Exploring the DNA-recognition potential of homeodomains

Abstract

The recognition potential of most families of DNA-binding domains (DBDs) remains relatively unexplored. Homeodomains (HDs), like many other families of DBDs, display limited diversity in their preferred recognition sequences. To explore the recognition potential of HDs, we utilized a bacterial selection system to isolate HD variants, from a randomized library, that are compatible with each of the 64 possible 39 triplet sites (i.e., TAANNN). The majority of these selections yielded sets of HDs with overrepresented residues at specific recognition positions, implying the selection of specific binders. The DNA-binding specificity of 151 representative HD variants was subsequently characterized, identifying HDs that preferentially recognize 44 of these target sites. Many of these variants contain novel combinations of specificity determinants that are uncommon or absent in extant HDs. These novel determinants, when grafted into different HD backbones, produce a corresponding alteration in specificity. This information was used to create more explicit HD recognition models, which can inform the prediction of transcriptional regulatory networks for extant HDs or the engineering of HDs with novel DNA-recognition potential. The diversity of recovered HD recognition sequences raises important questions about the fitness barrier that restricts the evolution of alternate recognition modalities in natural systems.

Global analysis of Drosophila Cys2-His2 zinc finger proteins reveals a multitude of novel recognition motifs and binding determinants

“…This is consistent with the evolutionary diversity within this family (Tadepally et al 2008;Emerson and Thomas 2009;Thomas and Emerson 2009), and with selection-based approaches to engineer zinc fingers with novel DNA-binding specificity that have generated fingers capable of recognizing a broad variety of sequences (Carroll et al 2006;Urnov et al 2010). The utilization of a broad range of DNA recognition preferences by naturally occurring ZFPs is in sharp contrast to homeodomains, the second most-common family of DNA-binding domains in metazoan genomes, which appear to utilize only a small fraction of their true recognition potential in natural systems (Chu et al 2012). In contrast to homeodomains, zinc fingers appear to function as highly malleable units that permit facile rewiring of regulatory systems by providing a wealth of new regulatory potential as trans-acting factors that can readily evolve novel recognition modalities.…”

“…We have employed a B1H system to determine the DNAbinding specificity of these zinc finger domains (Meng et al 2005(Meng et al , 2008Noyes et al 2008b;Chu et al 2012). We extracted a ''cluster'' of closely linked fingers (fewer than 20 amino acids between adjacent fingers) for analysis to minimize the amount of superfluous sequence expressed in the B1H system.…”

Overview of guide RNA design tools for CRISPR-Cas9 genome editing technology

“…Recently, efficient genome editing by the CRISPR-Cas9 system has been demonstrated in multiple organisms, including human, mouse, rat, zebrafish, Drosophila and C. elegans (Cong et al, 2013;Friedland et al, 2013;Gratz et al, 2013;Hou et al, 2013;Hwang et al, 2013;Jinek et al, 2013;Li et al, 2013;Mali et al, 2013b;Yang et al, 2013). In contrast to previous genome-editing techniques, such as zinc-finger nucleases (ZFNs) (Meng et al, 2008;Gupta et al, 2011;Chu et al, 2012;Enuameh et al, 2013) and transcription activator-like effector nucleases (TALENs) , the target specificity of CRISPRCas9 is primarily dictated by a Watson-Crick pairing of a 20-base sequence at the 5′-end of the sgRNA with the target site instead of protein-DNA recognition, providing a much easier system to target multiple genes simultaneously. It has been shown that compared with ZFNs and TALENs, CRISPR-Casmediated gene targeting has similar or greater efficiency in human cells, zebrafish and metazoan Nematostella vectensis (Ding et al, 2013;Ikmi et al, 2014;Smith et al, 2014).…”

Identification of a transcriptional activation domain in yeast repressor activator protein 1 (Rap1) using an altered DNA-binding specificity variant

“…To generate a Rap1 AS variant capable of binding the 3T4A UAS Rap1 site, we utilized a mutagenesis approach recently employed to create altered DNA-binding specificity variants of the Drosophila engrailed homeodomain (79). The six amino acids of the Rap1 DBD homeodomain-1 DNA recognition helix that face DNA (Fig.…”

“…Breakthroughs in the understanding of transcription mechanisms have been made through the generation and utilization of altered DNAbinding specificity mutants of other essential transcription factors, in both prokaryotes and eukaryotes. Examples include AS variants of bacteriophage cro protein (72); Escherichia coli Trp repressor (73) and sigma factor 70 (74); yeast TBP (75) and transcription factor Gcn4 (76); mammalian estrogen receptor (77,78); and Drosophila transcription factor Engrailed (79). These altered DNA-binding variant proteins proved key in unlocking the molecular mechanisms by which these disparate DNA-binding proteins operate.…”