DOTTORATO DI RICERCA IN BIOLOGIA CELLULARE E DELLO SVILUPPO

(da presentare entro il 15/06/2015)

Proposta di progetto di Dottorato Titolo della ricerca: Selezione di inibitori delle demetilasi istoniche e loro utilizzo per comprendere il ruolo della metilazione della lisina 4 dell’istone H3 nella regolazione trascrizionale. Docente guida proposto: Rodolfo Negri

DESCRIZIONE DELLA RICERCA Obiettivi della ricerca (max 4000 car.)

We recently discovered in S.cerevisiae an interesting conditional negative genetic interaction between the

unique JARID histone demethylase Jhd2, responsible for H3K4 demethylation, and Not4, a protein which is

involved in several different regulatory processes, including transcriptional regulation, RNA stability and

Jhd2 degradation. The double deletion mutant jhd2/not4 is hypersensitive to rapamycin and its sensitivity

is promptly suppressed by episomal Jhd2 expression in the double deletion strain. While this genetic

interaction could be useful for the understanding of the transcriptional role of Jhd2 in yeast, which is still

elusive, it is also an ideal system for in vivo screening of inhibitors specific for JARID demethylases. In a

pilot screening on 45 candidate small molecules, we identified a compound which specifically inhibits Jhd2

in vivo, leading to a consistent increase in trimethyl-H3K4. The compound inhibits human JARID 1B and

1D in vitro and shows a strong cytostatic effect, a mild cytotoxicity and a selective increase of trimethyl-

H3K4 in HeLa cells. We propose to better characterize the inhibitor’s effects in yeast and mammalian cells

(in particular for what concerns the cytostatic effect); to extend the screening to new candidate molecules

and to investigate on the meaning of the genetic interaction between Jhd2 and Not4 which could enlighten

some aspects of the role of Jhd2 in transcription.

Task 1: Analyzing the specificity and the effects of H3K4 methylation inhibitor 3195 in yeast and MCF7

breast cancer cells which over-express JARID 1B.

Transcriptomic effects will be evaluated by RNA seq in MCF7 cells at various times from inhibitor addition.

For this subtask we will use an RNA-Seq strategy to detect the complete coding and non-coding

transcriptome. For selected regulated targets we will analyze the main chromatin modifications and the occupancy of JARID

1B on promoters and coding regions at various time points following inhibitor addition by ChIP.

Task 2: Analyzing the role of S.cerevisiae Jhd2 in Diauxic and post-diauxic phase of growth:

One of the advantage of using chemical inhibitors rather than genetic mutants is that an enzymatic activity

can be promptly inhibited in particular physiological states. Jhd2 expression appears to be tightly regulated

in S.cerevisiae. Transcriptomic experiments (http://www.yeastgenome.org/cgi-

bin/expression/expressionConnection.pl?locus=YJR119C&type=summary ) show that the gene is

strongly induced during diauxic shift and in the post-diauxic phase. In order to understand the

possible role of its regulatory action in these conditions, we plan to analyze the transcriptome and

the pattern of H3K4 tri-methylation by ChIPseq in presence and absence of the inhibitor. These

experiments could complement the analysis of the jhd2 strain which could be biased by adaptive

phenomena.

Task 4: Screening for new JARID inhibitors.

A second generation group of molecules will be designed on the base of the structure of the 3195 compound.

Candidate molecules will be tested on purified Jarid 1B and 1D for enzyme inhibition. Successful candidates

will be analyzed together with the 3195 compound in Task 1.

Task 5: Biochemical characterization of candidate inhibitors

Molecules selected in task4 together with compound 3195 will be tested for association with purified Jarid

1B and 1D in presence or absence of peptides carrying H3K4 in different methylation states. The

experiments will be carried out with a Biacore apparatus (GE Healtcare) and the rates of complex formation

(ka) and dissociation (kd),will be determined.

Stato delle conoscenze e referenze (max 4000 car.)

Chromatin structure governs several aspects of cell metabolism. Histone N-terminal tails are

subjected to several covalent modifications which form a sophisticated combinatory code interpreted by a

plethora of regulatory protein complexes. Among the various modifications, Lysine (K) methylation is

particularly interesting, due to its widespread roles in transcriptional regulation, DNA repair and epigenetic

inheritance. For many years, histone lysine methylation has been considered irreversible and persisting

through cell division. Recently, two families of histone demethylating enzymes (HDMs) have been identified

in eukaryotes: the LSD1 family and the Jmjc-domain-containing family. The LSD1 HDMs are monoamine

oxidases that can demethylate mono- and di-methylated H3K4 and H3K9 and require flavin adenine

dinucleotide (FAD) for their function. On the other hand, Jumonji C domain-containing HDMs (JHDMs), 5

members in S.cerevisiae and at least 28 members in H.sapiens, are Fe2+ and -ketoglutarate-dependent

hydroxylases, and their reported substrate residues include H3K4, H3K9, H3K27, and H3K36 at all

methylation states. JHDMs are potential therapeutic cancer targets and among them, those capable to

demethylate specifically H3K4 (JARID 1A-1D,) look particularly interesting. Indeed, these enzymes are

strictly associated with human cancer and their mode of action seems to involve epigenetic mechanisms of

transcriptional regulation. Although it is relatively easy to screen for JHDMs inhibition in vitro, predicting in

vivo efficacy on the base of in vitro inhibitory activity is not straightforward.

In order to discover inhibitors specific for H3K4 histone demethylation we set up a screening system which

tests the effects of candidate small molecules inhibitors on a S.cerevisiae mutant strain which requires Jhd2

demethylase activity to efficiently grow in the presence of rapamycin. In order to validate the system we

screened a library of 45 structurally different compounds designed as competitive inhibitors of -

ketoglutarate (-KG) cofactor of the enzyme, and found that one of them, compound RS3195, inhibited Jhd2

activity in vitro and in vivo. The same compound effectively inhibits human JARID 1B and 1D in vitro and

increases H3K4 tri-methylation in HeLa cells nuclear extracts. When added in vivo to HeLa cells, the

compound leads to an increase of tri-methyl-H3K4 but does not significantly affect H3K9 and H3K27 tri-

methylation. On the same cells we observed a strong cytostatic effect at 30 M, concentration at which

around 47% of the cells remained blocked in G2/M. At the same concentration, compound RS3195 induced

a mild cytoxicity, and a moderate apoptogenic effect. Similar effects were observed in the MCF7 breast

cancer cell line which over-expresses JARID 1B whose K4-demethylase activity appears related to cancer

cells proliferation. In order to better understand the role of JARID 1B in oncogenesis we are currently testing

the transcriptomic effects of RS3195 and other demethylase inhibitors in the MCF7 cell line. In conclusion,

the inhibitor RS3195, differently from other known inhibitors which provoke a general increase of

methylation at all H3 lysine residues, appears to be specific for H3K4 demethylation in vivo. A direct

relationship between this inhibitory action and the observed cytostatic effect as well as RS3195 in depth

mechanism of action still remain to be fully elucidated. Our selection system may provide a new robust tool

for the discovery of effective H3K4-specific HDM inhibitors.

Lavori pubblicati negli ultimi 5 anni dal Docente Guida (2010-2015) 1 - MANNIRONI C, PROIETTO M, BUFALIERI F, CUNDARI E, ALAGIA A, DANOVSKA S, RINALDI T, FAMIGLINI V, COLUCCIA A, LA REGINA G, SILVESTRI R, NEGRI R. (2014) An High-Throughput In Vivo Screening System to Select H3K4-Specific Histone

Demethylase Inhibitors. PLoS One. 9(1):e86002. ISSN: 1932-6203 doi: 10.1371/journal.pone.0086002. eCollection 2014. PMID: 24489688. 2 - V. LICURSI, C. SALVI, V. DE CESARE, T RINALDI, B. MATTEI, C. FABBRI, G. SERINO, L. BRAMASOLE, J. ZIMBLER, E. PICK, B. BARNES, M. BARD, R. NEGRI (2014). The Cop9 signalosome is involved in the regulation of lipid metabolism and of transition metals uptake in S. cerevisiae. THE FEBS JOURNAL, vol. 281, p. 175-190, ISSN: 1742-464X, doi: 10.1111/febs.12584 PMID: 24164706. 3 - S. PIERSANTI, L. ASTROLOGO, V. LICURSI, R. COSTA, E. RONCAGLIA, A. GENNETIER, S. IBANES, M. CHILLON, R. NEGRI, E. TAGLIAFICO, EJ. KREMER, I. SAGGIO (2013). Differentiated neuroprogenitor cells incubated with human or canine adenovirus, or lentiviral vectors have distinct transcriptome profiles. PLoS One, vol. 8, p. e69808, ISSN: 1932-6203, doi: 10.1371 PMID: 23922808. 4 - F BUFALIERI, V LICURSI, M D'ANTONIO, T CASTRIGNANÒ, R AMENDOLA, R NEGRI (2012). The transcriptional response of mammalian cancer cells to irradiation is dominated by a cell cycle signature which is strongly attenuated in non-cancer cells and tissues. INTERNATIONAL JOURNAL OF RADIATION BIOLOGY, 88, 822-829. ISSN: 0955-3002 doi: 10.3109/09553002.2012.676230. Epub 2012 Apr 26.PMID: 22420862. 5 - Mardente S, Mari E, Consorti F, Di Gioia C, Negri R, Etna M, Zicari A, Antonaci A. (2012). HMGB1 induces the overexpression of miR-222 and miR-221 and increases growth and motility in papillary thyroid cancer cells. THE ONCOLOGY REPORT, vol. 28(6), p. 2285-2289, ISSN: 1548-5323, doi: 10.3892/or.2012.2058 PMID: 23023232. 6 - M. Cestelli Guidi, C. Mirri, E. Fratini, V. Licursi, R. Negri, A. Marcelli, R. Amendola (2012). In vivo skin leptin modulation after 14 MeV neutron irradiation: a molecular and FT-IR spectroscopic study. ANALYTICAL AND BIOANALYTICAL CHEMISTRY, 404, 1317-1326. ISSN: 1618-2642 doi: 10.1007/s00216-012-6018-3. Epub 2012 Apr 29. PMID: 22543714. 7 - FRATINI E, LICURSI V, ARTIBANI M, KOBOS K, COLAUTTI P, R. NEGRI, AMENDOLA R (2011). Dose-Dependent Onset of Regenerative Program in Neutron Irradiated Mouse Skin. PLoS One, vol. 6, p. e19242 -1-e19242 -12, ISSN: 1932-6203, doi: 10.1371/journal.pone.001924 8 – LICURSI V, CAIELLO I, LOMBARDI L, DE STEFANO ME, NEGRI R, PAGGI P (2012). Lack of dystrophin in mdx mice modulates the expression of genes involved in neuron survival and differentiation. EUROPEAN JOURNAL OF NEUROSCIENCE, 35, 691-701. ISSN: 0953-816X doi: 10.1111/j.1460-9568.2011.07984.x. Epub 2012 Feb 7. PMID: 22309284. 9 - BOSIO MC, NEGRI R., DIECI G (2011). Promoter architectures in the yeast ribosomal expression program. TRANSCRIPTION, vol. 2; p. 71-77, ISSN: 2154-1264, doi: 10.4161/trns.2.2.14486 10 - VINCENTI S, BRILLANTE N, LANZA V, BOZZONI I, PRESUTTI C, CHIANI F, ETNA MP, NEGRI R. (2011). HUVEC Respond to Radiation by Inducing the Expression of Pro-angiogenic MicroRNAs. RADIATION RESEARCH, vol. 175; p. 535-546, ISSN: 0033-7587, doi: 10.1667/RR2200.1 11 - Piccinni E., Chelstowska A., Hanus J., Widlak P., Loreti S., Tata A.M., Augusti-Tocco G., Bianchi M.M., Negri R. (2011). Direct interaction of Gas41 and Myc encoded by amplified genes in nervous system tumours. ACTA BIOCHIMICA POLONICA, vol. 58, p. 529-534, ISSN: 0001-527X PMID: 22068108. 12 - G. Romagnoli, E. Cundari, R. Negri, M. Crescenzi, L. Farina, A. Giuliani, M. Bianchi (2011). Synchronous protein cycling in batch cultures of the yeast Saccharomyces cerevisiae at log growth phase. EXPERIMENTAL CELL RESEARCH, 317. ISSN: 0014-4827 doi: 10.1016/j.yexcr.2011.09.007. Epub 2011 Sep 24. PMID: 21978910.

Fondi disponibili per svolgere il programma di ricerca. I fondi devono essere chiaramente indicati in modo da permettere alla commissione di valutare la loro effettiva disponibilità per svolgere il programma indicato.

- Istituto Pasteur Fondazione Cenci Bolognetti Grant 2014-2016 (60000 Euro) "In vivo selection of JARID histone demethylases inhibitors and their use to enlighten the biological role of these enzymes in yeast and mammalian cells with focus on transcriptional regulation."

- International FIRB: Molecular basis of diseases (2010-2015) (grant no. RBIN06E9Z8) –Coordinatore: Prof. Paolo Amati Budget per Unità operativa Negri: 54000 euro.

- Progetto di Ateneo 2014 “In vivo selection of JARID histone demethylases inhibitors and their use to enlighten the biological role of these enzymes in yeast and mammalian cells with focus on transcriptional regulation.” Budget: 7000 euro.

Pending

Progetto di Ateneo 2015 “In vivo selection of JARID histone demethylases inhibitors and their use to enlighten the biological role of these enzymes in yeast and mammalian cells with focus on transcriptional regulation.” Richiesta: 15000 euro.

Collaborazioni con laboratori nazionali ed internazionali Romano Silvestri – Professore ordinario - Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza

Università di Roma.

Cecilia Mannironi - ricercatrice – IBPM, CNR, Roma

Gianni Colotti – ricercatore – IBPM, CNR, Roma

Enrico Cundari – ricercatore – IBPM, CNR, Roma