Sequence specificity is obtained from the majority of modular C2H2 zinc-finger arrays

Abstract

C2H2 zinc fingers (C2H2-ZFs) are the most prevalent type of vertebrate DNA-binding domain, and typically appear in tandem arrays (ZFAs), with sequential C2H2-ZFs each contacting three (or more) sequential bases. C2H2-ZFs can be assembled in a modular fashion, providing one explanation for their remarkable evolutionary success. Given a set of modules with defined three-base specificities, modular assembly also presents a way to construct artificial proteins with specific DNA-binding preferences. However, a recent survey of a large number of three-finger ZFAs engineered by modular assembly reported high failure rates (∼70%), casting doubt on the generality of modular assembly. Here, we used protein-binding microarrays to analyze 28 ZFAs that failed in the aforementioned study. Most (17) preferred specific sequences, which in all but one case resembled the intended target sequence. Like natural ZFAs, the engineered ZFAs typically yielded degenerate motifs, binding dozens to hundreds of related individual sequences. Thus, the failure of these proteins in previous assays is not due to lack of sequence-specific DNA-binding activity. Our findings underscore the relevance of individual C2H2-ZF sequence specificities within tandem arrays, and support the general ability of modular assembly to produce ZFAs with sequence-specific DNA-binding activity.

Figure 1.

Clustering analysis of PBM data. Color scale reflects E-score, as indicated in the legend. All 8-mers that scored at E > 0.45 in at least one experiment are included.

Figure 2.

Sequence specificities of ZFAs constructed by modular assembly, as determined by PBM. ID for ZFA and results of assay for activity follow Ramirez et al. F1, F2 and F3 columns indicate the module numbers used for construction of the ZFA. The rank of the intended 9-mer target (out of all 131 072 possible 9-mers) is determined by E-score; ME and HK refer to the two array designs used. The last column shows the intended target (based on the modules used for assembly) compared to PBM results (the sequence motif shown is generated from the (up to) top 10 8-mers bound by the ZFA, as described in the main text).

Figure 3.

C2H2-ZF modules in functional ZFAs typically retain at least degenerate DNA-binding specificity in different contexts. The first row lists ZFAs yielding positive results on PBM, the first column lists the modules used to construct the ZFAs, and the second column indicates their three-base specificity. The remaining cells indicate which modules were used in the array, and what the specificity appeared to be based on the top ten most preferred 8-mers and 9-mers on the PBMs. Colors indicate whether the assayed specificity of the modules in the zinc-finger array was in agreement with its intended target specificity, as described in the main text. To the right is a condensed version of the same data. The source of the modules is also indicated.

Figure 4.

Comparison of the degeneracy of binding sites for artificial ZFAs constructed by modular assembly and natural ZFAs. Shown are the number of 8-mers with E > 0.45 (average for two array designs) for this study (top) and Badis et al. (36), which examined mouse transcription factors (bottom).

Figure 5.

Success rates of GNN and other C2H2-ZF module subtypes. (A) Proportion and number of ZFAs considered successful by PBM, as a function of the number of GNN modules. (B) Proportion and number of ZFAs of different XNN subtypes with the indicated outcome, considering only ZFAs that were successful by PBM. The number indicated reflects the proportion of times the module satisfies the condition indicated, i.e. gray bars indicate the proportion of instances in which the module gives a perfect match; black bars indicate the proportion of instances that are either a perfect or degenerate match.