Nam J and Davidson EH (2012), Barcoded DNA-tag reporters for multiplex cis-re...

ECB-ART-42421

PLoS One 2012 Jan 01;74:e35934. doi: 10.1371/journal.pone.0035934.

Show Gene links Show Anatomy links

Barcoded DNA-tag reporters for multiplex cis-regulatory analysis.

Nam J , Davidson EH .

???displayArticle.abstract???
Cis-regulatory DNA sequences causally mediate patterns of gene expression, but efficient experimental analysis of these control systems has remained challenging. Here we develop a new version of "barcoded" DNA-tag reporters, "Nanotags" that permit simultaneous quantitative analysis of up to 130 distinct cis-regulatory modules (CRMs). The activities of these reporters are measured in single experiments by the NanoString RNA counting method and other quantitative procedures. We demonstrate the efficiency of the Nanotag method by simultaneously measuring hourly temporal activities of 126 CRMs from 46 genes in the developing sea urchin embryo, otherwise a virtually impossible task. Nanotags are also used in gene perturbation experiments to reveal cis-regulatory responses of many CRMs at once. Nanotag methodology can be applied to many research areas, ranging from gene regulatory networks to functional and evolutionary genomics.

???displayArticle.pubmedLink??? 22563420
???displayArticle.pmcLink??? PMC3339872
???displayArticle.link??? PLoS One
???displayArticle.grants??? [+]

Species referenced: Echinodermata
Genes referenced: gata6 LOC100887844 LOC100888549 LOC115919910 LOC583082 nodall

???attribute.lit??? ???displayArticles.show???

	Figure 1. Architecture of Nanotags vectors and overview of workflow for the Nanotag-assisted cis-regulatory analysis.(A) Architecture of Nanotag vectors. Each Nanotag vector is composed of a basal promoter (BP) derived from gatae [7], a GFP open reading frame, a unique âˆ¼100 bp-long Nanotag sequence, and a core polyadenylation signal [8]. CRM::Nanotag constructs are built, pooled, and microinjected as described in Nam et al [1]. A pair of universal primers (orange coded) is designed for signal amplification. The forward universal primer contains T3 promoter sequence in its 5â€²-end for in vitro transcription of the sense strand of the entire pool of amplified Nanotags. Figure 1A is modified from Nam et al [1]. (B) Overview of the Nanotag-assisted cis-regulatory analysis. Two parallel protocols respectively for measuring the numbers of individual tags expressed (left panel) and those of incorporated (right panel) are shown. While QPCR is used to measure the total number of tags expressed per embryo from first strand cDNA or the total number of tags incorporated per cell from genomic DNA, the NanoString was used to measure the relative numbers of individual tags from amplified cDNA or amplified genomic DNA with much improved sensitivity. The numbers of individual tags incorporated per embryo are estimated by comparing results from QPCR and NanoString analyses. The level of tags expressed is corrected for DNA copy number, tag specific variations, and background BP activity specific to each sample.
	Figure 2. Variations among 130 Nanotags driven by the same CRM at four time points.Right panels show expression levels of the first half of Nanotag vectors driven by the same known active CRM, the nodal INT [14] and the other half driven by the same known inactive DNA fragment, the nodal 3P [14]. The Nanotag vector and driver pairs were swapped in left panels. Expressions of Nanotags were normalized to the numbers of DNA copies incorporated into the sea urchin genome. Note that four Nanotags 082, 083, 125, and 133 are missing due to problems in manufacturing probes.
	Figure 3. Hourly temporal profiles of CRM activities and gene expressions in developing sea urchin embryos.High resolution temporal profiles of genes and CRMs. Expression levels of the 46 genes (blue) and activities of CRMs (red) from each gene are shown in the order of 5â€² to 3â€² side of the gene. Temporal gene expressions were taken from Materna et al [17]. Scale of color at each time point (h) is proportional to the averages of three adjacent time points, hâˆ’1, h, and h+1. Numbers shown on the scale bar indicate the numbers of transcripts per embryo.
	Figure 4. Parallel measurement of CRM responses and gene responses to gene perturbations.The order of genes and CRMs are identical to that shown in Figure 3 . Fold change is shown in log2 scale as indicated in the scale bar. (A) Responses of the 46 genes and the 126 CRMs to five gene perturbations. Genes or CRMs that are up regulated in perturbed embryos at least two folds are red coded and those down regulated at least two folds are blue coded. All experiments were repeated in two different batches of embryos. â€œControl 1" is a negative control experiment, where two independent sets of control MASO (N-MASO) injected embryos in the same batch are compared, and â€œControl 2" is a repeat of â€œControl 1" with a different batch of embryos. N-MASO injected embryos in the control experiments are also used as controls for perturbed embryos. Note that any responses in the controls are false positives, and one set of the N-MASO injected embryos at 12 h (red coded time point) in â€œControl 1" showed overestimated activities for the majority of CRMs and is considered an outlier. (B) Reproducible responses of the 46 genes and the 126 CRMs to five gene perturbations in two batches of embryos. Only responses that were consistently observed in both experiments in Figure 3 are color coded. All of the false positives except for five in the control experiments were not reproduced, and four reproduced false positives are due to abnormality in one of the N-MASO injected embryos at 12 h. A significant fraction of responses in perturbed embryos were reproduced.

References [+] :

Amit, Unbiased reconstruction of a mammalian transcriptional network mediating pathogen responses. 2009, Pubmed