Identification of Proteins Associated with Stably Integrated Maize b1 Tandem Repeat Transgene Chromatin

The control of gene expression by cis-regulatory DNA sequences is a conserved genomic feature. The maize booster1 gene (b1) is a naturally occurring locus that serves as a mechanistic model for the control of gene expression from a distal cis element and a form of allelic interactions called paramutation. Two epi-alleles of b1 produce distinct pigmentation phenotypes correlated with transcriptional enhancement and the silencing of b1. These transcriptional dynamics depend on a hepta-tandem repeat sequence located 100 kb upstream of the b1 locus. In the heterozygous condition, the B' epi-allele paramutates B-I, heritably converting the B-I epi-allele to the epigenetic state and expression level of B', producing lightly pigmented plants. To identify b1TR-associated proteins, we used a targeted chromatin immunoprecipitation approach with a stably integrated transgenic b1TR locus. Applying a conservative filtering strategy, we detected several expected factors, including RNA Polymerase II, as well as the novel putative DNA-binding proteins ZAG4 and DDT4. ZAG4 and DDT4 activated GAL expression using b1TR as bait in yeast one-hybrid, supporting their potential interaction with this sequence. The identification of proteins uniquely associated with the UAS::b1TR chromatin provides insight into potential b1 regulatory factors and offers a foundation for future studies to investigate their roles in gene regulation.