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Prof. Claudio Schneider
Email: claudio.schneider@Lncib.it

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Prof. Roberto Gambari
Email: roberto.gambari@unife.it

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Elisabetta Lambertini
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Fax: 0532/974484
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Caiafa Paola Stampa
RESPONSABILE DELLA U. O.

Cognome e Nome Caiafa Paola
Qualifica Full Professor
Facoltà Medicine2, University of Rome ‘La Sapienza’, 00161
Dipartimento
Department of Cellular Biotechnology and Haematology
Settore Scientifico Disciplinare BIO/12Clinical Biochemistry and Clinical Molecular Biology
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PERSONALE STRUTTURATO

Cognome e Nome
CAIAFA PAOLA
Qualifica PO
Dipartimento Department of Cellular Biotechnology and Haematology
Ente di appartenenza Medicine2, University of Rome ‘La Sapienza’, 00161 Rome, Italy
Cognome e Nome
PASSANANTI CLAUDIO
Qualifica Technologist
Dipartimento
Molecular Biology and Pathology
Ente di appartenenza CNR, Rome, Italy
Cognome e Nome
REALE ANNA
Qualifica
PA
Dipartimento Department of Cellular Biotechnology and Haematology
Ente di appartenenza Medicine2, University of Rome ‘La Sapienza’, 00161 Rome, Italy
Cognome e Nome
Qualifica
Dipartimento
Ente di appartenenza
Cognome e Nome
Qualifica
Dipartimento
Ente di appartenenza
Cognome e Nome
Qualifica
Dipartimento
Ente di appartenenza

PERSONALE NON STRUTTURATO

Cognome e Nome CECCHINELLI BARBARA
Qualifica Post-doc yellow (Faculty of Medicine2)
Dipartimento Department of Cellular Biotechnology and Haematology
Ente di appartenenza
Medicine2, University of Rome ‘La Sapienza’, 00161 Rome, Italy
Cognome e Nome
MICHELE ZAMPIERI
Qualifica
PhD student
Dipartimento Department of Cellular Biotechnology and Haematology
Ente di appartenenza
Medicine2, University of Rome ‘La Sapienza’, 00161 Rome, Italy
Cognome e Nome
RIGGIO GIUSEPPE
Qualifica
Post-doc fellow (FIRB)
Dipartimento Department of Cellular Biotechnology and Haematology
Ente di appartenenza
Medicine2, University of Rome ‘La Sapienza’, 00161 Rome, Italy
Cognome e Nome
TIZIANA GUASTAFIERRO
Qualifica
PhD student
Dipartimento Department of Cellular Biotechnology and Haematology
Ente di appartenenza
Medicine2, University of Rome ‘La Sapienza’, 00161 Rome, Italy
Cognome e Nome
Qualifica
Dipartimento
Ente di appartenenza
Cognome e Nome
Qualifica
Dipartimento
Ente di appartenenza

LINEE DI RICERCA

Task 1: Poly(ADP-ribosyl)ation in the control of unmethylated state of CpG islands.
There is evidence in vivo that this modification plays a regulatory role in protecting genomic DNA methylation pattern (Zardo et al. 1997, de Capoa et al 1999, Zardo et al. 1999), particularly in maintaining the unmethylated state of CpG islands (Zardo and Caiafa 1998) which are located in the promoters of constitutively expressed housekeeping genes (Antequera and Bird 1993, Antequera and Bird 1994,Takai and Jones 2002). In fact, it has been found that competitive inhibition of poly(ADP-ribose) polymerases leads to introduction of anomalous methyl groups onto DNA and in particular onto CpG islands. The molecular mechanism involved in the interplay between poly(ADP-ribosyl)ation, and DNA methylation pattern is still enigmatic, but in vitro and in vivo experiments suggest two hypotheses to explain how these two epigenetic modifications are correlated. The first hypothesis is based on data (Zardo et al. 2002) showing that inhibition of PARP activity at different cell-cycle phases increases the mRNA and protein levels of the major maintenance DNA methyltransferase (Dnmt1) in G1/S phase and this increase of Dnmt1 results in increased PCNA-Dnmt1 complex formation, which allows maintenance as well as de novo DNA methylation processes during this anomalous phase. The early formation of the PCNA-Dnmt1 complex in G1/S phase could be able to modify the unmethylated state of the promoter regions in housekeeping genes (CpG islands) that are present in early replicating DNA (Delgado et al. 1998, Antequera and Bird 1999, Antequera 2003). A second hypothesis suggests that PARP-1 in its poly(ADP-ribosyl)ated form makes Dnmt1 catalytically inactive. Dnmt1 has two possible consensus aminoacid domains for non covalent binding with ADP-ribose polymers and in vitro experiments have shown that Dnmt1 links non covalently ADP-ribose polymers and that dsDNA 30 times more concentrated does not remove the link. In addition, ADPR-polymers, present on modified PARP-1, almost completely inhibit the catalytic activity of Dnmt1 while unmodified PARP-1 is not able to inhibit the enzyme. These data, added to the fact that the two proteins co-immunoprecipitate in vivo and that PARP-1 is in its modified form in this complex, suggest that modified PARP1, trapping Dnmt1 through ADP-ribose polymers, is responsible for the catalytic inactivation of the enzyme in chromatin (Reale et al. 2005). Through this mechanism the non-methylated state of CpG islands could be protected and a functional anomaly in the poly(ADP-ribosyl)ation process could therefore be responsible for new aberrant methylation. Thus, inhibition of PARP activity could allow new methyl groups to be inserted onto DNA both during replication and in chromatin: during replication because inhibition of PARP activity induces Dnmt1 overexpression in G1/S phase and increases the formation of the active complex PCNA-Dnmt1 in this anomalous phase and in chromatin because unmodified PARP1 is unable to inhibit Dnmt1. Our unpublished data (manuscript in preparation) have shown that poly(ADP-ribosyl)ation is involved in the control of Dnmt1 expression, and therefore in the control of level of the enzyme particularly responsible for DNA methylation process. In fact, expression and level of Dnmt1 are time-dependent on the treatment of cells with 3-aminobenzamide - a competitive inhibitor of PARP activity - so much so that short times of treatment induce an over-expression of Dnmt1 while longer times lead to its silencing. A parallel examination of all CpG islands showed that inhibition of PARP activity till is accompanied by a decrease in the “tiny DNA fragments” which are formed after HpaII digestion of genomic DNA, phenomenon which is reversed following prolonged treatment. In this scenario, which sees the spreading of anomalous methylation on CpG island regions, we found that the island that is present in the promoter of Dnmt1 undergoes methylation leading to the silencing of the enzyme which goes with reintroduction of unmethylated state on CpG islands. In this scenario our data show that a deregulation of PARP activity allows introduction of anomalous methyl groups on the promoter region of Dnmt1 and induces a deregulation of Dnmt1 activity that in turn could be responsible of a diffuse DNA hypomethylation. Although, at this stage, our research does not provide a direct correlation between poly(ADP-ribosyl)ation and cancer, it demonstrates that the deregulation of PARP activity is able to modify the methylation state of some CpG islands, an event that recalls what happens in promoter regions of tumour suppressor genes, which become methylated during tumourigenesis in cancer cells. Above all these data suggest that the hypomethylation, which is present on genomic DNA of some tumour cells, could be correlated to the observed spreading of methylation which causes the silencing of Dnmt1, the enzyme involved in maintenance methylation. The consequent silencing of Dnmt1 could in fact generate later the diffuse hypomethylation which is present in some tumours. Much research must be carried out before attributing such a role in carcinogenesis to poly(ADP-ribosyl)ation.
The aim is:
- to identify what genes, apart from Dnmt1, come under the control of this epigenetic modification and this in order to find a common denominator. Attention will be focused on some oncosuppressor genes that are anomalously methylated in cancer. The idea is to select genes that frequently undergo CpG island methylation in human cancer considering also their different biological functions: p16INK4a, p14ARF, p15 INK4b, hMLH1, MGMT, p73, ER, RASSF1, Rb, SOCS-1, DAPK and E-cad. Recently, subsets of CpG islands which are, regarding overexpression of DNMT1, methylation prone or methylation resistent for intrinsic differences in their sequences have been individuated. This allowed us to focus our attention on genes containing sequences which are methylation prone in condition of DNMT1 overexpression (i.e. as happens following PARP inhibition). One of them, CDX2, which is involved in growth arrest and is found down-regulated in colon carcinomas and in tumour cell lines, appears particularly useful to verify our model. Anyway genes to examine will be selected by Northen-Blot and gene microarray methods We will examine the methylation patterns of these genes on DNAs purified from mouse and human fibroblasts treated or not with competitive inhibitors of PARP activity or where PARP1 and/or PARP2 were previously silenced by (RNAi).
-- to establish if ADP-ribose polymers free or present on a specific member(s) of PARP family are by themselves involved in this control or if PARP activity acts through the modification of a proteic factor(s) that is involved in regulation of the promoter region of Dnmt1 gene or of another gene whose product is, in turn, involved in the regulation of Dnmt1 expression;
- to verify whether some repetitive sequences and /or oncogenes, which undergo hypomethylation in tumour cells, are demethylated following prolonged PARP activity inhibition.
Task 2: Are DNA methylation and poly(ADP-ribosyl)ation interconnected in the control of imprinting?
As study model for molecular mechanism(s) involved in the control of imprinting, the Hfg2/H19 locus is often used Here the two genes are adjacent and share the same enhancer region with the peculiarity that while H19 is expressed only by the paternal allele, Hfg2 is expressed only by the maternal one. Between the two genes there is a region of DNA termed insulator that, acting as boundary, plays a controlling role over the enhancer region. Inside insulator there is the imprinting control region (ICR) where consensus sequences for the ubiquitous CTCF protein are located. It is well known that in the regulation of imprinting an important role is played by CTCF protein through its link on consensus sequences that are located on insulator and in particular inside imprinting control region (ICR). It is accepted that the methylation pattern of these consensus regions allows or not the linking with CTCF as this protein is capable of binding only if this region is unmethylated. It has recently been shown that poly(ADP-ribosyl)ation also plays a role in the control of imprinting. In fact, when CTCF binds consensus sequences it is covalently poly(ADP-ribosyl)ated and polymers linked to it are such as to modify the molecular weight from 130 to 180 kDa. Following treatment of cells with 3-ABA, thus in condition of PARP activity inhibition, the binding is destabilized. Our awareness that DNA methylation and poly(ADP-ribosyl)ation are connected, leads us to verify if the two epigenetic modifications are interconnected also in the regulation of imprinting. Experiments will be performed using as a model of study the genic locus H19/Igf2 in MEF SD7/Dom cells which are heterozygotes for a lot of sequence variations (Pedone et al.FEBS Letters 458,45-50. 1999;Sperandeo et al., Am. J. Hum. Genet. 66, 841-847).
The aim is:
-to verify that inhibition of PARP activity does not lead to introduction of anomalous methyl groups into CTCF s consensus regions. Expression level of imprinted genes will be analyzed by Real Time PCR and Western-Blot while methylation pattern of consensus regions will be examined as described in METHODS which follow.
-to verify if ADP-ribose polymers covalently linked on CTCF are there with the purpose of binding non-covalently Dnmt1, thus inhibiting its enzymatic activity and guaranteeing the unmethylated state of consensus regions. Coimmunoprecipitation method will be used to verify this hypothesis. In preliminary experiments cellular level of CTCF will be increased by transfecting an expression vector for CTCF.
-to verify if CTCF units that are tied on ICR situs are kept together by non covalent binding with ADP-ribose polymers. This hypothesis stems from the fact that a consensus aminoacidic region required for non covalent association with ADP-ribose polymers, is present in C-terminal region of CTCF protein.

TECNOLOGIE IN POSSESSO DELL'U. O.

  • Determination of gene expression by Northern blot, RT-PCR, Real-Time PCR and Western-Blot
  • Over-expression and silencing of genes
  • PARP and Dnmts activity
  • Determination of methylation level by DNA sequencing and Methylation-Specific PCR (MSP)
  • Polymer-blot analysis
  • Protein coimmunoprecipitation
  • Chromatin immunoprecipitation (ChIP)
  • Analysis of “tiny DNA fragments” which are formed after HpaII digestion of genomic DNA
  • Biochemical techniques

STRUMENTAZIONE

Denominazione
Struttura ove la strumentazione è allocata
Responsabile della strumentazione
CO2 incubator for cell cultures, hood for cell culture, inverted microscope
Department of Cellular Biotechnology and Haematology
Sezione Biochimica Clinica
Beckman refrigerate ultracentrifuge
Department of Cellular Biotechnology and Haematology
Sezione Genetica Molecolare
Becton Dickinson FACSCalibur cytofluorimeter
Department of Cellular Biotechnology and Haematology
Sezione Genetica Molecolare
Fluorescence microscope
Department of Cellular Biotechnology and Haematology
Sezione Genetica Molecolare
Real Time PCR
Department of Cellular Biotechnology and Haematology
Sezione Biochimica Clinica

PUBBLICAZIONI

1. REALE A., DEMATTEIS G., GALLEAZZI G., ZAMPIERI M., CAIAFA P. (2005). MODULATION OF DNMT1 ACTIVITY BY ADP-RIBOSE POLYMERS. ONCOGENE. vol. 24 pp. 13-19 ISSN: 0950-9232
2. CAIAFA P., ZAMPIERI M. (2005). DNA methylation and Chromatin: the puzzling CpG islands. JOURNAL OF CELLULAR BIOCHEMISTRY. vol. 94 pp. 257-265 ISSN: 0730-2312
3. CORBI N, BRUNO T, DE ANGELIS R, DI PADOVA M, LIBRI V, DI CERTO MG, SPINALDI L, FLORIDI A, FANCIULLI M, PASSANANTI C. (2005) RNA polymerase II subunit 3 is retained in the cytoplasm by its interaction with HCR, the psoriasis vulgaris candidate gene product. J CELL SCI. :4253-4260.
4. CAIAFA P. (2005). PARP and epigenetic regulation. In ALEXANDER BURKLE Poly(ADP-ribosyl)ation (vol. Chaper 9).: Landes Bioscience, Eurekah.com.
5. ZARDO G., REALE A., DE MATTEIS G., BUONTEMPO S., CAIAFA P. (2003). A role for Poly(ADP-ribosyl)ation in DNA methylation. BIOCHEMISTRY AND CELL BIOLOGY-BIOCHIMIE ET BIOLOGIE CELLULAIRE. vol. 81 pp. 197-208 ISSN: 0829-8211
6. ZARDO G., REALE A., PASSANANTI C., PRADHAN S., BUONTEMPO S., DE MATTEIS G., ADAMS R.L.P., CAIAFA P. (2002). Inhibition of poly(ADP-ribosyl)ation induces DNA hypermethylation: a possible molecular mechanism. FASEB JOURNAL. vol. 16 pp. 1319-1321 ISSN: 0892-6638
7. DANGOND F., HENRIKSSON M., ZARDO G., CAIAFA P., EKSTROM T.J., GRAY S.G. (2001). Differential expression of HDACs: roles of cell density and cell cycle. INTERNATIONAL JOURNAL OF ONCOLOGY. vol. 19 pp. 773-777 ISSN: 1019-6439
8. KARIMOV M., TOMSCHIK M., LEUBA S.H., CAIAFA P., ZLATANOVA J. (2001). DNA methylation-dependent chromatin fiber compaction in vivo and in vitro: requirement for linker histone. FASEB JOURNAL. vol. 15 pp. 2631-2641 ISSN: 0892-6638
9. REALE A., MALANGA M., ZARDO G., STROM R., SCOVASSI A.I., FARINA B., CAIAFA P. (2000). In vitro induction of H1-H1 Cross-Linking by ADP-ribose polymers. BIOCHEMISTRY. vol. 39 pp. 10413-10418 ISSN: 0006-2960
10. ZLATANOVA J., CAIAFA P., VAN HOLDE K (2000). Linker histone binding and displacement: versatile mechanism for transcriptional regulation. FASEB JOURNAL. vol. 14 pp. 1697-1704 ISSN: 0892-6638

DOTTORATI DI RICERCA

Componente U.O. Dottorato di Ricerca Coordinatore Sede
Paola Caiafa
Human Biology and Genetics
Paolo Amati
University of Rome, “La Sapienza”
Anna Reale
Human Biology and Genetics
Paolo Amati
University of Rome, “La Sapienza”

CONGRESSI C.I.B.

Congressi Partecipazione
CNB4

CNB5

CNB6

CNB7

CNB8

CNB9
CNB10