BEN-solo proteins in genome architecture and function

Abstract

Eukaryotic genomes are organized in a complex manner inside the nucleus of each cell. Chromatin insulators are DNA elements that help to fold the linear DNA fiber in three-dimensional space and partition regulatory gene units. However, the mechanisms by which chromatin insulators and their associated proteins, insulator binding proteins (IBPs), determine genome organization and modulate gene expression remain elusive. This thesis investigates these mechanisms by examining the roles of a stage-specific family of insulator proteins in Drosophila, the BEN-solo proteins.

The BEN proteins regulate gene expression and cell specification in animal development. We characterized the genomic binding sites of the three BEN-solo proteins Elba1, Elba2 and Insensitive (Insv), and the Elba3 protein that acts as adapter for the heterotrimeric ELBA complex. ELBA and Insv share a set of common targets at the time of their ubiquitous expression – throughout the major wave of zygotic genome activation – but also regulate distinct loci. We found that ELBA and Insv not only repress their direct target genes, but also co-localize and act in redundancy with other known insulator binding proteins. Importantly, ELBA and Insv insulate closely-spaced transcription units ensuring the correct levels of promoter transcripts.

The regulatory state of paused RNA Polymerase II (Pol II) has been suggested to comprise a mechanism for insulator function. To understand how paused promoters may block enhancer interaction, we assessed a series of paused and nonpaused promoters in a transgenic reporter assay. Although we observed a high likelihood of paused promoters to act as enhancer blockers, there were several exceptions. Using a paused representative promoter, we dissected the contributions of Pol II and promoter-proximal IBPs to enhancer-blocking. We found that Pol II is dispensable for insulation at this site, while the IBPs and their motifs are essential, arguing against a general function of paused Pol II as enhancer-blocking mechanism.

Insulators are known to set barriers between active and silent chromatin domains. We found that chromatin accessibility at chromatin boundaries in elba and insv mutants is frequently altered, suggesting an involvement of the proteins in chromatin boundary formation. Remarkably, shifted boundaries are accompanied by a deregulation of nascent transcripts. We further assessed the initial establishment of heterochromatin domains. Using a reporter embedded in the heterochromatin of the Drosophila Y chromosome, and the GFP-tagged heterochromatin protein 1a (HP1a), we found that ELBA contributes to heterochromatin silencing while Insv appears to limit HP1a clustering on a global scale. We hypothesize that ELBA and Insv contribute to boundary formation at some common sites, while diverging in their contributions to heterochromatin domain formation.

Taken together, our results identify multiple roles of the BEN-solo family as chromatin factors, and highlight the intrinsic properties of insulator elements and IBPs as the main mechanism in enhancer blocking.

Keywords: gene regulation, transcription, insulators, genome architecture, Polymerase II pausing, chromatin, Drosophila development.

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