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Evaluating the specificity spectra of DNA binding molecules is a nontrivial challenge that hinders the ability to decipher gene regulatory networks or engineer molecules that act on genomes. Here we compare the DNA sequence specificities for different classes of proteins and engineered DNA binding molecules across the entire sequence space. These high-content data are visualized and interpreted using an interactive 'specificity landscape' which simultaneously displays the affinity and specificity of a million-plus DNA sequences. Contrary to expectation, specificity landscapes reveal that synthetic DNA ligands match, and often surpass, the specificities of eukaryotic DNA binding proteins. The landscapes also identify differential specificity constraints imposed by diverse structural folds of natural and synthetic DNA binders. Importantly, the sequence context of a binding site significantly influences binding energetics, and utilizing the full contextual information permits greater accuracy in annotating regulatory elements within a given genome. Assigning such context-dependent binding values to every DNA sequence across the genome yields predictive genome-wide binding landscapes (genomescapes). A genomescape of a synthetic DNA binding molecule provided insight into its differential regulatory activity in cultured cells. The approach we describe will accelerate the creation of precision-tailored DNA therapeutics and uncover principles that govern sequence-specificity of DNA binding molecules. chemical genomics | Cognate Site Identification | DNA binders | genomescapes | Energy Landscapes www.pnas.org/cgi/doi/10.1073/pnas.0914023107

Fumarate and nitrate reduction regulatory (FNR) proteins are bacterial transcription factors that coordinate the switch between aerobic and anaerobic metabolism. In the absence of [O.sub.2], FNR binds a [[4Fe-4S].sup.2+] cluster (ligated by Cys-20, 23, 29, 122) promoting the formation of a transcriptionally active dimer. In the presence of [O.sub.2], FNR is converted into a monomeric, non-DNA-binding form containing a [[2Fe-2S].sup.2+] cluster. The reaction of the [[4Fe-4S].sup.2+] cluster with [O.sub.2] has been shown to proceed via a 2-step process, an [O.sub.2]-dependent 1-electron oxidation to yield a [[3Fe-4S].sup.+] intermediate with release of I [Fe.sup.2+] ion, followed by spontaneous rearrangement to the [[2Fe-2S].sup.2+] form with release of 1 [Fe.sup.3+] and 2 [S.sup.2-] ions. Here, we show that replacement of Ser-24 by Arg, His, Phe, Trp, or Tyr enhances aerobic activity of FNR in vivo. The FNR-S24F protein incorporates a [[4Fe-4S].sup.2+] cluster with spectroscopic properties similar to those of FNR. However, the substitution enhances the stability of the [[4Fe-4S].sup.2+] cluster in the presence of [O.sub.2]. Kinetic analysis shows that both steps 1 and 2 are slower for FNR-S24F than for FNR. A molecular model suggests that step 1 of the FNR-S24F iron-sulfur cluster reaction with [O.sub.2] is inhibited by shielding of the iron ligand Cys-23, suggesting that Cys-23 or the cluster iron bound to it is a primary site of [O.sub.2] interaction. These data lead to a simple model of the FNR switch with physiological implications for the ability of FNR proteins to operate over different ranges of in vivo [O.sub.2] concentrations. gene regulation | iron-sulfur | oxygen sensing | oxidation | transcriptional regulation

Electrochemistry measurements on DNA-modified electrodes are used to probe the effects of binding to DNA on the redox potential of SoxR, a transcription factor that contains a [2Fe-2S] cluster and is activated through oxidation. A DNA-bound potential of +200 mV versus NHE (normal hydrogen electrode) is found for SoxR isolated from Escherichia coil and Pseudomonas aeruginosa. This potential value corresponds to a dramatic shift of +490 mV versus values found in the absence of DNA. Using Redmond red as a covalently bound redox reporter affixed above the SoxR binding site, we also see, associated with SoxR binding, an attenuation in the Redmond red signal compared with that for Redmond red attached below the SoxR binding site. This observation is consistent with a SoxR-binding-induced structural distortion in the DNA base stack that inhibits DNA-mediated charge transport to the Redmond red probe. The dramatic shift in potential for DNA-bound SoxR compared with the free form is thus reconciled based on a high-energy conformational change in the SoxR-DNA complex. The substantial positive shift in potential for DNA-bound SoxR furthermore indicates that, in the reducing intracellular environment, DNA-bound SoxR is primarily in the reduced form; the activation of DNA-bound SoxR would then be limited to strong oxidants, making SoxR an effective sensor for oxidative stress. These results more generally underscore the importance of using DNA electrochemistry to determine DNA-bound potentials for redox-sensitive transcription factors because such binding can dramatically affect this key protein property. DNA electrochemistry | iron-sulfur proteins | oxidative stress

Alterations in tissue-specific gene expression greatly affect cell function. Transcription factors (TFs) interact with cis-acting binding sites in noncoding enhancer promoter regions. Transposable elements (TEs) are abundant and similarly represented among mammalian genomes. TEs are important in gene regulation, but their function is not well understood. We have characterized a TE containing functional TF-binding sites for the carcinogen-activated dioxin receptor xenobiotic responsive element (XRE) and the epithelial-mesenchymal transition regulator Slug (Slug site). A Mus promoter database was scanned for XREs to predict coregulation with other TFs. We identified an overrepresented (1,398 genes) B1 retrotransposon containing XRE and Slug sites within 35 bp of each other (designated as B1-X35S). This B1-X35S retrotransposon differed from classic Bls by the presence of the Slug site and by its differential nucleotide conservation outside the X35S region. Phylogenetically, B1-X35S appeared recently in evolution, close to the B1-B subfamily. Comparative gene expression in 61 mouse tissues revealed that B1-X35S-containing genes had lower median expression levels than those with canonical B1 TEs, suggesting a repressive role for X35S. Indeed, X35S was functional and able to bind aryl hydrocarbon (dioxin) receptor (AhR) and Slug and, importantly, to repress cis-reporter genes. Moreover, AhR and Slug were recruited to X35S in vivo and repressed the endogenous expression of X35S-containing genes. Our results demonstrate the existence of a widely present B1 subfamily in the mouse. Because AhR and Slug are relevant in tumor development and differentiation, X35S may represent a genome-wide regulatory mechanism and a tool to modulate gene expression.

The myocardin family proteins (myocardin, MRTF-A, and MRTF-B) are serum response factor (SRF) cofactors and potent transcription activators. Gene-ablation studies have indicated important developmental functions for myocardin family proteins primarily in regulation of cardiac and smooth muscle development. Using Xenopus genome and cDNA databases, we identified a myocardin-related transcription factor expressed specifically in the skeletal muscle lineage. Synteny and sequence alignments indicate that this gene is the frog orthologue of mouse MASTR [Creemers EE, Sutherland LB, Oh J, Barbosa AC, Olson EN (2006) Coactivation of MEF2 by the SAP domain proteins myocardin and MASTR. Mol Cell 23:83-96]. Inhibition of MASTR function in the Xenopus embryo by using dominant-negative constructions or morpholino knockdown results in a dramatic reduction in expression of skeletal muscle marker genes. Overexpression of MASTR in whole embryos or embryonic tissue explants induces ectopic expression of muscle marker genes. Furthermore, MASTR cooperates with the myogenic regulatory factors MyoD and Myf5 to activate transcription of skeletal muscle genes. An essential function for MASTR in regulation of myogenic development in the vertebrate embryo has not been previously indicated. Myf5 | serum response factor | Xenopus

Fusion of macrophages is an essential step in the differentiation of osteoclasts, which play a central role in the development and remodeling of bone. Osteoclasts are important mediators of bone loss, which leads, for example, to osteoporosis. Macrophage fusion receptor/signal regulatory protein [alpha] (MFR/SIRP[alpha]) and its ligand CD47, which are members of the Ig superfamily (IgSF), have been implicated in the fusion of macrophages. We show that CD200, which is not expressed in cells that belong to the myeloid lineage, is strongly expressed in macrophages at the onset of fusion. By contrast, the CD200 receptor (CD200R), which, like CD200, belongs to the IgSF, is expressed only in cells that belong to the myeloid lineage, including osteoclasts, and in CD4+ T cells. Osteoclasts from CD200-/- mice differentiated at a reduced rate. Activation of the NF-[kappa]B and MAP kinase signaling pathways downstream of RANK, a receptor that plays a central role in the differentiation of osteodasts, was depressed in these cells. A soluble recombinant protein that included the extracellular domain of CD200 rescued the fusion of CD200-/- macrophages and their activation downstream of RANK. Conversely, addition of a soluble recombinant protein that included the extracellular domain of CD200R or short-hairpin RNA-mediated silencing of the expression of CD200R prevented fusion. Thus CD200 engagement of the CD200R at the initiation of macrophage fusion regulated further differentiation to osteoclasts. Consistent with in vitro observations, CD200-/- mice contained fewer osteoclasts and accumulated more bone than CD200+/+ mice. The CD200-CD200R axis is therefore a putative regulator of bone mass, via the formation of osteoclasts. fusion | macrophage | RANK | MAPK

Hexim1 is a cellular protein that associates with the positive transcription elongation factor b (P-TEFb) to regulate RNA polymerase II elongation of nascent mRNA transcripts. It directly binds to Cyclin T1 of P-TEFb and inhibits the kinase activity of Cdk9, leading to an arrest of transcription elongation. Here, we report the solution structure of the Cyclin T binding domain (TBD) of Hexim1 that forms a parallel coiled-coil homodimer composed of two segments and a preceding alpha helix that folds back onto the first coiled-coil unit. NMR titration, fluorescence, and immunoprecipitation experiments revealed the binding interface to Cyclin T1, which covers a large surface on the first coiled-coil segment. Electrostatic interactions between an acidic patch on Hexim1 and positively charged residues of Cyclin T1 drive the complex formation that is confirmed by mutagenesis data on Hexim1 mediated transcription regulation in cells. Thus, our studies provide structural insights how Hexim1 recognizes the Cyclin T1 subunit of P-TEFb, which is a key step toward the regulation of transcription elongation. Cyclin T1 | NMR spectroscopy | transcription elongation

We have identified a conserved region in the C-terminal domain of bromodomain-containing protein 4 (BRD4) that mediates its specific interaction with positive transcription elongation factor b (P-TEFb). This domain is highly conserved in testis-specific bromo-domain protein (BRDT) and Drosophila fs(1)h. Both BRDT and fs(1)h specifically interact with P-TEFb in mammalian cells, and this interaction depends on their C-terminal domains. Overexpression of the BRD4 P-TEFb-interacting domain disrupts the interaction between the HIV transactivator Tat and P-TEFb and suppresses the ability of Tat to transactivate the HIV promoter. Incubation of cells with a synthetic peptide containing the C-terminal domain of BRD4 interferes with transactivation of the HIV promoter by the Tat protein. cyclin-dependent kinase 9

The mechanisms by which DNA-damaging agents trigger the induction of the stress response protein p53 are poorly understood but may involve alterations of chromatin structure or blockage of either transcription or replication. Here we show that transcription-blocking agents can induce phosphorylation of the Ser-15 site of p53 in a replication-independent manner. Furthermore, microinjection of anti-RNA polymerase II antibodies into the nuclei of cells showed that blockage of transcription is sufficient for p53 accumulation even in the absence of DNA damage. This induction of p53 occurs by two independent mechanisms. First, accumulation of p53 is linked to diminished nuclear export of mRNA; and second, inhibition specifically of elongating RNA polymerase II complexes results in the phosphorylation of the Ser-15 site of p53 in a replication protein A (RPA)- and ATM and Rad3-related (ATR)-dependent manner. We propose that this transcription-based stress response involving RPA, ATR, and p53 has evolved as a DNA damage-sensing mechanism to safeguard cells against DNA damage-induced mutagenesis. antibody microinjection | DNA damage response | RNA polymerase II | nuclear export | phosphorylation

The homotetrameric tumor suppressor p53 consists of folded core and tetramerization domains, linked and flanked by intrinsically disordered segments that impede structure analysis by x-ray crystallography and NMR. Here, we solved the quaternary structure of human p53 in solution by a combination of small-angle x-ray scattering, which defined its shape, and NMR, which identified the core domain interfaces and showed that the folded domains had the same structure in the intact protein as in fragments. We combined the solution data with electron microscopy on immobilized samples that provided medium resolution 3D maps. Ab initio and rigid body modeling of scattering data revealed an elongated cross-shaped structure with a pair of loosely coupled core domain dimers at the ends, which are accessible for binding to DNA and partner proteins. The core domains in that open conformation closed around a specific DNA response element to form a compact complex whose structure was independently determined by electron microscopy. The structure of the DNA complex is consistent with that of the complex of four separate core domains and response element fragments solved by x-ray crystallography and contacts identified by NMR. Electron microscopy on the conformationally mobile, unbound p53 selected a minor compact conformation, which resembled the closed conformation, from the ensemble of predominantly open conformations. A multipronged structural approach could be generally useful for the structural characterization of the rapidly growing number of multidomain proteins with intrinsically disordered regions. DNA binding | intrinsically unfolded | modular | natively disordered | protein

Messenger RNA transcripts are coated from cap to tail with a dynamic combination of RNA binding proteins that process, package, and ultimately regulate the fate of mature transcripts. One class of RNA binding proteins essential for multiple aspects of mRNA metabolism consists of the poly(A) binding proteins. Previous studies have concentrated on the canonical RNA recognition motif-containing poly(A) binding proteins as the sole family of poly(A)-specific RNA binding proteins. In this study, we present evidence for a previously uncharacterized poly(A) recognition motif consisting of tandem CCCH zinc fingers. We have probed the nucleic acid binding properties of a yeast protein, Nab2, that contains this zinc finger motif. Results of this study reveal that the seven tandem CCCH zinc fingers of Nab2 specifically bind to polyadenosine RNA with high affinity. Furthermore, we demonstrate that a human protein, ZC3H14, which contains CCCH zinc fingers homologous to those found in Nab2, also specifically binds polyadenosine RNA. Thus, we propose that these proteins are members of an evolutionarily conserved family of poly(A) RNA binding proteins that recognize poly(A) RNA through a fundamentally different mechanism than previously characterized RNA recognition motif-containing poly(A) binding proteins. CCCH zinc finger | poly(A) binding protein | RNA binding

NF-[kappa]B and STATs regulate multiple cellular processes through the transcriptional activation of genes with diversified functions. Although the molecular mechanisms that can turn on/off the overall NF-[kappa]B/STAT signaling have been extensively studied, how NF-[kappa]B/ STAT-target genes can be differentially regulated is poorly understood. Here we report that PIASy, a member of the PIAS (for protein inhibitor of activated STAT) protein family, is a physiologically important transcriptional repressor of NF-[kappa]B and STAT1. Piasy deletion in dendritic cells resulted in enhanced expression of a subset of NF-[kappa]B and STAT1-dependent genes in response to LPS or IFN-[gamma], treatment, respectively. Consistently, Piasy null mice are hypersensitive to the LPS-induced endotoxic shock. Furthermore, PIASy and PIAS1 display specific as well as redundant effects on the regulation of NF-[kappa]B/STAT1 signaling. [Pias1.sup.-/-][Piasy.sup.-/-] embryos died before day 11.5. The disruption of one allele of Pias1 in the [Piasy.sup.-/-] background significantly enhanced the effect of Piasy deletion on the transcriptional induction of NF-[kappa]B/STAT1-dependent genes, and vice versa. Our results demonstrate that PIASy cooperates with PIAS1 to regulate the specificity and magnitude of NF-[kappa]B/STAT1-mediated gene activation. transcriptional regulation

p63 is a p53-related transcription factor. Utilization of two different promoters and alternative splicing at the C terminus lead to generation of six isoforms. The a isoforms of TAp63 and [DELTA]Np63 contain a transactivation-inhibitory (TI) domain at the C termini, which can bind to the transactivation (TA) domain and inhibit its transcriptional activity. Consequently, TAp63[alpha] can directly inhibit its activity through an intramolecular interaction; similarly, [DELTA]Np63[alpha] can inhibit the activity of the active TAp63 isoforms through an intermolecular interaction. In this work, we demonstrate that after induction of apoptosis, the TI domain of the p63[alpha] isoforms is cleaved by activated caspases. Cleavage of [DELTA]Np63[alpha] relieves its inhibitory effect on the transcriptionally active p63 proteins, and the cleavage of TAp63[alpha] results in production of a TAp63 protein with enhanced transcriptional activity. In agreement with these data, generation of the N-terminal TAp63 fragment has a role in apoptosis because stable cell lines expressing wild-type TAp63 are more sensitive to apoptosis compared with cells expressing the noncleavable mutant. We also used a model system in which TAp63 expression was induced by trichostatin-A treatment in HCT116 cells. Trichostatin-A sensitized these cells to apoptosis, and this sensitization was associated with cleavage of up-regulated p63. cancer | p53 | transcription | histone deacetylase inhibitor | trichostatin A

Translational initiation factor 2 (IF2) is a guanine nucleotide-binding protein that can bind guanosine 3',5'-(bis) diphosphate (ppGpp), an alarmone involved in stringent response in bacteria. In cells growing under optimal conditions, the GTP concentration is very high, and that of ppGpp very low. However, under stress conditions, the GTP concentration may decline by as much as 50%, and that of ppGpp can attain levels comparable to those of GTP. Here we show that IF2 binds ppGpp at the same nucleotide-binding site and with similar affinity as GTP. Thus, GTP and the alarmone ppGpp can be considered two alternative physiologically relevant IF2 ligands, ppGpp interferes with IF2-dependent initiation complex formation, severely inhibits initiation dipeptide formation, and blocks the initiation step of translation. Our data suggest that IF2 has the properties of a cellular metabolic sensor and regulator that oscillates between an active GTP-bound form under conditions allowing active protein syntheses and an inactive ppGpp-bound form when shortage of nutrients would be detrimental, if not accompanied by slackening of this synthesis. fast kinetics | GTP | translation regulation | nutritional stress