A holistic perspective of gene expression during the E. coli growth cycle. DNA structure and nucleosome placement

Transcription

1 A holistic perspective of gene expression during the E. coli growth cycle. Andrew Travers DNA structure and nucleosome placement Haifa, 13/05/12 1

2 A/T rich base-steps are more deformable than G/C-rich base-steps Adapted from Olson et al. (2007) Sfe/mfe values from Protozanova et al

3 Nucleosome occupancy in vitro correlates negatively with deformability Nucleosome occupancy positively correlated with stacking energy i.e nucleosome occupancy is positively correlated with DNA stiffness Window = 125 bp Cédric Vaillant, LJC, Lyon 3

4 DNA stacking energy correlates with nucleosome occupancy in vitro AND genetic organisation Window = 125 bp 4

5 In vitro data Kaplan et al Read density = nucleosome occupancy? (Nuc/bp) Series1 Series Sfe_Mfe/basestep -kcal/mol/basestep Series 1 = melting Series 2 = stacking 5

6 In vivo data Cole et al Read density = nucleosome occupancy? (Nuc/bp) Series1 Series Sfe_Mfe/basestep -kcal/mol/basestep Series 1 = melting 6 Series 2 = stacking

7 Caveats: 1. Selective recovery of nucleosomes; Some methods, e.g. paired-end sequencing, likely select for canonical nucleosomes, i.e. with full length DNA. This selects against nucleosomes with signiicant transient unwrapping from one end. 2. Occupancy measures based on recovery selects for nucleosome stability, which,in the dynamic environment of in vivo chromatin is not necessarily quantitiatively the same as real occupancy. 3. Partial MNase digestion analysis of chromatin identifies linker DNA directly for limit MNase digestion linker DNA position is inferred, and can differ between protocols. 4. Are nuclesome positions actively maintained in vivo? If so energy deprivation (for example) could perturb positioning to default of more stable positions bydisabling remodelers (Moshkin et al. 2012). 7

8 BUT. the opposite conclusion DNA binding to histone octamer is directly proportional to DNA flexibility (with one exception) or inversely correlated with DNA stiffness Virstedt et al.,

9 9

10 K2/K1 ~ 5 10 Churcher & Hiriart

11 Relative affinity to ADY2-1 nucleosome in vitro 4 Salt dilution K1/K ADY ADY M NaCl Stacking energy (-kcal/mol/basestep) 11

12 Building a nucleosome Trapping Trapping correlates directly with P a but wrapping is inversely proportional to P a Entropy/enthalpy balance likely depends in part on k1/k2 for initial binding (Travers et al., Phil Trans A, 2012) Same principle for DNA circularisation 12

13 Configurational Space Highly flexible - isotropic bending Stiffer - anisotropic bending WLC model does not apply 13

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15 Origins of DNA replication ARS elements: 3 categories in database: 1. confirmed 2. highly likely 3. dubious hits of these 506 (87.8% have stacking energy minimum) value 15

16 Stacking energy profile of the ADH2 gene (kcal/mole/bs) 16

17 Sequence organisation of +1 nucleosomes is asymmetric Mavrich et al

18 The most unstable region in the Saccharomyces cerevisiae genome Chr 15 from to DCP1/YOL149W -> AATATCTTCTGGAAAATGAGCCAAAAGATTCTTTTGCT TGAGCACAGAGGTGAGATGCCC AAGTGAGAATTTTTTTTA AATAATGATGTACTTTAATACAA TATATATATATATATATAT ATATATATATATATATATATA AGGAATGATAACTC TATTTAAGTAGATTATGACATTGTA GTAGAAGAGGGCG TGCTACTTGCGCTCTGTTTCTTCTTAGTCATTCTTTTCTTTTTAGGA <- SPT20/YOL148C TATATA AATAA TGAå unstable region processing signal stop codon 18

19 The 2nd most unstable DNA region in the Saccharomyces genome LDB18 -> AAAAAGAGGCTGTTGAATTTACAAAAGGTAATTTATAGTACG TGAACATGTGTGC GTATATACATATATATATATATATATATATATATATATATATATATA CTTTAGCA TCGAAACAGCTAGTCCTTTTTAGAGTTCAATTTTTCCA CAAAGGCTTTTTTAGCC <- YBT1 19

20 20 Liu & Wang, PNAS, 1987

21 Possible roles of TA box A topological sink would work for one pol only 21

22 A 300 chromosome analysis high OriC Melting energy low Gamma-Proteobacteria Average (50kb) Gamma-Proteobacteria Average (500kb) Ter 22

23 Thanks to: E. Coli chromosomes Patrick Sobetzho Jacob s University, Bremen Georgi Muskhelishvili Yeast nucleosomes Boris Adryan Dept. of Genetics, Cambridge Graeme Mitchison Cédric Vaillant Mark Churcher Edwige Hiriart Hope Cole David Clark Michael Thompson DAMTP, Cambridge ENS, Lyon MRC-LMB NICHMD, NIH DAMTP, Cambridge and all who went before 23