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What is the mechanism through which transcription factors (TFs) assemble specifically along the enhancer DNA? The IFN-β enhanceosome provides a good model system: it is small; its components' crystal structures are available; and there are biochemical and cellular data. In the IFN-β enhanceosome, there are few protein-protein interactions even though consecutive DNA response elements (REs) overlap. Our molecular dynamics (MD) simulations on different motif combinations from the enhanceosome illustrate that cooperativity is achieved via unique organization of the REs: specific binding of one TF can enhance the binding of another TF to a neighboring RE and restrict others, through overlap of REs; the order of the REs can determine which complexes will form; and the alternation of consensus and non-consensus REs can regulate binding specificity by optimizing the interactions among partners. Our observations offer an explanation of how specificity and cooperativity can be attained despite the limited interactions between neighboring TFs on the enhancer DNA. To date, when addressing selective TF binding, attention has largely focused on RE sequences. Yet, the order of the REs on the DNA and the length of the spacers between them can be a key factor in specific combinatorial assembly of the TFs on the enhancer and thus in function. Our results emphasize cooperativity via RE binding sites organization.
Author Summary
An enhanceosome is a functional unit that consists of DNA segment called enhancer; its transcription factors (TFs); and their interacting cofactors. To function, the TFs must assemble on their corresponding response elements (REs) cooperatively. Understanding how TFs assemble is important because the TF combination on the enhancer spells gene-specific activation (or repression). Traditional studies focused mainly on the derivation of consensus DNA sequences, and the TF interaction with its respective RE. This yielded limited success in deciphering the mechanism of selective TF binding. Here, in addition to the conventional roles of protein and DNA, we studied the organization of REs. The IFN-β enhanceosome is a good example because there are limited protein-protein interactions between consecutive TFs. Our molecular dynamics simulations revealed that cooperativity is achieved via overlap of REs, in addition to sparse protein-protein interactions. That is, because the REs overlap, binding of neighboring TFs affect each other through DNA conformation perturbation. In addition, alternation of consensus and non-consensus REs along the enhancer allows more efficient binding of TFs, while the order of the REs excludes unwanted TFs, and enhances selective TF binding. Our findings emphasize the overlooked role of the order and organization of REs, and the length of spacers between consecutive REs.
Citation: Pan Y, Nussinov R (2011) The Role of Response Elements Organization in Transcription Factor Selectivity: The IFN-β Enhanceosome Example. PLoS Comput Biol 7(6): e1002077. https://doi.org/10.1371/journal.pcbi.1002077
Editor: Emad Tajkhorshid, University of Illinois, United States of America
Received: December 14, 2010; Accepted: April 19, 2011; Published: June 16, 2011
Copyright: © 2011 Pan, Nussinov. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under contract number HHSN261200800001E. This research was supported (in part) by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Cellular response to environmental signals relies on tight gene regulation. Specific recognition of response elements (REs) by transcription factors (TFs) [1]–[4] and their combinatorial assembly [1], [5], [6] on promoters and enhancers is crucial for functional, gene-specific transcription initiation [7]. However, how TFs recognize specific REs along the genome which contains hundreds of thousands of similar RE sequences, how the TFs and their co-regulators assemble to form the enhanceosome which is the functional unit, and how the RE organization on the enhancer DNA (the order of the REs on the DNA stretch and the spacer sizes between consecutive REs) play a role in the specificity are still open questions. It has been argued that the cell is populated by a large number of copies of the TF [1], [4], [8]. Consequently, all chromatin-exposed REs will be bound by their corresponding TF, if the TF can be favorably accommodated on the enhanceosome [1], [6], out-competing other TFs. Conformational ensembles of the RE-bound TFs will undergo allosteric, DNA-induced population shifts, which would alter the TFs' co-factor binding sites to binding-favored states [1], [9]. Whether the RE-bound TF will affect function depends on factors such as co-factor availability and post-translational modification state, which relate to the cellular environment. RE availability is governed by chromatin packaging and re-modeling [10], which is determined by the organism's developmental state and cellular environment.
Selective RE recognition and TF activation on chromatin-exposed DNA were proposed to reflect three factors [1]: (i) the cellular network (or environment) which determines the post-translational modification states, co-factor concentration, etc; (ii) protein and DNA which exist as dynamic conformational ensembles that re-distribute allosterically upon binding, post-translational modification, external conditions, etc; and (iii) tight packing of multiple TFs and co-regulators in enhanceosomes (or promoters). This last factor relates to TFs shapes and sizes, and lengths of intervening DNA stretches between neighboring REs [1]. Although dubbed in the literature as 'combinatorial assembly', the implications as specificity-determining factor in RE recognition have largely been overlooked.
Enhanceosomes often involve tens of TFs [1], [2], [11] packed along a DNA stretch of several hundreds of bps [1], [2], [12], [13]. REs typically occur in clusters with spacers of variable lengths where REs can also overlap [6], [14]. Given the large number of possible REs, and RE nucleotide sequence redundancy, the question of how specific TFs prevail over others for given REs is crucial since each RE is associated with a different gene and thus a different function [1], [2], [9].
The IFN-β enhanceosome has been a model system for transcription regulation due to its small size. While a typical enhanceosome functions through long-range interactions [15], the IFN-β enhanceosome sits only tens of bps upstream of the IFN-β gene transcription initiation site and recruits co-factors such as p300 [16] which acetylates histone H1 [17]. The acetylation of histone 'loosens' the nucleosomes at the TATA box region, exposing the promoter, thus promoting assembly of the general transcription factor TFIIB and RNA polymerase II [18] which leads to transcription initiation [8]. IFN-β gene expression requires a minimal number of 8 proteins on the enhancer (Figure 1): ATF-2/c-Jun dimer, four IRF-3 and/or IRF-7 proteins, and an NFκB dimer (typically p50 and p65) [19], that are activated through three different pathways [20]–[22]. The synergistic [23], thus orderly [8] assembly is assisted by the HMG I (Y) protein [24], [25]. Once the IFN-β protein is expressed to a certain level, it dramatically increases IRF-7 expression, which further promotes the re-assembly of the enhanceosome with the IRF-7 incorporated [26].
(A) The four crystal structures used in the simulations. The PDB codes and the protein components are labeled. The three structures are rendered in a same orientation with respect to the DNA sites. The full lengths of DNAs in each of the structures are not shown for clarity. The structure of 2PI0, which is very similar to that of 2O6G, is not shown. (B) The DNA sequence of the IFN-β enhancer, which was divided into four positive regulatory domains PRDIV, III, I and II from upstream to downstream. Core binding site for each protein is underlined separately. Consensus binding sites are in green. Base positions are labeled with respect to the transcription initiation site.
The IFN-β enhancer is composed of four positive regulatory domains (PRDs), IV, III, I, and II from positions −99 to −55 with respect to the transcription initiation site (Figure 1). Several crystal structures are available [27]–[30], each of which encompasses part of the enhanceosome (Figure 1). p50 has been shown to bind to the IFN-β enhancer prior to viral entry, while completion of the assembly of all 8 TFs on the DNA occurs after infection [31]. Of interest, binding of IRF-3 at PRDIII depends on the ATF-2/c-Jun heterodimer orientation on the DNA [32]. PRDIV is composed of two components, the consensus for ATF-2 binding and non-consensus for c-Jun (Figure 1); similarly, PRDII is also divided into two non-symmetric parts: the 5′ site is recognized by p50 and the 3′ site by RelA [33]. The four IRF-3 binding sites within PRDI and PRDIII are also arranged in alternative consensus and non-consensus motifs (Figure 1). Crystal structures of the DNA/IRF-3/IRF-7 complex indicated that IRF-3 binds site C (and/or A) and IRF-7D (and/or B). Understanding how these loosely packed TFs communicate with each other and the role of the REs organization in TF selectivity is important for deciphering the mechanism of cooperative assembly. Using MD simulations and modeling we show that despite the sparseness of protein-protein interactions within the enhanceosome, packing along the DNA is already maximized: binding of each of the four enhanceosome TF dimers to their respective REs cooperatively influences the association of a neighboring pair, by partially pre-configuring the overlapped segment of the neighboring binding sites. We also show that the arrangement of consensus and non-consensus binding sites on the DNA facilitates the optimization of the binding of TF partners. The emerging picture from our results is that overlap of REs leads to specificity by enhancing binding of one TF and restricting others. Together, our results can provide an explanation for how specific assembly on enhancer DNA can be achieved despite the limited protein-protein interactions within the assembly.
To gain insight into TFs-REs binding selectivity and the role of the REs organization on enhancer DNA, MD simulations and structural analysis were performed on complexes derived from three crystal structures (Figure 1) of the virus-inducible IFN-β gene enhanceosome. These structures are incomplete entities of the enhanceosome. A striking structural feature of this enhanceosome is the sparseness of interactions among the proteins which to date has not been observed for other systems. Figure 1 in Text S1 shows all interactions within 4.5 Å between the proteins. Since nonetheless information has to be communicated among the TFs, we focus on potential allosteric conformational changes in the DNA upon protein binding within and outside the binding sites. In addition, because IRF-7 prefers sites B and D while IRF-3 prefers A and C, we closely monitored their interaction energy differences. Using interaction energy instead of binding free energy to assess the association is based on the assumption that the trend of interaction energy parallels that of the binding free energy. This generally holds for such systems since the DNA binding domains are fairly well structured; the binding motifs of ATF-2 and c-Jun and of IRF-3 and IRF-7 are very similar to each other; and the entropy term differences are often negligible. However, it should be kept in mind that these are large systems. While based on the structural fluctuation properties the MD simulation results were interpreted with the assumption that steady-state equilibrium was reached, it is possible that much longer simulations may reveal further dynamic changes not captured in this work.
Dynamics of the 1t2k complex reveals high flexibility
Molecular dynamics (MD) simulations were performed on various combinations of the structural motifs from the 1t2k crystal structure (Table 1). Figure 2 shows the conformational changes of each simulated system with average structures from the respective trajectories superimposed onto the crystal structure. Several observations were made: 1) the full complex was unexpectedly flexible, with the DNA deviating significantly from the crystal structure (Figure 2a). However, the local DNA conformations at the sites where the proteins were bound were relatively stable (Figure 2b); 2) when the two IRF-3 proteins were removed, the DNA bent toward the ATF-2/c-Jun motif with large magnitude, while the DNA conformation in the ATF-2/c-Jun region was reasonably retained (Figure 2c). When the ATF-2/c-Jun motif was removed, the DNA conformation deviated less from the crystal (Figure 2d); 3) when one IRF-3 was removed, the conformation of the DNA at that IRF-3 site drifted away while the IRF-3 bound region still conserved the crystal conformation (Figure s 2e, f). As expected, when simulated alone, the DNA relaxed and lost its unique conformational features such as kinks present in the crystal structure (Figure 2j), and the ATF-2/c-Jun hetero dimer demonstrated high flexibility during the 60-ns trajectory (data not shown). Further analysis showed that IRF-3A anchored well into the major groove throughout the trajectory while IRF-3B was ejected from the major groove to some extent (data not shown). This may have to do with binding specificity and tightness of each IRF molecule. IRF-3A binding was more specific (more hydrogen bonds (HBs) with bases) while IRF-3B was less so, as further discussed later. These results show that the overall complex is quite flexible due to the sparse protein-protein interactions, and in the absence of protein binding the DNA conformation easily deviates from the protein-bound crystal structure.
(A–F) Superposition of average structures over the last 10 ns with crystal conformation of 1t2k complex. (A and B) full complex; (C) DNA/ATF-2/c-Jun; (D) DNA/IRF-3a/IRF-3b complex; (E) DNA/ATF-2/c-Jun/IRF-a; and (F) DNA/ATF-2/c-Jun/IRF-3b complexes. The crystal conformation is in gray. Other coloring schemes are as in Figure 1. Structural motifs used for partial superimpositions are highlighted in circles. (G–J) DNA minor groove width dynamics during the 60 ns trajectory for simulations of the full complex, DNA/ATF-2/c-Jun, DNA/IRF-3a/IRF-3b and DNA, respectively. DNA regions that correspond to binding sites of proteins were boxed in red and labeled with protein names.
Structural comparison and dynamic data reveal the importance of binding site order
Although it is expected that the DNA conformation will fluctuate due to the lack of significant interactions between the proteins, the extent of DNA bending in the DNA/ATF-2/c-Jun simulation was still surprising. Inspection of the crystal structure revealed that the DNA conformation at the c-Jun site appeared unusual as it had few contact with the c-Jun arm on the right hand-side (Figure 1, Figure 2a in Text S1). To quantitatively characterize the DNA conformation, we calculated groove parameters. Because the four DNA groove parameters are inter-correlated (larger major groove width corresponds to smaller major groove depth; smaller minor groove width to larger minor groove depth), table 2 presents only the minor groove depths. The largest are at -93T and -87A, where His40 and Leu42 from IRF-3A and IRF-3B interact with the minor groove. Comparison with a similar crystal structure illustrates that the uniqueness of this conformation (Figures 2a, b in Text S1) is due to the presence of IRF-3A. This explains the dramatic DNA conformational change in the DNA/ATF-2/c-Jun complex simulation, because upon removal of IRF-3A, the DNA/ATF-2/c-Jun motif had to adjust its conformation to optimize the interactions, resulting in large changes.
Further analysis of the binding specificity and experimental biochemical data shed some light on the nature of the cooperativity. The interaction of ATF-2 with the consensus site TGAC (Figure 1) involved specific HBs with bases and electrostatic interactions with the DNA backbone, with an Asn344 side-chain HB with T-99 and G-98 (C of the complementary strand), and Arg352 HB with C (G of the complementary strand). On the other hand, c-Jun interacts with (non-consensus) DNA backbone without any specific HB with the bases. Interestingly two other similar structures involving c-Jun (1JNM and 2H7H) were found to have no HBs with bases either, suggesting that indeed binding of c-Jun could be of lower DNA sequence stringency compared to ATF-2. Combined, these results suggest that ATF-2/c-Jun binding orientation and DNA conformational change were dominated by the requirement to selectively favor IRF-3 binding because IRF-3a and c-Jun share two nucleotides. This also explains the previous experimental observation that in the absence of IRF-3, c-Jun/ATF-2 were able to bind their respective sites even when the order of the two sites was reversed [32]. However, when IRF-3 was present, the ternary complex was formed only when the two sites had the wild type sequence. Reversing the order of the DNA binding sites for ATF-2 and c-Jun will put the ATF-2 binding sequence next to the IRF site, hampering native IRF-3 binding.
Factors dictating binding specificity and cooperativity in the 1t2k complex
Although the sequence of binding events between ATF-2/c-Jun and IRF-3 dimers is unclear, MD simulations revealed that the effect of dimer binding on the DNA conformation is local and limited. It does not appear that one dimer binding pre-configures the entire adjoining RE for the next dimer binding except the overlapped segments. This is evidenced by the relaxation of DNA conformations following removal of either ATF-2/c-Jun or the IRF-3 dimer. Details of DNA conformational changes upon removal of the proteins are given in Figures 2g–j. In the full complex, the groove parameters were dramatically different from site to site (Figure 2g). Upon removal of IRF-3A and IRF-3B, the minor groove next to the ATF-2/c-Jun binding site immediately became larger (Figure 2h) although it partially recovered later in the trajectory. When ATF-2 and c-Jun were removed, minor groove widths at IRF sites were reasonably retained, and the c-Jun binding site conformation was partially preserved, particularly near the IRF-3A end (Figure 2i), suggesting that IRF-3 binding can keep the DNA in favorable conformation for c-Jun binding. Although the DNA organization seems to be loose which allows very limited protein-protein interactions between the ATF-2/c-Jun and the IRF-3 motifs, modeling a conformation with IRF-3 binding one-bp upstream revealed that there would be extensive steric clashes between IRF-3 and ATF-2 and c-Jun (Figure 3 in Text S1). This clarifies why IRF-3A binds to the non-consensus AAAA site, particularly in the presence of ATF-2/c-Jun, even though a consensus site is available one-bp upstream (GAAA). This result shows that binding site overlap was already maximized. Taken together, this suggests that binding cooperativity is achieved largely via overlapped DNA and via limited protein-protein interactions, as evidenced in Figure 4 in Text S1.
IRF-3 binding specificities are different at consensus and non-consensus sites
As revealed in crystal structures 2O6G and 2PI0, the apparent conformations of the four IRF-3 (IRF-3A, -3B, -3C and -3D) bound to PRDIII and I, respectively, are very similar and are similarly bound to DNA (Figures 1, 4). Only one protein-protein interaction occurred among the IRFs (between IRF-3A and IRF-3C) (Figure 1 in Text S1). However, interestingly the protein-DNA interactions are distinct: for example, those for IRF-3A and IRF-3C (chains e and g from 2O6G) were more extensive, involving both HBs with bases and electrostatic interactions with DNA backbones (Table 3), while those for IRF-3B and -3D were mainly with the DNA backbone (Table 3). Each monomer interacted similarly with the DNA at the minor groove via conserved residues His40 and Leu42 [11]. The significance of the minor groove interaction by these two residues is that the base pairs involved were the two central pairs of the upstream IRF binding site; that is, the two consecutive IRF-3 proteins shared part of the binding site, with one binding from the major groove side and the other from the minor groove. The differences between sites A/C and B/D with respect to the association with DNA lie in the interactions of IRF-3 at the major groove. Arg78 of IRF-3A and IRF-3C formed 3-center HBs with two consecutive G bases at positions −91 and −90 relative to the transcription initiation site (Figure 3a, c); by interacting with these two G bases, IRF-3A also shared a couple of bps with c-Jun. Arg86 formed HBs with the next two A bases and a C on the complementary chain; Arg81 interacted with the DNA backbone; and interestingly, of the three arginines, only Arg81 was conserved in the IRF-3 family. Ser8 also formed HB with the T that forms a bp with one of the two consecutive As bound to Arg86. In contrast, in IRF-3B, Arg78 interacted with the DNA backbone, Arg81 interacted weakly with a G base without forming a HB, and Arg86 formed a HB with an A base. For IRF-3D, Arg78 formed HB with a T base; Arg81 interacted with DNA backbone while Arg86 was not in close contact with any DNA bases or backbone. These data show that while all IRF-3 proteins were able to form some HBs with DNA bases, and thus render some specificity, the extent of the specificity varied due to differences in HBs. IRF-3A and -3C were more specific and IRF-3B and -3D were less so.
(A), (B), (C) and (D) show conformations at binding sites IRF-3a, -3b, -3c and -3d, respectively. Bases G and A are shown in cyan and magenta, respectively while other bases are colored based on atom type. Arg78 binds similarly through HB interactions with 2 consecutive G bases in sites A and C while Arg78 pointed away to interact with a T base. Arg78 failed to form HB with the G base in sites B and D due to the presence of a T base next to the G base.
The binding patterns for crystal structure 2PI0 [28], which differ by one base pair each in sites A and C and by having a 3 bp spacer instead of 2 between binding sites IRF-3C and -3D, were essentially the same in terms of the general specificity trend (Table 3); that is, interactions for IRF-3A and -3C were more extensive and specific than for IRF-3B or -3D. Alignment of partial structures revealed that both the protein and DNA segment involved in direct contact matched very well between the two structures (Figures 2D, E in Text S1). The only difference is that Leu42 and His40 interacted at the minor groove with two terminal bps instead of the two central ones.
Dynamics and cooperativity revealed by simulations of DNA/IRF-3 (2O6G/2PI0)
MD simulations were performed on both 2O6G and 2PI0 which are only slightly different in DNA sequence and complex conformation as described earlier. Simulations of the full complex 2O6G revealed that as expected, DNA fluctuation was smaller in the IRF-3 bound region than at the terminal (Figures 4a, b). When only the DNA/IRF-3A/-3B or the DNA/IRF-3C/-3D complex were simulated, DNA conformation at the IRF-3 bound region was again relatively conserved (Figures 4c, d); however, the DNA region now deprived of IRF-3 relaxed and deviated from the starting structure. The complexes with the motif combinations DNA/IRF-3A/IRF-3C and DNA/IRF-3B/IRF-3D were also simulated to evaluate whether binding of a dimer on the same DNA side (AC or BD) would be different from that on opposite sides (AB, or CD) since experimentally, cooperative binding of IRF-3 dimers exists only when both PRDI and PRDIII sites are present [34]. The results from these simulations were similar in terms of DNA conformational dynamics (data not shown). Since binding of two IRF-3 molecules at sites A and C and two IRF-7 molecules at sites B and D is the functionally relevant mode, the DNA/IRF-3A,-3C/IRF-7B,-7D was modeled and simulated as well. While the global conformational changes of the full complex were similar to those of the DNA/IRF-3A/-3B/-3C/-3D simulation results, the protein-DNA interaction profile did reveal some differences. In the DNA/IRF-3ABCD complex, the IRF-3 interaction energies with DNA were more spread while for the DNA/IRF-3AC-7BD complex these interactions were closer to each other (Figures 5A, B), although this feature is not obvious for the 2PI0 complex simulation (Figures 6A, B). Furthermore, the interaction energy for IRF-3BD with DNA was less favorable than that of the IRF-7BD for both 2O6G and 2PI0 complexes (Figures 5C, D, E and 6C, D). Other interaction energies were also calculated and presented in Figure 5 in Text S1. These results indicate that positions B and D prefer IRF-7 while A and C favor IRF-3.
(A–D) Superposition of averaged structures over the last 10 ns trajectories for the 2O6G full complex (A and B), the DNA/IRF-3a/IRF-3b (C), and DNA/IRF-3c/IRF-3d (D) complex, respectively. Structural motifs used for superposition are highlighted with circles. Crystal structure is shown in gray. Coloring code as in Figure 1. (E–H) DNA groove parameter changes over the 60-ns trajectory for the full complex (E), the DNA/IRF-3a/IRF-3b (F), the DNA/IRF-3c/IRF-3d (G), and the DNA alone (H), respectively. DNA regions that correspond to binding sites of proteins were boxed in red and labeled with protein names.
(A)–(E) are for the 2O6G full complex, the full complex with IRF-7 at the b and d positions, DNA/IRF-3a/IRF-3c, DNA/IRF-3b/IRF-3d, and DNA/IRF-7b/IRF-7d respectively.
(A)–(D) are for the 2PI0 full complex, the full complex with IRF-7 at the b and d positions, DNA/IRF-3b/IRF-3d, and DNA/IRF-7b/IRF-7d, respectively.
Analysis of the DNA groove parameters confirmed the limited impact of one IRF binding on the other. In the 2O6G complex, there was significant minor groove narrowing between binding sites (Figure 4e). After removal of IRF-3C and IRF-3D or IRF-3A and IRF-3B, these structural features completely disappeared in the region where IRF-3 was removed, whereas the IRF-3 bound region still remained close to that of the crystal structure (Figures 4f, g). In the DNA/IRF-3C/-3D complex, the minor groove for the spacer region between sites B and C remained narrow, suggesting that much of the binding site for IRF-3B was in a 'ready' state because the sites overlap (Figure 4g). Comparison of DNA parameters between the full and partial complexes shows that there is some impact on the overall DNA conformation when the two dimers were bound together (Figures 4e, f and g). In the absence of proteins, the groove parameters were characteristic of free DNA (Figure 4h).
The structural basis for the cooperativity and preferences of the IRF-3/IRF-7 proteins for specific DNA sites
Similar to the 1T2K complex, the simulations of 2O6G did not show that the binding of one IRF-3 dimer was able to keep the neighboring DNA full sites in the crystal structure conformation. However, it did show that the DNA conformations in the IRF-3 bound region were well retained. Because the binding sites for the two IRF-3 dimers (or monomers) overlap significantly, cooperativity can take place through a pre-organization of the overlapped DNA concomitant with the binding of one dimer. The DNA conformation in the full complex differed from that of the DNA/IRF-3 dimer, suggesting cooperative strengthening of the interaction of each with the DNA.
Above, we showed that the interactions of Arg78 were different at the four IRF binding sites, with sites A and C similar to each other, and different from B and D (Table 3, Figure 3). The main reason why Arg78 oriented differently at sites B and D relates to the T base preceding the consensus sequence (Figure 1). Due to the protruding methyl group from the T, Arg78 could not form stable HB with the G within the binding sites and was forced to turn away (Figures 3b, d). When IRF-7 was bound at these two positions, such steric conflict did not exist, fitting snugly at the sites. Figure 7 shows that the binding of IRF-7 at the B site was different from that of IRF-3 at the same site because the residue at the Arg78 position was Thr93 which has a shorter side chain and thus able to make hydrophobic interactions with the otherwise unfavorable methyl group of T (Figures 7a, b). As a result, IRF-7 binds DNA more tightly at sites B or D than IRF-3 (Figures 7c, d).
(A) and (B) Average structures of DNA/IRF-3 and DNA/IRF-7 motifs at the binding sites c and b, respectively, from the 2PI0 complex simulations trajectories for the last 10 ns. Residues T93 and R96 in IRF-7 corresponding to R78 and R81 are shown. (C) and (D) Superposition of the averaged structures IRF-3 (C) and IRF-7 (D) (in green and orange) bound at binding site b onto the crystal structure (in purple), showing the different binding tightness of IRF-3 and IRF-7.
Analysis of the interactions in the 2o61 (DNA/IRF-3C/IRF-7D/p50/RelA) structure
In the 2o61 crystal structure, interactions between IRF-3C and IRF-7D are sparse, with only one HB between Arg60 of IRF-3C and Ser125 of IRF-7D, which is the C-terminal residue (Figure 1 in Text S1). Interactions between p50 and RelA are extensive (Figure 6 in Text S1). Analysis of the protein-DNA interactions again revealed an interesting phenomenon. IRF-3C interacts with DNA in a pattern similar to what was described for the 2O6G complex. However, the IRF-7D interaction is more extensive and specific than IRF-3B and IRF-3D in 2O6G (Table 3). Thr, which replaced the IRF-3 Arg78, did not need to bend or re-orient to avoid the steric conflict with the underneath T base. Instead, it made van der Waals/hydrophobic contact through the methyl group.
Dynamics of the DNA/IRF-3C/IRF-7D/p50/RelA complex
In the full complex simulation, both the local conformations and the overall structure were retained relatively well compared with the 1t2k complex, although the conformational difference from the crystal structure was still noticeable (Figures 8a, b): the simulations of DNA/IRF-3C/IRF-7D and DNA/p50/RelA complexes show that DNA conformations were minimally perturbed at the binding sites (Figures 8c, d) while the overall structures significantly drifted from the crystal conformation, which was expected. This result illustrated again that the DNA conformation fluctuation and the relatively large movement between the segments was the consequence of the sparseness of protein-protein interactions on different DNA segments.
Superimposition of average structures over the last 10 ns of the trajectories for the full complex (A–B), DNA/IRF-3c/IRF-7d (C), and DNA/p50/RelA (D) on to the crystal structure. The superimposed motifs were highlighted in red circles. Protein-DNA interactions for the full complex (E), DNA/IRF-3c (F) and DNA/IRF-7d (F). (G–J) are the DNA minor groove parameters for the full complex, DNA/IRF3c/IRF-7d, DNA/p53/RelA, and DNA respectively.
Details of the protein-DNA interaction energies are presented in Figures 8e, f. A few interesting observations can be noted: 1) The interaction energies between the DNA and p50/RelA were very similar in the full complex and in the p50/RelA-DNA motif, indicating stable interactions for this association (data not shown); 2) the interaction energies for IRF-3 and IRF-7 with DNA were very similar to each other (Figure 8f), suggesting that the IRF-7 binding at the D (and the B) site was more favorable than the IRF-3 binding at the same sites. This observation is consistent with the simulations results of the 2O6G and 2PI0 full complexes with IRF-7 bound at the B and D sites. DNA groove parameter analysis also revealed limited yet observable DNA conformational impact by protein binding at the neighboring site (Figures 8g–j). When the p50/RelA dimer was removed from the complex, the minor groove width downstream of IRF-7D did not change significantly (Figure 8h). However, the conformation in the IRF-3C and IRF-7D bound region did not maintain well in the crystal structure, suggesting that IRF binding was not as tight in the absence of p50/RelA. On the other hand, when IRF-3C and IRF-7D were removed, the p50 and RelA bound portion retained well the crystal structure conformation, highlighting the stability of this protein-DNA motif (Figure 8i). In this case, the DNA conformation for the IRF-7D binding site was also similar to the crystal structure, confirming the impact of p50/RelA binding on the DNA conformation at the IRF-7 site.
High flexibility is characteristic of this enhanceosome
The combinatorial assembly mechanism of TFs in the enhanceosome is of paramount importance. Even for the small IFN-β enhanceosome, despite considerable cell biology, biophysics, and structural characterization work, it is still unclear how the three modules are selectively recognized and come together to lead to transcription initiation. From the functional standpoint, the IFN-β enhancesome complex can be roughly divided into three modules: ATF-2/c-Jun, IRF, and p50/RelA sites listed from upstream to downstream (Figure 1). While we have shown that packing has reached maximum tightness, the complexes demonstrated high flexibility, higher than typically observed in protein-DNA complexes where there exist extensive protein-protein interactions. DNA can be very flexible, capable of forming sharply looped DNA-protein complexes [35]. However, complexes where two proteins bind shoulder to shoulder on a DNA segment with high specificity and extensive protein-protein interactions, allow very limited DNA fluctuations. For example, the complex of the p53 tetramer with DNA presents very limited DNA conformational change or DNA bending, with a maximum of 30 degrees of curvature only when the DNA sequence is optimized [36], which is evidenced in low resolution experiments. Such dynamic properties can be demonstrated through MD simulations, and is not always captured in crystal structures possibly due to crystal effects.
Overlap of REs leads to cooperative, thus selective TF binding on enhancer DNA
The salient feature that the IFN-β enhanceosome harbors few protein-protein interactions suggests that assembly cooperativity could stem from DNA conformational changes following protein binding; that is, TF binding-induced conformational changes may propagate along the DNA, pre-configure neighboring REs for optimal binding by a second TF, and this could be a key factor in RE recognition. Yet, our results show that the direct effect on DNA conformation by binding of a TF dimer is limited to only the neighboring sites. This is supported by our simulation results that removing a protein molecule from the complexes will cause the DNA conformation to drift away from that in the crystal structure, with only a few bps next to the binding sites reasonably retaining the crystal conformation. Thus, instead of long range DNA allosteric effects, our results suggest that overlap of binding sites is the mechanism of enhanceosome binding cooperativity, between ATF-2/c-Jun and IRF-3A, among IRFs, and between IRFs and p50/RelA proteins. Overlap of binding sites is reasonable and likely to be a broadly utilized enhanceosome mechanism. Constructs with different overlaps of REs and abolished protein-protein interactions may help in delineating the impact of these conformational factors on transcription.
Dimer binding and interactions between different dimers
Hetero-dimerization of TFs is widely recognized and known to be important for binding specificity and consequently function [37]. Experimental data show that pairs of the enhanceosome TFs are often expressed together. For example, the RelA/p50 and RelB/p50 data suggest that they are synthesized at the same time, and are found in complex with p100 in the nucleus [38], [39] and bind DNA first [40]. The question is why unique combinations of ATF-2/c-Jun, IRF-3/IRF-7 and p50/p65?
NF-kB (p50/RelA) is a ubiquitous eukaryotic TF which plays critical roles in transcription of numerous genes [41] and is often modified [42]. Like the ATF-2/c-Jun dimer, it is present in most cells and involved in many biological processes including proliferation, differentiation, and apoptosis [43]–[45]. p50/RelA dimerization is important for transcription. Since the binding specificity is high and the dimerization interface is stable, the binding of this motif is expected to contribute significantly to the stability of the enhanceosome. Interestingly, when the spacer between p50/RelA and IRF-7D changes from 2 to 3 nucleotides the transcriptional activity is only slightly affected. Because the two binding sites still overlap by 3–4 bps with the 3-bp spacer, it is understandable that cooperativity, and thus function, is only minimally changed.
ATF-2 and c-Jun belong to a super-family of TFs that share the basic-region Leucine-zipper motif but have different DNA binding specificities. The ATF-2/c-Jun heterodimer is more populated and binds DNA tighter than either homodimer [46]. c-Jun by itself recognizes the so-called AP-1/TRE site with the symmetrical sequence TGACTCA while ATF-2 recognizes the ATF/CRD consensus site TGACGTCA, which is also symmetric [47]. The difference is in one bp. This difference may suggest that c-Jun dimer binding is not as specific as the ATF-2 since it binds to smaller sites (TGA) while ATF-2 needs two TGAC sites. Combining previous work which shows that the assembly of ATF-2/c-Jun/IRF-3 complex occurs only when the DNA sites were in the 'right' order [32] and our simulation results, it is likely that the non-consensus site is only for c-Jun binding since structural analysis demonstrates that it has few specific interactions with the DNA. Thus, nature has designed the DNA sequence and the ATF-2/c-Jun dimer for optimized binding specificity of each TF and cooperativity between neighboring partners.
IRF-3 activation requires dimerization through phosphorylation [48] which appears controlled by acetylation [49]. However, the IRF-3 dimerization benefit is not obvious, as there is almost no interaction between the DNA binding domains either on the same or opposite sides of the DNA. In addition, it seems that IRF-3 at sites B and D can be easily replaced by IRF-7, since IRF-7 binding at these two positions is more stable than IRF-3 binding. Therefore, the initial binding mode of dimeric IRF-3 (same- or opposite-side of the DNA) may not be as important as previously thought and IRF-3/IRF-7 dimerization should also be favorable. Because the binding of the IRF DNA binding domain was weak when the other proteins were absent [29], dimerization may allow concurrent binding, which enhances not only the binding affinity, but also the specificity, excluding other TFs from binding to the same sites. Interestingly, the IRF-5/IRF-7 dimer is a repressor of IFN genes [50]. Further study is needed to gain insight into the structural basis of this difference between IRF-3 and IRF-5 binding.
Environment affects TF-RE specificity
Assembly of a unique enhanceosome depends on factors such as the chromatin state, i.e., whether the enhancer is available, the TFs concentration and post-translational modification states, and TFs affinity to their respective REs [1]–[3], [51], [52]. Specificity also relates to binding of partners (and cofactors) since allostery and structural reorganization are always involved in conformational perturbation during binding [53]. A recent analysis of 8mer REs [54] suggested that while each TF has sequence preferences, just about half of the TFs bind to distinct DNA motifs. TFs from even the same family may show large differences in affinity and site preference [2], [3], [9]. Related to our case, IRF-4 and IRF-5 both bind strongly to DNA containing CGAAAC segments but weakly to TGAAAG and CGAGAC; and specifically, IRF-3 prefers sites A and C while IRF-7 has higher affinity toward B and D. Although there is distinct sequence preference [55] and some correlation between binding affinity and specificity [56], RE sequences are not the only factor that determines what will bind. As shown in table 3, various binding patterns were observed in complexes with similarities at specific positions. For example, binding patterns of Arg78 and Arg86 were different in two crystal structures (PDB 1T2K and 2PI0) at identical non-consensus sites, while other residues including Arg81, Ser82 and Ala83 interacted with DNA in almost the same way. In one case (2PI0), both arginines formed HB with respective bases, while in the other (1T2K) Arg78 only interacted with the methyl groups of two thymines. One of the major differences between the two complexes is that in 1T2K, ATF-2/c-Jun dimer bound upstream of the IRF-3A, which forced Arg78 to point inward and to interact with bases within its own binding sites. As a result, Arg86 adjusted its interactions as well. Similarly, although IRF-3 binding at sites B and D was not optimal relative to IRF-7, it was able to bind at these sites with adjusted orientations, resulting in transcription upon viral infection. Of interest, TFs from the same family that share similar DNA binding domains often have different functions [57]. These could reflect altered cofactor binding sites, the outcome of RE-induced allosteric propagation.
To conclude, our work emphasizes the crucial, yet largely overlooked role of the organization of successive REs along regulatory DNA stretches, such as enhancers and promoters, in specifying TF binding selectivity. To date, efforts have largely focused on analysis of binding sites and derivation of consensus sequences. Yet, the order of REs and the spacers between consecutive REs can also play a critical role (Figure 9). Spacer sizes determine the TF shape and dimensions: TFs which are too large or too small are disfavored due to either steric effects (Figure 9a) or lack of interactions with the adjoining TFs (Figure 9a). Overlapping REs (Figure 9b) can function via cooperative effects through the binding of TFs to complementary bases, excluding disfavored TFs or enhancing those with relatively low affinity. We propose that overlap of REs is a general mechanism in enhanceosome assembly, beyond the IFN-β. Finally, the order of the binding sites can also be expected to have a functional significance, with a reversed order (Figure 9c) functioning as a repressor. It will be interesting to test the role of spacers by in vivo experiments, where other TFs are also present. Genome searches for identical binding sites but with reversed order are expected to uncover additional occurrences of such a functional mechanism which could be tested experimentally. Combined with current experimental data, our results lead us to propose key factors in RE selectivity and functional TF assembly: exposed (i.e. not covered by nucleosomes) enhancer DNA, available for TF binding; RE sequence and order; the length (positive or negative) of spacers between REs; the TFs concentration and post-translational modification states; and proteins and DNA conformational ensembles. Here, our study emphasizes the key role of cooperativity in making the REs a functionally unique gene regulation site. RE organization along the DNA and the intervening spacers play a key role in selective combinatorial assembly, and as such, in the regulation of gene expression.
(A) A certain size spacer between the two REs excludes the binding of two proteins that are too small (lacking favorable contacts), or too large (will have steric clash). (B) Two proteins bind partially overlapped REs on different DNA faces. Binding of the first protein reconfigures the overlapped DNA conformation (opposite side), leading to more favorable binding of the second. (C) Switching the order of neighboring REs impacts the overlapped binding site, disfavoring binding of the corresponding protein.
Materials and Methods
MD simulation protocol
MD simulations were performed on four partial enhanceosome crystal structures and their components [28]–[30]. The composition of each simulation is listed in table 1. Each system was solvated with a TIP3P water box [58] with a margin of at least 10 Å from any edge of the box to any protein or DNA atom. Solvent molecules within 1.6 Å of the DNA or within 2.5 Å of the protein were removed. The systems were then neutralized by adding sodium ions. The resulting systems were subjected to a series of minimizations and equilibrations using the CHARMM program (academic version) [59], [60] and the CHARMM 22 and 27 force field for the protein [61] and nucleic acid [62], [63], respectively. The production MD simulations were performed at temperatures of 300 degrees Kelvin using the NAMD program [64] and the CHARMM force field. Periodic boundary conditions were applied and the non-bonded lists were updated every 20 steps. The NPT ensemble [65] was applied and the pressure kept at 1 atom using Langevin-Nose-Hoover coupling [66]. SHAKE constraints [67] on all hydrogen atoms and a time step of 2 fs and a nonbonded cutoff of 14 Å with force shift algorithm were used in the trajectory production. Electrostatic interactions were treated with particle mesh Ewald algorithm [68], [69]. The sizes of the systems were about 110,000 atoms and the duration for each simulation was 60 ns.
Modeling of enhanceosome complexes
Two complexes were modeled that constituted the DNA IRF-3ac/IRF-7bd with both the 2O6G and 2PI0 templates. In addition, because some of the residues were missing in the crystal structure of 2PI0, IRF-3 structure at position B was used to model IRF-3 at positions A and D. These complexes were constructed by superimposing the backbone of IRF-3 or IRF-7 onto the proteins that were originally there. The systems were minimized for 2000 steps with the ABNR algorithm. The obtained structures were then solvated and further minimized as described in the previous procedures. DNA parameters were calculated with the CURVES program [70], [71].
Supporting Information
Text S1.
Supplemental information file.
This study utilized the high-performance computational capabilities of the Biowulf PC/Linux cluster at the National Institutes of Health, Bethesda, MD (http://biowulf.nih.gov).
Author Contributions
Conceived and designed the experiments: YP RN. Performed the experiments: YP. Analyzed the data: YP. Contributed reagents/materials/analysis tools: YP. Wrote the paper: YP RN.
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Frontiera E Confini Della Fisica Di Molte Particelle
R. A. Broglia, John Robert Schrieffer, Societŕ italiana di fisica
Amsterdam, Jan 1, 1988 - Science - 460 pages
Subjects at the forefront of many-particle research, like high-temperature superconductivity, the quantum Hall effect, heavy fermions, tunneling of quantal systems with many degrees of freedom, Monte Carlo calculations on a lattice etc., were presented. The meeting was well timed as it coincided with a very active period of research in the study of superconductivity and thus provided an excellent opportunity to assess some of the major developments that have taken place in the last years.
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text: Just because you run a small business, doesn't mean your business credit card should be without large rewards. Whether you're looking for capital to get up and running or long-term cash back rewards for the office supplies you purchase the most, small business credit cards can be a major tool for all small business owners. Small business credit cards often offer features that help business owners better manage their expenses and cash flow, too, keeping your day-to-day business as usual.


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If you'd like to try something different and healthier than wheat pasta, oats, farfel, and brown rice, consider protein-rich quinoa ("KEEN-wah"). Because this grain is so mild-flavored and versatile, you'll find lots of easy quinoa recipes to suit your taste. This roundish sand-colored grain has a mild, nutty flavor and light texture. Quinoa rates highest in nutritive value amongst the grains. It is the only grain that is considered a full protein, and is a great source of iron, all the B vitamins, phosphorus and fiber, yet quinoa is easy to digest and prepare. It is as versatile as rice or farfel and can be used in many dishes, from entrées to desserts, for breakfast, lunch or dinner.
Quinoa is good served hot or at room temperature. It works well in pilafs, stews, burgers soups, salads and as a morning cereal drizzled with maple syrup. To cook quinoa for a pilaf or salad, you can use water or broth. For your morning cereal, try apple or orange juice. To make the cooked grains fluffier, first rinse and roast them in a dry non-stick pan over medium-low heat, stirring until they become fragrant and pop. In about three minutes, when the popping stops, add the liquid, cover and cook until the quinoa is done, about 15 minutes.
Nature protects each grain of quinoa with a coating of saponin, a bitter, soap-like substance that acts as a natural insect repellent, so it is important to rinse quinoa well before cooking. Unlike other whole grains, the germ — the nutrient-rich middle layer of a grain — covers the entire kernel of quinoa. As quinoa cooks, the germ separates from the kernel, creating little white rings. When you see these rings, you know the grains are fully cooked.
Here is a selection of great dishes to help you get acquainted with this super grain.
Colorful Quinoa Tabbouleh
- 1 cup Quinoa, washed and drained well
- 2 ½ cups water
- 4 medium tomatoes diced
- 1 medium cucumber, peeled if waxed and chopped
- 1 cup fresh parsley, washed well and finely minced
- 1/3 cup fresh mint leaves, finely minced (optional)
- 1/2 cup pitted black olives
- 1/2 cup red onion, finely minced
- 1/3 cup lemon juice, freshly squeezed
- 1 Tbs Eden Ume Plum Vinegar (a salty liquid extracted from Japanese plums available in health food stores)
- 3 Tbs Extra Virgin Olive Oil
- 4 ounces roasted sunflower or pumpkin seeds
Bring the water to a boil in a medium saucepan, add the quinoa, cover, reduce the flame to medium-low and simmer for 15 minutes or until all water has been absorbed. Place the quinoa in a large mixing bowl and fluff up with a spoon until grain is separated. Add all remaining ingredients and mix thoroughly before transferring to a serving bowl. Decorate with some parsley leaves and olives
Preparation 15 minutes
Serves 8
Cooking time 20 minutes
Quinoa and pomegranate salad
Quinoa is so versatile that it lends itself to sweet or salty recipes. Here it is presented with pomegranate seeds that are now appearing in local markets. Have this recipe in mind when planning your Rosh Hashanah menu. It is easy to make and it is nutritious as it is delicious!
Special note:
There is coriander also known as cilantro, in this recipe which has a sort of lemony taste. In my experience I found that people have an interesting relationship with this herb; namely either they love it or hate it. People hailing from Mediterranean countries appreciate it's pungent taste, while those of Eastern European descent are not accostumed to it's taste. Feel free to use your discretion when making this recipe which is great with it or without it.
Furthermore, mint adds another dimension to the dish. I pluck some mint out of my backyard garden and use it sparingly, not to overpower the dish with the mint flavor.
- 1 cup (150g) quinoa
- 1 ½ cups water
- ½ tsp salt
- ½ tsp garlic powder (optional)
- ½ cup pine nuts
- 1 pomegranate, seeds removed
- Small handful mint, chopped (optional)
- Small handful coriander/cilantro, chopped (optional)
- 1 lime juiced
- 1 Tbs extra-virgin olive oil
Wash quinoa very thoroughly. Cook according to package instructions (about 20 minutes) adding salt and garlic powder to the cooking water. allow to cool for a few minutes, then fluff up with a fork.
Meanwhile, toast the pine nuts in a dry frying pan until lightly golden. Mix the pine nuts, pomegranate seeds, herbs, lime juice and 4 Tbs oil with the quinoa. Serve as is, or as a side dish to fish or chicken.
Recipe extra
Feel free to add chopped dried apricots instead of the pomegranate. Or try adding some broiled shredded chicken.
Preparation 15 minutes
Serves 6
Special Quinoa burgers
Burgers are always ever so popular and sure to please all ages. Why not try these delightful quinoa burgers! Whenever I cook quinoa I end up making too much. This is a good way of using up leftover cooked quinoa. They are great served for lunch the next day!
- 2 cups cooked quinoa
- 1 cup minced onions
- ½ cup minced parsley
- 2 eggs
- 1 clove garlic, minced
- 1 tsp soy sauce
- 1 Tbs (homemade) mayonnaise
- ¼ tsp. basil (optional)
- 2 Tbs sesame or olive oil (or what is available)
- 2 Tbs whole wheat pastry flour
- Techina
Place first eight ingredients into a large bowl. Mix well with a wooden spoon. Add just enough pastry flour to hold the mixture together. If the mixture gets too dry, add a little water. Form 1-inch thick patties with wet hands.
Bake patties at 350 on both sides until golden brown.
Serve on lettuce leaves with red onion rings. Top each burger with 1 tsp. of Techina.
Makes 8 medium sized patties
Extra Note For a wonderful presentation put quinoa mixture into cupcake holders in a muffin tin. Bake an extra ten minutes on 400 degrees Fahrenheit or 180 degrees centigrade then serve one or two muffins per person.
Preparation: 30 minutes
Serves 4 to 6
Creamy Quinoa Pudding
- 1 cup Quinoa, rinsed
- 2 ½ cups Almond Milk
- 1/8 tsp Sea Salt
- 1 tsp organic sesame tahini
- 2 Tbs Maple Syrup
- 2 Tbs corn starch, dissolved in 3 T. cold water
- 1 Tbs pure vanilla extract
- ½ tsp nutmeg, freshly grated (optional)
- ½ tsp ground cinnamon
- 4 cinnamon sticks for garnish
In a heavy saucepan cook quinoa, Almond milk, and salt until it comes to a boil. Cover and simmer for 20 minutes. Add sesame butter and Maple syrup. Mix well. Add corn starch, stirring constantly until mixture thickens. Add vanilla and spices. Top with your choice of chopped nuts, roasted seeds or fruit and a dash of cinnamon. Decorate with a cinnamon stick. Delicious warm or chilled.
Preparation 5 minutes
Serves 4
Cooking Time 0:25 minutes


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Are you ready for the greatest two minutes in sports? You better be! The 136th Kentucky Derby is here. That's right -- that amazing cavalry charge down the racing strip known as Churchill Downs.
This is always the most difficult race of the year to handicap. After all, how do you sift through all the tidbits associated with 20 horses who are still in their infancy. Remember, some of these three year-olds have raced only five times in their career.
In past years, I have handicapped this race from every conceivable angle. I have given you the exacta, trifecta and everything else. I admit, I have not always won. Heck, no one wins all the time. But this year, I have decided just to give you the "cold winner.
And the answer is: Lookin At Lucky.
Now, I know what you are thinking. Beck is picking the favorite. How lame is that. But have you looked a how often the favorite wins this race ? Thank you.
And I must admit, I liked this horse before the real favorite, Eskendereya, bowed out with an injury.
Lookin At Lucky is trained by Bob Baffert, who has won this race three times before.
The white-haired professor has in the past guided Silver Charm, Real Quiet and War Emblem to the winner's circle in the Run for the Roses! And the bay colt has never been off the board. He's eight for eight lifetime -- and that includes six victories. And he has excuses for his only two losses.
Plus, he's the only multiple Grade 1 stakes winner in the field -- and he has the capable Garrett Gomez in the irons. Let's face it, he's a battle tested horse who is ready for a mile and a quarter.
Yes, no horse has won from the rail since Ferdinand in 1986, but that doesn't bother me. It's better to be on the inside, than all the way in the middle of the second starting gate.
If you like the girls, I am sure you are rooting for the filly Devil May Care. If you are a fan of trainer Todd Pletcher, you can cheer for four different horses and hope that the nine-time Eclipse Award winning trainer, finally wins the Kentucky Derby.
If you revere Joe Torre then I'm sure you will rally behind Homeboykris, who is a 50-1 long shot. Torre, the former Yankees skipper, is the co-owner of that colt.
And if you think a 20-year-old kid can pilot a horse to victory -- go with jockey Joe Talamo and the talented chestnut colt, Sidney's Candy.
But the pick here is Lookin At Lucky. And if you get "lucky," you might get odds of 4 to 1. He's simply the best horse in the field! So sip a mint julep, put on a derby hat -- and root for the number one horse.
And don't worry if it rains -- his grandsire, Mr. Prospector was a monster in the mud.
Got to run, I'm heading to the windows!
Handicapping the Derby: Are You Feeling "Lucky?"
With Eskendereya out, there's only one choice
Are you ready for the greatest two minutes in sports? You better be! The 136th Kentucky Derby is here. That's right -- that amazing cavalry charge down the racing strip known as Churchill Downs.


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LOS ANGELES, Aug. 18 (UPI) -- Aziz Ansari is reportedly getting serious with girlfriend Courtney McBroom.
The 31-year-old Parks and Recreation star and McBroom have been dating since earlier this year. McBroom is a trained pastry chef who has worked at Momofuku Noodle Bar and Momofuku Milk Bar in New York.
"Courtney has pretty much moved into Aziz's place in LA," a source tells Us Weekly. "They've been visiting the city the past few days. He's writing a book about relationships as well lately, and is on deadline for that."
Aziz Ansari Dating Courtney McBroom: Exclusive Details https://t.co/wgQGJJbsqH pic.twitter.com/jUCcr0VSnL— ※†ㅏㅓⓔ ♡ ! π ⓔ™ ※ (@MYKE_ROSS) August 17, 2014
The couple keep their relationship out of the spotlight, but the actor did provide some details to Billboard in May.
"I met her years ago when she was seeing someone," Ansari revealed. "I thought she was really cool. Then I ran into her randomly, and she was single. I've kind of settled down. I was the guy eating Skittles and having lots of fun, and then I was like, 'I need a nice nutritious salad' -- she's the salad."
Ansari will reprise his role as Tom Haverford on season seven of Parks and Recreation. The NBC comedy returns for its final season in spring 2015.


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Cardinal Keith O'Brien, the Archbishop of St Andrews and Edinburgh, has made controversial claims that women should be shown scans of their unborn child before proceeding with an abortion.
The leader of Scotland's Catholic Church said he thinks the NHS should introduce the measure which is currently used in America in order to reduce terminations in Scotland. In the US currently, there are seven states which require women to receive ultra sound scans and descriptions of the foetus before proceeding with an abortion.
Anti-abortion campaigners are in agreement with Cardinal O'Brien saying that if these procedures are in place they may help persuade women not to go through with the abortion after seeing their unborn baby and how developed they are. Approximately 200,000 abortions are carried out in Scotland every year.
These comments come on the 15th anniversary of the Glasgow-based Cardinal Winning pro-life initiative which offers help for women facing crisis pregnancies. Sister Roseann Rweddy who runs the initiative said that around 120 babies are alive today because their mothers availed of their service.
A Marie Stope International Spokesperson said that the comments made by Cardinal O'Brien are deeply worrying.
"We believe the Catholic Church in Scotland's desire for women to be forced to have - and look at – a scan of the foetus before being granted an abortion is deeply worrying. This is something we've increasingly seen in the US over the previous year, and in several states this has in fact passed into law.
We do not want to see a situation like this in UK, where a woman's right to choose and access this procedure is gradually eroded. Women invariably know whether it is the right time in their life to have a child, and the decision to choose to terminate a pregnancy must be theirs to make without any further barriers being introduced."
What do you think?


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Sunday, May 21st 2017
May: Loch Lomond Shores Market Day
10:00 - 16:00
Loch Lomond Shores welcomes the newly combined open air Farmers' and Craft's Market on the first and third Sunday of each month.
The farmers' market with fresh and local produce piled high on every stall you really must come along. There's free parking, lots to do for kids and families and plenty of local shopping – all in all, a great day out. The market producers vary depending on the seasonal availability of products, but at most markets you will find fresh cheese, home-baked breads and cakes, seasonal vegetables, chutneys and oils, freshly baked pies, fresh meats and fabulous smoked fish.
The artisan craft market will provide beautiful handmade gifts, including art prints, jewellery, cards and more. Foodies can also snap up lots of fresh produce, handmade food items including cakes and fudge, plus lots more from local food and drink producers. This will be the last market weekend at Loch Lomond Shores before the new year, so visitors are encouraged to stock up on their favourite items. All the produce comes from local Scottish businesses.
The market kicks off at 10am and runs till around 4pm, but it's best to arrive early as stock is limited to keep everything as fresh as possible.
For information on the market visit the Loch Lomond Shores Farmers' Market facebook page or Loch Lomond Shores Artisan Craft Market facebook page.BACK


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Portland declares yellow zone parking ban
Parking ban begins at 10 p.m. Monday
The city of Portland has issued a yellow zone parking ban beginning at 10 p.m. Monday. The parking ban will run until 6 a.m. Tuesday.
Sunday night, city crews were able to clear more than half of the downtown area and are hoping to clear and remove snow from the remaining streets during Monday night's ban. The Yellow Zone is an area within the downtown business district that is geographically bounded by Commercial Street on the south, Cumberland Avenue on the north, Franklin Street Arterial on the east and State Street on the west.
The parking ban is in effect within the square area of these four streets. As winter operations are still under way, the city asks residents and visitors to park off-street.
Vehicles left on the street during a ban will be towed at the owner's expense
There are a number of places people can park at no charge during a parking ban, including:
-Deering Oaks - on the left hand side of Tennis Court Road only
-State Street gravel lot - abutting Deering Oaks
-Fitzpatrick Stadium parking lot
-Hadlock Field parking lot
-All Portland Public School lots except the lots at Reiche School which abuts the school and playground
- Parking lot at Nathan Clifford School
-Parking lot on Munjoy Street, formerly known as the Adams School lot
-Cutter Street lot (off the Eastern Prom)
-Beach Street lot accessible from Commercial Street (This lot is available after 8 p.m.)
-Lot across the street from the International Marine Terminal on Commercial Street (corner of Park and Commercial Streets)
-On Peaks Island, parking is available at the Welch Street parking lot, the St. Christopher's Church parking lot, the Brackett Memorial Church parking lot, and the CMP back lot at Island Avenue and City Point Road
Vehicles can begin to park in these designated areas at 5 p.m. Vehicles must be moved from the city lots by 7 a.m.


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One of the benefits of teaching on a collegiate level is that "fun" can frequently go out the window (as one of my professors routinely told a class full of students regarding the silent films the class watched, "I don't care if you like the film, that's not the point."); still, most of us don't want to bore the pants off students and try reasonably hard to make things interesting. It seems "fun" of the educational variety is even more important at the elementary and secondary levels, where I vaguely recall masses of activities designed to get us "engaged" and "interested."
In light of "serious games" and "edutainment" discussions, I'm always interested to see what regular retail games are listed as good "educational" titles. College@Home has a pretty thorough list covering a variety of topics; some of the titles are expected (Civilization, Carmen Sandiego) to some slightly more interesting titles (Spore, Age of Mythology, a lot of regular retail sims). Makes me glad I just get to take the fun out of learning for undergraduates — I'm not sure I'd want to herd kids through Roller Coaster Tycoon.
Virtual Learning: 25 Best Sims and Games For the Classroom
[College@Home via Water Cooler Games]


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Historical:Java software
- There is an Open Source java applet in the original Panorama Tools distribution. It works in most java enabled browser, including those with the real JVM from Sun and those with the bad Microsoft implementation.
- PTViewer 2.7L2 is a version of the original PTViewer applet with many improvements. Some of them are: sharper image, faster panning, progressive image loading, integrated toolbar.
Immervision has a free java applet with directionnal sound, animation and other advanced features which work with all JVM since version 1.1 (including IE and netscape ones). At this time this viewer is in Beta test version. (Applet Page)Does not exist any more.
- Ipix Expensive! Many users in the community feel that Ipix has done a lot of harm to the panorama community in the past years. Read more on this FFII page
- Panorado Applet - a free java viewer applet - "flat" view, but very fast, thus suited for larger images.
Stitching Software
PTEditor helps to extract rectilinear images from spherical panoramas and insert them again after they have been editied.


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The David Sheldrick Wildlife Trust is a bright light of hope in an often dangerous and murky political environment that is Africa. Through the years and no matter what has been happening with the government or other agencies, Sheldrick Trust has been there to offer life to wildlife that would have surely died without it.
The vision of the Sheldricks is strong as is their tireless pursuit of solutions to the problems of elephants and rhinos created mostly by man. Daphne Sheldrick and her daughter Angela are 100% committed to saving and protecting African wildlife, especially elephants and rhinos. Their whole life is built around doing this important work.Fortunately there are many elephant lovers around the world who are helping to support their mission.
But even with an abundance of monetary aid, the project could not be successful without the hands on administration and the unwavering dedication of the amazing, tireless, compassionate and saintly men who are the keepers for the orphans and who have made their care their life's vocation. (At this time there are no women elephant keepers, and the longest one has lasted through training is six months.) It is an austere life but for the love and closeness one develops with the young pacyderms.
These elephant guardians, who in fact become the foster mothers for the babies, live full time at the orphanage and only spend 4 days a month free where many of them must travel long distances to visit their human families.
Edwin Lusichi says he sleeps well at night, knowing that he is doing something important and useful with his life. He also reaps the rewards for it daily by being able to watch and learn and share a remarkable bond with an animal as challenging, as interesting, as special and as intelligent as an elephant.
He grew up in the western part of the country and one of his most important early experiences was that of scout leader, where he learned and taught others to respect and do no harm to any animal, even an insect. His early aspirations took led him to the priesthood and he spent more than a year in the seminary. This career was unexpectedly interrupted and he carried on by studying computers. Only problem was, there were no jobs. He had come to Nairobi to be with his dad. Out of work, he visited the Sheldrick orphanage often and loved observing and being near the baby elephants. One day by chance there was a big rescue operation and they needed someone to stay at the orphanage and help mind the store. This led to a full time position.
Now, 12 years later, he still enjoys sleeping with the baby elephants and caring for them, but besides that, he coordinates and reports daily to the Sheldrick family about each animal and new foster parent, plays tour guide at the daily tourist visits, recruits new foster parents, trains new caretakers, administers veterinary operations, and hosts the plethora of stars and political dignitaries who stop by and want to be involved. (Recent visitors include Natalie Portman and Kristin Davis.)
While visiting our two baby orphans in Nairobi, Turkwel (about 2 yrs. old), and Kainuk (about 1 year old), it was very sweet to see how bonded they were to each other. I was told that Kainuk will scream and cry if she is separated from Turkwel. Shy Turkwel meanwhile proudly ushers Kainuk around and comforts her. They are in stables at night where they can see and touch each other. They were both rescued, a year apart, from the Lake Turkana area. Perhaps they are part of the same extended family.
Keeper Samuel Lotukoi who has worked for Sheldricks for 9 years, is also from the Lake Turkana area. He was taking care of Turkwel on our last visit to the orphanage and the three of them were like a happy trio. Samuel, like all the caretakers I had the privilege to meet, is very friendly and a true elephant lover. Like the others he only gets off 4 days a month to travel and see his family. He was lucky that for several years he worked at Tsavo where his family has moved to. But he is not complaining about being in Nairobi either.
Mishak Nzimbi, at the Sheldricks for 23 years, is the longest serving elephant keeper at the orphanage. He has a reputation for understanding them all and for being, without exception, every elephant's favorite keeper who has had wonderful responses from even the trickiest elephants. He also does innumerable radio shows in his native Wkumba tribe to foster respect and caring for wildlife.
I wish caretakers of elephants at zoos and circuses around the world, could come and see the level of caring and love that is present here. They would see what it takes to truly let elephants thrive. Perhaps by comparison they could see what paltry lives they are giving to their poor caged and mistreated charges. Wouldn't it be wonderful if there were a massive raising of consciosness regarding the respect due to animals. If it were to begin anywhere, it would have to be at the elephant and rhino orphanage
Benjamin Kyalo, a 12 year veteran of the Trust, is the chief caretaker at the Ithumba camp in Tsavo East National Park. He oversees the nurturing of the older orphans and keeps track of them, watches over their transitions back to the wild. Ex- orphans often come back to visit and if they are sick they come back to the camp, using it as a kind of elephant clinic. There they are given supplements and any needed medication or veterinary care.
Benjamin was not happy when he worked in the auto industry. One day he went to visit the orphanage and found his true calling. He just couldn't believe that some people could spend their time working with elephants. He applied and got trained to be a caretaker but in his group more people passed than they actually needed. The administration said they would let the elephants decide who would be their caretakers. "An elephant can tell when you have love in your heart." The elephants chose him. Now he is project manager at Ithumba. Despite all Benjamin's responsibilities, when you visit Ithumba, he stays with you and gently and patiently answers all your questions.
It is telling of their dedication to find out that all the caretakers know all the elephant faces and names by sight. That includes the babies as well as those who have gone into the wild for a number of years.
Please see Part 1 of this article:
Sheldrick Wildlife Trust - How To Save An Elephant (Part 1 of 3)
and Part 3 - Sheldrick Wildlife Trust - I Have Two Families - My Family at Home and My Elephant Family (Part 3 of 3)
Photos by Georja Umano
Georja Umano is an actress/comedienne and animal advocate.
To learn more about the work of the Sheldrick Wildlife Trust and its projects, or to foster an orphan elephant, visit:
From the USA to make a tax deductible contribution, send check to:
201 North Illinois Street
16th Floor - South Tower
Indianapolis, IN 46204


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Hilton Head Island is home to some of the game's most beautiful golf holes. Every year the PGA TOUR comes to "Golf Island" to tee it up in the RBC Heritage and every year, they leave with a greater appreciation for the legendary Lowcountry locale. While every hole on and around Hilton Head Island offers its own unique challenge, here is a collection of the most picturesque par threes, fours, and fives bound to take your breath away:No. 17, Harbour Town Golf LinksWhile the 18th hole at the iconic Harbour Town Golf Links is often the most talked about, the par three 17th silently steals the show. Measuring 185 yards from the back tees (174 yards during the RBC Heritage), it plays out to the Calibouge Sound and provides a spectacular vista, especially in the setting sunlight. The undulated green is guarded by a lagoon, large bunker, and Pete Dye's signature railroad ties.No. 6, Oyster Reef Golf ClubRees Jones' signature hole at Oyster Reef Golf Club, the par three 6th is one of the most exciting on the island; it's a huge reason why Oyster Reef was named among the Top 20 in the Carolinas and the "Golf Club of the Year" by the Lowcountry Golf Course Owners Association. Tipping out at 192 yards from the back tees, its green is set against the Port Royal Sound and gives many golfers pause to enjoy the natural beauty of its routing.Click here to view all the beautiful holes of HHI. For Golf Packages - Check OutThe Sea Pines Resort Villa Golf Package4 DAYS + 3 ROUNDS OF CHAMPIONSHIP GOLFTake advantage of The Sea Pines Resort's Villa Golf Package and enjoy world-class golf. Experience our private villas which provide the perfect living space for golfers after a great day out on the course. Enjoy having your own fully-equipped kitchen, dining room and relaxing living room. INCLUDED IN YOUR PACKAGE* One round of golf at Harbour Town Golf Links* One round of golf at Heron Point by Pete Dye* One round of golf at Atlantic Dunes by Davis Love III* Complimentary green fees for afternoon replays on Heron Point and Atlantic Dunes by Davis Love III (Based on availability. Cart fee applies.)* View More Info >>
View All Top HHI Holes
2017 Stay and Play Information:Stay & Play Packages available starting as low as $128 per person. All Stay & Play Packages include hotel stay, all golf fees including complimentary range balls, full breakfast buffet and taxes included.To book your package or receive a quote, contact Kevin Grantz at 812-246-8880, ext. 4 or email@example.com Events:Sunday, March 12th: Selection Sunday Shamble12:00pm Shotgun Start at Champions Pointe.Join us for a great day of golf, then hang out and watch to see where your team will be placed in the NCAA Tournament. Sign up your team of two before we fill up! This event is open to public and members.Call the Champions Pointe Pro Shop for more information or to sign up: (812) 294-1800, ext. 4. Champions Pointe Friday Night 2-Person LeagueNew League coming to Champions Pointe in 2017*League is open to Members & Public*This league is a 2-person team format, played on Friday evenings, May-September. Each team will play a match against another 2-person team every week. To sign up or for more information, call Chuck Paskovics at (502) 552-36662017 Covered Bridge Ladies LeagueSpring is just around the corner and it is time to sign up for Ladies League! Ladies League play begins on Wednesday, April 5th and plays every Wednesday at 5:30pm. This league is open to Public and Members. Sign up by March 31st to receive the Early Bird Special Rate of $35. After March 31st: $75 For more information and to sign up, contact: Anetha McCarty at 502-533-8234 Or Darlene Austin at 502-417-5246
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The 117th U.S. Open Championship will be played in the state of Wisconsin for the first time this June at Erin Hills. Don't miss your opportunity to see the world's greatest players compete on the game's grandest stage in golf's ultimate test as they vie for the coveted U.S. Open trophy. Limited tickets are on sale now and can be purchased online.Looking to come to Milwaukee for the US Open check out our travel and golf guide at golftrips.com.
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With excellent accommodations, magnificent golf courses, and true southern hospitality all nestled in the heart of the beautiful South Carolina Lowcountry, it's no wonder that Santee has become a golfer's preferred vacation destination.With over a decade of experience preparing golf package vacations in the area, Golf Santee's warm and inviting staff welcomes the opportunity to make your golf vacation plans a reality.Offering over a dozen championship golf courses and several different type accommodation properties your golf vacation plans can be customized to fit your specific needs.
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UPCOMING EVENTS1st Friday of the Month: LIVE EntertainmentThrough March: Happy Hour (Daily, 2-4pm)Through 3/17: Lucky Lunch Box Giveaway2/17-2/28: Mardi Gras Menu4/16: Easter Brunch2/27: Golf Fever Ladies Night3/14: Vino Van Gogh Painting Event Featuring Drink Specials
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One of the Top 10 Ranked Golf Courses in WisconsinThe Arnold Palmer Signature Course at Geneva National is a big favorite. The course winds through the rolling woods of Geneva National, with three spectacular finishing holes along Lake Como. Arnold Palmer himself calls the Signature 17th hole one of his favorites, placing it as #11 in his personal Dream 18 in the eastern part of the states. The Palmer Course has also been named Top 10 Wisconsin Golf Courses by both GOLFWEEK and Golf Digest. A tough course featuring elevated tees and challenging greens, The King gives you golf at its best.Please call the resort at (262) 245-7000 or make a tee time online.
The Lodge at Ventana Canyon sits against the backdrop of the Santa Catalina Mountains. Nestled within this spectacular setting is a semi-private country club and Lodge with just 50 suites and 36 holes of golf . Winding through the canyons and arroyos of this 600-acre desert preserve are Tom Fazio's two 18 hole championship courses (Mountain and Canyon), which have won numerous awards as the best in the country. The Lodge has been a prestigious AAA Four Diamond property for 20 consecutive years, and our many awards reflect our commitment to the highest standards in service excellence. The Lodge reflects the spirit of warmth and comfort cherished by seasoned travelers. For groups over four, future dates, or custom packages, please call reservations at 520-577-4092.
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MidwestGolf.com ||
NCGolf.com ||
NewHampshireGolf.com ||
NJGolf.com ||
NorthDakotaGolf.com ||
NYGolf.com ||
OhioGolf.com ||
PEIGolf.com ||
PennsylvaniaGolfer.com ||
RhodeIslandGolf.com ||
SCGolf.com ||
ScottlandGolfer.com ||
SouthDakotaGolf.com ||
TexasGolf.com ||
UtahGolf.com ||
WalesGolf.com ||
WashingtonGolf.com ||