a specific nucleosome is shifted away from the nucleosome, information technology volition still be accurately counted as missing regardless of where it has been shifted to. This differs from our test of the supershift fragments because at higher concentrations of TP53, college-order complexes are created, further shifting the nucleosome sequence and removing them from the supershift count.

To confirm the differences seen in our nucleosome binding experiments are not due to the placement of the p53BS at various locations within 601, we performed a control binding experiment to the pool of 16 DNA sequences (Supplemental Fig. S3). In this assay, all sixteen Deoxyribonucleic acid templates were mixed at equal amounts and so added to TP53 protein. The TP53–DNA supershift band was then excised, quantified, and sequenced. The results indicate that at that place are relatively minor differences in binding to templates containing the aforementioned p53BS. The high-analogousness p53BS is bound stronger than the low-affinity p53BS beyond all templates as expected.

The two approaches higher up are unable to judge the nonspecific binding of TP53 to nucleosomes; therefore to decide both specific and nonspecific binding straight, we performed a more than in-depth analysis using the Dna amounts determined by qPCR. Briefly, after isolating the Dna from the native PAGE, the amount of Dna was determined past qPCR using a standard curve generated from a control Deoxyribonucleic acid fragment. This allows usa to determine the absolute number of DNA molecules in a shifted or nucleosome ring before distension for NGS library generation. Subsequently sequencing, we and so convert the relative counts from the sequencing library to an absolute number. These absolute counts were and then used to determine the percentage of nucleosomes jump for each template across all TP53 concentrations (Equation ii). The G _D was then determined past nonlinear regression (Heffler et al. 2012). As seen with the previous methods, significant supershift occurs for templates with p53BS at the edge or outside of the nucleosome (Fig. iiiA,B). In both the loftier-affinity p53BS and low-analogousness p53BS groups, it is clear that p53BS located near the nucleosome edges have the smallest K _D values. As the p53BS moves closer into the nucleosome dyad, Chiliad _D value gradually increases (Fig. iiiC) and eventually shows no significant difference from the control non-BS-containing nucleosomes. Interestingly, p53BS affinity only matters when it is placed at nucleosome edges or in the linker; at that place is no significant difference betwixt high-affinity p53BS and depression-affinity p53BS when they are located within 50 bp of the nucleosome dyad (Fig. threeC). All analysis approaches consistently demonstrate that just p53BSs located within the nucleosome edge are bound. Furthermore, bounden site analogousness merely affects TP53 binding for TFBSs located at the same nucleosomal positions. When nosotros wait at the same site in the linker region, the high-affinity p53BS has a lower K _D than low-affinity p53BS, but when we compare the high-affinity p53BS in the edges (SHL 7) with the low-analogousness BS in the linker (SHL viii), the low-affinity group displays smaller K _D values, indicating higher binding analogousness. This effect shows that nucleosome translational position takes precedent over binding site affinity.

TP53 has higher analogousness to p53BS near nucleosome boundaries. (A–C) The sequence results for the nucleosome bands were used to determine the binding affinities, K _D, with nonlinear regression. (A) The percentage of nucleosomes bound by TP53 equally TP53 is titrated. The location of the high-affinity p53BS is indicated. (B) The percentage of nucleosomes bound by TP53 as TP53 is titrated. The location of the low-analogousness p53BS is indicated. (C) K _D values with standard errors were plotted for each nucleosome.

TP53 has two Dna-binding domains: a cadre domain (DBD), which shows sequence-specific binding power; and a highly bones C-concluding domain (CTD), which binds without specificity (Lee et al. 1995; Reed et al. 1995; Selivanova et al. 1996). To evaluate the role of the DBD and the CTD, nosotros repeated our nucleosome binding assay with mutant or truncated proteins. The DBD mutant TP53C135S and the truncated TP53 (TP53ΔCTD: Gly108-Lys370) were compared to the total-length normal TP53 with 0.25 pmol of nucleosomes (Supplemental Figs. S4, S5). For TP53C135S there is a smear on the EMSA gel suggestive of nonspecific bounden. For TP53ΔCTD there is piffling to no smearing, suggesting limited binding. TP53C135S appears to bind nonspecifically as axiomatic by the low relative supershift for all nucleosome fragments. These results are consistent with previous finding showing that both Deoxyribonucleic acid-binding domains are required for TP53 binding (Sullivan et al. 2018). The purified TP53 variant proteins were obtained from different sources, which could affect the presented interpretation.

TP53 binds nucleosome edges in human lung fibroblasts

To validate the relevance of our in vitro bounden results, we examined TP53-induced binding in IMR-90 human being lung fibroblasts combined with steady-state nucleosome occupancy every bit measured by MNase-seq (Kelly et al. 2012; Sammons et al. 2015). To perform this analysis, we reanalyzed the published MNase-seq and ChIP-seq data sets. The TP53 Scrap-seq results are later TP53 is induced and define the specific sites bound by TP53. The MNase-seq data were from the same cell line in a steady state without TP53 activation and define the nucleosome position/occupancy before TP53 binds. TP53 ChIP-seq experiments were performed on IMR-ninety cells treated with a DMSO command or subsequently TP53 activation with nutlin. For each bound site, the p53BS was determined from a predefined listing of TP53 motif locations. This footstep ensures that we are examining direct binding by TP53 and have accurate binding locations but volition miss ill-defined or nonconsensus binding sequences. The raw MNase-seq information were and so extracted, standardized, and visualized centered at the p53BS located within the TP53 ChIP-seq peaks. Equally shown previously, boilerplate nucleosome occupancy peaks at TP53-induced binding sites (Fig. 4A). Closer examination of individual sites later on clustering the MNase-seq results shows that p53BSs are located within the nucleosome edge flanked by a region of lower nucleosome occupancy (Fig. 4B). These results suggest that TP53-induced binding in vivo occurs at the edges of nucleosomes, in a way consistent with our in vitro findings.

Nucleosome occupancy at TP53 nutlin-induced binding sites. (A) Average nucleosome occupancy at induced TP53 binding sites determined from MNase-seq. TP53 Chip-seq results from Sammons et al. (2015) with MNase-seq data from Kelly et al. (2012; Sammons et al. 2015). The MNase-seq reads were extracted, standardized (1 billion reads), and extended with ArchTEx (Lai et al. 2012). (B) Nucleosome occupancy at each p53BS (±m-bp) later nutlin-induced TP53 binding. Standardized and extended MNase-seq reads were clustered by symmetry (Lai and Cadet 2010).

Discussion

To identify the rules defining TF targeting in chromatin, nosotros developed a new approach to examine the binding characteristics of TFs to nucleosome DNA. Our arroyo combines traditional proven nucleosome binding assays with NGS. This approach allowed the straight comparison of multiple nucleosomes in a single experiment. Each nucleosome template sequence was designed allowing the positioning of high or low-analogousness p53BS in diverse translational and rotational settings. Nucleosomes were so generated from the pool of nucleosome sequences, purified, and competitively jump to TP53. Each experiment is controlled at multiple steps. The starting nucleosomes or input to each bounden reaction is quantified and sequenced after Folio (TP53-null lane). This input measurement ensures correction for any variation in nucleosome formation efficiency or starting quantities. Within this input lane, bare bands corresponding to the supershifted TP53–nucleosome circuitous are too quantified and sequenced. This measurement represents the background for each particular supershift. As can exist seen in Figure ii, for the 0 nM sample (gray bar), at that place is just slight differences between different nucleosome sequences. In addition to examining the supershifted fragments, nosotros besides analyzed the nucleosome bands and compared the quantities of each template to the input nucleosomes. In this example nosotros are determining the nucleosome sequences that accept shifted out of the nucleosome band later on being bound. This analysis does not crave us to make up one's mind the appropriate supershift band and is resilient to the formation of higher-order TP53–nucleosome complexes seen at higher TP53 concentrations. To ensure the accurate accented quantification of our results, we measure the DNA concentration of each gel-excised sample past qPCR. By using this corporeality with the NGS results, we tin can calculate the percentage of spring nucleosomes for each nucleosome type beyond all TP53 concentrations. The resulting information were and so fitted using nonlinear regression to obtain One thousand _D values, assuasive the examination of both specific and nonspecific binding. The methodology we have proposed is not express to TP53–nucleosome bounden but can be applied to understand how other pioneer factors demark nucleosomal Deoxyribonucleic acid.

Regardless of the assay methods we used, all approaches consistently showed that TFBS positioning within a nucleosome affects TP53-binding capability. TP53 displays a strong preference to sites outside the 100 bp surrounding the nucleosome dyad. These results are consistent with a dynamic partial unwrapping near nucleosome edges (Polach and Widom 1995; Li and Widom 2004). In this model, DNA near the entry–exit region is unwrapped from the histone proteins exposing the DNA to TF binding. Once bound, the nucleosome tin so rewrap, thus kicking off the TF (Luo et al. 2014). Our results for TP53 suggest that TP53 can access the partially unwrapped nucleosome and remain stably bound. Binding by TP53 to nucleosomes differs when compared to FOXA, which can target its binding sites about nucleosome dyads (McPherson et al. 1993; Chaya et al. 2001). FOXA binds nucleosomal DNA on one side as a monomer (Cirillo and Zaret 2007), whereas TP53 binds as a tetramer complex that partially wraps around the Dna (Malecka et al. 2009; Emamzadah et al. 2011). These differences in how a TF contacts its binding site may explain the ability of some TFs to bind within the nucleosome core.

TP53 displays a potent preference to the nucleosome structure itself and binds nucleosomes in a consensus sequence-independent manner with relative high affinity. Our experiments show that command nucleosomes are bound with a K _D of 38.12–38.54 nM compared with 19.04 nM for the high-analogousness p53BS located in the linker. Fluorescence recovery after photobleaching (FRAP) supports the model that TP53 binds nonspecifically to chromatin in the nucleus. Hinow et al. (2006) compared wild-type TP53 with a Dna binding mutant and showed indistinguishable nucleus diffusion properties, suggesting that TP53 had significant sequence-contained binding to chromatin (Hinow et al. 2006). Our experiments with mutated TP53C135S and truncated TP53ΔCTD suggest that the sequence-contained nucleosome binding is due to TP53's CTD. Our findings are in agreement with recent studies that show the CTD to be a crucial role for maintaining TP53 full function and ensuring binding stability in vivo (Rodriguez et al. 2000; Espinosa and Emerson 2001; McKinney et al. 2004; Tafvizi et al. 2011; Kim et al. 2012; Laptenko et al. 2015).

Our in vitro results combined with the in vivo binding patterns suggest that TP53 is not a FOXA-like canonical pioneer factor; its specific binding capabilities are express to nucleosome edges flanked past a nucleosome-gratuitous region. This presents a model for TP53 nucleosome binding where TP53 encroaches on nucleosomes from an exposed linker region where TP53'due south CTD binds nonspecifically and slides along the DNA (Tafvizi et al. 2008, 2011; Khazanov and Levy 2011; Murata et al. 2015). It so gains admission to its bounden site within the nucleosome edge by the nucleosome partially unwrapping (Fig. five). Once TP53 binds, information technology can recruit histone acetyltransferases, further activating the spring enhancer or promoter region (Gu and Roeder 1997; Gu et al. 1997). Our model does non take into consideration the consequences of the role of histone variants like H2A.Z, which has been shown to associate with TP53 binding in vivo (Gevry et al. 2007). All members of the TP53/TP63/TP73 family share a similar Deoxyribonucleic acid-binding domain with high sequence and structural homology (Levrero et al. 2000). Structural studies of Deoxyribonucleic acid-binding domains for TP53, TP63, and TP73 cocrystallized with Deoxyribonucleic acid target sequences reveal an overall conserved conformation and DNA–protein contact sites for the iii proteins (Chen et al. 2011; Ethayathulla et al. 2012; Ethayathulla et al. 2013). These structural similarities between family unit members suggest that TP63 and TP73 will also demark nucleosomal Deoxyribonucleic acid in a similar mode. Genomic studies on TP63 have suggested that TP63 also acts as pioneer TF during epidermal development by binding at regions of the genome with high encoded nucleosome occupancy (Sethi et al. 2014).

Model of TP53 binding to targets within nucleosomes. TP53 scans DNA in a sequence-independent manner and targets p53BS located at the nucleosome edge. One time bound, TP53 can recruit cofactors and histone acetyltransferases (HATs).

Chromatin accessibility has been recognized every bit a prerequirement for functional activity at regulatory elements (Tsompana and Buck 2014). Our results demonstrate a more meticulous understanding of how positioning within a nucleosome can bear on TF bounden. In particular, our results demonstrate that positioning within a nucleosome is more of import than the analogousness of the underlying binding site and that small differences in positioning, 50 bp, can dramatically affect TF bounden. Therefore, to accurately place the earliest events during gene activation, loftier-resolution nucleosome maps volition be needed with an understanding of which TFs can target their binding sites within the nucleosome. The methodology we have presented hither provides a comprehensive approach to examine the rules dictating nucleosome binding past pioneer factors; further studies on all pioneer factors will allow the identification of general themes driving gene regulation by pioneer factors.

Methods

Design of the nucleosome positioning templates

Nucleosomal templates were derived from the Widom 601 strong nucleosome-positioning sequence (Lowary and Widom 1998; Anderson and Widom 2000). The 601 sequence was first scanned for the presence of sequences like to the p53BS. The original 601 sequence contains a TP53 half-site core (CATG) located but outside the nucleosome edge. To ensure that this sequence does not touch on the binding assays, we modified this sequence to AGGT. We called it "601-modified," which was regarded as an additional control sequence. The original Widom 601 Dna (601) was however used in the written report and had indistinguishable results compared with 601-modified. Two p53BS were used: an adapted high-analogousness ideal sequence, 5′-GGGCATGTCCGGGCATGTCC-three′ (Veprintsev and Fersht 2008; Noureddine et al. 2009); and a natural lower-affinity sequence from the CDKN1A promoter, five′-AGACTGGGCATGTCTGGGCA-3′ (Westfall et al. 2003). Within each p53BS, at that place are two cores (underlined above), which are straight bound by TP53. We design different fragments to ensure that the cores are in either an exposed or curtained orientation as adamant past the crystal structure of a nucleosome with the Widom 601 sequence (Makde et al. 2010). Therefore, 14 templates were designed starting from the 217-bp Widom 601 sequence and compared with nonspecific bounden to two control sequences (Supplemental Table S1).

In vitro nucleosome reconstitution and purification

The 217-bp nucleosome sequences for these experiments were obtained equally double-stranded DNA fragments from Integrated Dna Technologies. All 16 synthesized Deoxyribonucleic acid templates were amplified via PCR and column-purified (Qiagen) and quantified. In vitro nucleosomes were generated from H2A/H2B dimer and H3.1/H4 tetramer (Pecker). All 16 nucleosome sequences were mixed at equal tooth amounts. Mixed DNAs were so added to histones at octamer/DNA tooth ratios of 1.5:one in two M NaCl. Nucleosomes were reconstituted through salt slope dialysis as previously described (Hayes and Lee 1997), which were further purified past 7%–20% sucrose slope centrifuge (Fang et al. 2016) and concentrated past l,000 centrifugal filter units (Millipore, Amicon ultra).

Dna bounden assay followed by EMSA

The protein-nucleosome binding assays were carried out four times with the purified nucleosomes mentioned above and man full-length recombinant TP53 protein (Abcam catalog no. ab84768) in seven µL DNA binding buffer (10 mM Tris-Cl at pH 7.five, l mM NaCl, 1 mM DTT, 0.25 mg/mL BSA, 2 mM MgCl₂, 0.025% Nonidet P-forty substitute, and 5% glycerol) and then incubated for 10 min on ice and so for thirty min at room temperature. Increasing concentrations of TP53 (0–286 nM; 0–2 pmol) were added to 0.16 pmol purified nucleosomes. Poly peptide bounden was detected past mobility shift assay on 4% (west/v) native polyacrylamide gels (acrylamide/bisacrylamide, 29:1, w/w, 7 × 10 cm) in 0.5× Tris borate-EDTA buffers at 100 Five at four°C. Afterwards electrophoresis, DNA was imaged past staining with SYBR green (Lonza).

Library construction and sequencing

All visual bands were excised from the gel, as well as the bands at the same locations in the other lanes. Each gel slice was processed separately for a total of 104 samples from six TP53 full-length and two TP53C135S experiments. The DNA concentration for each sample was adamant past qPCR using a standard curve generated from the control 601 sequence. Illumina sequencing libraries were generated using standard two-stride PCR amplicon methodology with indexing (encounter Supplemental Methods). The number of cycles for PCR step 1 was adamant by the sample concentration determined by qPCR, and ranged from 8 to 12 cycles. All samples were multiplexed and sequenced on a MiSeq using 2 × 150-bp paired-end sequencing.

Data analysis

Quality sequence reads were mapped to each specific starting sequence using BLAT (Kent 2002). Processed results after mapping are in Supplemental Table S2. The results were then analyzed relative to control/nonspecific bounden (relative supershift) or past determining the bounden affinity by fitting a binding curve. Relative supershift is determined from the supershift bands and controls technical variability introduced past gel-excision, PCR, NGS library construction, or NGS sequencing. In this method, each specific nucleosome sequence is measured relative to nonspecific binding (control 601 fragment):

R e l a t i five e S u p e r s h i f t = log 2 ( r e a d southward south u p e r s h i f t N r e a d s south u p e r southward h i f t 601 r e a d due south due north u c l e o s o m e b a n d p 53 n u fifty 50 N r e a d s n u c l e o s o m e b a northward d p 53 n u fifty fifty 601 ) ,

(1)

where N is 1 of the 16 nucleosome sequences, 601 is the control nucleosome sequence, reads supershift is the supershift band, and reads nucleosome band is the nucleosome band in TP53-nix lane.

By using the DNA concentrations after gel-excision with the number of each nucleosome sequence in a sample, the accented number of a particular nucleosome tin can be determined for each sample. These absolute nucleosome counts when applied to the nucleosome only bands at each TP53 concentration are used to decide the pct of leap nucleosomes for each nucleosome fragment with the following:

% B o u northward d N u c l east o s o k due east N = 1 − # North u c l east o southward o m e N i due north northward u c l e o s o grand due east b a n d # N u c l east o s o chiliad e Due north i due north due north u c 50 eastward o southward o m due east b a due north d i northward p 53 n u l fifty .

(2)

The K _D values are and then estimated with nonlinear regression in Prism (Heffler et al. 2012).

Assay of TP53-induced binding in IMR-90 cells

TP53 ChIP-seq bounden sites identified after nutlin-induced bounden in IMR-90 cells were obtained from GEO accession {"blazon":"entrez-geo","attrs":{"text":"GSE58740","term_id":"58740"}}GSE58740. MNase-seq data from proliferating IMR-xc were obtained from GEO accretion {"type":"entrez-geo","attrs":{"text":"GSE21823","term_id":"21823"}}GSE21823 and aligned to hg19 with Bowtie 2 (Langmead and Salzberg 2012). Adjustment to GRCh38 (hg38) does non change the presented results. p53BS from HOMER (Heinz et al. 2010) were intersected to the TP53-induced binding sites to define the verbal location of bounden. TP53 ChIP-seq binding sites without a previously defined p53BS were excluded from the analysis. The MNase-seq reads were extracted, standardized (1 billion reads), and extended (120 bp) as done previously with ArchTEx (Lai et al. 2012; Rizzo et al. 2012). Symmetry of resulting MNase-seq information set at p53BS was and so determined with ArchAlign with 0-bp shifts and region reversal enabled (Lai and Buck 2010; Givens et al. 2012).

Supplementary Material

Acknowledgments

Nosotros thank Dr. Jeffrey Hayes and Dr. Laxmi Mishra at University of Rochester Medical Center for suggestions of sucrose slope centrifuge to purify nucleosomes, and UB Genomics and Bioinformatics Core for adjacent-generation sequencing services. This work was partially supported by Mark Diamond Research Fund FA-sixteen-22 to X.Y. and past the New York Land Department of Health C026714.

Footnotes

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