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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

Allopolyploid species form through the fusion of two differentiated genomes and, in the earliest stages of their evolution, essentially all genes in the nucleus are duplicated. Because unique mutations occur in each ancestor prior to allopolyploidization, duplicate genes in these species potentially are not interchangeable, and this could influence their genetic fates. This study explores evolution and expression of a simple duplicated complex--a heterodimer between RAG1 and RAG2 proteins in clawed frogs (Xenopus). Results demonstrate that copies of RAG1 degenerated in different polyploid species in a phylogenetically biased fashion, predominately in only one lineage of closely related paralogs. Surprisingly, as a result of an early deletion of one RAG2 paralog, it appears that in many species RAG1/RAG2 heterodimers are composed of proteins that were encoded by unlinked paralogs. If the tetraploid ancestor of extant species of Xenopus arose through allopolyploidization and if recombination between paralogs was rare, then the genes that encode functional RAG1 and RAG2 proteins in many polyploid species were each ultimately inherited from different diploid progenitors. These observations are consistent with the notion that ancestry can influence the fate of duplicate genes millions of years after duplication, and they uncover a dimension of natural selection in allopolyploid genomes that is distinct from other genetic phenomena associated with polyploidization or segmental duplication.

An analysis on differential role of positional isomers of symmetric bis-2-(pyridyl)-1H-benzimidazoles as DNA binding agents was carried out. It was found that some minor chemical alterations in a set of isomeric bis-2-(n-pyridyl)-1H-benzimidazoles change their molecular structure and geometry.

The synthesis of three types of met- and zinc- myoglobin dyads, ZnMbAc(4)[Me.sup.+], ZnMbAc(6)[Me.sup.+], and metMbAc(6) is described by incorporating chemically modified metalloporphyrin cofactor appending an acridine(Ac) or a methylacridium ion ([[AcMe].sup.+]) into apo-Mb. It is shown that synthetic manipulation at the Mb surface with the use of an artificial DNA binder along with photoinduced reaction, can give insight to build new Mb-DNA complex and sensitive fluorescent for DNA.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.039 Byline: Meredith Howard-Ashby, Stefan C. Materna, C. Titus Brown, Lili Chen, R. Andrew Cameron, Eric H. Davidson Keywords: Transcription factor; Sea urchin; Homeobox; Development Abstract: A set of 96 homeobox transcription factors was identified in the Strongylocentrotus purpuratus genome using permissive blast searches with a large collection of authentic homeodomain sequences from mouse, human and fly. A phylogenetic tree was constructed to compare the sea urchin homeobox gene family to those of vertebrates, with the result that with the only a few exceptions, orthologs of all vertebrate homeodomain genes were uncovered by our search. QPCR time course measurements revealed that 65% of these genes are expressed within the first 48 h of development (late gastrula). For genes displaying sufficiently high levels of transcript during the first 24 h of development (late blastula), whole mount in situ hybridization was carried out up to 48 h to determine spatial patterns of expression. The results demonstrate that homeodomain transcription factors participate in multiple and diverse developmental functions, in that they are used at a range of time points and in every territory of the developing embryo. Author Affiliation: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA Article History: Received 18 April 2006; Revised 4 August 2006; Accepted 16 August 2006

A genome-wide screen was carried out to identify GATA-3 as the most highly enriched transcription factor in the mammary ductal epithelium of pubertal mice and show that GATA-3 is localized to the body cells of terminal end buds (TEBs) and to the mature luminal cells of mammary ducts. The findings suggest that GATA-3 actively maintains luminal epithelial differentiation in the adult mammary gland, which raises important implications for the pathogenesis of breast cancer.

A new transcription factor of the CCAAT-binding family, MtHAP2-1, is identified in the model legume Medicago truncatula, for which RNA interference (RNAi) and in situ hybridization experiments have indicated a key role during nodule development. The complementary expression pattern of miR169 and MtHAP2-1 and the phenotype of miR-resistant MtHAP2-1 nodules have suggested that the miR169-mediated restriction of MtHAP2-1 expression to the nodule meristematic zone is necessary for the differentiation of nodule cells.

The extent to which DNA ejection is incomplete at zero osmotic external pressure when phage is opened with its receptor in vitro is investigated. It is demonstrated that DNA could be 'pulled' out from the capsid by DNase I acting as a DNA binding protein or spermine acting as a DNA condensing agent.

The C-terminal fragment of [Ca.sub.v]1.2, an L-type voltage-gated calcium channel (LTC), associated transcription regulator (CCAT) binds to a nuclear protein, associates with an endogenous promoter and regulates the expression of a wide variety of endogeneous genes important for neuronal signaling and excitability. The results have provided evidence that voltage-gated calcium channels can directly activate transcription and have described a mechanism linking voltage-gated channels to the function and differentiation of excitable cells.

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

The functions and mechanism of action of the myocardin family of coactivators and the physiological significance of transcriptional coactivation in the context of signal-dependent and cell-type-specific gene regulation are reviewed. Members of the myocardin family of coactivators activate genes involved in cell proliferation, migration, and myogenesis by associating with serum response factor (SRF).

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.02.020 Byline: Lorenza Penengo (1), Marina Mapelli (1), Andrea G. Murachelli (1), Stefano Confalonieri (1), Laura Magri (1), Andrea Musacchio (2), Pier Paolo Di Fiore (1)(2)(3), Simona Polo (1)(2), Thomas R. Schneider (1)(2) Abstract: The interaction between ubiquitinated proteins and intracellular proteins harboring ubiquitin binding domains (UBDs) is critical to a multitude of cellular processes. Here, we report that Rabex-5, a guanine nucleotide exchange factor for Rab5, binds to Ub through two independent UBDs. These UBDs determine a number of properties of Rabex-5, including its coupled monoubiquitination and interaction in vivo with ubiquitinated EGFRs. Structural and biochemical characterization of the UBDs of Rabex-5 revealed that one of them (MIU, motif interacting with ubiquitin) binds to Ub with modes superimposable to those of the UIM (ubiquitin-interacting motif):Ub interaction, although in the opposite orientation. The other UBD, RUZ (Rabex-5 ubiquitin binding zinc finger) binds to a surface of Ub centered on Asp58.sub.Ub and distinct from the 'canonical' Ile44.sub.Ub-based surface. The two binding surfaces allow Ub to interact simultaneously with different UBDs, thus opening new perspectives in Ub-mediated signaling. Author Affiliation: (1) IFOM, the FIRC Institute for Molecular Oncology Foundation, Via Adamello 16, 20139 Milan, Italy (2) European Institute of Oncology, Via Ripamonti 435, 20141 Milan, Italy (3) University of Milan, 20122 Milan, Italy Article History: Received 7 December 2005; Revised 6 February 2006; Accepted 14 February 2006 Article Note: (miscellaneous) Published online: February 16, 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2005.10.042 Byline: Outi Hallikas (1), Kimmo Palin (2), Natalia Sinjushina (3), Reetta Rautiainen (1), Juha Partanen (3), Esko Ukkonen (2), Jussi Taipale (1) Abstract: Understanding the regulation of human gene expression requires knowledge of the 'second genetic code,' which consists of the binding specificities of transcription factors (TFs) and the combinatorial code by which TF binding sites are assembled to form tissue-specific enhancer elements. Using a novel high-throughput method, we determined the DNA binding specificities of GLIs 1-3, Tcf4, and c-Ets1, which mediate transcriptional responses to the Hedgehog (Hh), Wnt, and Ras/MAPK signaling pathways. To identify mammalian enhancer elements regulated by these pathways on a genomic scale, we developed a computational tool, enhancer element locator (EEL). We show that EEL can be used to identify Hh and Wnt target genes and to predict activated TFs based on changes in gene expression. Predictions validated in transgenic mouse embryos revealed the presence of multiple tissue-specific enhancers in mouse c-Myc and N-Myc genes, which has implications for organ-specific growth control and tumor-type specificity of oncogenes. Author Affiliation: (1) Molecular and Cancer Biology Program, Biomedicum Helsinki, PO Box 63, FIN-00014 University of Helsinki, Finland (2) Department of Computer Science, PO Box 68, FIN-00014 University of Helsinki, Finland (3) Developmental Biology Program, Institute of Biotechnology, PO Box 56, FIN-00014 University of Helsinki, Finland Article History: Received 14 June 2005; Revised 21 September 2005; Accepted 21 October 2005 Article Note: (miscellaneous) Published: January 12, 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.07.023 Byline: Patrick Allard (a)(b), Qin Yang (b), William F. Marzluff (c), Hugh J. Clarke (a)(b) Keywords: Oocyte; Histones; Translational control; SLBP; Meiotic maturation Abstract: Although messenger RNAs encoding the histone proteins are among the most abundant in mammalian oocytes, the mechanism regulating their translation has not been identified. The stem-loop binding protein (SLBP) binds to a highly conserved sequence in the 3'-untranslated region (utr) of the non-polyadenylated histone mRNAs in somatic cells and mediates their stabilization and translation. We previously showed that SLBP, which is expressed only during S-phase of proliferating cells, is expressed in growing oocytes at G2 of the cell cycle and accumulates substantially during meiotic maturation. We report here that elevating the amount of SLBP in immature (G2) oocytes is sufficient to increase translation of a reporter mRNA bearing the histone 3'-utr and endogenous histone synthesis and that this effect is not mediated through increased stability of the encoding mRNAs. We further report that translation of the reporter mRNA increases dramatically during meiotic maturation coincident with the accumulation of SLBP. Conversely, when SLBP accumulation during maturation is prevented using RNA interference, both translation of the reporter mRNA and synthesis of endogenous histones are significantly reduced. This effect is not mediated by a loss of the encoding mRNAs. Moreover, following fertilization, SLBP-depleted oocytes also show a significant decrease in pronuclear size and in the amount of acetylated histone detectable on the chromatin. These results demonstrate that histone synthesis in immature and maturing oocytes is governed by a translational control mechanism that is directly regulated by changes in the amount of SLBP. Author Affiliation: (a) Department of Biology, McGill University, Montreal, QC, Canada (b) Department of Obstetrics and Gynecology, McGill University, Montreal, QC, Canada (c) Program in Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA Article History: Received 21 April 2005; Revised 22 June 2005; Accepted 19 July 2005

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2005.08.012 Byline: Rashu B. Seth, Lijun Sun, Chee-Kwee Ea, Zhijian J. Chen Abstract: Viral infection triggers host innate immune responses through activation of the transcription factors NF-[kappa]B and IRF3, which coordinately regulate the expression of type-I interferons such as interferon-[beta] (IFN-[beta]). Herein, we report the identification of a novel protein termed MAVS (mitochondrial antiviral signaling), which mediates the activation of NF-[kappa]B and IRF3 in response to viral infection. Silencing of MAVS expression through RNA interference abolishes the activation of NF-[kappa]B and IRF3 by viruses, thereby permitting viral replication. Conversely, overexpression of MAVS induces the expression of IFN-[beta] through activation of NF-[kappa]B and IRF3, thus boosting antiviral immunity. Epistasis experiments show that MAVS is required for the phosphorylation of IRF3 and I[kappa]B and functions downstream of RIG-I, an intracellular receptor for viral RNA. MAVS contains an N-terminal CARD-like domain and a C-terminal transmembrane domain, both of which are essential for MAVS signaling. The transmembrane domain targets MAVS to the mitochondria, implicating a new role of mitochondria in innate immunity. Author Affiliation: Howard Hughes Medical Institute, Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390 Article History: Received 8 June 2005; Revised 8 August 2005; Accepted 11 August 2005 Article Note: (miscellaneous) Published online: August 25, 2005

Electrophoretic mobility shift assay (EMSA) and affinity particle purification are used for examining the cis elements and trans-acting factors responsible for the gene expression of methyl coenzyme M reductases (MCRs). The results have shown that IMP dehydrogenase-related protein VII (IMPDH VII) encoded by MTH126 is a good candidate for the transcriptional regulator of the mcr operon in this methanogen.

A truncated version of the GCN4 coiled-coil peptide was investigated by ultraviolet resonance Raman (UVRR) spectroscopy with 198 nm excitation, where amide modes are optimally enhanced. The hydrated regions of the coiled-coil peptide structure was found to melt at much lower temperature reflecting stability of the tightly bound regions from which water was excluded.

A comparative bioinformatic analysis of four publicly available Lactobacillus genomes and the genome of Lactobacillus salivarius subsp. salivarius strain UCC118 is performed to identify secreted proteins and those linked to the cell wall. The role in adhesion of the sortase protein and sortase-dependent proteins are examined by using transcriptional analysis and functional characterization of targeted gene knockout mutants.