Najafova, Z. | Search bioRxiv

bioRxiv2025

PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

Polycomb Repressive Complex 2 (PRC2) is a key regulator of chromatin architecture and transcriptional repression, playing essential roles in development and disease. While its enzymatic activity is well characterized, the molecular factors governing PRC2 subnuclear organization, particularly in cancer cells, are largely unknown. Here, we integrate high-resolution in situ spatial proteomics, imaging, and functional genomics to investigate PRC2 compartmentalization in triple-negative breast cancer (TNBC) cells. We identify PHF19, a sub-stoichiometric PRC2 accessory subunit upregulated in TNBC, as a key factor driving the formation of micron-sized nuclear PRC2 bodies. These structures act as functional hubs that stabilize PRC2 occupancy and reinforce H3K27me3 domain organization. Mechanistically, we identify an intrinsically disordered region (IDR) within PHF19 that is essential for its clustering behavior in cells and link this property to the role of PHF19 in promoting cancer cell motility. Our findings uncover a non-enzymatic layer of PRC2 regulation, whereby its local compartmentalization through accessory subunits directly impinges on cellular behavior. These insights expand our understanding of PRC2 spatial biology with implications for both normal development and disease progression.

molecular biology↗

bioRxiv2024

CRISPR gene and transcriptome engineering (CRISPRgate) improves loss-of-function genetic screening approaches

The CRISPR/Cas9 technology has revolutionized genotype-to-phenotype assignments through large-scale loss-of-function (LOF) screens. However, limitations like editing inefficiencies and unperturbed genes cause significant noise in data collection. To address this, we introduce CRISPR Gene and Transcriptome Engineering (CRISPRgate), which uses two specific sgRNAs to simultaneously repress and cleave the target gene within the same cell, increasing LOF efficiencies and reproducibility. CRISPRgate outperforms conventional CRISPRko, CRISPRi, or CRISPRoff systems in suppressing challenging targets and regulators of cell proliferation. Additionally, it efficiently suppresses modulators of EMT and impairs neuronal differentiation in a human iPSC model. In a multiplexed chromatin-focused phenotypic LOF screen, CRISPRgate exhibits improved depletion efficiency, reduced sgRNA performance variance, and accelerated gene depletion compared to individual CRISPRi or CRISPRko, ensuring consistency in phenotypic effects and identifying more significant gene hits. By combining CRISPRko and CRISPRi, CRISPRgate increases LOF rates without increasing genotoxic stress, facilitating library size reduction for advanced LOF screens. MotivationThe CRISPR technology (CRISPRko/CRISPRi) enables the specific depletion of target genes with fewer off-target effects, facilitating precise investigations of gene function. Despite its benefits, CRISPR applications have limitations. Residual active protein expression mediated by in-frame DNA repair or alternative splicing1-8 as well as strong epigenetic regulation and difficulties in sgRNA design to the transcription start site (TSS)9-12 hinder the full potential of loss-of-function studies using CRISPRko or CRISPRi. We aimed to achieve robust target gene reduction in order to improve the reproducibility of the CRISPR technology by integrating the widely used CRISPRko and CRISPRi approaches into a single application.

biochemistry↗