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

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