Dynamic Protein Interactions of the Polycomb Repressive Complex 2 during Differentiation of Pluripotent Cells

Polycomb proteins assemble to form complexes with important roles in epigenetic regulation. The Polycomb Repressive Complex 2 (PRC2) modulates the di- and tri-methylation of lysine 27 on histone H3, each of which are associated with gene repression. Although three subunits, EZH1/2, SUZ12, and EED, form the catalytic core of PRC2, a wider group of proteins associate with low stoichiometry. This raises the question of whether dynamic variation of the PRC2 interactome results in alternative forms of the complex during differentiation. Here we compared the physical interactions of PRC2 in undifferentiated and differentiated states of NTERA2 pluripotent embryonic carcinoma cells. Label-free quantitative proteomics was used to assess endogenous immunoprecipitation of the EZH2 and SUZ12 subunits of PRC2. A high stringency data set reflecting the endogenous state of PRC2 was produced that included all previously reported core and associated PRC2 components, and several novel interacting proteins. Comparison of the interactomes obtained in undifferentiated and differentiated cells revealed candidate proteins that were enriched in complexes isolated from one of the two states. For example, SALL4 and ZNF281 associate with PRC2 in pluripotent cells, whereas PCL1 and SMAD3 preferentially associate with PRC2 in differentiating cells. Analysis of the mRNA and protein levels of these factors revealed that their association with PRC2 correlated with their cell state-specific expression. Taken together, we propose that dynamic changes to the PRC2 interactome during differentiation may contribute to directing its activity during cell fate transitions

Identification and characterisation of a novel Polycomb Repressive Complex 2 associated protein that is an alternatively spliced product of the LCOR gene locus

THESIS 10942Polycombs are evolutionary conserved epigenetic regulators crucial for specification of cell types during development. They assemble in multiprotein complexes to modify amino terminal tails of histone H3 at lysine 27 to regulate the expression of underlying genes. PRC2 mediates repression by catalysing trimethylation of histone H3 at lysine 27 (H3K27me) at the promoters of master transcriptional regulators, such as HOX and cell cycle and proliferation regulating genes. Recently, it emerged that PRC2 also mediates the mono-and dimethylation of H3K27 and that these histone marks are associated with gene activation and enhancer silencing, respectively

A chromatin-independent role of Polycomb-like 1 to stabilize p53 and promote cellular quiescence

Polycomb-like proteins 1-3 (PCL1-3) are substoichiometric components of the Polycomb-repressive complex 2 (PRC2) that are essential for association of the complex with chromatin. However, it remains unclear why three proteins with such apparent functional redundancy exist in mammals. Here we characterize their divergent roles in both positively and negatively regulating cellular proliferation. We show that while PCL2 and PCL3 are E2F-regulated genes expressed in proliferating cells, PCL1 is a p53 target gene predominantly expressed in quiescent cells. Ectopic expression of any PCL protein recruits PRC2 to repress the INK4A gene; however, only PCL2 and PCL3 confer an INK4A-dependent proliferative advantage. Remarkably, PCL1 has evolved a PRC2- and chromatin-independent function to negatively regulate proliferation. We show that PCL1 binds to and stabilizes p53 to induce cellular quiescence. Moreover, depletion of PCL1 phenocopies the defects in maintaining cellular quiescence associated with p53 loss. This newly evolved function is achieved by the binding of the PCL1 N-terminal PHD domain to the C-terminal domain of p53 through two unique serine residues, which were acquired during recent vertebrate evolution. This study illustrates the functional bifurcation of PCL proteins, which act in both a chromatin-dependent and a chromatin-independent manner to regulate the INK4A and p53 pathways

A chromatin-independent role of polycomb-like 1 to stabilize p53 and promote cellular quiescence

Polycomb-like proteins 1-3 (PCL1-3) are substoichiometric components of the Polycomb-repressive complex 2 (PRC2) that are essential for association of the complex with chromatin. However, it remains unclear why three proteins with such apparent functional redundancy exist in mammals. Here we characterize their divergent roles in both positively and negatively regulating cellular proliferation. We show that while PCL2 and PCL3 are E2F-regulated genes expressed in proliferating cells, PCL1 is a p53 target gene predominantly expressed in quiescent cells. Ectopic expression of any PCL protein recruits PRC2 to repress the INK4A gene; however, only PCL2 and PCL3 confer an INK4A-dependent proliferative advantage. Remarkably, PCL1 has evolved a PRC2- and chromatin-independent function to negatively regulate proliferation. We show that PCL1 binds to and stabilizes p53 to induce cellular quiescence. Moreover, depletion of PCL1 phenocopies the defects in maintaining cellular quiescence associated with p53 loss. This newly evolved function is achieved by the binding of the PCL1 N-terminal PHD domain to the C-terminal domain of p53 through two unique serine residues, which were acquired during recent vertebrate evolution. This study illustrates the functional bifurcation of PCL proteins, which act in both a chromatin-dependent and a chromatin-independent manner to regulate the INK4A and p53 pathways

Delineating transcriptional networks of prognostic gene signatures refines treatment recommendations for lymph node-negative breast cancer patients.

The majority of women diagnosed with lymph node-negative breast cancer are unnecessarily treated with damaging chemotherapeutics after surgical resection. This highlights the importance of understanding and more accurately predicting patient prognosis. In the present study, we define the transcriptional networks regulating well-established prognostic gene expression signatures. We find that the same set of transcriptional regulators consistently lie upstream of both 'prognosis' and 'proliferation' gene signatures, suggesting that a central transcriptional network underpins a shared phenotype within these signatures. Strikingly, the master transcriptional regulators within this network predict recurrence risk for lymph node-negative breast cancer better than currently used multigene prognostic assays, particularly in estrogen receptor-positive patients. Simultaneous examination of p16(INK4A) expression, which predicts tumours that have bypassed cellular senescence, revealed that intermediate levels of p16(INK4A) correlate with an intact pRB pathway and improved survival. A combination of these master transcriptional regulators and p16(INK4A), termed the OncoMasTR score, stratifies tumours based on their proliferative and senescence capacity, facilitating a clearer delineation of lymph node-negative breast cancer patients at high risk of recurrence, and thus requiring chemotherapy. Furthermore, OncoMasTR accurately classifies over 60% of patients as 'low risk', an improvement on existing prognostic assays, which has the potential to reduce overtreatment in early-stage patients. Taken together, the present study provides new insights into the transcriptional regulation of cellular proliferation in breast cancer and provides an opportunity to enhance and streamline methods of predicting breast cancer prognosis

Gustav Mahler: Iz čudesnog dječakovog roga (studenti Muzičke akademije Sveučilišta u Zagrebu, 15. 2. 2019.)

Snimka izvedbe održane 15. 2. 2019. u Koncertnoj dvorani "Blagoje Bersa". U realizaciji projekta sudjelovale su Muzička akademija, Akademija likovnih umjetnosti i Akademija dramske umjetnosti Sveučilišta u Zagrebu te Hochschule der Künste Bern. Izvođači: komorni ansambl Muzičke akademije Sveučilišta u Zagrebu (Lara Rimac (violina), Urban Jerman (violina), Kristina Knežević (viola), Stin Lebar (violončelo), Nives Županić Tica (flauta, piccolo), Antonio Haller (oboa, engleski rog), Matej Fridl (rog), Peter Firšt (truba), Hrvoje Štimac (harmonika), Brin Bernatović (harfa), Šimun Matišić (udaraljke), Rupert Čunko (udaraljke)), solisti Nikolina Hrkać (sopran), Tjaša Zidanič (mezzosopran), Mia Gazdović (sopran), Jurica Jurasić Kapun (bariton), Tena Lončarević (sopran), Marko Antolković (kontratenor), Maja Sremec (sopran), Patricia Žudetić (sopran), Marin Čargo (bariton), Rafaela Čanžar (mezzosopran), Ivana Miletić (sopran), Melis Vlahović (sopran), Lucija Ercegovac (mezzosopran) i Emilia Rukavina (mezzosopran). Dirigent: izv. prof. art. Pavle Zajcev. Mentori: red. prof. art. Dunja Vejzović, izv. prof. art. Pavle Zajcev, red. prof. art. Mladen Tarbuk. Suradnica za scenski pokret: nasl. v. pred. Mateja Pučko Petković. Kostimografija: Barbara Repe. Redatelj: red. prof. art. Mathias Behrends. Program: Gustav Mahler: Iz čudesnog dječakovog roga, 18 scena za glas i komorni ansambl (priredio: Mladen Tarbuk). 1. Kako se ćutim (solistice: Nikolina Hrkać (uloga: Luda), Tjaša Zidanič (uloga: Doktor)); 2. Jaka moć uživljavanja (solisti: Mia Gazdović (uloga: Djevojka), Jurica Jurasić Kapun (uloga: Dječak)); 3. Utjeha u nesreći (solisti: Tena Lončarević (uloga: Ona), Jurica Jurasić Kapun (uloga: On)); 4. Pjesma progonjenog u tornju (solisti: Marko Antolković (uloga: Zatočenik), Maja Sremec (uloga: Djevojka)); 5. Zaludu trud (solisti: Patricia Žudetić (uloga: Ona), Marin Čargo (uloga: On)); 6. Dok hodah zelenom šumom (solisti: Maja Sremec (uloga: Ona), Marko Antolković (uloga: On)); 7. Ne doviđenja! (solisti: Marko Antolković (uloga: On), Rafaela Čanžar (uloga: Komentatorica), Nikolina Hrkać (uloga: Ona)); 8. Noćna pjesma stražara (solisti: Jurica Jurasić Kapun (uloga: On), Tjaša Zidanič (uloga: Djevojka), Maja Sremec (uloga: Komentatorica)); 9. Pohvala visokoumne procjene (solisti: Patricia Žudetić (uloga: komentatorica), Nikolina Hrkać (uloga: Kukavica), Marin Čargo (uloga: Magarac)); 10. Mala priča o Rajni (solisti: Jurica Jurasić Kapun (uloga: On), Ivana Miletić (uloga: Ona)); 11. Zemaljski život (solistice: Ivana Miletić (uloga: Dijete), Rafaela Čanžar (uloga: Majka), Tena Lončarević (uloga: Komentatorica)); 12. Tko je smislio ovu pjesmicu (solistica: Patricia Žudetić (uloga: Ona)); 13. Tamo gdje lijepe trublje sviraju (solisti: Melis Vlahović (uloga: Djevojka), Mia Gazdović (uloga: Komentatorica), Marin Čargo (uloga: Vojnik)); 14. Rastanak u ljeto (solistica: Nikolina Hrkać); 15. Nebeski život (solistice: Mia Gazdović, Patricia Žudetić, Tena Lončarević, Maja Sremec, Lucija Ercegovac); 16. U boj (solisti: Jurica Jurasić Kapun (uloga: Vojnik), Rafaela Čanžar (Komentatorica)); 17. Tri anđela pjevahu (solistica: Lucija Ercegovac); 18. Prasvjetlo (solistica: Emilia Rukavina)

Gustav Mahler: Iz čudesnog dječakovog roga (studenti Muzičke akademije Sveučilišta u Zagrebu, 15. 2. 2019.)

Snimka izvedbe održane 15. 2. 2019. u Koncertnoj dvorani "Blagoje Bersa". U realizaciji projekta sudjelovale su Muzička akademija, Akademija likovnih umjetnosti i Akademija dramske umjetnosti Sveučilišta u Zagrebu te Hochschule der Künste Bern. Izvođači: komorni ansambl Muzičke akademije Sveučilišta u Zagrebu (Lara Rimac (violina), Urban Jerman (violina), Kristina Knežević (viola), Stin Lebar (violončelo), Nives Županić Tica (flauta, piccolo), Antonio Haller (oboa, engleski rog), Matej Fridl (rog), Peter Firšt (truba), Hrvoje Štimac (harmonika), Brin Bernatović (harfa), Šimun Matišić (udaraljke), Rupert Čunko (udaraljke)), solisti Nikolina Hrkać (sopran), Tjaša Zidanič (mezzosopran), Mia Gazdović (sopran), Jurica Jurasić Kapun (bariton), Tena Lončarević (sopran), Marko Antolković (kontratenor), Maja Sremec (sopran), Patricia Žudetić (sopran), Marin Čargo (bariton), Rafaela Čanžar (mezzosopran), Ivana Miletić (sopran), Melis Vlahović (sopran), Lucija Ercegovac (mezzosopran) i Emilia Rukavina (mezzosopran). Dirigent: izv. prof. art. Pavle Zajcev. Mentori: red. prof. art. Dunja Vejzović, izv. prof. art. Pavle Zajcev, red. prof. art. Mladen Tarbuk. Suradnica za scenski pokret: nasl. v. pred. Mateja Pučko Petković. Kostimografija: Barbara Repe. Redatelj: red. prof. art. Mathias Behrends. Program: Gustav Mahler: Iz čudesnog dječakovog roga, 18 scena za glas i komorni ansambl (priredio: Mladen Tarbuk). 1. Kako se ćutim (solistice: Nikolina Hrkać (uloga: Luda), Tjaša Zidanič (uloga: Doktor)); 2. Jaka moć uživljavanja (solisti: Mia Gazdović (uloga: Djevojka), Jurica Jurasić Kapun (uloga: Dječak)); 3. Utjeha u nesreći (solisti: Tena Lončarević (uloga: Ona), Jurica Jurasić Kapun (uloga: On)); 4. Pjesma progonjenog u tornju (solisti: Marko Antolković (uloga: Zatočenik), Maja Sremec (uloga: Djevojka)); 5. Zaludu trud (solisti: Patricia Žudetić (uloga: Ona), Marin Čargo (uloga: On)); 6. Dok hodah zelenom šumom (solisti: Maja Sremec (uloga: Ona), Marko Antolković (uloga: On)); 7. Ne doviđenja! (solisti: Marko Antolković (uloga: On), Rafaela Čanžar (uloga: Komentatorica), Nikolina Hrkać (uloga: Ona)); 8. Noćna pjesma stražara (solisti: Jurica Jurasić Kapun (uloga: On), Tjaša Zidanič (uloga: Djevojka), Maja Sremec (uloga: Komentatorica)); 9. Pohvala visokoumne procjene (solisti: Patricia Žudetić (uloga: komentatorica), Nikolina Hrkać (uloga: Kukavica), Marin Čargo (uloga: Magarac)); 10. Mala priča o Rajni (solisti: Jurica Jurasić Kapun (uloga: On), Ivana Miletić (uloga: Ona)); 11. Zemaljski život (solistice: Ivana Miletić (uloga: Dijete), Rafaela Čanžar (uloga: Majka), Tena Lončarević (uloga: Komentatorica)); 12. Tko je smislio ovu pjesmicu (solistica: Patricia Žudetić (uloga: Ona)); 13. Tamo gdje lijepe trublje sviraju (solisti: Melis Vlahović (uloga: Djevojka), Mia Gazdović (uloga: Komentatorica), Marin Čargo (uloga: Vojnik)); 14. Rastanak u ljeto (solistica: Nikolina Hrkać); 15. Nebeski život (solistice: Mia Gazdović, Patricia Žudetić, Tena Lončarević, Maja Sremec, Lucija Ercegovac); 16. U boj (solisti: Jurica Jurasić Kapun (uloga: Vojnik), Rafaela Čanžar (Komentatorica)); 17. Tri anđela pjevahu (solistica: Lucija Ercegovac); 18. Prasvjetlo (solistica: Emilia Rukavina)