"Ig gene class switch or

aberrant translocation…:
who is the master?"
Michel Cogné
CNRS UMR 7276, Limoges University, France

UMR CNRS 7276. (CRIBL)

Contrôle de la réponse immune
B et lymphoproliférations
 IgH locus regulation
VDJ Cµ Cd Cg3 Cg1 Ca1 Cg2 Cg4 Ce Ca2
Em Sm Sg3 Sg1 Sa1 Sg2 Sg4 Se Sa2
LCR 3’
 IgH genes in immature B cells
Cellule B immature: IgM seulement

VDJ Cµ Cd Cx
ADN Em Sm Sx

transcription

VDJ Cµ
ARN
translation

m Heavy chain=> IgM

 IgH in mature IgM+IgD+ cells
Cellule B mature: IgM + IgD

VDJ Cµ Cd Cx
ADN Em Sm Sx

transcription

VDJ Cµ VDJ Cd
ARN ARN
translaton

m + d heavy chains=> IgM + IgD

 IgH in stimulated B-cells
VDJ Cµ Cd Cx
ADN Em Sm Sx

transcription transcription
IgH in stimulated B-cells 1. Cleavage by AID + UNG + Exo1 +????

VDJ Cx
Em Sm Sx

Repair by Non Homologous End Joining (+ some Alternate End-Joining)

Masters of gene regulation

Navneet Matharu et al,

PLOS GENET 2015 https://doi.org/10.1371/journal.pgen.1005640
A model of super-enhancer…

• Super-enhancers as defined in 2013 by Richard Young and coll.:

= chromosomal regions differing from enhancers in
- primary structure: “ clusters of enhancers”, spanning regions
over several kb,
- chromatin structure and binding by trans-acting factors: one
order of magnitude higher density of H3K27ac and H3K4me1 ,
resulting in strong binding by Mediator, dense occupation by
master regulators ( i.e. TF governing cell lineage commitment)
- function: higher transcriptional strength, architectural role
supporting loops and long-distance interactions, importance for
cell lineage identity
A model of super-enhancer: the
IgH 3’ regulatory region (3’RR)
• a cluster of 4 weak enhancers associated in a 30kb locus
control region
• bound by B lineage and/or GC-commitment (Pax5, Bach2 Oct)
& activation responsive transcription factors (NFkB, Ets1, YY1)
• dyad symmetry of hs3 enhancers and flanking « junk DNA »

3’RR super-enhancer
hole

Eµ hs3a hs1,2 hs3b hs4 hs5, 6, 7

 GC Dark
Peripheral B cell maturation zone
SHM

CXCL12
naive B cell CXCR4+
Centroblast

CXCL13,BAFF,
HGF, IL-15, IL-6

CXCR5+
Centrocyte
IL-21
CXCL13
IL-4

CXCR5+
Plasma cell
TFH CSR

GC
Ig secretion light zone
 Peripheral B cell maturation
SHM

naive B cell CXCR4+

Eµ Eµ
Centroblast

CXCR5+
Centrocyte
Sµ

Eµ
Eµ

Plasma cell
CSR
Ig secretion
Wuerffel et al, Immunity 2007
 The 3’RR super-enhancer has little role in
immature and resting B-cells
 Heterozygous 3’RR deletion
preserves the expression ratio
naive B cell
Eµ
of both alleles
(in IgHb (wt)/ IgHa (3’RR-KO) heterozygous mice)

13.6% 14.6%
B220

IgMa IgMb
Rouaud et al, JBC, 2012
 The 3’RR super-enhancer has little role in
immature and resting B-cells

naive B cell
Eµ
Rouaud et al, JEM 2013

3’RR deletion barely affects

VDJ transcription…
 The 3’RR impacts somatic hypermutation
SHM

naive B cell CXCR4+

Eµ Eµ
Centroblast
Rouaud et al, JEM 2013

3’RR deletion barely affects …but alters AID recruitment

VDJ transcription… on rearranged JH segments
 The 3’RR impacts somatic hypermutation
SHM

naive B cell CXCR4+

Eµ Eµ
Centroblast
Rouaud et al, JEM 2013

3’RR deficient mice

3’RR deficient
mice
The 3’RR superenhancer controls class switching

naive B cell CXCR4+

Eµ Eµ
Centroblast

wt/wt N/N D/D

CD-19 CD-19

7.0% 0.1% 0.1%

IgG3

CXCR5+
CD-19

2.5% 0.2% 0.6% Centrocyte

Eµ

IgE
CD-19

7.8%
3.4% 0.4%
0.1% 0.3%
0.1%
CSR

IgA
 The 3’RR impacts the boost of IgH locus
expression normally occuring in plasma cell

naive B cell CXCR4+

Eµ Eµ
Centroblast
Vincent-Fabert et al, Blood 2010

283.5 23.7
± 52.4 ± 2.4
Serum IgM
1000

IgM (mg/ml)
100 P=0.001

10
levels
CXCR5+
Wt D3’RRCentrocyte
Sµ

mice
Eµ
Eµ
9940
± 691
12000

In vitro IgM
IgM (ng/ml)

Plasma cell 8000

1366
± 1022
secretion
4000

0
D3’RR
Wt
mice
Ig secretion
Are the IgH 5’ (Eµ) and 3’ superenhancers
controlling each other?

CXCR5+

Saintamand et al, Sci Rep 2017

Eµ
Are the IgH 5’ (Eµ) and 3’ superenhancers
controlling each other?

CXCR5+

Saintamand et al, Sci Rep 2017

Eµ
26 Ln(10) 3.106

Wt
0 0 0
mice

21 20 25
26 Ln(10) 3.106

Eµ
deficient 0 0 0
mice

21 20 25
26 Ln(10) 3.106

3’RR
deficient 0 0 0
mice
21 20 25

113220K 113255K 113255K 113425K 113425K 113428K

MAR MAR

hs4 hs3a hs1.2 hs3b α ε γ2a γ2b γ1 γ3 δ µ Eµ JH4

Iµ
3’RR structure helps recruit AID

3’RR super-enhancer
hole

Eµ hs3a hs1,2 hs3b hs4 hs5, 6, 7

3’RR structure helps recruit AID

hs1,2
U

hs3a hs3b

hs4
3’RR structure helps recruit AID
cEµ m d g3 g1 g2b g2a e a 3’ RR
VDJ

hs3a 1,2 3b 4

Progressively
Increasing CSR/SHM defects

DIR

c3’RR

Saintamand A et al., Nature Comm. 2016

Le Noir S et al., Nucleic Acids Research. 2017

V D J Eµ Sµ Cµ

A B
 AID, CSR, and legitimacy of recombination…

VDJ Cµ Cd Cg3 Cg1 Ca1 Cg2 Cg4 Ce Ca2

Em Sm Sg3 Sg1 Sa1 Sg2 Sg4 Se Sa2
3’ RR

SHM (SSBs
m/d ARNm germline transcript g
 3’ RR
Deletional CSR
(DSBs)
Interféron g…
AID CD40 x CD40L

VDJ Cg2 3’RR

Em Sm/Sg
mRNA g2
bi-allelic pathways for CSR in mouse and human
Major pathway:
« cis CSR »
(often biallelic)
Major pathway VDJ Sµ/Sx Cx

VDJ Sµ Cµb Sx Cx
Cµ
Wild type allele
b allotype Cµ
Cµ
a
Mutated allele
a allotype

Cµ

Alternate pathway: CSR foci ? single location?

CSR machinery
Alternative pathway
interallelic « iCSR »
VDJ Sµ Cµ b Sx Cx
Wild type allele
Cµ Cµ « iCSR »
b allotype joining both
Cµ
a Sµ/Sx alleles (10%
Mutated allele
a allotype
to 25% of all
Cµ CSR events)
14 14
 Model of the IgH locus
conformation as suggested by 3C

Wuerffel et al, Immunity 2007

3’IgH loops during « iCSR »?
CSR factories?
 The 3’RR promotes legitimate
interactions between IgH locus segments
both in cis (loops) and in trans

100
>> 55µm
µm

– 5µm
11 to 5 µm
Allele wtb
80

V DH J Eμ μδ γ3 γ1 γ2b γ2a α ε 3’R ≤ 11µm

µm ns
H H 60 R ** ***

D3’RRa
100
100
RP23-109B20
Allele 40
3’R
R 80
80

% IgH-IgH distances
20
RP23-109B20
0 60
60

b
DAPI DAPI t
DAPI

R
w

t
/w
'R

a
IgH IgH IgH
D3

R
40
40

'R
D3 20
20

00

D3’RR D3’RRa/wtb

b
wt

R
w

t
/w
'R

a
D3

R
'R
D3
wt D3’RR D3’RRa/wtb

Le Noir et al, Oncotarget 2017

 The 3’RR promotes CSR on the opposite allele * *

IgG2a in supernatant (µg/mL)

1500 *** *** 5 *** *** *
150 *** ***
4
Serum IgG2a (µg/mL) 1000 3

IgG2a spots
2
100
500 1
60 0.4

0.3
40
50
0.2
20
0.1

0 0.0
0
O ero a****
/b b/b KO ***
o
er *** /b ** /b ns
45 K ***
et *** et Ta t b

ro

/b
KO
T t

a/
ns ns

té

tb
R 5000R
H W W H W W

hé
R

wt
'R

RR
IgEa in supernatant (A.U.)

w
340 3' 20

3'
4000
Serum IgAa (A.U.)

35 3000

30 2000 15

In vitro CSR In vitro CSR

IgEa spots
sera
25 1000

supernatants ELISPOTS
20 60 10
15
40
10
5
20
5
0 0
0
o a/b****bb ns

ro

/b

b
KO ét ér

KO

b/
ns

ta
té
t
* **** wt ns

ro

/b
**

KO
wt
hé
RR

w
w

a/
2000 R

té

tb
h 500 ns *
IgG1a in supernatant (A.U.)
'3 R
3'

wt
hé
RR

w
150

3'
1500 400
Serum IgG1a (A.U.)

1000 300
200
500

IgG1a spots
100
100
15 15

10 10
50
5 5

0 0
0
b

b
ro

ro

/b

/b
KO

KO
a/

tb
té

te

Ta

tb

ro

/b
KO
wt

a/
w

té

tb
hé

He
RR

RR

W
W

wt
hé
RR
Le Noir et al, 2017

w
3'
3'

3'
 The 3’RR promotes legitimate
interactions between IgH locus segments
both in cis (loops) and in trans
Allele ΔEm
3’RR
JH Eμ µ δ γ3 γ1 γ2b γ2a α ε hs3 hs1, hs3 hs4 hs5,6,7
a 2 b
XbaI
hs4

Allele Δ3’RR
Eμ 3’RR

HindIII Eμ
Eµ-hs4 Eµ-calreticulin
66 6
6

Crosslinling frequency 4
Crosslinling frequency

44 4

22 2
2

n.d. n.d. n.d. n.d. n.d. n.d

00 0
0 .
S

S
S

S
S

S
S

S
LP

LP
LP

LP

LP

LP
LP

LP
KO

KO

Le Noir et al, Oncotarget 2017

R

R
KO

KO
T

T
'R

'R
W

W
AR

AR
/3

/3

R
3 'R
Eµ

3 'R
Eµ
-M

-M
Eµ

Eµ
The 3’RR promotes SHM on the opposite allele
How to understand the regulation of the nucleus
by loops, TADs and superenhancers?

In insects, polytene chromosomes were described in 1881 by BALBIANI in salivary glands; their large size is due to many
longitudinal copies (hence the name polytene = many stranded), formed after repeated division of the chromosome without
cell division. Polytene chromosomes contain dark bands and inter bands (lightly stained with nuclear stains and containing less
DNA and more RNA. 2]
The bands become enlarged at certain times to form “puffs” (BALBIANI’s rings).. In the puffs, the chromonemata uncoil
and open out to form many loops. Puffing is caused by the uncoiling of individual chromomeres in a band and indicates the
site of active genes where mRNA synthesis takes place.[6As the expanded loops appear as rings, they are called Balbiani
rings after the name of the researcher who discovered them. They are formed of DNA, RNA and a few proteins ‘RNA pol).
They are sites of transcription
How to understand the regulation of the nucleus
by loops, TADs and superenhancers?
How to understand the regulation of the nucleus
by loops, TADs and superenhancers?

Molecular Cell 74, 212–222, April 4, 2019

How to understand the regulation of the nucleus
by loops, TADs and superenhancers?

Molecular Cell 74, 212–222, April 4, 2019

How to understand the regulation of the nucleus
by loops, TADs and superenhancers?

https://www.youtube.com/watch?v=Tn5qgEqWgW8
Can we molecularly act on super-enhancers?
“mediator” complex helps forming loops between promoters and enhancers

Head
Center
tail
Can we molecularly act on super-enhancers?
“mediator” complex helps forming loops between promoters and enhancers

Pol II

cohesin

AID
Can we molecularly act on super-enhancers?
“mediator” complex helps promoter/enhancer interactions within loops
Does “med1” contribute in B-cell to all long-distance IgH
locus regulations (including recombination and SHM)
Med1 disruption affects VDJ recombination and usage of distal genes

Dalloul I et al, submitted

Does “med1” contribute in B-cell to all long-distance IgH
locus regulations (including recombination and SHM)
Med1 disruption affects somatic hypermutation

Dalloul I et al, submitted

Does “med1” contribute in B-cell to all long-distance IgH
locus regulations (including recombination and SHM)
Med1 disruption affects class switch recombination in vitro

Dalloul I et al, submitted

Does “med1” contribute in B-cell to all long-distance IgH
locus regulations (including recombination and SHM)
Med1 disruption affects class switch recombination in vivo

Dalloul I et al, submitted

Does “med1” contribute in B-cell to all long-distance IgH
locus regulations (including recombination and SHM)
Med1 disruption also affects Ig secretion by plasma cells

Dalloul I et al, submitted

Can we pharmacologically act on superenhancers?
MED1 interacts with BRD4
through their intrinsically
disordered regions (IDRs)

=> form phase-separated

droplets, concentrating and
compartmentalizing the
superenhancer-associated
apparatus
Richard Young and coll.
Science 2018 ;361(6400).

BRD4 is a bromodomain
protein with good
specificity for
superenhancers

+ specifically druggable
by BET-inhibitors such
as JQ1
Can we pharmacologically act on superenhancers?

BET-inhibitor
JQ1
reduces class
switching
in vitro

Dalloul Z et al, in preparation

Can we pharmacologically act on superenhancers?

JQ1 decreases class-switched (g or e) transcripts while increasing pre-

switch germline transcripts

JQ1 decreases AID recruitment to SWITCH regions

Dalloul Z et al, in preparation

Can we pharmacologically act on superenhancers?

JQ1 decreases in vivo production of class-switched Abs (IgG and IgE)

Dalloul Z et al, in preparation

Can we pharmacologically act on superenhancers?

JQ1 decreases the number of Ag-specific TFH cells & changes their polarity

Dalloul Z et al, in preparation

Can we pharmacologically act on superenhancers?

JQ1 increases the production of Th2 cytokines in broncho alveolar fluid

Dalloul Z et al, in preparation

Can we pharmacologically act on superenhancers?

JQ1 increases the severity of Ag-induced asthma

Dalloul Z et al, in preparation

More to understand about the regulation of the
nucleus by loops, TADs and superenhancers?

Alt FW and coll, The fundamental role of chromatin loop extrusion, Nature 2019, in press
A role for the 3’RR superenhancer in cancer?
IgH rearrangements in lymphoma (t 8;14)
IgH rearrangements in lymphoma (t 8;14)
3’ RR
IgH rearrangements in lymphoma (t 8;14)

3’ RR

3’ RR
Other IgH rearrangements in lymphomas

From He & Levine, Blood 2016, 127: 3004

3’RR-driven mouse lymphoma models
3’RR-driven c-myc transgene  « Burkitt like »
lymphoma model (2/3)

(1/3)
 « Anaplastic plasmacytoma»
model

Truffinet et al., J.Immunol 2007

Fiancette et al., J.Immunol 2011
 3’RR-driven mouse lymphoma models
Triple/Quadruple KO models (collaboration FW Alt, Boston)

B-lineage tumors
lymphoma model through
● p53 defect
● NHEJ early defect (Ligase 4)
V(D)J-like
+/- IgH 3’RR mutation (hs3b-4) c-myc translocations

lymphoma model through

● p53 defect switch-like

other
other
● NHEJ late defect (floxed XRCC4)
c-myc translocations
● CD21-cdre (conditional late B cells) T
+/- IgH 3’RR mutation (hs3b-4) T

Gostissa, Nature 2009: Oncogenicity of switch-like translocations is 3’RR-dependent!

 Physiological regulation of the 3’RR
superenhancer: more to understand?

Dalloul Z et al, in preparation

AID

VDJ switch 3’super enhancer

 AID & the IgH 3’ regulatory region (3’RR)
as partners for long-distance CSR….
and maybe more?
AID
only a silent parner?
3’RR super-enhancer
« The Target »
Eµ hs3a hs1,2 hs3b hs4
When the mastermind becomes the target….!

ChIP evaluation of
QPCR evaluation of transcription
RNA Pol2 load

eRNA Transcription from mouse 3’RR enhancers

Péron et al, Science, 2012
When the mastermind becomes the target….!

RNA seq: Human IgH a2 gene and 3’RR2

RNAseq (mouse IgH a gene and 3’RR)

eRNA Transcription from human 3’RR enhancers

Dalloul I et al, Plos Genetics, 2019
 When the mastermind becomes the target….!
Dot plot analysis of mouse IgH 3’RR
Sa
region
Ca
mb
Hs3a

hs1.2

Hs3b

hs4

Sa
region Ca mb hs3a hs1.2 hs3b hs4

Like-Switch (LS) regions

3’RR superenhancers include « like-switch » direct repeats

 When the mastermind becomes the target….!
Dot plot analysis of mouse IgH 3’RR
Sa
region
Ca
mb
Hs3a

hs1.2

Hs3b

hs4

region Ca
Sa
Ca mb hs3a hs1.2 hs3b hs4

G4-DNA / non-template strand

G4-DNA / template strand

3’RR superenhancers include « like-switch » direct repeats

 When the mastermind becomes the target….!
Dot plot analysis of mouse IgH 3’RR Dot plot human 3’RR1 Dot plot Human 3’RR2
Sa
region
Ca
mb
Hs3a

hs1.2

Hs3b

hs4

region Ca
Sa
Ca
Sa
Ca
Ca mb hs3a hs1.2 hs3b hs4 Sa
region region

G4 / NTS
G4 / TS

3’RR superenhancers include « like-switch » direct repeats

Dalloul I et al, Plos Genetics, 2019
Sµ-LS recombination (locus suicide recombination)
joins Sµ to the 3’RR

Péron et al, Science, 2012

Locus suicide recombination in human B-cells
(LSR-seq assay in human samples)
(by NGS / Ion Proton sequencing)

Dalloul I et al, Plos Genetics, 2019

Positions of breaks in human 3’RRs (joined to Sµ)

Cµ Cd Cg3 Cg1 Ca1 Yg Cg2 Cg4 Ce Ca2

Sm Sg3 Sg1 Sa1 Sg2 Sg4 Se Sa2

3’ RR1 3’ RR2
Dalloul I et al, Plos Genetics, 2019
 Locus suicide recombination
Class switch recombination (CSR)
(LSR)
cytokine-dependent B cell activation B cell activation
transcription factors
3’RR 3’RR
+ + activators activators
VDJ Cµ Cx Cε Cα VDJ Cµ Cx Cε Cα

Sµ Sx Sα LS LS LS LS Sµ Sx Sα LS LS LS LS

Partial
deletion complete deletion of C
genes
AID AID AID AID
and co- and co-factors and cofactors and cofactors
factors
3’RR
VDJ Cx Cε Cα VDJ

IgX

IgX

Survival as Apoptosis
memory B cell
IgX
 CNRS UMR7276
(CRIBL) Contrôle de la
Réponse immune B
et Lymphoproliférations
Jeanne Cook-Moreau
Yves Denizot
Sophie Péron
Eric Pinaud

Hend Boutouil
Collaborations with: Nicolas Denis-Lagâche
Fred Alt Iman Dalloul
Amy Kenter Zeinab Dalloul
Jane Skok Brice Laffleur
Sandrine Le Noir
Alexis Saint-Amand