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Bacterial functional genomics at Joint Genome Institute. Pacific Biosciences User Meeting 09/18/13 Matthew Blow. DNA base modifications can be directly detected by PacBio sequencing. U nmodified DNA template. Time. Delay in base incorporation opposite methyl adenine. - PowerPoint PPT Presentation
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Bacterial functional genomics at
Joint Genome Institute
Pacific Biosciences User Meeting09/18/13
Matthew Blow
DNA base modifications can be directly detected by PacBio sequencing
Unmodified DNA template
Time
Modified DNA template
Delay in base incorporation opposite methyl adenine.
Time
Detection of DNA base modifications is coverage dependent
Detectable with ~50x coverage
Require high coverage (100s x) for detection
These modifications are important in microbial biology~50x generated for microbe genome assembly
DNA base modification sequencing at JGI
x 2Sufficient for >500 bacterial epigenomes / year
- CSP call for bacterial epigenome projects (2 accepted)
- JGI ‘Grand Challenge’ project:
“Epigenomic landscape of Bacteria”
Functional roles of DNA modification in Bacteria
MTRE
Me
Me
Me
Me
Me
Me
Me
Me5 ’ … G A AT T C … 3 ’3 ’ … C T TA A G … 5 ’
Me
Me
Methylase (MT) protects host DNA
5 ’ … G A AT T C … 3 ’3 ’ … C T TA A G … 5 ’
Challenge:Sequence specificities of restriction systems
are hard to read
1. Restriction systems
2. Regulation
Gene regulation
GATC in E.Coli and other
γ-proteobacteria
Me
Cell cycle regulation
GANTC in α-proteobacteria
Me
‘Orphan’ MT
Me
Me
Me
Many Others?Previously no easy way to look!
The Epigenomic Landscape of Bacteria
Modified motifs
100+ Diverse microbes
100+ Methylomes
Microbial genome program
Microbes with ‘interesting’ methylases
Rich Roberts (NEB)
Jonas Korlach, Khai luong,
Luke Hickey (Pacific Biosciences)
50x PacBio
sequencing
2. New insights into roles of
methylation in Restriction
systems Known (GATC, GANTC)
3. Identification and characterization
of regulatory DNA modifications
Novel
1. Descriptive tables of ‘Methylation landscape’
Results
Global patterns of DNA modifications in bacteria
Total analyzed genomes = 198
Genomes with modified motifs = 169 (>90%)
Types of modification = 6mA (80%), 4/5mC (19%), ?T(1%)
Total modified motifs = ~500
Average # modified motifs / genome = 3 (Max = 12)
Novel motifs = ~20%
New insights into restriction systems
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Kn
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n D
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met
hyl
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spec
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itie
s Type I methylaseType III methylase
New insights into restriction systems
• Analysis in progress by Rich Roberts at NEB
• Many new patterns and specificities (e.g. novel protection of typeIIG and typeIII enzymes with m4C rather than 6mA)
• Deep data may enable predictive models of DNA methylase specificity for type I enzymes
Identification of putative regulatory DNA modifications
1) Are there any widely evolutionarily conserved modifications?
2) Are there unusual clusters of motifs in any organisms?
3) Are there unmodified sites in the genome?
Evolutionarily conserved
DNA modifications
Evolutionarily conserved
DNA modifications
The known GATC regulatory modification in γ-proteobacteria
Broad smattering of GATC modifications in other bacteria
2/2 thermococci
2/2 cyanobacteria
5/11 clostridia
Evolutionarily conserved
DNA modifications
The known GANTC regulatory modification in α-proteobacteria
No GANTC outside of α-proteobacteria
No GANTC outside of α-proteobacteria
Evolutionarily conserved
DNA modifications
2/2 Halobacteria species
2/6 δ-proteobacteria species
Evolutionarily conserved
DNA modifications
All other modifications observed in 4 or less organisms
Conclusions:
-No new major class of widely conserved modification
-Several modifications conserved across more restricted sets of organisms
-Need further evidence that they have some regulatory function
Are there unusual clusters of motifs in any organisms?
Known example: gAtc sites in E.coli
Origin of replicationGATC motif cluster
Competition between methylase (dam) and other proteins for binding to GATC controls DNA replication
Most enriched 1kb regions across all
modifications / genomes
Ctag not gAtc
Conservation
Genes
Natrialba magadiiHalopiger xanaduensis
CTAG motifsorc1/cdc6 family replication
initiation protein
Conserved cluster of modified CTAG at replication
origin in H. turkmenica
Conserved CTAG in non-coding sequence
Conclusions:-Several genomes contain enriched clusters of modified motifs -May be indicators of novel regulatory functions for methylation-E.g Putative Ctag methylation control of DNA replication
Are there unmodified sites in the genome?
Unmethylated motif
Non-methylated sites pose problem if part of restriction systems!
Methylated motif
Are there unmodified sites in the genome?
Unmethylated motif
Methylase
DNA-binding protein
Methylated motifNon-methylated sites may be due to ‘protection’ by competing binding proteins
1. Non methylated is just footprint of DNA binding protein (no regulation)
2. Competition between Methylase and DNA binding protein forms regulatory mechanism (multiple published examples support this)
Can we detect known non-methylated GATC
sites in E. Coli?
GATC
GATC
Unmethylatedregulatory?
Includes 3 / 3 known regulatory sites upstream of antigen 43!
Antigen43 (Waldron et al Mol microbiol 2002, Wallecha et al J. bacteriol 2002)
Novel cluster of 3 unmodified sites upstream of tonB
GATCMe
GATCMe
Reproducible evidence for non-methylated GATC in
gamma-proteobacteriaFour γ-proteobacteria with regulatory GATC modification
E.coli S.oneidensis Alteromonas sp. S.bongori
Four clostridia with restriction system associated GATC modification C.thermocellum (a) C.thermocellum (b) C.clariflavum (b) P.ferrophilus (b)
(Archaea)
What are the properties of non-methylated sites? -Enriched at 5’ end of genes / operons-One example reproducible across 2 genomes (tonB dependent receptor)-Multiple sites upstream of transcriptional regulators
Evidence for non-methylated GANTC sites in
alpha-proteobacteriaBradyrhizobium elkani Rhizobium etli
Agrobacterium tumefaciens Methylobacterium extorquens
Are there examples of regulatory signatures at
novel motifs?
Spirochaeta smaragdinae raAtty
4/9 unmodified sites clustered upstream of GntR transcriptional regulatorConclusions:
-Analyses of non-methylated sites identifies known regulatory sites in gamma-proteobacteria-Candidate novel regulatory sites associated with known regulatory methylases-Evidence for regulatory sites involving ‘novel’ methylases
Bacterial Epigenomics Summary
We have generated ~200 bacterial DNA base modification datasets on the PacBio platform
New insights into role of methylation in restriction systems and gene regulation
JGI is soliciting additional projects to explore the functions of DNA base modification(CSP call)
Acknowledgements
JGIJean Zhao
Katy Munson
Feng Chen
Chris Daum
Christa Pennacchio
Matt Bendall
Rex Malmstrom
Len Pennacchio
PacBioJonas KorlachKhai LuongLuke Hickey
NEB
NEBRich Roberts
Dana Macelis
Pac BioJonas Korlach
Khai Luong
Luke Hickey