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. 2010 Feb 23;107(8):3930-5.
doi: 10.1073/pnas.1000147107. Epub 2010 Feb 8.

Functional cis-regulatory genomics for systems biology

Jongmin Nam  1 , Ping Dong, Ryan Tarpine, Sorin Istrail, Eric H Davidson
Affiliations

Functional cis-regulatory genomics for systems biology

Jongmin Nam et al. Proc Natl Acad Sci U S A. .

Abstract

Gene expression is controlled by interactions between trans-regulatory factors and cis-regulatory DNA sequences, and these interactions constitute the essential functional linkages of gene regulatory networks (GRNs). Validation of GRN models requires experimental cis-regulatory tests of predicted linkages to authenticate their identities and proposed functions. However, cis-regulatory analysis is, at present, at a severe bottleneck in genomic system biology because of the demanding experimental methodologies currently in use for discovering cis-regulatory modules (CRMs), in the genome, and for measuring their activities. Here we demonstrate a high-throughput approach to both discovery and quantitative characterization of CRMs. The unique aspect is use of DNA sequence tags to "barcode" CRM expression constructs, which can then be mixed, injected together into sea urchin eggs, and subsequently deconvolved. This method has increased the rate of cis-regulatory analysis by >100-fold compared with conventional one-by-one reporter assays. The utility of the DNA-tag reporters was demonstrated by the rapid discovery of 81 active CRMs from 37 previously unexplored sea urchin genes. We then obtained simultaneous high-resolution temporal characterization of the regulatory activities of more than 80 CRMs. On average 2-3 CRMs were discovered per gene. Comparison of endogenous gene expression profiles with those of the CRMs recovered from each gene showed that, for most cases, at least one CRM is active in each phase of endogenous expression, suggesting that CRM recovery was comprehensive. This approach will qualitatively alter the practice of GRN construction as well as validation, and will impact many additional areas of regulatory system biology.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Parallel measurement of in vivo cis-regulatory activities of many DNA sequences by using the DNA-tag reporters. (A) The structure of a DNA-tag reporter construct. Each basic unit of DNA-tag reporter construct is composed of a basal promoter (BP) from the sea urchin gatae gene (35), a GFP ORF (ORF), a pair of DNA-tags flanking a 145-bp-long fragment of human CD4 cDNA, and a core poly-adenylation (A) signal (39). For a later set of 129 DNA-tag reporters, a pair of primer sites (orange coded regions) were introduced to amplify the entire pool of DNA-tag reporters and a part of the GFP ORF (magnified region) either from cDNA or genomic DNA isolated from injected embryos. (B) A pool of many DNA constructs is injected with seven molar excess amount of randomly sheared genomic DNA as carrier/spacer (40). Coinjected linear DNA molecules form random concatenates and are incorporated into chromosomes in a mosaic fashion (17). Expression of each tag reporter is measured by QPCR following the method developed by Revilla-i-Domingo et al. (21).
Fig. 2.
Fig. 2.
Cis-regulatory modules for the foxn2/3 locus. (A) Conserved DNA patches and candidate CRMs selected for the foxn2/3 locus. Black horizontal line with ticks represents genomic sequence of the locus. Interval between ticks on black line is 5 kb. Color-coded vertical lines above the black horizontal line indicate sequence conservation between Sp and Lv for each 25- bp-long window: green, 0 mismatch; blue, 1 mismatch; pink, 2 mismatches; yellow, 3 mismatches; red, 4 mismatches. Thick and thin pink horizontal lines below black line indicate exons and introns of foxn2/3 from 5′ to 3′, respectively. Horizontal lines with labels indicate candidate CRMs: red, active; yellow, marginally active; black, inactive; gray, cloning failed. (B) Cis-regulatory activities of 17 candidate CRMs from the foxn2/3 locus. Normalized expression level after background correction is shown for each time point measured. The names of candidate CRMs are shown at the bottom. Expression level significantly higher (≥2.5) than the background level for each time point is red coded. Note that expression levels >10 are shown as 10. (C) High-resolution time course activities of CRMs measured using the 129 DNA-tag reporters over 27 different time points. CRM activities are corrected for background activities, and the relative expression level of foxn2/3 is shown (SI Appendix).
Fig. 3.
Fig. 3.
Distribution of the numbers of CRMs discovered for each gene. Genes are categorized by the number of CRMs discovered, and the number of genes in each category is presented as a bar.
Fig. 4.
Fig. 4.
Temporal sufficiency scores (St) of CRMs. The St value of the entire set of CRMs discovered for each gene (crm_all; orange coded) and the St_crm value of each CRM (crm_1st, crm_2nd, and crm_3rd; gray coded) are presented as bar graphs. Genes are ordered by the St values. When there are more than three CRMs discovered for a gene, only the top three CRMs ranked by St_crm value are shown.
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