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.
Highlights
We recently demonstrated in the sea urchin embryo that multiplexed, quantitative cis-regulatory analysis can be performed by use of ‘‘barcoded’’ DNA-tag reporters, in which the activity of each cis-regulatory modules (CRMs) in a co-injected mixture is monitored by measuring the number of transcripts produced that include its unique tag sequence [1]
The vectors into which the CRMs or putative CRMs are inserted contain a gatae basal promoter (BP) [7], a GFP open reading frame, a unique Nanotag sequence of about 100 bp in length that can be used for either NanoString or quantitative PCR (QPCR) detection, and a core polyadenylation signal [8] (Fig. 1A)
The multiplexed power of the Nanotag system makes prior criteria such as interspecific sequence conservation less important as a search tool, with the payoff of a more unbiased analysis. Initial screens such as we used in this work to obtain the average 3 kb fragments in the 130 construct library can be followed by secondary rounds of screens in which those large fragments shown to be active are further dissected to obtain the active CRMs per se
Summary
We recently demonstrated in the sea urchin embryo that multiplexed, quantitative cis-regulatory analysis can be performed by use of ‘‘barcoded’’ DNA-tag reporters, in which the activity of each CRM in a co-injected mixture is monitored by measuring the number of transcripts produced that include its unique tag sequence [1]. Individual CRMs or putative CRMs are cloned into each tag vector, the whole set mixed together, and delivered into sea urchin eggs After their random incorporation into the embryo genomes and development to the desired stage, RNA is extracted from the embryos, and the transcripts deriving from each of the individual tag reporters are identified by using PCR primer pairs specific to each tag. Due to the large size of current sea urchin gene regulatory networks (GRNs) [3,4], and the ancillary requirement for multiple cis-regulatory analyses of various kinds, the need arose for a vastly expanded methodology for high throughput experimental CRM analysis We demonstrate such a methodology, which will be applicable to a wide range of problems in addition to GRN analysis
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