A comprehensive HA-tagged PRC1 toolkit enables standardized chromatin profiling and proteomic analysis in Drosophila melanogaster
TL;DR
It is shown that endogenous HA tagging preserves protein expression, chromatin localization, and genome-wide binding profiles, enabling direct comparison using a common antibody to analyze distinct PcG subunits, and provides a platform for standardized analysis of Polycomb function and chromatin regulation.
Abstract
Polycomb group (PcG) proteins are evolutionarily conserved epigenetic regulators that maintain transcriptional states during development and are frequently misregulated in disease. Here, we generated a comprehensive collection of endogenously HA-tagged alleles for the core components of Polycomb Repressive Complex 1 (PRC1) and additional chromatin regulators in Drosophila melanogaster using CRISPR/Cas9-mediated genome engineering. We show that endogenous HA tagging preserves protein expression, chromatin localization, and genome-wide binding profiles, enabling direct comparison using a common antibody to analyze distinct PcG subunits. Proteomic analyses recovered known components of canonical and non-canonical PRC1 and PRC2 complexes and identified additional PcG-associated factors. Notably, multiple components of the nuclear pore complex (NPC) were detected as PcG interactors, and genetic analyses confirmed a functional interaction between them. This resource provides a platform for standardized analysis of Polycomb function and chromatin regulation that can easily be extended to other chromatin regulatory complexes and applied across different developmental stages. Article Summary Polycomb group (PcG) proteins are essential regulators of development and genome function. Comparative studies of individual PcG components are often limited by the availability and quality of specific antibodies. Here, we generated a collection of Drosophila melanogaster lines carrying endogenous HA epitope tags in core components of the canonical Polycomb Repressive Complex 1 (PRC1), along with additional chromatin regulators. These tagged proteins faithfully reproduce native chromatin binding and can be used for chromatin mapping and protein interaction studies using a single, highly specific antibody. This standardized resource provides a practical tool for investigating PcG function and chromatin regulation in vivo.