A step‐by‐step protocol for formaldehyde‐assisted isolation of regulatory elements from Arabidopsis thaliana

Abstract

The control of gene expression by transcriptional regulators and other types of functionally relevant DNA transactions such as chromatin remodeling and replication underlie a vast spectrum of biological processes in all organisms. DNA transactions require the controlled interaction of proteins with DNA sequence motifs which are often located in nucleosome-depleted regions (NDRs) of the chromatin. Formaldehyde-assisted isolation of regulatory elements (FAIRE) has been established as an easy-to-implement method for the isolation of NDRs from a number of eukaryotic organisms, and it has been successfully employed for the discovery of new regulatory segments in genomic DNA from, for example, yeast, Drosophila, and humans. Until today, however, FAIRE has only rarely been employed in plant research and currently no detailed FAIRE protocol for plants has been published. Here, we provide a step-by-step FAIRE protocol for NDR discovery in Arabidopsis thaliana. We demonstrate that NDRs isolated from plant chromatin are readily amenable to quantitative polymerase chain reaction and next-generation sequencing. Only minor modification of the FAIRE protocol will be needed to adapt it to other plants, thus facilitating the global inventory of regulatory regions across species.

Activation of YUCCA5 by the Transcription Factor TCP4 Integrates Developmental and Environmental Signals to Promote Hypocotyl Elongation in Arabidopsis

“…Recombinant TCP4D3-MBP protein, a fusion between the TCP4 DNA binding domain and maltose binding protein tag (Aggarwal et al, 2010), strongly and specifically retarded synthetic oligonucleotides corresponding to BS2, but not to other motifs in the electrophoretic mobility shift assay ( Figure 4D). Induction of TCP4 resulted in increased DNA accessibility at the YUC5 locus in the chromatin context compared with the mock control (Supplemental Figure 7), as estimated by the FAIRE (formaldehyde-assisted isolation of regulatory element) experiment (Simon et al, 2012;Omidbakhshfard et al, 2014). Taken together, these results strongly suggest that TCP4 brings about changes in the chromatin of the YUC5 locus, possibly by binding to the upstream regulatory sequences directly, and promotes its transcriptional activity within hours of its induction.…”

“…FAIRE assays were performed as described earlier (Simon et al, 2012;Omidbakhshfard et al, 2014) with some modifications. Ten-day-old Pro35S:mTCP4:GR seedlings were treated with mock or 12 mM DEX for 3 h, and 2 g of tissue was fixed with formaldehyde and regulatory elements were isolated as described (Omidbakhshfard et al, 2014). TA3 was used as an internal control and TUB2 was used as a negative control.…”

The TCP4 transcription factor regulates trichome cell differentiation by directly activating GLABROUS INFLORESCENCE STEMS in Arabidopsis thaliana

“…In short, 2 g of 14‐day‐old 35S :: mTCP4 : GR lines were treated with Mock and DEX (20 μ m ) solution for 4 h and cross‐linked with formaldehyde. The regulatory elements were isolated as mentioned (Omidbakhshfard et al ., ). TA3 was used as internal control.…”

“…FAIRE protocol was followed as mentioned in the (Omidbakhshfard et al ., ) with slight modifications. In short, 2 g of 14‐day‐old 35S :: mTCP4 : GR lines were treated with Mock and DEX (20 μ m ) solution for 4 h and cross‐linked with formaldehyde.…”

LEAFY is a pioneer transcription factor and licenses cell reprogramming to floral fate

“…0.8-1.2 gram of root explants was used for each biological replicate. FAIRE-qPCR was conducted as previously described 116 . Primer sequences for FAIRE-qPCR were designed to query published DNase I hypersensitivity sites near each candidate locus 117 and are listed in Table S1.…”