Mutator transposons in Zea mays impact transcriptional regulatory networks and underlying gene expression

Abstract

Eukaryotic gene expression is transcriptionally regulated by cis- and trans-regulatory inputs. The interactions between cis-regulatory elements (CREs) and transcription factor (TF) trans-regulators that recognize CREs coordinate regulation of gene expression. Much work has been done to identify these protein-DNA interactions because they represent interactions that can directly change transcriptional activity. Interactions between TFs and target genes can be predicted by representative gene-regulatory networks (GRNs) to derive these causal relationships. In crop species, the rewiring of GRNs by either selecting for existing variants or introducing new genetic variants is a potential strategy for trait improvement. However, the predictive power of inferred GRNs must be determined experimentally. One method to test whether GRN predicted TF-target gene interactions represent functionally important interactions is to perturb the networks. In maize, a family of DNA transposons, Mutator, can be used to disrupt gene function and are known to contain cis-regulatory sequences that may influence gene expression. Transposable elements (TEs) or transposons are mobile repetitive DNA sequences that have proliferated in the genomes of many crop species. To maintain gene function, host genomes have found ways to silence TEs and TEs have adapted to persist regardless of this silencing. TEs may carry potential cryptic regulatory sequences that were once used in their ancestral state to facilitate their own transposition. It is important to determine how these TE-derived CREs have contributed to TE evolution and gene regulation via cis-trans interactions. In this thesis we use Mutator in maize to study TF-target gene interactions and transposon biology. The central questions we ask in these thesis chapters are (1) what is the relative accuracy of predicted GRNs in maize (2) can we determine the value of GRN predictions by perturbation with TF loss-of-function mutants in maize (3) what is the frequency and function of putative cryptic promoters in Mutator (4) can we study Mutator to learn more about how the effects of TE insertion can be masked when regulatory sequences within TEs are co-opted by the host genome. To address these thesis questions, I first present background information on both Mutator transposons and their use for reverse genetics studies in maize (Chapter I). It is pertinent to understand why Mutator is used in maize for insertional mutagenesis and how properties of Mutator can impact downstream analyses. In addition, I will provide brief background information on why it is necessary to determine the functional relevance of inferred GRNs in vivo. I will then present two research studies that were conducted in maize for this thesis. TF mutant alleles were isolated from a Mutator transposon-indexed population in maize, UniformMu. We utilized these mutant alleles to study how Mutator may provide novel alternative promoters that can impact gene expression patterns and levels and influence TE-host genome evolution (Chapter II). We also used these mutant alleles to perturb predicted GRNs in maize and test expression of predicted targets (Chapter III). To summarize these studies, we provide insight into what should be known when using Mutator insertion stocks for future reverse genetics studies in maize.