Edward N. Trifonov University of Haifa, Israel Prague, Brno 2013 Nucleosome positioning sequence code: 33 years of agony and final picture Lab of G. Bunick, 2000 DNA in the nucleosome is severely deformed. Neighboring base pairs become partially unstacked. Some of the dinucleotide stacks may be more deformable than others. This also depends on their rotational orientations. 4069BFAB DISTANCE ANALYSIS (Autocorrelation) 5’…RRRYYYYYRRRRRYYY… First matrix of nucleosome DNA bendability Mengeritsky and ENT, 1983 Pattern of 1980-1983 yrRRRryYYYyr xxAAAxxTTTxx Trifonov, Sussman , 1980 Trifonov, 1980 Mengeritsky, Trifonov, 1983 dna-1 5’ 5’…YYYRRRRRYYYYYRRR… 89981E58 This achievement in the single-base accuracy mapping of the nucleosomes has not been accepted by chromatin research community. The reasons: 1.Mistrust. The physics of the phenomenon and multiple alternative positions of the nucleosome centers are hard to grasp for non-physicists, and the sequences did not show any obvious periodicity 2. The chromatin research community was not ready yet to conduct high resolution experimental studies History of the chromatin code. Pre-genomic studies 1980-2006 ~10.5 base periodicity of some dinucleotides Trifonov, Sussman (1980) ...T T A A A A A T T T T T A A A A A T T... Mengeritsky, Trifonov (1983) ...Y Y R R R R R Y Y Y Y Y R R R R R Y Y... Mengeritsky, Trifonov (1983) ...x Y R x x x R Y x x x Y R x x x R Y x... Zhurkin (1983) ...W W W W x S S S S x W W W W x S S S S... Satchwell et al. (1986) ...x W W W x x S S S x x W W W x x S S S... Shrader, Crothers(1989), Tanaka et al.,(1992) ...C C x x x x x C C C C C x x x x x C C... Bolshoy (1995) ...V W G x x x x x x x V W G x x x x x x... Baldi et al. (1996) ...x x G G R x x x x x x x G G R x x x x... Travers, Muyldermans (1996) ...C T A T A A A C G C C T A T A A A C G... Widlund et al. (1997) ...C T A G x x x x x x C T A G x x x x x... Lowary, Widom (1998) ...S S A A A A A S S S S S A A A A A S S... Fitzgerald, Anderson (1998) ...C C G G G G G C C C C C G G G G G C C... Kogan et al. (2006) dna-1 5’ TA AA TT GG CC GC CG Suggestion of an approximate pattern by Segal,…,Widom, Nature 442, 772 2006 The work of Segal et al., 2006, was the first high throughput whole-genome analysis. It drew a lot of attention, and the approach became very fashionable in the chromatin community. But the emphasis was still on low resolution studies, maps of “occupancy”, where the alternative positions of the nucleosomes and rotational setting of DNA are not seen. No attempts were made to derive an exact nucleosome positioning sequence pattern from the whole genome sequences. When we joined the high througput efforts our primary task was to derive the detailed nucleosome positioning sequence pattern This involved three original techniques A.Signal regeneration from its parts B. Shannon N-gram extension C. Extraction and analysis of strong nucleosomes Nucleosome positioning patterns, species: species authors method C GRAAA TTTYC G C. elegans Gabdank, 2009 A C AAAAA TTTTT G C. elegans Rapoport, 2011 B C AAAAA TTTTT G A. gambiae same B C AAAAA TTTTT G C. albicans same B C AAAAA TTTTT G D. melanogaster same B C AAAAA TTTTT G S. cerevisiae same B T AAAAA TTTTT A A. mellifera same B T AAAAA TTTTT A A. thaliana same B T AAAAA TTTTT A D. discoideum same B T AAAAA TTTTT A D. rerio same B T AAAAA TTTTT A G. gallus same B T AAAAA TTTTT A H. sapiens same B T AAAAA TTTTT A M. musculus same B c GGGGG CCccc G C. reinhardtii same B Y RRRRR YYYYY R consensus A – signal regeneration, nucleosomes B – Shannon N-gram extension, whole genome Structural and sequence periodicity of nucleosome DNA DNase I digestion of chromatin 10.30-10.40 bp Prunell, Kornberg, Lutter, Klug, Levitt, Crick, 1979 Beat effect, DNase I 10.33-10.40 bp Bettecken, 1979 Analytical geometry of nucl. DNA 10.30-10.50 bp Ulanovsky, 1983 DNA path in nucleosome crystals 10.36-10.44 bp Cohanim, 2006 CG periodicity, honey bee 10.36-10.44 bp Bettecken, 2009 DNase I digestion of chromatin 10.36-10.44 bp Duke University, 2013 Common range 10.36-10.40 bp Magic distances, 10.4•n bases nearest integers 10.4 10 20.8 21 31.2 31 41.6 42 52.0 52 62.4 62 72.8 73 83.2 83 93.6 94 104.0 104 114.4 114 The ideal nucleosome positioning sequence would contain some periodically repeating motif, and all the distances between the same dinucleotides would be magic distances. Strong nucleosome DNA would show many magic distances. Lowary and Widom (1998) took large ensemble of synthetic DNA fragments with random sequences, and selected those of them which formed strong nucleosomes The sequences demonstrated very strong periodicity of TA dinucleotides Clone 601, from collection of Lowary and Widom (1998) ...CAGCGCGTACGTGCGTTTAAGCGGTGCTAGAGCTGTCTAC... TACGTGCGTTTA TAAGCGGTGCTA TAGAGCTGTCTA We took all TAnnnnnnnnTA segments from the collection of Lowary/Widom, and analysed which dinucleotides are most frequently located in the interval between TA, and in which positions Regeneration of signal from its incomplete versions: AA positional autocorrelation AAnnnnnnnnAA regeneration (all occurrences of AAnnnnnnnnAA are aligned, and other dinucleotides counted within the period) AAnnnnCCnnAA Gabdank, 2009 ↓ ↓ Bendability matrix for strong nucleosome DNAs of Lowary and Widom collection 0 1 2 3 4 5 6 7 8 9 0 AA 0 16 3 0 0 1 0 0 0 0 0 AC 0 5 2 5 2 3 5 3 1 0 0 AG 0 25 11 9 2 4 1 1 1 0 0 AT 0 2 0 3 1 1 3 1 2 0 0 CA 0 0 1 0 2 4 3 1 0 0 0 CC 0 0 0 0 5 4 7 3 6 0 0 CG 0 0 4 4 4 4 4 5 3 0 0 CT 0 0 0 2 1 2 1 9 11 22 0 GA 0 0 12 4 3 3 0 0 0 0 0 GC 0 0 4 7 6 7 5 10 5 0 0 GG 0 0 7 4 3 3 7 0 1 0 0 GT 0 0 2 7 6 4 5 6 2 6 0 TA 48 0 1 1 4 1 2 3 0 0 48 TC 0 0 0 0 1 1 1 4 10 0 0 TG 0 0 0 1 8 6 4 2 1 0 0 TT 0 0 1 1 0 0 0 0 5 20 0 22.5 min T A G A G x x x x C T A – manually T A G A G G C C T C T A – by dynamic programming Y R R R R R Y Y Y Y Y R T A G A G G C C T C T A The periodical pattern hidden in the sequences of Lowary and Widom is selfcomplementary, and manifests alternation of RRRRR and YYYYY TAAACTCTTTAAAAATCTTTTAAAAACCCTTGTACATATCTTAAAACCCTTTTAAAATCTCTTGTAAATCTTTAAAACCCTTTTAAAATCCCTTGTAAA TCTTTTAAAACCCTTT AAATATTTTAAAACACTTTTCAAACAATTTTGAACCCTTTAAAAATCTTTATAAAACCTTTGTAAATCTTTTAAAGCCCTTTAAAATCTCTTATAAATC TTTTAAAACCCTTTTA CCCTGTAAAACTTTTAAAACCCTTTTAAAATCCCTTGTAAATCTTTTTAAACCCTTTTAAAATCCTTGTAAATATTTTAAAATCCCGTGTAATTCTTTT AAAACTCTTTTAAAAT AAATTTTAAAAAGGTTTTATAAGATTTGCAAGGGATTTTAAAGGGATTTAAAAGATTTACAAAAGTTTTTTAAAGGTTTAAAATTGTTTTAAAAGGATT TTAAAATATTTACAAG TTTTAAAAGGGTTTTAAAATATTTACATATGTTTTTTAAAGTTTTTTAAAGGGTTTAAAAGTGTTTTGCAAGATTTACAAGAGATTTTAAAAGGGTTTT AAGAGATTTACAAGAG ATCCTTTAAAAAATCATGTAAATCTTTTTAAAACCTTTTAAAATCCCTTGTAAATCTTTTAAAATCCTTTTAAAATCTCTTGTAAATGTTTAAAAACCC TTTTAAAATCTCTTGT AAGGGTTTTAAAATATTTACAAGGGATTTTAAAAGGGTTTTAAAAAATTTACAAGTGATTTTAAAAGATTTACAAGGGATTTTAAAAGGTTTTAAAAAA ATTTACAAAAGTTTAT AAATCTTTTAAAACCCTTTTAAAATCCCTTGTAAATCTTTTAAAACACTTTTAAACCCTTTAAAAATCTTTAAAAAAACCTTTATAAATCTTTTAAAAC TCTTTAAAATCTCTTG AAATGTTTTAAAACCTTTTTAAAATAATTTTAAACCCTTTAAAAATCGTTAAAAAACTTTTGTAAATCTTTTAAAGCCCTTTAAAATCCCTTGTAAATA TTATAAAACCCTTTTA TGATTTTAAAAGGGTTTAAAAAGATTTACAAGGGATTTTAAAAGGGTTTTAAAAAATTTACAAGAGATTTTAAAAGGTTTTAAAAAGATTTACAAGAGT TTTAAAGGGTCTTCTT ATCTTTTAAAAATCCTTGTACATCTTTTAAAACCCTTTCAAACCCTTTAAAAATCTCTTGTAAATCTTTTAAAACCCTTTTAAAATCCCTTGTAAATCT TTCAAAACACTTTAAA CCTTTAAAATCCCTTGTAAATCTTTTAAAACCCTTTTCAAATCCCTTGTAAATGTTTTAAAACCCTTTTAGAACAATTTTAAACCCTTTAAAAATCTTT AAAAACCCTTTGTAAA TTTACAAAGGTTTTTAAAAGATTTTGAAAGGGTTTAAAAGTGTTTTAAAAGATTTACAAGGGATTTTAAAAGGGTTTTAAAGATTTACAAGAGATTTTA AAAGGGTTTTAAAAGA CTTGTAAATCTTTTAAAACCCTTTTAAAATCCTTTGTAAATATTTTAAAAGCCTTTTAAAATCCATTGTAAATCTTTTAAAATCCTTTGTAAATCTTTT AAAACCCTTTTAAAAT AGGATTTTAAAAATGTTTTAAAAGATTTACAATGGATTTTAAAAGGGTTTAAAATATTTATAAGGGATTTTGAAGGGCTTTCAAAGATTTATAAAGGTT TTTTAAAAATTTTTAA TTGTAAATTATTTAAAAATCTTTTAAAACTCCTTGTACATCTTTTAAAACTCTTTTAAAATTTCTTGTAAATCTTTAAAACCCTTTAAAATCCCTTGTA AATCTTTTAAAATACT ACCCTTTAAAAATCTTTTAAAAATCTTTGTAAATCTTTTAAAGCCCTTTGAAATCCCTTGTAAATATTTTAAAATCTTTTAAAATTCCTTGTAAATGTT TTAAAACCCTTTTAAA GATTTGCAAAAGATTTTAAAAGATTTACAAAGGATTTTAAAAGATTTACAATGGATTTTAAAGGGGTTTAAAAGATTTACAAAGGTTTTTTAAAGATTT TTAAAGGGTTTTAAAT The strongest nucleosomes of A. thaliana display very clear though still imperfect periodicity The ideal pattern for A.thaliana is repetition of TAAAAATTTTTA, again, alternation of RRRRR and YYYYY, and complementary symmetry Before this picture was generated (Dec. last year) nobody ever had seen that the nucleosome sequences look, indeed, periodical From the bendability matrices for the strong nucleosomes: T AGAGG CCTCT A Lowary and Widom T AAAAA TTTTT A A.thaliana T AAAAA TTTTT A C.elegans T AAAAA TTTTT A H.sapiens T AAAAA TTTTT A isochores L1, L2, H1 and H2 C GGGGG CCCCC G isochores H3 Y RRRRR YYYYY R common for all A. thaliana T AAAAA TTTTT A strong nucleosomes T AAAAA TTTTT A Shannon extension C. elegans T AAAAA TTTTT A strong nucleosomes c grAAA TTTyc g signal regeneration isochores L1, L2 T AAAAA TTTTT A strong nucleosomes T AAAAA TTTTT A Shannon extension isochores H1 T AAAAA TTTTT A strong nucleosomes c AgAAA TTTcT g Shannon extension isochores H2 T AAAAA TTTTT A strong nucleosomes c ggggA Tcccc g Shannon extension isochores H3 C GGGGG CCCCC G strong nucleosomes C aGGGG CCCCt G Shannon extension Y RRRRR YYYYY R – all, and all with complementary symmetry dna-1 5’ 5’…YYYRRRRRYYYYYRRR… TA CG TG CA AT GC AC GT Contact with arginines Exposed The rest of the period is occupied by RR (AA,AG,GA,GG) and YY (TT, TC, CT, CC) dinucleotides, in their optimal partial unstacking positions Nucleosome positioning pattern 2013 The dinucleotide stacks are placed in such positions within the nucleosome DNA period to ensure best possible bending. The better the bending – the stronger the nucleosome. But the bulk of the nucleosomes are only marginally stable. Only a fraction of properly positioned dinucleotides is present in any given nucleosome DNA sequence. CGGAAATTTTCCGGAAATTTCCGGAAATTTCCGGGAAATTTCCGGAAATTTCCGGAAATTTTCCGGAAATTTCCGGAAATTTCCGGGAAATTTCCGGAA ATTTCCGGAAATTTTCC CagaggagcttcctggggaTCCaGAcATgataagatacaTTgatGAgtTTggacaAAccacaactagAATgcagtGAAAaaaatgctttATTTgtgaAA tTTgtgatgctaTTgct YRRRRRagYYYYctRRRgaYYYRRRcRYgataRRRtacaYYgatRRRtYYggacRRRccacaactRRRRYgcagtRRRRaaaaYRctttRYYYgtRRRR tYYgtgatgctaYYgYY Match of the BamHI nucleosome (typical semistable nucleosome) to the standard nucleosome probe (GAAAATTTTC)n TAAACTCTTTAAAAATCTTTTAAAAACCCTTGTACATATCTTAAAACCCTTTTAAAATCTCTTGTAAATCTTTAAAACCCTTTTAAAATCCCTTGTAAA TCTTTTAAAACCCTTT AAATATTTTAAAACACTTTTCAAACAATTTTGAACCCTTTAAAAATCTTTATAAAACCTTTGTAAATCTTTTAAAGCCCTTTAAAATCTCTTATAAATC TTTTAAAACCCTTTTA CCCTGTAAAACTTTTAAAACCCTTTTAAAATCCCTTGTAAATCTTTTTAAACCCTTTTAAAATCCTTGTAAATATTTTAAAATCCCGTGTAATTCTTTT AAAACTCTTTTAAAAT AAATTTTAAAAAGGTTTTATAAGATTTGCAAGGGATTTTAAAGGGATTTAAAAGATTTACAAAAGTTTTTTAAAGGTTTAAAATTGTTTTAAAAGGATT TTAAAATATTTACAAG TTTTAAAAGGGTTTTAAAATATTTACATATGTTTTTTAAAGTTTTTTAAAGGGTTTAAAAGTGTTTTGCAAGATTTACAAGAGATTTTAAAAGGGTTTT AAGAGATTTACAAGAG ATCCTTTAAAAAATCATGTAAATCTTTTTAAAACCTTTTAAAATCCCTTGTAAATCTTTTAAAATCCTTTTAAAATCTCTTGTAAATGTTTAAAAACCC TTTTAAAATCTCTTGT AAGGGTTTTAAAATATTTACAAGGGATTTTAAAAGGGTTTTAAAAAATTTACAAGTGATTTTAAAAGATTTACAAGGGATTTTAAAAGGTTTTAAAAAA ATTTACAAAAGTTTAT AAATCTTTTAAAACCCTTTTAAAATCCCTTGTAAATCTTTTAAAACACTTTTAAACCCTTTAAAAATCTTTAAAAAAACCTTTATAAATCTTTTAAAAC TCTTTAAAATCTCTTG AAATGTTTTAAAACCTTTTTAAAATAATTTTAAACCCTTTAAAAATCGTTAAAAAACTTTTGTAAATCTTTTAAAGCCCTTTAAAATCCCTTGTAAATA TTATAAAACCCTTTTA TGATTTTAAAAGGGTTTAAAAAGATTTACAAGGGATTTTAAAAGGGTTTTAAAAAATTTACAAGAGATTTTAAAAGGTTTTAAAAAGATTTACAAGAGT TTTAAAGGGTCTTCTT ATCTTTTAAAAATCCTTGTACATCTTTTAAAACCCTTTCAAACCCTTTAAAAATCTCTTGTAAATCTTTTAAAACCCTTTTAAAATCCCTTGTAAATCT TTCAAAACACTTTAAA CCTTTAAAATCCCTTGTAAATCTTTTAAAACCCTTTTCAAATCCCTTGTAAATGTTTTAAAACCCTTTTAGAACAATTTTAAACCCTTTAAAAATCTTT AAAAACCCTTTGTAAA TTTACAAAGGTTTTTAAAAGATTTTGAAAGGGTTTAAAAGTGTTTTAAAAGATTTACAAGGGATTTTAAAAGGGTTTTAAAGATTTACAAGAGATTTTA AAAGGGTTTTAAAAGA CTTGTAAATCTTTTAAAACCCTTTTAAAATCCTTTGTAAATATTTTAAAAGCCTTTTAAAATCCATTGTAAATCTTTTAAAATCCTTTGTAAATCTTTT AAAACCCTTTTAAAAT AGGATTTTAAAAATGTTTTAAAAGATTTACAATGGATTTTAAAAGGGTTTAAAATATTTATAAGGGATTTTGAAGGGCTTTCAAAGATTTATAAAGGTT TTTTAAAAATTTTTAA TTGTAAATTATTTAAAAATCTTTTAAAACTCCTTGTACATCTTTTAAAACTCTTTTAAAATTTCTTGTAAATCTTTAAAACCCTTTAAAATCCCTTGTA AATCTTTTAAAATACT ACCCTTTAAAAATCTTTTAAAAATCTTTGTAAATCTTTTAAAGCCCTTTGAAATCCCTTGTAAATATTTTAAAATCTTTTAAAATTCCTTGTAAATGTT TTAAAACCCTTTTAAA GATTTGCAAAAGATTTTAAAAGATTTACAAAGGATTTTAAAAGATTTACAATGGATTTTAAAGGGGTTTAAAAGATTTACAAAGGTTTTTTAAAGATTT TTAAAGGGTTTTAAAT The strongest nucleosomes of A. thaliana display very clear though still imperfect periodicity The ideal pattern for A. thaliana is repetition of TAAAAATTTTTA, again, alternation of RRRRR and YYYYY, and complementary symmetry Cat in bushes. Courtesy of I. Gabdank Example of the output from the nucleosome mapping server http://www.cs.bgu.ac.il/~nucleom Mapping of sharply positioned nucleosomes Nucleosomes around the GT splice junctions GT AG Dots • - N9 atoms of guanines Guanines of GT- and AG-ends of introns are oriented towards the surface of the histone octamer, away from exterior. Such orientation is the best for guanines to minimize spontaneous depurination and oxidation The most frequent spontaneous damages to DNA bases: depurination of G (N9 atoms) oxidation of G deamination of C A description... Nucleosome DNA which carries promoter TATAAA box has two rotational settings encoded in the sequence (two peaks within one period) TATA-switch Two alternative positions of TATAAA box in the promoter nucleosomes are separated by 140 (220) degrees, which closely correspond to exposed and inaccessible orientations of the box. By shifting the DNA along its path by 4(6) bases, the promoter is switched ON or OFF. The switch (shift) may be triggered by remodelers or transcription factors. Today the single-base resolution nucleosome mapping is the only practical tool to study fine structure of chromatin and its role in factor binding, transcription, replication, DNA repair, transposition, recombination, apoptosis, chromatin domains, and more Immediate questions: Where in genomes the strong nucleosomes are located? What they are doing there? Tentative answer: Strong nucleosomes are chromatin organizers. ACKNOWLEDGEMENTS Recent contributions (2009-2013): Idan Gabdank (Beer Sheva, Israel) Zakharia Frenkel (Haifa, Israel) Alexandra Rapoport (Haifa, Israel) Thomas Bettecken (München, Germany) Jan Hapala (Brno, Czech Republic) Bilal Salih (Haifa, Israel) Vijay Tripathi (Haifa, Israel) Earlier contributions (1980-2008) Thomas Bettecken Joel Sussman Galina Mengeritsky Levy Ulanovsky Alex Bolshoy Ilya Ioshikhes Amir Cohanim Fadil Salih Simon Kogan Funding (2009-2012) Israel Science Foundation, and South Moravian Program Why DNA binds to histone octamers by one side? It could be either intrinsic DNA curvature or better bending in one specific direction (deformational anisotropy of DNA) Both should be sequence-dependent 645F23AE The purine-purine•pyrimidyne-pyrimidyne stacks (RR•YY) are very asymmetric Nucleosome positioning sequence pattern is very weak (as the nucleosomes should be easy to unfold) The weak pattern overlaps with other messages (“noise”). That makes the signal/noise ratio very low. VERY large database of the nucleosome DNA sequences is needed, to extract and fully describe the signal It is easy, however, to detect the signal DISTANCE ANALYSIS (Autocorrelation) T.Bettecken, E.N.T., 2009 Whole-genome periodicities (distance analysis) AA TT CG GC CA TG AG CT AT GG CC GA TC AC GT TA S. cerevisiae ● ● ● ● ● ● ● ● ● ● ● ● ● - - ● C. elegans ● ● ● ● ● ● ● ● ● - - ● ● ● ● - A. thaliana ● ● - ● ● ● - - ● ● - - - - - - D. rerio ● ● - ● - - - - - ● ● - - - - - C. albicans ● ● - - ● ● - - - - - - - - - - A. mellifera ● ● ● ● - - - - - - - - - - - - D. melanogaster ● ● ● ● - - - - - - - - - - - - G. gallus - - - - - - ● ● - - - - - - - - A. gambiae ● ● - - - - - - - - - - - - - - C. reinhardtii ● ● - - - - - - - - - - - - - - D. discoideum - - ● - - - - - - - - - - - - - H. sapiens - - ● - - - - - - - - - - - - - M. musculus - - - - - - - - - - - - - - - - AAnnnnnnnnAA repeat structure (C. elegans) Regenerated pattern (AAATTTCCGG)(AAAT… Positional matrix of bendability(C.elegans) 1 2 3 4 5 6 7 8 9 0 1 2 C G C G G G G A G A A A A A A A T T T T T T T C T C C C C G LINEAR FORM OF THE POSITIONAL MATRIX OF BENDABILITY (C.elegans): CGRAAATTTYCG (YRRRRRYYYYYR) Trinucleotides of C. elegans genome counts 1 AAA 4162266 2 TTT 4160750 3 ATT 2488998 4 AAT 2486813 5 GAA 1873844 6 TTC 1871673 7 CAA 1667120 8 TTG 1663842 9 TCA 1498069 10 TGA 1496493 ....... ....... TOPMOST TRINUCLEOTIDES MAKE TOGETHER THE DOMINANT PATTERN GAAAATTTTC: GAAAATTTTC GAAAATTTTC GAAAATTTTC GAAAATTTTC GAAAATTTTC GAAAATTTTC GAAAATTTTC GAAAATTTTC This technique is known since 1948 – Shannon N-gram extension It has been very helpful in further studies of the nucleosome positioning patterns Human isochores Lab of G. Bernardi, 2006 Nucleosome positioning patterns of various isochores (Frenkel et al., 2011) by N-gram extension isochores G+C % C AGGGG CCCCT G C GGGGA TCCCC G C AGAAA TTTCT G T AAAAA TTTTT A T AAAAA TTTTT A Y RRRRR YYYYY R Fig1_1 Nucleosome positioning patterns for human isochores L1 and H3 derived by signal regeneration from apoptotic nucleosomes: L1: T AAAAA TTTTT A H3: C AGGGG CCCCT G Frenkel et al., 2011 Nucleosome positioning patterns, isochores (Frenkel, 2011, 2012) isochore method T AAAAA TTTTT A L1 (<37% G+C) B T AAAAA TTTTT A same A T AAAAA TTTTT A L2 (37-41% G+C) B C AGAAA TTTCT G H1 (41-46% G+C) B C GGGGA TCCCC G H2 (46-53% G+C) B C AGGGG CCCCT G H3 (>53% G+C) B C AGGGG CCCCT G same A Y RRRRR YYYYY R consensus A signal regeneration, nucleosomes B Shannon N-gram extension, whole genome Shannon N-gram reconstruction of linkers TTTTATTTTAAAATAAAA human linkers AAAATAAAATATTTTATTTT yeast linkers TAAAgTAcTTTA human, apoptotic cuts consensus: TAxxxTAxxxTAxxx (B. Salih, T. Bettecken, Z. Frenkel) TTAAAAATTTTTAAAAATTTTTAA human L1 isochores, nucleosomes 98CBFEE3 BamHI nucleosome of Ponder and Crawford, 1977 BamHI fragments of BamHI nucleosome DNA Calculated Observed in the gel 24 34 43 54 ~53 | 64 ~63 | misfit (73) (~73) | ± 1 base 82 ~83 | 92 ~93 | 103 112 122 Example of the nucleosomes at and around GT splice junction Hapala, 2011 Plenty of various other nucleosome positioning patterns have been suggested during 30 years since the first observation of sequence periodicity. At the best they provide occupancy maps (resolution of ~15 bases). The (GRAAATTTYC)n and (RRRRRYYYYY)n are the only patterns that generate maps with single-base resolution, verified by crystal data. The future of the chromatin structure/function is with the high resolution studies.