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

1970

1972

1974

1976

1978

1980

1982

1984

1986

1988

1990

1992

1994

1996

1998

2000

2002

2004

2006

2008

2010

2012

2013

a0

50

100

150

200

250

Year

Kn

ow

n D

NA

met

hyl

ase

spec

ific

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