Tali E. Haran

Sequence-dependent cooperative binding of p53 to DNA targets and its relationship to the structural properties of the DNA targets

The prime mechanism by which p53 acts as a tumor suppressor is as a transcription factor regulating the expression of diverse downstream genes. The DNA-binding domain of p53 (p53DBD) interacts with defined DNA sites and is the main target for mutations in human primary tumors. Here, we show that the CWWG motif, found in the center of each consensus p53 half-site, is a key player in p53/DNA interactions. Gel-mobility-shift assays provide a unique opportunity to directly observe the various oligomeric complexes formed between p53DBD and its target sites. We demonstrate that p53DBD binds to p53 consensus sites containing CATG with relatively low cooperativity, as both dimers and tetramers, and with even lower cooperativity to such sites containing spacer sequences. p53DBD binds to sites containing CAAG and CTAG with measurable affinity only when imbedded in two contiguous p53 half-sites and only as tetramers (with very high cooperativity). There are three orders-of-magnitude difference in the cooperativity of interaction between sites differing in their non-contacted step, and further two orders-of-magnitude difference as a function of spacer sequences. By experimentally measuring the global structural properties of these sites, by cyclization kinetics of DNA minicircles, we correlate these differences with the torsional flexibility of the binding sites.

TBP flanking sequences: asymmetry of binding, long-range effects and consensus sequences.

We carried out in vitro selection experiments to systematically probe the effects of TATA-box flanking sequences on its interaction with the TATA-box binding protein (TBP). This study validates our previous hypothesis that the effect of the flanking sequences on TBP/TATA-box interactions is much more significant when the TATA box has a context-dependent DNA structure. Several interesting observations, with implications for protein–DNA interactions in general, came out of this study. (i) Selected sequences are selection-method specific and TATA-box dependent. (ii) The variability in binding stability as a function of the flanking sequences for (T-A)4 boxes is as large as the variability in binding stability as a function of the core TATA box itself. Thus, for (T-A)4 boxes the flanking sequences completely dominate and determine the binding interaction. (iii) Binding stabilities of all but one of the individual selected sequences of the (T-A)4form is significantly higher than that of their mononucleotide-based consensus sequence. (iv) Even though the (T-A)4 sequence is symmetric the flanking sequence pattern is asymmetric. We propose that the plasticity of (T-A)n sequences increases the number of conformationally distinct TATA boxes without the need to extent the TBP contact region beyond the eight-base-pair long TATA box.

The coexistence of the nucleosome positioning code with the genetic code on eukaryotic genomes

It is known that there are several codes residing simultaneously on the DNA double helix. The two best-characterized codes are the genetic code—the code for protein production, and the code for DNA packaging into nucleosomes. Since these codes have to coexist simultaneously on the same DNA region, both must be degenerate to allow this coexistence. A-tracts are homopolymeric stretches of several adjacent deoxyadenosines on one strand of the double helix, having unusual structural properties, which were shown to exclude nucleosomes and as such are instrumental in setting the translational positioning of DNA within nucleosomes. We observe, cross-kingdoms, a strong codon bias toward the avoidance of long A-tracts in exon regions, which enables the formation of high density of nucleosomes in these regions. Moreover, long A-tract avoidance is restricted exclusively to nucleosome-occupied exon regions. We show that this bias in codon usage is sufficient for enabling DNA organization within nucleosomes without constraints on the actual code for proteins. Thus, there is inter-dependency of the two major codes within DNA to allow their coexistence. Furthermore, we show that modulation of A-tract occurrences in exon versus non-exon regions may result in a unique alternation of the diameter of the ‘30-nm’ fiber model.

Low-level p53 expression changes transactivation rules and reveals superactivating sequences

Transcriptional activation by the tumor suppressor p53 is considered to depend on cellular level, although there are few systems where this dependence on cellular level of p53 has been directly addressed. Previously, we reported that transactivation from p53 targets was sensitive to both p53 amount and DNA sequence, with some sequences being responsive to much lower p53 levels than others when examined in yeast model systems or human cells. Because p53 is normally present at low levels and perturbations might lead to small increases, we examined transactivation under limiting p53. Unlike the positive relationship between transactivation and binding affinity from target sequences at high cellular levels of human p53 in yeast, no such relationship was found at low levels. However, transactivation in the yeast system and the torsional flexibility of target sequences were highly correlated, revealing a unique structural relationship between transcriptional function and sequence. Surprisingly, a few sequences supported high transactivation at low p53 levels in yeast or when transfected into human cells. On the basis of kinetic and flexibility analyses the “supertransactivation” property was due to low binding off rates of flexible target sites. Interestingly, a supertransactivation response element can differentiate transcriptional capacities of many breast cancer-associated p53 mutants. Overall, these studies, which are relevant to other transcription factors, address the extent to which transactivation properties of p53 target sequences are determined by their intrinsic physical properties and reveal unique rules of engagement of target sequences at low p53 levels.

Nearest-neighbor non-additivity versus long-range non-additivity in TATA-box structure and its implications for TBP-binding mechanism

TBP recognizes its target sites, TATA boxes, by recognizing their sequence-dependent structure and flexibility. Studying this mode of TATA-box recognition, termed ‘indirect readout’, is important for elucidating the binding mechanism in this system, as well as for developing methods to locate new binding sites in genomic DNA. We determined the binding stability and TBP-induced TATA-box bending for consensus-like TATA boxes. In addition, we calculated the individual information score of all studied sequences. We show that various non-additive effects exist in TATA boxes, dependent on their structural properties. By several criterions, we divide TATA boxes to two main groups. The first group contains sequences with 3–4 consecutive adenines. Sequences in this group have a rigid context-independent cooperative structure, best described by a nearest-neighbor non-additive model. Sequences in the second group have a flexible, context-dependent conformation, which cannot be described by an additive model or by a nearest-neighbor non-additive model. Classifying TATA boxes by these and other structural rules clarifies the different recognition pathways and binding mechanisms used by TBP upon binding to different TATA boxes. We discuss the structural and evolutionary sources of the difficulties in predicting new binding sites by probabilistic weight-matrix methods for proteins in which indirect readout is dominant.

RNA-dependent chromatin localization of KDM4D lysine demethylase promotes H3K9me3 demethylation

The JmjC-containing lysine demethylase, KDM4D, demethylates di-and tri-methylation of histone H3 on lysine 9 (H3K9me3). How KDM4D is recruited to chromatin and recognizes its histone substrates remains unknown. Here, we show that KDM4D binds RNA independently of its demethylase activity. We mapped two non-canonical RNA binding domains: the first is within the N-terminal spanning amino acids 115 to 236, and the second is within the C-terminal spanning amino acids 348 to 523 of KDM4D. We also demonstrate that RNA interactions with KDM4D N-terminal region are critical for its association with chromatin and subsequently for demethylating H3K9me3 in cells. This study implicates, for the first time, RNA molecules in regulating the levels of H3K9 methylation by affecting KDM4D association with chromatin.

DNA binding and 3′–5′ exonuclease activity in the murine alternatively-spliced p53 protein

In this study we show that the naturally occurring C-terminally alternative spliced p53 (referred to as AS-p53) is active as a sequence-specific DNA binding protein as well as a 3′–5′-exonuclease in the presence of Mg2+ ions. The two activities are positively correlated as the sequence-specific DNA target is more efficiently degraded than a non-specific target. In contrast, a mutated AS-p53 protein that is deficient in DNA binding lacks exonuclease activity. The use of modified p53 binding sites, where the 3′-phosphate is replaced by a phosphorothioate group, enabled the inhibition of DNA degradation under the binding conditions. We demonstrate that AS-p53 interacts with its specific DNA target by two distinct binding modes: a high-affinity mode characterized by a low-mobility protein-DNA complex at the nanomolar range, and a low-affinity mode shown by a high-mobility complex at the micromolar range. Comparison of the data on the natural and the modified p53 binding sites suggests that the high-affinity mode is related to AS-p53 function as a transcription factor and that the low-affinity mode is associated with its exonuclease activity. The implications of these findings to a specific cellular role of AS-p53 are discussed.

How mutations shape p53 interactions with the genome to promote tumorigenesis and drug resistance

The tumor suppressive transcription factor p53 regulates a wide array of cellular processes that confer upon cells an essential protection against cancer development. Wild-type p53 regulates gene expression by directly binding to DNA in a sequence-specific manner. p53 missense mutations are the most common mutations in malignant cells and can be regarded as synonymous with anticancer drug resistance and poor prognosis. The current review provides an overview of how the extraordinary variety of more than 2000 different mutant p53 proteins, known as the p53 mutome, affect the interaction of p53 with DNA. We discuss how the classification of p53 mutations to loss of function (LOF), gain of function (GOF), and dominant-negative (DN) inhibition of a remaining wild-type allele, hides a complex p53 mutation spectrum that depends on the distinctive nature of each mutant protein, requiring different therapeutic strategies for each mutant p53 protein. We propose to regard the different mutant p53 categories as continuous variables, that may not be independent of each other. In particular, we suggest here to consider GOF mutations as a special subset of LOF mutations, especially when mutant p53 binds to DNA through cooperation with other transcription factors, and we present a model for GOF mechanism that consolidates many observations on the GOF phenomenon. We review how novel mutant p53 targeting approaches aim to restore a wild-type-like DNA interaction and to overcome resistance to cancer therapy.

Diverse p53/DNA binding modes expand the repertoire of p53 response elements

Significance The tumor suppressor protein p53 acts as a transcription factor by binding as a tetramer to response elements made of two DNA half-sites separated by a variable spacer, leading to diverse cellular outcomes. Fifty percent of all p53 response elements contain spacers, but their significance is unclear at present. Here, we show that p53 binds to response elements containing long spacers in two different modes: fully specific and hemispecific. In the latter, only one p53 dimer is specifically bound to a DNA half-site, whereas the other dimer is bound to the spacer DNA. Nonetheless, the two modes have comparable binding affinity and specificity. Our findings expand and diversify the p53 regulatory network. The tumor suppressor protein p53 acts as a transcription factor, binding sequence-specifically to defined DNA sites, thereby activating the expression of genes leading to diverse cellular outcomes. Canonical p53 response elements (REs) are made of two decameric half-sites separated by a variable number of base pairs (spacers). Fifty percent of all validated p53 REs contain spacers between 1 and 18 bp; however, their functional significance is unclear at present. Here, we show that p53 forms two different tetrameric complexes with consensus or natural REs, both with long spacers: a fully specific complex where two p53 dimers bind to two specific half-sites, and a hemispecific complex where one dimer binds to a specific half-site and the second binds to an adjacent spacer sequence. The two types of complexes have comparable binding affinity and specificity, as judged from binding competition against bulk genomic DNA. Structural analysis of the p53 REs in solution shows that these sites are not bent in both their free and p53-bound states when the two half-sites are either abutting or separated by spacers. Cell-based assay supports the physiological relevance of our findings. We propose that p53 REs with long spacers comprise separate specific half-sites that can lead to several different tetrameric complexes. This finding expands the universe of p53 binding sites and demonstrates that even isolated p53 half-sites can form tetrameric complexes. Moreover, it explains the manner in which p53 binds to clusters of more than one canonical binding site, common in many natural REs.

29 Structural and binding properties of DNA response elements bound to p53 proteins and the role of spacer sequences in p53-DNA interactions.

27 Analysis of DNA deformations in transcription factor–DNA complexes Jared Sagendorf*, Helen M. Berman and Remo Rohs Molecular and Computational Biology Program, Department of Biological Sciences, Southern California, Los Angeles, CA 90089, USA; Center for Integrative Proteomics Research, Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA *Email: sagendor@usc.edu