1. pRB AND CELLULAR SENESCENCE
Leu X Cys X Glu
Luca Michetti AA

2. 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

3. 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

4. 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

5. 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?

6. 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?

7. 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?

8. 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?

9. 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

10. 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),
Talluri, S., & Dick, F. A. (2012). Regulation of transcription and chromatin structure by pRB: here, there and everywhere. Cell Cycle, 11(17),