The catalytically inactive KDM5B-NTT isoform associates with chromatin and increases H3K4 trimethylation in breast cancer cells

Background. Histone lysine demethylases are key regulators of cancer-related processes. Beyond their enzymatic activity, catalytically independent functions are increasingly recognized. We previously identified a N-terminal truncated and catalytically inactive isoform of KDM5B, called NTT, stably expressed in several human cell lines. Overex-pression of NTT-FLAG in MCF7 breast cancer cells was shown to increase global H3K4me3 levels and modulate transcription of several genes. Methods. In this study, we used the CUT&RUN assay to investigate the genomic lo-calization and the local effects on histone methylation of ectopically expressed NTT-FLAG. Moreover, phenotypic assays were performed to assess the effects of NTT-FLAG over-expression on cell migration and invasion. Results. NTT-FLAG preferentially associates with actively transcribed genes enriched in H3K4me3 at promoter regions, contributing to increased H3K4me3 levels at target sites. Functionally, NTT overexpression enhances MCF7 cell migration and invasiveness. Conclusions. Our findings support a model in which NTT and PLU-1 scan chromatin via PHD1 in a transient, low-affinity manner, while H3K4me3 stabilizes their binding by interacting with PHD3. However, catalytic activity likely depends on corepressor en-gagement, whose availability is influenced by endogenous KDM5B levels. Functionally, NTT appears to enhance cell migration and invasiveness by regulating genes such as PLAUR and CSF1, supporting its role in driving tumour-associated phenotypes inde-pendently of catalytic activity.