Mutagenesis by reversible promoter insertion to study the activation of NF-kappaB

Abstract

Genetic dissection of signaling pathways in mammalian cells involves screening or selecting phenotypic mutants obtained by a variety of techniques. Limitations in current methods include inadequate genome coverage and difficulty in validating the link between mutation and phenotype. We describe an improved method for insertional mutagenesis with retroviral vectors and show that the ability to induce mutations increases greatly if a randomly inserted promoter directs transcription into the host DNA. The mutant phenotype is due to the expression of a hybrid transcript derived from the vector and the insertion site. Because other alleles of the affected gene remain intact, the phenotype is dominant, but is reversible by inactivating the promoter, for example, by site-specific recombination. Importantly, in mutant clones with multiple inserts, limited excision yields progeny with different patterns of inserts remaining. Characterizing these progeny allows the mutant phenotype to be associated with a specific target gene. Relative simplicity and robust target validation make the method suitable for a broad range of applications. We have used this technique to search for proteins that regulate NF-kappaB-dependent signaling in human cells. Two validated targets are the relA gene, which codes for the NF-kappaB p65 subunit, and the NF-kappaB regulator act1. Overexpression of the corresponding proteins, caused by insertion of a promoter into the first intron of each gene, leads to NF-kappaB-dependent secretion of factors that activate NF-kappaB through cell-surface receptors, establishing an autocrine loop.

Fig. 1.

The effect of the LTR promoter on the efficiency of retroviral mutagenesis. (A) The retroviral vector. The dLTR variant carries an inactivating deletion in the 3′ LTR that is copied into the 5′ LTR upon reverse transcription and integration. SV40, simian virus 40 immediate early promoter and origin of replication; Neo^r, neomycin phosphoribosyl transferase gene; Ψ, packaging signal. (B) Yield of GCV-resistant colonies among HCT9 cells (6) infected with promoter-competent (LTR) or promoter-deficient (dLTR) retroviruses or mock-infected (mock). For each treatment, colonies were counted on 10 plates of ≈7.5 × 10⁵ cells.

Fig. 2.

Vectors used and examples of reversible phenotypes among zeocin-selected mutants. (A) The vector within a plasmid. (B) The vector as integrated provirus. tet-RP, tetracycline-regulated promoter (TORE); pUC ori, the origin of replication from pUC19. The LTR modification (⊠) includes a LoxP site and deletion of the promoter. Dashed lines, predicted transcripts. Filled boxes, host DNA. Other abbreviations are as in Fig. 1. A short ORF and an unpaired splice donor site follow the regulated promoter, as described in the text. (C) Clones 3B37, 3B22/35, 3B311, and the parental cell line were tested for resistance to zeocin or GCV after the introduction of Cre or empty vector. One 6-cm plate (3.5 × 10⁵ cells) was treated for 10 days. Surviving cells were visualized with Methylene blue. Mutants with an altered response to both drugs are scored as trans, and those with an altered zeocin response only are scored as cis.

Fig. 3.

Reversible NF-κB activation in the mutant clones. (A) EMSA. Cells from the mutant clones (C, treated with Cre; V, treated with empty vector) were assayed in comparison with the parental cell line, untreated or treated with IL-1. (B) IL-8 expression in the mutant clones. The expression of this NF-κB target gene was assayed by the Northern method in cells treated as indicated. Dox, doxycycline.

Fig. 4.

The causative insertional event in mutant 3B22/35. (A) Integration patterns in clone 3B22/35 and derivatives as determined by Southern analysis of ApoI-digested DNA. The phenotype correlates with the presence of the band marked by an arrow. (B) Schematic representation of the integrated provirus. Recognition sites for ApoI, the probe for Southern blotting (probe), and the fragment recovered by means of iPCR are shown with respect to the LTRs (open arrows) and the regulated promoter (filled arrow). Hatched boxes represent host genomic DNA. (C) RT-PCR assay of the fusion transcript. Samples from parental cells and the 3B22/35 clone, treated with vector or Cre, were compared by using RT-PCR, with primers that anneal within the insert and within exon 3 of relA. Stat3 primers were used for the control amplification. (D) Reversible overexpression of p65. The levels of total p65 and p65 phosphorylated on Ser-536 were measured by using the Western method.

Fig. 5.

Properties of the mutant clones. (A) Reversible expression of relA and act1in the mutant clones. Northern analyses were on RNA from parental and mutant cells and treated as indicated. Equal loading was verified by using a gapdh probe. (B) Expression of the hybrid act1 transcript in clone 3B311. RNA from the parental cell line and 3B311, treated with Cre or empty vector, was assayed by using RT-PCR, with one primer annealing within the insert and the other within the common exon 2 of act1.A cdc2-specific primer set was used for the control amplification. (C) NF-κB-inducing activity in conditioned media. Media conditioned overnight by confluent cultures of 3B311, 3B22/35, and the parental cell line was applied to an indicator cell line (13). Luciferase activity was measured 18 h later. Untreated, Cre-treated, and vector-treated variants of each cell line were analyzed. The NF-κB-inducing activity is shown as a fold increase over that in the medium from untreated parental cells. Averages of triplicate experiments are shown.