Abstract
BackgroundChromatin proteins control gene activity in a concerted manner. We developed a high-throughput assay to study the effects of the local chromatin environment on the regulatory activity of a protein of interest. The assay combines a previously reported multiplexing strategy based on barcoded randomly integrated reporters with Gal4-mediated tethering. We applied the assay to Drosophila heterochromatin protein 1a (HP1a), which is mostly known as a repressive protein but has also been linked to transcriptional activation.ResultsRecruitment to over 1000 genomic locations revealed that HP1a is a potent repressor able to silence even highly expressing reporter genes. However, the local chromatin context can modulate HP1a function. In pericentromeric regions, HP1a-induced repression was enhanced by twofold. In regions marked by a H3K36me3-rich chromatin signature, HP1a-dependent silencing was significantly decreased. We found no evidence for an activating function of HP1a in our experimental system. Furthermore, we did not observe stable transmission of repression over mitotic divisions after loss of targeted HP1a.ConclusionsThe multiplexed tethered reporter assay should be applicable to a large number of chromatin proteins and will be a useful tool to dissect combinatorial regulatory interactions in chromatin.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0096-y) contains supplementary material, which is available to authorized users.
Highlights
Chromatin proteins control gene activity in a concerted manner
As an addition to this original thousands of reporters integrated in parallel (TRIP) protocol, we inserted five copies of the Gal4UAS motif upstream of the promoter of our integrated reporters. This makes it possible to tether a fusion protein consisting of GalDBD and a chromatin protein of interest, here heterochromatin protein 1a (HP1a). (Fig. 1a)
Various studies in Drosophila have previously shown that tethered heterochromatin protein 1 (HP1) can cause silencing of a reporter gene [17,18,19,20]
Summary
Chromatin proteins control gene activity in a concerted manner. We developed a high-throughput assay to study the effects of the local chromatin environment on the regulatory activity of a protein of interest. Eukaryotic genomes are packaged in various types of chromatin that each has specific roles in the regulation of gene expression and other nuclear functions. These chromatin types (or “states”) are defined by their distinct but sometimes partially overlapping protein compositions [1, 2]. More than 1000 random integration sites throughout the genome offer the required statistical power to infer how various chromatin environments may influence the regulatory activity of the protein of interest. We illustrate this approach using Drosophila HP1a as a model
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