Local sequence features that influence AP-1 cis-regulatory activity

In the genome, most occurrences of transcription factor binding sites (TFBS) have no cis-regulatory activity, which suggests that flanking sequences contain information that distinguishes functional from nonfunctional TFBS. We interrogated the role of flanking sequences near Activator Protein 1 (AP-1) binding sites that reside in DNase I Hypersensitive Sites (DHS) and regions annotated as Enhancers. In these regions, we found that sequence features directly adjacent to the core motif distinguish high from low activity AP-1 sites. Some nearby features are motifs for other TFs that genetically interact with the AP-1 site. Other features are extensions of the AP-1 core motif, which cause the extended sites to match motifs of multiple AP-1 binding proteins. Computational models trained on these data distinguish between sequences with high and low activity AP-1 sites and also predict changes in cis-regulatory activity due to mutations in AP-1 core sites and their flanking sequences. Our results suggest that extended AP-1 binding sites, together with adjacent binding sites for additional TFs, encode part of the information that governs TFBS activity in the genome.</jats:p

PTRE-seq reveals mechanism and interactions of RNA binding proteins and miRNAs

A large number of RNA binding proteins (RBPs) and miRNAs bind to the 3′ untranslated regions of mRNA, but methods to dissect their function and interactions are lacking. Here the authors introduce post-transcriptional regulatory element sequencing (PTRE-seq) to dissect sequence preferences, interactions and consequences of RBP and miRNA binding

High-throughput functional testing of ENCODE segmentation predictions

The histone modification state of genomic regions is hypothesized to reflect the regulatory activity of the underlying genomic DNA. Based on this hypothesis, the ENCODE Project Consortium measured the status of multiple histone modifications across the genome in several cell types and used these data to segment the genome into regions with different predicted regulatory activities. We measured the cis-regulatory activity of more than 2000 of these predictions in the K562 leukemia cell line. We tested genomic segments predicted to be Enhancers, Weak Enhancers, or Repressed elements in K562 cells, along with other sequences predicted to be Enhancers specific to the H1 human embryonic stem cell line (H1-hESC). Both Enhancer and Weak Enhancer sequences in K562 cells were more active than negative controls, although surprisingly, Weak Enhancer segmentations drove expression higher than did Enhancer segmentations. Lower levels of the covalent histone modifications H3K36me3 and H3K27ac, thought to mark active enhancers and transcribed gene bodies, associate with higher expression and partly explain the higher activity of Weak Enhancers over Enhancer predictions. While DNase I hypersensitivity (HS) is a good predictor of active sequences in our assay, transcription factor (TF) binding models need to be included in order to accurately identify highly expressed sequences. Overall, our results show that a significant fraction (∼26%) of the ENCODE enhancer predictions have regulatory activity, suggesting that histone modification states can reflect the cis-regulatory activity of sequences in the genome, but that specific sequence preferences, such as TF-binding sites, are the causal determinants of cis-regulatory activity

Functional characterization of enhancer activity during a long terminal repeat\u27s evolution

Many transposable elements (TEs) contain transcription factor binding sites and are implicated as potential regulatory elements. However, TEs are rarely functionally tested for regulatory activity, which in turn limits our understanding of how TE regulatory activity has evolved. We systematically tested the human LTR18A subfamily for regulatory activity using massively parallel reporter assay (MPRA) and found AP-1- and CEBP-related binding motifs as drivers of enhancer activity. Functional analysis of evolutionarily reconstructed ancestral sequences revealed that LTR18A elements have generally lost regulatory activity over time through sequence changes, with the largest effects occurring owing to mutations in the AP-1 and CEBP motifs. We observed that the two motifs are conserved at higher rates than expected based on neutral evolution. Finally, we identified LTR18A elements as potential enhancers in the human genome, primarily in epithelial cells. Together, our results provide a model for the origin, evolution, and co-option of TE-derived regulatory elements

Isolation and characterization of indole acetic acid (IAA) producing <i style="">Klebsiella</i> <i style="">pneumoniae</i> strains from rhizosphere of wheat (<i>Triticum aestivum</i>) and their effect on plant growth

993-1000The present study was undertaken for isolation of Klebsiella strains from rhizosphere of wheat (T. aestivum), screening and characterization of these strains for in vitro indole acetic acid (IAA) production and studying the effect of these strains on plant growth under gnotobiotic conditions. Nine strains of Klebsiella were isolated from rhizosphere of wheat (var. Lokwan) and identified as K. pneumoniae by 16S rRNA gene sequencing. Six K. pneumoniae strains showed in vitro IAA production. Colorimetric analysis showed that K8 produced maximum IAA (27.5 mg l-1) in the presence of tryptophan (1 mg ml-1) at 72 h of incubation with optimum conditions as pH 8.0, 37°C and 0.5% (w/v) NaCl concentration. GC-MS analysis and IR studies confirmed presence of IAA in the cell filtrates of strain K8. Effect of six IAA producing Klebsiella strains on plant growth was studied by performing series of seed germination tests using moth bean seeds under axenic conditions and pot experiments using sterilized soil and wheat seeds (var. Lokwan). Strain K11 and K42 demonstrated increase in root length of inoculated moth beans (~ 92.71% over the control). Results of pot experiments indicated that almost all the six IAA producing Klebsiella strains significantly increased the root length and shoot height of inoculated wheat seedlings over the control. The results suggest that these are promising isolates from wheat rhizosphere and merits research on appliance of these strains in agriculture