Cis-regulatory Motif Analysis Research Articles

Arabidopsis HSFA9 Acts as a Regulator of Heat Response Gene Expression and the Acquisition of Thermotolerance and Seed Longevity.

Heat-shock transcription factors (HSFs) are crucial for regulating plant responses to heat and various stresses, as well as for maintaining normal cellular functions and plant development. HSFA9 and HSFA2 are two of the Arabidopsis class A HSFs and their expressions are dramatically induced in response to heat shock (HS) stress among all 21 Arabidopsis HSFs. However, the detailed biological roles of their cooperation have not been fully characterized. In this study, we employed an integrated approach that combined bioinformatics, molecular genetics and computational analysis to identify and validate the molecular mechanism that controls seed longevity and thermotolerance in Arabidopsis. The acquisition of tolerance to deterioration was accompanied by a significant transcriptional switch that involved the induction of primary metabolism, reactive oxygen species and unfolded protein response, as well as the regulation of genes involved in response to dehydration, heat and hypoxia. In addition, the cis-regulatory motif analysis in normal stored and controlled deterioration treatment (CDT) seeds confirmed the CDT-repressed genes with heat-shock element (HSE) in their promoters. Using a yeast two-hybrid and molecular dynamic interaction assay, it is shown that HSFA9 acted as a potential regulator that can interact with HSFA2. Moreover, the knock-out mutants of both HSFA9 and HSFA2 displayed a significant reduction in seed longevity. These novel findings link HSF transcription factors with seed deterioration tolerance and longevity.

Identification of Cis-Regulatory Sequences Controlling Pollen-Specific Expression of Hydroxyproline-Rich Glycoprotein Genes in Arabidopsis thaliana.

Hydroxyproline-rich glycoproteins (HRGPs) are a superfamily of plant cell wall structural proteins that function in various aspects of plant growth and development, including pollen tube growth. We have previously characterized protein sequence signatures for three family members in the HRGP superfamily: the hyperglycosylated arabinogalactan-proteins (AGPs), the moderately glycosylated extensins (EXTs), and the lightly glycosylated proline-rich proteins (PRPs). However, the mechanism of pollen-specific HRGP gene expression remains unexplored. To this end, we developed an integrative analysis pipeline combining RNA-seq gene expression and promoter sequences to identify cis-regulatory motifs responsible for pollen-specific expression of HRGP genes in Arabidopsis thaliana. Specifically, we mined the public RNA-seq datasets and identified 13 pollen-specific HRGP genes. Ensemble motif discovery identified 15 conserved promoter elements between A. thaliana and A. lyrata. Motif scanning revealed two pollen related transcription factors: GATA12 and brassinosteroid (BR) signaling pathway regulator BZR1. Finally, we performed a regression analysis and demonstrated that the 15 motifs provided a good model of HRGP gene expression in pollen (R = 0.61). In conclusion, we performed the first integrative analysis of cis-regulatory motifs in pollen-specific HRGP genes, revealing important insights into transcriptional regulation in pollen tissue.

Opposite physiological effects upon jasmonic acid and brassinosteroid treatment on laticifer proliferation and co-occurrence of differential expression of genes involved in vascular development in rubber tree.

During growth of woody plant-trunk, the secondary meristem functions in giving rise the xylem and phloem. Rubber tree (Hevea brasiliensis Muell. Arg.), in addition, contains laticifers (latex producing vessels) in the vicinity of phloem. Insights into regulatory mechanisms of gene networks underlying laticifer proliferation in rubber tree has remained very limited. The candidate vascular development-related genes were selected to investigate for expression profile in phloem and xylem tissues of high latex yield- and high wood yield-clones of rubber tree. The differential gene expression between the mature branch-xylem and -phloem tissues was clearly observed. The cis-regulatory motif analysis revealed the existent of putative jasmonic acid (JA)- and brassinosteroid (BR)-responsive regulatory motifs in promoter regions of these genes, and consequently the effect of exogenous application of JA, BR or their respective signaling inhibitors, on the formation of laticifers in rubber tree was demonstrated. Interestingly, the laticifer numbers were significantly increased in JA-treatment, correlated with up-regulation of phloem development-related genes in both rubber tree clones. On the contrary, the laticifers were decreased in BR-treatment accompanying by up-regulation of xylem development-related genes, especially in high wood yield-rubber tree clone. BR-inhibitor treatment also enhanced laticifer numbers, while JA-inhibitor suppressed laticifer differentiation. Taken together, this study unveils the molecular interplay between JA/BR on vascular development in rubber tree and how this impacts the appearance of laticifers in this plant. This process isvital for a better understanding on laticifer differentiation and its impact in the manipulation of wood and latex yield in rubber tree improvement program.

Overexpression of both AcSVP1 and AcSVP4 delays budbreak in kiwifruit A. chinensis var. deliciosa, but only AcSVP1 delays flowering in model plants

Kiwifruit SHORT VEGETATIVE PHASE (AcSVP) genes have been implicated in regulation of dormancy and flowering. Previously, we have shown that AcSVP2 and AcSVP3 have different effects on dormancy and flowering in transgenic kiwifruit and model plants. The role of two homologous genes, AcSVP1 and AcSVP4, remained unclear. Here, AcSVP1 and AcSVP4 were functionally characterized by ectopic expression in Actinidia species which show different chilling requirements for budbreak, as well as the model plant tobacco. Overexpression of AcSVP1 and AcSVP4 delayed spring budbreak in a high-chill A. chinensis var. deliciosa, but did not affect budbreak and flowering in a low-chill A. eriantha. Overexpression of AcSVP1 delayed flowering and seed germination in tobacco, while overexpression of AcSVP4 had only minor effects. Analysis of cis-regulatory motifs in the promoter regions of AcSVP1 and AcSVP4 and promoter transactivation assays revealed that AcSVP1 and AcSVP4 may be controlled by similar transcription factors, suggesting a conserved regulatory mechanism and a synergistic role for AcSVP1 and AcSVP4 in preventing premature kiwifruit budbreak during winter.

RGBM: regularized gradient boosting machines for identification of the transcriptional regulators of discrete glioma subtypes.

We propose a generic framework for gene regulatory network (GRN) inference approached as a feature selection problem. GRNs obtained using Machine Learning techniques are often dense, whereas real GRNs are rather sparse. We use a Tikonov regularization inspired optimal L-curve criterion that utilizes the edge weight distribution for a given target gene to determine the optimal set of TFs associated with it. Our proposed framework allows to incorporate a mechanistic active biding network based on cis-regulatory motif analysis. We evaluate our regularization framework in conjunction with two non-linear ML techniques, namely gradient boosting machines (GBM) and random-forests (GENIE), resulting in a regularized feature selection based method specifically called RGBM and RGENIE respectively. RGBM has been used to identify the main transcription factors that are causally involved as master regulators of the gene expression signature activated in the FGFR3-TACC3-positive glioblastoma. Here, we illustrate that RGBM identifies the main regulators of the molecular subtypes of brain tumors. Our analysis reveals the identity and corresponding biological activities of the master regulators characterizing the difference between G-CIMP-high and G-CIMP-low subtypes and between PA-like and LGm6-GBM, thus providing a clue to the yet undetermined nature of the transcriptional events among these subtypes.

Corrigendum.

Corrigendum.

Genome-Wide Analysis of the Musa WRKY Gene Family: Evolution and Differential Expression during Development and Stress.

The WRKY gene family plays an important role in the development and stress responses in plants. As information is not available on the WRKY gene family in Musa species, genome-wide analysis has been carried out in this study using available genomic information from two species, Musa acuminata and Musa balbisiana. Analysis identified 147 and 132 members of the WRKY gene family in M. acuminata and M. balbisiana, respectively. Evolutionary analysis suggests that the WRKY gene family expanded much before the speciation in both the species. Most of the orthologs retained in two species were from the γ duplication event which occurred prior to α and β genome-wide duplication (GWD) events. Analysis also suggests that subtle changes in nucleotide sequences during the course of evolution have led to the development of new motifs which might be involved in neo-functionalization of different WRKY members in two species. Expression and cis-regulatory motif analysis suggest possible involvement of Group II and Group III WRKY members during various stresses and growth/development including fruit ripening process respectively.

Computational Analysis of “-omics” Data to Identify Transcription Factors Regulating Secondary Metabolism in Rauvolfia serpentina

Rauvolfia serpentina has been known to produce therapeutically important indole alkaloids used in treatment of various diseases. Despite its medicinal importance, complete understanding of its secondary metabolism is challenging due to complex interplay among various transcription factors (TFs) and genes. However, weighted co-expression analysis of transcriptome along with integration of metabolomics data has proficiency to elucidate topological properties of complex regulatory interactions in secondary metabolism. We aimed to implement an integrative strategy using “-omics” data to identify TFs of “unknown function” and exemplify their role in regulation of valuable metabolites as well as metabolic traits. A total of 69 TFs were identified through significant thresholds and removal of false positives based on cis-regulatory motif analysis. Network-biology inspired analysis of co-expression network lead to generation of four statistically significant and biologically robust modules. Similar to known regulatory roles of WRKY and AP2-EREBP TF families in Catharanthus roseus, this study presented them to regulate synthesis of alkaloids in R. serpentina as well. Moreover, TFs in module 4 were observed to be regulating connecting steps between primary and secondary metabolic pathways in the synthesis of terpenoid indole alkaloids. Integration of metabolomics data further highlight the significance of module 1 since it was statistically predicted to be involved in synthesis of specialized metabolites, and associated genes may physically clustered on genome. Importantly, putative TFs in module 1 may modulate the major indole alkaloids synthesis in response to various environmental stimuli. The methodology implemented herein may provide a better reference to identify and explore functions of transcriptional regulators.

DMINDA: an integrated web server for DNA motif identification and analyses.

DMINDA (DNA motif identification and analyses) is an integrated web server for DNA motif identification and analyses, which is accessible at http://csbl.bmb.uga.edu/DMINDA/. This web site is freely available to all users and there is no login requirement. This server provides a suite of cis-regulatory motif analysis functions on DNA sequences, which are important to elucidation of the mechanisms of transcriptional regulation: (i) de novo motif finding for a given set of promoter sequences along with statistical scores for the predicted motifs derived based on information extracted from a control set, (ii) scanning motif instances of a query motif in provided genomic sequences, (iii) motif comparison and clustering of identified motifs, and (iv) co-occurrence analyses of query motifs in given promoter sequences. The server is powered by a backend computer cluster with over 150 computing nodes, and is particularly useful for motif prediction and analyses in prokaryotic genomes. We believe that DMINDA, as a new and comprehensive web server for cis-regulatory motif finding and analyses, will benefit the genomic research community in general and prokaryotic genome researchers in particular.

Unraveling Regulation of the Small Heat Shock Proteins by the Heat Shock Factor HvHsfB2c in Barley: Its Implications in Drought Stress Response and Seed Development

The rapid increase in heat shock proteins upon exposure to damaging stresses and during plant development related to desiccation events reveal their dual importance in plant development and stress tolerance. Genome-wide sequence survey identified 20 non-redundant small heat shock proteins (sHsp) and 22 heat shock factor (Hsf) genes in barley. While all three major classes (A, B, C) of Hsfs are localized in nucleus, the 20 sHsp gene family members are localized in different cell organelles like cytoplasm, mitochondria, plastid and peroxisomes. Hsf and sHsp members are differentially regulated during drought and at different seed developmental stages suggesting the importance of chaperone role under drought as well as seed development. In silico cis-regulatory motif analysis of Hsf promoters showed an enrichment with abscisic acid responsive cis-elements (ABRE), implying regulatory role of ABA in mediating transcriptional response of HvsHsf genes. Gene regulatory network analysis identified HvHsfB2c as potential central regulator of the seed-specific expression of several HvsHsps including 17.5CI sHsp. These results indicate that HvHsfB2c is co-expressed in the central hub of small Hsps and therefore it may be regulating the expression of several HvsHsp subclasses HvHsp16.88-CI, HvHsp17.5-CI and HvHsp17.7-CI. The in vivo relevance of binding specificity of HvHsfB2C transcription factor to HSE-element present in the promoter of HvSHP17.5-CI under heat stress exposure is confirmed by gel shift and LUC-reporter assays. Further, we isolated 477 bp cDNA from barley encoding a 17.5 sHsp polypeptide, which was predominantly upregulated under drought stress treatments and also preferentially expressed in developing seeds. Recombinant HvsHsp17.5-CI protein was expressed in E. coli and purified to homogeneity, which displayed in vitro chaperone activity. The predicted structural model of HvsHsp-17.5-CI protein suggests that the α-crystallin domain is evolutionarily highly conserved.

An integrated toolkit for accurate prediction and analysis ofcis-regulatory motifs at a genome scale

We present an integrated toolkit, BoBro2.0, for prediction and analysis of cis-regulatory motifs. This toolkit can (i) reliably identify statistically significant cis-regulatory motifs at a genome scale; (ii) accurately scan for all motif instances of a query motif in specified genomic regions using a novel method for P-value estimation; (iii) provide highly reliable comparisons and clustering of identified motifs, which takes into consideration the weak signals from the flanking regions of the motifs; and (iv) analyze co-occurring motifs in the regulatory regions. We have carried out systematic comparisons between motif predictions using BoBro2.0 and the MEME package. The comparison results on Escherichia coli K12 genome and the human genome show that BoBro2.0 can identify the statistically significant motifs at a genome scale more efficiently, identify motif instances more accurately and get more reliable motif clusters than MEME. In addition, BoBro2.0 provides correlational analyses among the identified motifs to facilitate the inference of joint regulation relationships of transcription factors. The source code of the program is freely available for noncommercial uses at http://code.google.com/p/bobro/. xyn@bmb.uga.edu Supplementary data are available at Bioinformatics online.