Brr6 Plays a Role in Gene Recruitment and Transcriptional Regulation at the Nuclear envelope

Correlation between transcriptional regulation and positioning of genes at the nuclear envelope is well established in eukaryotes but the mechanisms involved are not well understood. We provide evidence that Brr6 interacts with chromatin, helping to maintain normal chromatin architecture and transcriptional regulation of certain loci at the nuclear envelope. Specifically, our results showed that brr6-1 impairs PAB1 transcript levels and disrupts PAB1 and GAL1-10 locus positioning. In addition, RNAseq analysis revealed misregulation of both coding and non-coding transcription across the FUR4-GAL1,10,7 region as well as changes at numerous other genes. The discovery that brr6-1 is disomic for CHIII raised the possibility that some of these effects stemmed from increased copy number of CHIII genes. However, disomy was not present in wild type cells where the NLS-Brr6N fragment was expressed, therefore we were able to use the Brr6N fragment to link many of the brr6-1 effects at the PAB1 and FUR4-GAL1,10,7 loci to Brr6 function, including: 1) decreased PAB1 expression, 2) defective PAB1 locus positioning, and 3) misregulation across the FUR4-GAL1-10 region. Together with our ChIPseq results showing zinc finger-dependent association of the Brr6N fragment with the FUR4 gene, these results strongly argue for a role for Brr6 in gene recruitment and regulation at the nuclear envelope. For RNAseq analysis: RNA was analyzed from 2 biological replicates of wild type or brr6-1 mutant (arg to lys point mutation in putative zinc finger domain of Brr6) samples grown in 2% glucose or in 2% raffinose/0.04% sucrose followed by 2hr induction in 2% galactose. RNA levels are compared between wild type and mutant for each condition. For ChIPseq analysis: extracts were prepared from wild type cells carrying empty vector, or non-membrane FLAG-tagged Brr6N and Brr6∆C4N fragment constructs (+/- putative zinc finger domain). r ChIP was done with FLAG antibody beads. IP'd DNA was normalized against DNA in total extract. The vector sample served as a control for non-specific background in the IP. The ∆C4N samples showed whether interaction was dependent on the zinc finger domain.