%0 Book Section %B Methods Mol Biol %D 2017 %T DNase I SIM: A Simplified In-Nucleus Method for DNase I Hypersensitive Site Sequencing %A Filichkin, S. A. %A Megraw, M. %X

Identifying cis-regulatory elements is critical in understanding the direct and indirect interactions that occur within gene regulatory networks. Current approaches include DNase-seq, a technique that combines sensitivity to the nonspecific endonuclease DNase I with high-throughput sequencing to identify regions of regulatory DNA on a genome-wide scale. Yet, challenges still remain in processing recalcitrant tissues that have low DNA content. Here, we describe DNase I SIM (for Simplified In-nucleus Method), a protocol that simplifies and facilitates generation of DNase-seq libraries from plant tissues for high-resolution mapping of DNase I hypersensitive sites. By removing steps requiring the use of gel agarose plugs in DNase-seq, DNase I SIM reduces the time required to perform the protocol by at least 2 days, while also making possible the processing of difficult plant tissues including plant roots.

%B Methods Mol Biol %V 1629 %P 141-154 %@ 1940-6029 (Electronic)1064-3745 (Linking) %G eng %U https://link.springer.com/protocol/10.1007/978-1-4939-7125-1_10 %! Methods in molecular biology (Clifton, N.J.) %0 Journal Article %J Dev Cell %D 2016 %T Establishment of Expression in the SHORTROOT-SCARECROW Transcriptional Cascade through Opposing Activities of Both Activators and Repressors %A Sparks, E. E. %A Drapek, C. %A Gaudinier, A. %A Li, S. %A Ansariola, M. %A Shen, N. %A Hennacy, J. H. %A Zhang, J. %A Turco, G. %A Petricka, J. J. %A Foret, J. %A Hartemink, A. J. %A Gordan, R. %A Megraw, M. %A Brady, S. M. %A Benfey, P. N. %K Arabidopsis Proteins/ genetics/ metabolism %K Arabidopsis/ genetics/growth & development/ metabolism %K Computer Simulation %K Gene Expression Regulation, Plant %K Gene Regulatory Networks %K Genes, Plant %K Genes, Reporter %K Genes, Synthetic %K Models, Genetic %K Plant Roots/cytology/metabolism %K Plants, Genetically Modified %K Promoter Regions, Genetic %K Repressor Proteins/genetics/metabolism %K Trans-Activators/genetics/metabolism %K Transcription Factors/ genetics/ metabolism %K Two-Hybrid System Techniques %X

Tissue-specific gene expression is often thought to arise from spatially restricted transcriptional cascades. However, it is unclear how expression is established at the top of these cascades in the absence of pre-existing specificity. We generated a transcriptional network to explore how transcription factor expression is established in the Arabidopsis thaliana root ground tissue. Regulators of the SHORTROOT-SCARECROW transcriptional cascade were validated in planta. At the top of this cascade, we identified both activators and repressors of SHORTROOT. The aggregate spatial expression of these regulators is not sufficient to predict transcriptional specificity. Instead, modeling, transcriptional reporters, and synthetic promoters support a mechanism whereby expression at the top of the SHORTROOT-SCARECROW cascade is established through opposing activities of activators and repressors.

%B Dev Cell %V 39 %P 585-596 %8 12/2016 %@ 1878-1551 (Electronic)1534-5807 (Linking) %G eng %U https://doi.org/10.1016/j.devcel.2016.09.031 %! Developmental cell