Phd in CELL AND DEVELOPMENTAL BIOLOGY

Thesis title: Functional characterization of the catalytically inactive KDM5B-NTT isoform in breast cancer cell lines

KDM5B is histone demethylase acting as a reader and an eraser of H3K4me3, mainly having a repressive role. It is upregulated in breast cancer, where it contributes to tumor progression. Recent findings highlighted the importance of catalytic independent roles of KDM5B, demonstrating that an inactive isoform is compatible with life in mice. The existence of a truncated, catalytically inactive, but highly stable isoform, termed NTT (N-terminal truncated), were described in breast cancer, but its functional role remained to be further investigated. In this study, we characterized the molecular and functional features of NTT in comparison with the canonical PLU1 isoform in MCF7 cells, using overexpression of FLAG-tagged proteins as experimental approach. CUT&RUN analyses demonstrated that NTT isoform binds the chromatin, preferentially associating with promoters of actively transcribed genes enriched in H3K4me3 marks, likely through its PHD domains. It shares more than half of the genomic targets with PLU1, suggesting a common chromatin recruitment mechanism. NTT-FLAG peaks were found on several DEGs previously identified by RNA-seq in NTT-FLAG overexpressing MCF7 cells, Moreover, a subset of these genes displayed a spreading of H3K4me3 across their promoters, suggesting a regulatory effect on their transcription. Functionally, NTT-FLAG overexpression promoted cellular motility with more directed migration patterns than PLU1, specifically in MCF7 but also in MDA-MB-231. Both isoforms induced upregulation of PLAUR and CSF1, linking them to invasion and migratory pathways, even though NTT seems less sensitive to cellular background, including differences in the intrinsic migratory potential of MCF7 and MDA-MB-231. Furthermore, NTT and PLU1 modulated LAMA2 and LAMC2 in MDA-MB-231, reshaping the extracellular matrix. Conversely, neither isoform markedly affected DNA repair, probably due to compensation by other members of KDM5 family. These results emphasized that non-enzymatic mechanisms significantly contribute to tumor progression