pRB AND CELLULAR SENESCENCE Leu X Cys X Glu Luca Michetti AA 2017-18

Senescence «Stable form of cell cycle arrest wherein cells exit the cell cycle and can remain post-mitotic for an indefinite period of time» (Campisi, 2005) Integration of different general pRB functions 3. Global chromatin condensation 2. Local chromatin repression 1. Transcriptional

Senescence Specific Interactor: PML PML nuclear bodies General PML Role in senescence Multiple isoforms: PML-IV as senescence-inducing factor pRB PML body LXCXE-like interaction between pRB and PLM Specific pRB-associated functions PML

Aims and Approach Aims Approach Define pRB-PML specific On DNA regulation Define pRB-PML specific joint functions On senescence onset and mantaining Aims Characterize protein-protein interaction proliferation Compare wt Rb1 and Rb1ΔL/ ΔL Approach during Ras-induced Model system: MEFs senescence PML-IV induced

Results Real-time on E2F target genes ChIP on E2F target genes for 1. DNA interaction and regulation ChIP on E2F target genes for pRB Real-time on E2F target genes PML H3K9me3 Recovered activation of E2F target genes, but only in a over-expressed PML background PML needs pRB to localize on E2F target genes Demonstrate pRB needs PML to repress gene expression pRB needs PML to organize heterochromatin Suggest Open Questions Activation of E2F target genes when PML is not present? Repression mechanism?

Results Flow-cytometry to detect staining of BrdU Immuno- 2. Senescence-associated markers γ-H2AX Immuno- fluorescence against Flow-cytometry to detect staining of BrdU PML nuclear bodies Positive in a Rb1ΔL background (before day 8) also after E2F reintroduction during senescence: sustained DNA synthesis DNA damage signalling and PML nuclear bodies are mantained Demonstrate Senescence-associated PML function is not impaired by loss of pRB interaction Loss of pRB-PML interaction renders senescence less «robust» Suggest Open Questions Do E2F target genes still localize in PML bodies?

Results GST Pull-down and CO-IP 3. Characterize proteins presence and interactions GST Pull-down and CO-IP Senescence-specific and LXCXE-dependent interaction between GST-Rb and PML; Decreased interaction in the Rb1ΔL background Demonstrate pRB is able to interact with multiple PML isoforms PML is post-translational modified and this modifications are needed for its interaction with pRB Senescence induces specific LXCXE-dependent interaction between pRB and PML Suggest Open Questions How does interaction work? Why are modifications needed and which ones?

Conclusive Points PML is directed in a Rb1-LXCXE-motif-dependent manner on E2F target genes and helps repression Rb1ΔL/ ΔL cells are senescent but reversibly arrested in their cell cycle Incomplete senescence? PML is post-translational modified during senescence, that is when it specifically interacts with Rb1 Modify PML structure to render it LXCXE-compatible?

Future Analysis ChIP-seq Genome-wide analysis H3K9me3 RNA-seq Demonstrate direct or indirect interaction pRB and PML interactome analysis Understand Suv39h1 recruitment Chip-seq: On H3K9me3 combined with the proteins, to understand a more general role of pRB in its employment on DNA PML modifications: which, where and why Biochemical relevance of the modification: no LXCXE motif in PML

References Talluri, S., & Dick, F. A. (2014). The retinoblastoma protein and PML collaborate to organize heterochromatin and silence E2F-responsive genes during senescence. Cell Cycle, 13(4), 641-651. Talluri, S., & Dick, F. A. (2012). Regulation of transcription and chromatin structure by pRB: here, there and everywhere. Cell Cycle, 11(17), 3189-3198.