Balancing Somatic Hypermutation and g ypDNA Repair in Immunoglobulin Genes

Patricia GearhartLaboratory of Molecular Gerontologyy gyNational Institute on Aging, NIH

Baltimore

ANTIBODY (B cells)Light chain

ANTIBODY (B cells)

Heavy chain

Variable regions

Constant regions

Bind pathogen; Eliminate pathogen;Bind pathogen;hypermutation

Eliminate pathogen;switch recombination

Activation-Induced Deaminase (AID)

deaminates cytosine to uracildeaminates cytosine to uracil

T C G A A G C T AID

T C G A A G U T

U:G is mutagenic: strand breaks & mutations

A G U A G C TT C G T C G A

5’ 3’AID

BER MMR

A G A G C TT C G T C G A

5’ 3’UNG MSH2-MSH6

A G U A G C TT C G T C G A

5’ 3’2

AP endonuclease

A G A G C TT C G T C G A

5’ 3’

Exonuclease 16

A G U A G C T5’ 3’2

DNA polymerase Rev1

T C G T C G A

A G N A G C T5’ 3’

DNA polymerase η

T C G T C G A6

A G C N G C T5’ 3’

Poleta

A G N A G C TT C N T C G A

G:C mutationsSt d b k

A G C N G C TT C G T C G A

A:T mutationsSt d b k

NeubergerStavnezerJacobs

Strand breaksGearhartScharffReynaud

Strand breaks

I The Master CatalystI. The Master Catalyst

G II. Going Rogue

III. The Targeting Enigma

I. AID - the Master Catalyst

Protein loop that recognizes the AID hot spot: WRC (W = A/T; R = A/G)

Rahul Kohli, JHU

JBC 284:22898-04 (2009)

DNA cytosine deaminases

Antibody diversity: AID WRCAntibody diversity: AIDHIV retroviruses: APOBEC 3F

APOBEC 3G

WRCTTCCCC

How do they recognize different motifs?

Structural alignment suggests a “h t t iti l ”“hotspot recognition loop”

H th i G fti l f th APOBEC3 Hypothesis: Grafting loops from the APOBEC3 enzymesinto AID will lead to predictable changes in specificity

Purify variant deaminases from bacterial cultures. Incubate with oligonucleotides containing XXC (X = A, mC, G, or T).

X-2 X-1C5’ 60 nt 3’

Deaminase

XXU

Uracil DNA glycosylase g y y

XX

Abasic endonuclease ^

24 nt 36 nt

AID/APOBEC loop graft variants change AID/APOBEC loop graft variants change sequence motifs as predicted

AID WRC

AID 3FL TTCAID-3FL TTC

AID-3GL CCC

Are motifs altered in E. coli?

rpoBrifampin deathrpoB death

rifampin Resistant colonies;sequence rpoB gene

rpoBAID

deaminaserpoBX

WRC

9 amino acid loop in AID is responsible for recognizing hot spot motif WRC.

Similar loops in APOBEC3F/G can be transferredto change specificity of AID. to change specificity of AID.

II Going Rogue Uracils in DNAII. Going Rogue – Uracils in DNA

AID proposed to deaminate RNA, whereas genetic and biochemical D propos to am nat N , wh r as g n t c an och m ca data support DNA deamination

…but, no evidence for uracils in immunoglobulin loci.

Uracils in variable region DNA from B cells

R b MaulRob Maul

Digest DNA (Ung-/- Peyer’s patch B cells) with l l l ( ) d d l ( E)uracil glycosylase (UDG) and abasic endonuclease (APE)

ConstantVariable U UU U Constant

UDG

APE

PCR amplify

XXHypothesis: more uracils = less amplification of V gene

Location of primers for nested PCR

VDJ 8 kbCμ

VDJ Cμ

k5 kbVκ Cκ

Vκ Cκ

PCR assay

pg DNA 300 300 100 30

Ung-/- Ung-/-Aid-/-

y

300 300 100 30UDG, APE - + + + - + + +

VDJ

Cμ

Vκ

More UDG-sensitive sites in V genes than C genes;

Cκ

More UDG sensitive sites in V genes than genes;AID dependent.

III The Targeting EnigmaIII. The Targeting Enigma

Setting up the Sμ locus for AID

Rob Maul Deepa Rajagopal

JEM 206:1237-44 (2009)

Peaks of hypermutation after V and Iμ promoters

10-2

4

10-6

10-4

10-8

Does location of RNA pol II correlate with this pattern?

H w d es DNA structure in S affect transcripti n?

5 kb0 2 4 1 3

How does DNA structure in Sμ affect transcription?

E H H H

S7 S8 S9S1 S2 S3 S4 S5

Eμ Sμ RR Cμ1

S7 S8 S9S1 S2 S3 S4 S5

GGGGAGCTGGGGAGCTGGGClusters of G and AGCT hotspots for AIDp

R-Loop RegionRNA pol II

DNA-RNA hybrid

Lieber

Map the position of RNA pol II by nuclear run-on

Stimulate Ung-/- splenic B cells with LPS + IL4. After 2 days, isolate nuclei.

Addition of

g p y ,

ll

Gentle Lysis[32P]UTP, ATP,

CTP, GTP

cells nuclei transcription

Position of RNA pol II molecules

S7 S8 S9S1 S2 S3 S4 S5

Eμ Sμ RR Cμ1

tens

ity Ung-/-

Day 06

8

10S1S2S3

β-actCD3δ

Rel

ativ

e in

t y

0

2

4S3S4S5S7 S8

S9

S1 S2 S3 S4 S5 S7 S8 S9 γ-act β-act CD3δ

Ung-/-

Day 2

ativ

e in

tens

ity

2

4

6

8

10S1S2S3S4

β-actCD3δ

γ-act

Rel

a

0

2

S1 S2 S3 S4 S5 S7 S8 S9 γ-act β-actCD3δ

S5S7 S8

S9

P l f l h d f h Pileup of polymerases on either side of the repetitive region.Does not depend on cell activation.

Aid-/-Ung-/-y10

S1 β-actAid UngDay 2

Rel

ativ

e in

tens

ity

2

4

6

8S2

S3

S4S5

CD3δ

S8

S9

R 0 S7 S8S1 S2 S3 S4 S5 S7 S8 S9 γ-act β-act CD3δ

C

S1 β-actSμ dely 8

10

S1 S2 ∆S Cμ

Eμ Cμ1

S2

∆S

Cμ

CD3δ

t

Sμ delDay 2

Rel

ativ

e in

tens

it

0

2

4

6

8

Pileup does not depend on AID. Depends on DNA sequence of Sμ.

Cμ γ-act0S1 S2 ∆S Cμ γ-act β-act CD3δ

p p p q

RNA pol II location confirmed by ChIP

RNA pols pause in R-loops

GGGGAGCT GGGGAGCT GGGGAGCT

p p p

CCCCTCGA CCCCTCGA CCCCTCGA

CμSμ coreiEμR-loop

RNAP RNAP RNAP

Fast TranscriptionSlow Transcription- High pol densityFast Transcription

- Low pol densityHigh pol density

How does DNA structure affect somatic hypermutation?

Sequenced JH4 intron DNA from immunized B cells from Ung-/- miceSequenced JH4 intron DNA from immunized B cells from Ung mice

8

10

y (x

10-3

)

4

6

on F

requ

ency

0

2

Mut

ati

not sequenced

Sμ RREμ

Mutations increase before the repetitive region Mutations increase before the repetitive region and decrease after

DNA structure pauses RNA pol II and affects mutationDNA structure pauses RNA pol II and affects mutation

CμRNA pol II

CμAID

AID may associate with RNA pol II. R-loop regions pause RNA AID may associate with RNA pol II. R loop regions pause RNA polymerases, increasing AID binding to ssDNA and mutations. After core, AID falls off or is diluted out, and mutation stops.

What targets AID to V region?g g

Cμ CγS S

DSB DSB

V

NHEJIgM → IgGgM g

Rob MaulHuseyin SaribasakRhonda McClure

Rahul KohliJim StiversDavid WilsonRhonda McClure

Zheng CaoWilliam Yang

David WilsonRanjan SenRoger Woodgate