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Università degli Studi di Milano.
Member of LERU (League of European Research Universities). 1° University in Italy for scientific productivity (Leiden ranking 2012). 1° University in Italy in Biomedical Sciences. Dipartimento di Bioscienze 90 Faculty members, 6 EMBO Members. Genetics, Molecular Biology, Structural Biology, Biochemistry, Bioinformatics, Developmental Biology, Physiology, Plant Biology, Ecology. PhD program in Biomolecular Sciences. 50 students. Joint PhD program with EMBL, Heidelberg.
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ES cell Progenitor cells Pancreatic cell Endoderm Hepatocyte Myoblast
Mesoderm Ectoderm Progenitor cells Totipotent cell Neuron Hepatocyte Myoblast Pancreatic cell B cell Epithelial cell Pluristratified ES cell
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NF-Y-DNA structure NF-YA/NF-YB/NF-YC interface
A1 helix contacts extensively NF-YB/NF-YC (1760 Å2) heterotrimerization through side-chain and backbone polar interactions A1A2-linker: extended conformation that directs A2 helix towards the DNA A1 A2 a1(YC) a2(YB) aC(YC) T A C K241 Q242 R245 I246 R249 R250 R253 K261 P263 R266 N239 V238 S121 T122 L123 F125 F94 S97 E98 E101 E90 S93 F50 Y56 E86 Q85 E52 E82 F113 D112 D109 Q110 D116 I117 A1 helix 4
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The genomic strategy of NF-Y:
The ENCODE Project. ChIp-Seq/Mass-Spec in 9 cell types of 10 histone PTMs and 72 -and counting- DNA-binding Proteins (Mostly TFs). Ok 5 5
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Clustering NF-Y with 80 nuclear proteins.
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40/50 TFs show correlations,
P value E-40
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# Promoters with Factor
NF-Y friends # Peaks # Peaks in promoters # Promoters with Factor Expected Overlap w YB Actual Overlap w YB (a) Hypergeo. Prob. RPC155 2075 282 278 53 87 3.11E-08 RAD21 17401 898 883 169 195 XRCC4 201 7 1 0.3825 TR4 612 241 45 78 3.22E-08 ORC2 15401 3522 3404 664 1286 3.18E-199 TFIIIC 10004 2310 2229 436 732 2.52E-71 Pol3 563 65 61 12 15 TFIIF 10662 5241 5127 989 1540 1.80E-131 TBP 9933 6068 5945 1145 2295 0.00E+00 SPT5 1839 249 245 47 108 2.32E-21 pS2Pol2 29410 9741 9466 1838 3126 Pol2 10316 9954 1946 3347 TFIIB 2475 1445 1424 273 620 9.13E-119 NFE2 3477 380 375 72 122 4.55E-12 ZNF274 794 9 4 2 0.4502 ZNF263 8128 1327 1298 250 299 5.84E-07 Ifna6hStat2 2173 341 332 64 88 3.40E-05 Ifna6hStat1 1245 211 208 40 49 GATA2 9025 633 621 119 173 3.66E-10 GATA1 3182 225 224 42 55 YY1 5250 2534 2510 478 878 1.61E-104 E2F6 20609 9516 9187 1795 3037 E2F4 9862 7163 7025 1351 2615 cMYC 13693 6506 6300 1227 2248 MAX 6402 2500 1006 8.17E-172 cJUN 18480 3394 3277 640 1138 8.36E-139 cFOS 14404 2966 2881 560 2279 JUND 945 308 307 58 124 2.20E-20 ATF3 939 496 495 94 181 3.34E-23 SIRT6 1794 141 27 33 SETDB1 8379 997 979 188 287 5.13E-19 BRF2 67 0.2534 BRF1 317 46 44 13 BRD4 10746 3203 3139 604 982 1.16E-85 BDP1 520 89 17 18 GTFs TFs Cofactors
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NF-Y and friends stay together...
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Function (p-value <1e-05)
...to make life and death decisions cFos NF-YB cJun Function (p-value <1e-05) Yes No 2884 Cellular Assembly and Organization 6345 None 10719 Cell Death, Protein Synthesis, Gene Expression, Cellular Growth and Proliferation, Molecular Transport, Protein Trafficking, Cell Cycle 4472 Cell Cycle, Gene Expression, DNA Replication, Recombination, Repair, Cellular Assembly and Organization 5980 683 1155 Cell Cycle, Cellular Function and Maintenance, Cell Death cFos cJun NFYB RefSeq
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The amazing precision of ChIP-Seq
1 -1 2 -2 4 -4 C G 5 -5 3 -3 6 -6 7 -7 12 -12 10 -10 11 -11 13 -13 14 -14 15 -15 16 -16 17 -17 18 -18 19 -19 20 -20 21 -21 22 -22 23 -23 24 -24 25 -25 5’ 8 -8 9 -9 Lys 78 Ala 77 Ile 76 Lys 81 Ser 273 His 271 Gln 53 Ala 46 Lys 109 Ala 63 Ser 62 Lys 49 Ile 61 Lys 53 Lys 50 Leu 45 L1 (B) L2 (B) a2 (B) a1 (C) L1 (C) A2 Asn 61 a1 (B) Met 279 Gln 97 Arg 94 Thr 95 Arg 93 Lys 75 Arg 288 Ser 291 Pro 44 Arg 281 His 277 Arg 47 Met 60 Arg 64 Ile 59 Ala 60 Pro 58 Leu 270 His 275 Arg 283 Gly 287 Gly 286 Phe 289 Arg 274 A1A2-linker L2 (C)
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Where do the friends sit at the table?
CCAAT ? E2F E box TBP AP1
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We can tell
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Reconstructing promoters architecures
10 50 CCAAT E2F E box TBP AP1
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Protein transfections of TAT-NF-YA in vivo.
Domashenko, Blood, 2010. Progenitor cells Pancreatic cell Endoderm Hepatocyte ES cell Myoblast Mesoderm Hematopoietic cells HSCs Totipotent cell Ectoderm Neuron Epithelial cell Pluristratified
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-LIF +TAT-NF-YAs UNTREATED -LIF +TAT -LIF VINCULIN TAT-NF-YAs NF-YAl NF-YAs SOX2 NANOG KLF5 Fold enrichment
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PROMOTERS WITH CCAAT PSFM SCORE>0.8
GENOME PROMOTERS PROMOTERS WITH CCAAT PSFM SCORE>0.8 % ChIP-Seq data NF-YA promoters BACKGROUND 21747 5493 25.26 2400 TFs PROMOTERS ( ) P-value E2F1 6102 2183 35.78 5.11E-106 1472 NANOG 343 163 47.52 9.61E-020 144 E-50 KLF4 2966 900 30.34 6.52E-012 505 E-25 SOX2 178 78 43.82 2.07E-008 41 E-06 ESRRB 1240 383 30.89 1.76E-006 169 STAT3 320 101 31.56 4.35E-003 64 E-07 OCT4 660 193 29.24 8.07E-003 131 E-12 CTCF 1308 301 23.01 9.72E-001 228 E-13 TFBs analysis on Nanog core promoters Nanog peaks de novo discovery analysis TF NAME MATRIX ID P-VALUE NFYA MA0060.1 4.86E-27 MIZF MA0131.1 1.92E-17 ELK4 MA0076.1 2.36E-16 GABPA MA0062.2 1.18E-14 ELK1 MA0028.1 1.50E-13 E2F1 MA0024.1 1.22E-11 HIF1A::ARNT MA0259.1 TF NAME MATRIX ID P-VALUE GABPA MA0062.2 1.83E-234 ELK4 MA0076.1 2.94E-220 ELK1 MA0028.1 5.17E-168 E2F1 MA0024.1 9.10E-156 MIZF MA0131.1 2.36E-152 TFAP2A MA0003.1 7.63E-111 NFYA MA0060.1 3.65E-108 Mouse promoters Nanog peaks Ranking positions third Human promoters Nanog peaks Ranking positions first Mouse Human 82.2 ChIP:NF-YB 89.3 Fold enrichment NF-YAs ChIP:NANOG NF-YC NF-YAsm29 Fold enrichment NF-YC ChIP-Seq: NANOG - + ChIP-Seq: NF-YA
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A growing network of TFs in ESC.
NF-YB NF-YA NF-YC 37 Short KLF4 SALL4 MYC STAT3 OCT4 SOX2 NANOG 19
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Past Present Collaborators DSBB U. Milano Post-Docs PhDs
Khalid Zemzoumi Alberto Di Silvio (Axxam I) Fabienne Cocchiarella (UniMo) Marià Pitarque Martì Anna Testa Nicoletta Cordani (HSR, I) Silvia Pozzi (Reg Emilia) Alessandra Viganò (U. Basel, CH) PhDs Maria Carla Motta (U. Manchester UK) Patricia Lievens Chiara Liberati (Axxam, I) Giuseppina Caretti (UniMi I) Mattia Frontini (MRC UK) Carol Imbriano (UniMo I) Barbara Testoni (CNRS F) Claudia Forni (Farmigea I) Giacomo Donati (MRC UK) Michele Ceribelli (NIH USA) Daniele Merico (U. Toronto CA) Andrea Fossati (UniMi I) Marianna Bellorini Chiara Vecchi (UniMo I) Valentina Salsi (UniMo I) Giulia Petrovich (U. Edimburgh UK) Present Collaborators Serena Borrelli Raffaella Gatta Diletta Dolfini Elena Martynova Mario Minuzzo Nerina Gnesutta Mariangela Lorenzo Federico Zambelli Giulio Pavesi Marco Nardini Martino Bolognesi C Imbriano U Modena (I) L Tora IGBMC (F) C Romier IGBMC (F) C Castagnoli B.Cute Torino (I) G Melino MRC Leicester (I) A Terrinoni IDI (I) E Candi U. Roma (I) D Aberdam INSERM-Nice (F) GP Dotto U. Lausanne (CH) E Berti U. Mi-Bicocca (I) M d’Incalci Ist Mario Negri (I) D. Hochhauser UCL (UK) J. Fleming/K. Struhl Harvard (USA) Active Motif (USA)
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