The Catalytically Inactive KDM5B-NTT Isoform Associates with Chromatin and Increases H3K4 Trimethylation in Breast Cancer Cells

Histone lysine demethylases are mainly known as transcriptional regulators for their demethylase activity, but catalysis-independent functions are increasingly recognized. This work focuses on lysine-specific demethylase 5B (KDM5B), targeting H3K4me3. The canonical functional isoform is PLU-1. A catalytically inactive N-terminally truncated isoform (NTT) was previously identified in human cell lines. NTT lacks the Jumonji N-terminal (JmjN) domain, responsible for demethylase activity, and part of the AT-Rich Interaction Domain (ARID), but retains the plant homeodomains (PHDs) involved in chromatin interaction. The study aimed to investigate NTT chromatin association, its potential regulatory role, and its phenotypic effects. NTT-FLAG was overexpressed in MCF7 cells, using PLU-1-FLAG and empty vector for comparison. Chromatin association and H3K4me3 distribution were analyzed by Cleavage Under Targets and Release Using Nuclease (CUT&RUN), while migration and invasion were assessed by wound-healing, live-imaging microscopy, and invasion assays. NTT-FLAG and PLU-1-FLAG displayed similar genomic localization, preferentially associating with active promoters enriched in H3K4me3. Both isoforms increased promoter-associated H3K4me3, suggesting that chromatin binding alone is insufficient to promote demethylation. Functionally, NTT overexpression was associated with increased directional migration and invasive capacity compared with PLU-1. Overall, these findings support a catalysis-independent regulatory role for NTT and suggest a model in which chromatin interaction is driven by PHD domains, while co-interactor availability may be required for proper PLU-1 demethylase activity.