Context-dependent effects of HP1 assessed in high throughput. Drosophila melanogaster

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. The assay combines a previously reported multiplexing strategy based on barcoded randomly integrated reporter genes (Akhtar, Cell, 2013; PMID: 23953119) with Gal4-mediated tethering of the protein of interest. We applied the assay to Drosophila Heterochromatin protein 1a (HP1a), which is mostly known as a repressive protein working by a chromatin compaction mechanism but has also been linked to transcriptional activation. Probing the effects of HP1a in more than one thousand genomic locations revealed that HP1a is a potent repressor able to silence even highly expressing loci. However, the local chromatin context can modulate HP1a function. In pericentromeric regions, HP1a-induced repression was enhanced by two-fold. In regions marked by a H3K36me3-rich chromatin signature linked to transcriptional elongation, 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 recruitment in any of the loci examined. The multiplexed tethered reporter assay will be a useful tool to dissect combinatorial regulatory interactions in chromatin. Overall design: TRIP assay in drosophila Kc167 cells combined with targeted recruitment of Gal4-HP1 to integrated 5xGa4UAS-pMT-GFP reporters. Two technical replicate experiments of TRIP pool transfected with Gal4-HP1, Gal4 or HP1 performed by day 2 and day 16 after transient transfection.

染色质蛋白以协同方式调控基因活性。本研究开发了一种高通量检测方法，用于探究局部染色质环境对目标蛋白调控活性的影响。该检测方法结合了此前报道的基于条形码随机整合报告基因的多重化策略（Akhtar, Cell, 2013; PMID: 23953119）与目标蛋白的Gal4介导锚定技术。我们将该检测方法应用于果蝇异染色质蛋白1a（Heterochromatin protein 1a, HP1a）：该蛋白通常被认为是通过染色质压缩机制发挥作用的阻遏蛋白，但也有研究将其与转录激活相关联。在超过一千个基因组位点检测HP1a的作用后发现，HP1a是一种强效阻遏蛋白，即便对于高表达位点也能实现沉默。不过，局部染色质环境可调控HP1a的功能。在着丝粒周边区域，HP1a介导的阻遏作用被增强了两倍。在与转录延伸相关的富含H3K36me3的染色质特征区域中，HP1a依赖的沉默效应显著降低。在本实验系统中，我们未发现HP1a具有激活功能的证据。此外，在所有检测的位点中，当靶向HP1a的招募消失后，我们未观察到阻遏效应可通过有丝分裂稳定传递。这种多重锚定报告基因检测方法将成为解析染色质中组合式调控相互作用的实用工具。整体实验设计：在果蝇Kc167细胞中开展TRIP检测，同时将Gal4-HP1靶向招募至整合的5xGa4UAS-pMT-GFP报告基因。分别在瞬时转染后第2天和第16天，对转染了Gal4-HP1、Gal4或HP1的TRIP混合样本完成两次技术重复实验。