Integrating bioinformatic resources to predict transcription factors interacting with cis-sequences conserved in co-regulated genes

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.[Background] Using motif detection programs it is fairly straightforward to identify conserved cis-sequences in promoters of co-regulated genes. In contrast, the identification of the transcription factors (TFs) interacting with these cis-sequences is much more elaborate. To facilitate this, we explore the possibility of using several bioinformatic and experimental approaches for TF identification. This starts with the selection of co-regulated gene sets and leads first to the prediction and then to the experimental validation of TFs interacting with cis-sequences conserved in the promoters of these co-regulated genes.[Results] Using the PathoPlant database, 32 up-regulated gene groups were identified with microarray data for drought-responsive gene expression from Arabidopsis thaliana. Application of the binding site estimation suite of tools (BEST) discovered 179 conserved sequence motifs within the corresponding promoters. Using the STAMP web-server, 49 sequence motifs were classified into 7 motif families for which similarities with known cis-regulatory sequences were identified. All motifs were subjected to a footprintDB analysis to predict interacting DNA binding domains from plant TF families. Predictions were confirmed by using a yeast-one-hybrid approach to select interacting TFs belonging to the predicted TF families. TF-DNA interactions were further experimentally validated in yeast and with a Physcomitrella patens transient expression system, leading to the discovery of several novel TF-DNA interactions.[Conclusions] The present work demonstrates the successful integration of several bioinformatic resources with experimental approaches to predict and validate TFs interacting with conserved sequence motifs in co-regulated genes.The work of Z.K., G.H., A.S., F.S., C.B., and L.B. was supported by the STREG project. W.X.’s work was supported by the China Scholarship Council (CSC). The authors would like to acknowledge the support of Jasmin Huebner and Nina Schmidt in the generation and analysis of transgenic A. thaliana.Peer Reviewe

Analysis of the DNA-binding activities of the Arabidopsis R2R3-MYB transcription factor family by one-hybrid experiments in yeast

22 pags.- 4 Figs.The control of growth and development of all living organisms is a complex and dynamic process that requires the harmonious expression of numerous genes. Gene expression is mainly controlled by the activity of sequence-specific DNA binding proteins called transcription factors (TFs). Amongst the various classes of eukaryotic TFs, the MYB superfamily is one of the largest and most diverse, and it has considerably expanded in the plant kingdom. R2R3-MYBs have been extensively studied over the last 15 years. However, DNA-binding specificity has been characterized for only a small subset of these proteins. Therefore, one of the remaining challenges is the exhaustive characterization of the DNA-binding specificity of all R2R3-MYB proteins. In this study, we have developed a library of Arabidopsis thaliana R2R3-MYB open reading frames, whose DNA-binding activities were assayed in vivo (yeast one-hybrid experiments) with a pool of selected cis-regulatory elements. Altogether 1904 interactions were assayed leading to the discovery of specific patterns of interactions between the various R2R3-MYB subgroups and their DNA target sequences and to the identification of key features that govern these interactions. The present work provides a comprehensive in vivo analysis of R2R3-MYB binding activities that should help in predicting new DNA motifs and identifying new putative target genes for each member of this very large family of TFs. In a broader perspective, the generated data will help to better understand how TF interact with their target DNA sequences.Part of this work was supported by the French National Research Agency (CERES, Grant ANR-BLAN-1238) and the PLANT-KBBE Initiative (STREG, ANR-08-KBBE-011-01). http:// www.agence-nationale-recherche.fr/. The work of Z.K., F.S. and A.S. was supported by the STREG project. W.X. was supported by a fellowship from the China Scholarship Council (CSC).Peer reviewe

The transcription factor BELLRINGER modulates phyllotaxis by regulating the expression of a pectin methylesterase in Arabidopsis

Plant leaves and flowers are positioned along the stem in a regular pattern. This pattern, which is referred to as phyllotaxis, is generated through the precise emergence of lateral organs and is controlled by gradients of the plant hormone auxin. This pattern is actively maintained during stem growth through controlled cell proliferation and elongation. The formation of new organs is known to depend on changes in cell wall chemistry, in particular the demethylesterification of homogalacturonans, one of the main pectic components. Here we report a dual function for the homeodomain transcription factor BELLRINGER (BLR) in the establishment and maintenance of the phyllotactic pattern in Arabidopsis. BLR is required for the establishment of normal phyllotaxis through the exclusion of pectin methylesterase PME5 expression from the meristem dome and for the maintenance of phyllotaxis through the activation of PME5 in the elongating stem. These results provide new insights into the role of pectin demethylesterification in organ initiation and cell elongation and identify an important component of the regulation mechanism involved

TRANSPARENT TESTA 16 and 15 act through different mechanisms to control proanthocyanidin accumulation in Arabidopsis testa

Flavonoids are secondary metabolites that fulfil a multitude of functions during the plant life cycle. In Arabidopsis proanthocyanidins (PAs) are flavonoids that specifically accumulate in the innermost integuments of the seed testa (i.e. endothelium), as well as in the chalaza and micropyle areas, and play a vital role in protecting the embryo against various biotic and abiotic stresses. PAs accumulation in the endothelium requires the activity of the MADS box transcription factor TRANSPARENT TESTA (TT) 16 (ARABIDOPSIS B-SISTER/AGAMOUS-LIKE 32) and the UDPglycosyltransferase TT15 (UGT80B1). Interestingly tt16 and tt15 mutants display a very similar flavonoid profiles and patterns of PA accumulation. By using a combination of genetic, molecular, biochemical, and histochemical methods, we showed that both TT16 and TT15 act upstream the PA biosynthetic pathway, but through two distinct genetic routes. We also demonstrated that the activity of TT16 in regulating cell fate determination and PA accumulation in the endothelium is required in the chalaza prior to the globular stage of embryo development. Finally this study provides new insight showing that TT16 and TT15 functions extend beyond PA biosynthesis in the inner integuments of the Arabidopsis seed coat

Binding specificities of selected R2R3-MYBs in relation with their biological roles.

<p>Heat map representation of the Y1H results observed with selected R2R3-MYBs involved in (<b>A</b>) biotic and abiotic stress responses, (<b>B</b>) cell fate determination and flavonoid biosynthesis (in TTG1-dependent complexes) and (<b>C</b>) cell wall biosynthesis (cellulose and xylan <i>vs</i> lignins). Yellow: yeast growth on selective media, blue: no yeast growth on selective media. Stars indicate the DNA sequences that form an <i>AC</i>-rich element in between two consecutive DNA motifs (*: group IIa, **: group IId). Double head arrows indicate the most discriminating DNA motifs between the R2R3-MYB groups.</p

Summary of Y1H results for all R2R3-MYB DNA binding domain (DBD) tested.

<p>On the left a maximum likelihood tree that defines a total of 35 DBD subgroups is displayed with collapsed branches. This tree was computed based on a multiple alignment of the R2R3 domains as defined [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141044#pone.0141044.ref003" target="_blank">3</a>]. On top a cladogram of the 16 <i>cis</i>-elements assayed is plotted. In pink and blue are highlighted group I and group II DNA motifs, respectively. Binding scores in the matrix take values from 0 to 100% when all members of a subgroup bind to a given DNA sequence with high affinity (++ in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141044#pone.0141044.s010" target="_blank">S4 Table</a>). Yellow square: low binding scores within DNA group I. Orange and red squares highlight the preferential binding of some DBDs toward the DNA motifs from groups IIa and IIc or groups IIa and IId, respectively.</p

R2R3-MYBs binding activity

<p><b>(A)</b> Heat map representation (using the EPCLUST Tool) of the Y1H results. DNA motifs are grouped accordingly to their selectivity against the different R2R3-MYB subgroups. Yellow: yeast growth on selective media (<i>i</i>.<i>e</i>. interaction between a given DNA motif and a R2R3-MYB), blue: no yeast growth on selective media (<i>i</i>.<i>e</i>. no <i>trans</i>-activation). In pink and blue are highlighted group I and group II DNA motifs, respectively. <b>(B)</b> Box plot representation of the number of DNA motifs recognised per R2R3-MYB. <b>(C)</b> Number of DNA motifs recognised per R2R3-MYB subgroup. Error bars: binding variation amongst the R2R3-MYBs within each subgroup. Numbers above each column indicate the number of R2R3-MYB of each subgroup. S: subgroup. U: ungrouped.</p

Sequence LOGOs analysis.

<p>(<b>A</b>) Sequence LOGOs of the R2 and R3 DNA-recognition α-helices of <i>Arabidopsis thaliana</i> R2R3-MYB DBD subgroups (left) and of the bound <i>cis</i>-elements assayed on the Y1H experiments, classified into two groups (right). The bottom LOGOs are the calculated <i>consensi</i> for both groups. Boxed columns within the recognition helices highlight residues that most likely contact DNA nitrogen bases based on alignments to MYB protein data bank structures. Red arrowheads indicate key amino acid residues involved in the interaction with DNA that are conserved in almost all plant and animal MYB proteins ([<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141044#pone.0141044.ref050" target="_blank">50</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141044#pone.0141044.ref054" target="_blank">54</a>]). Red squares highlight key amino acid residues that are associated with specific DBD subgroups and for which some experimental evidences (<i>in vitro</i> and/or <i>in vivo</i>) on their role in the interaction with DNA targets are available. <b>(B)</b><i>AC</i>-rich sequence LOGOs associated with the <i>trans</i>-activation activity of DBD subgroup 1, 2, 3, 13 and 24.</p

Hierarchical clustering analysis of the binding scores associated with the 35 DNA-binding domains toward the DNA motifs belonging to group II.

<p>Preferential binding of DBDs toward groups IIa and IIc or groups IIa and IId are highlighted by orange and red squares, respectively. Stars indicate the DNA sequences that form an <i>AC</i>-rich element in between two consecutive DNA motifs (*:group IIa, **: group IId). Arrowheads indicate R2R3-MYB DBDs that were found to be the most strongly associated with group IIb (<i>AC</i>-elements) DNA motifs in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141044#pone.0141044.g003" target="_blank">Fig 3</a>.</p