Bonnet et al., Supplementary Fig.

S1
EE3
EE4
EE5
EE6
EE7
KLF
N
a
k
e
d
W
T
T
c
ETS
EE2/1
EE3
EE4
N
a
k
e
d
E
E
1
6
9
/
1
6
9 T
c
Hypersensitive nucleotides
Protected nucleotides
EE1/2
Fig. S1: In vivo DMS genomic footprinting profiles throughout the EE region in WT and
EE
169/169
Tcs. Dimethylsulfate treatment of thymocytes and genomic DNA, DNA preparation,
and ligation-mediated PCR were performed as described previously [1]. A set of specific
primers was designed to analyze footprint patterns on the bottom strand over the entire EE
region. EE1-7 sites are indicated on the left. Protected (filed circles) and hypersensitive (open
circles) nucleotides are shown on the right. Naked: genomic DNA treated in vitro by DMS.
Due to the differences in size between the EE
wt
and EE
169
sequences, the WT and mutant
samples were analyzed in separate acrylamide gels. The dotted line indicate the
corresponding regions in the EE
wt
and EE
169
sequences. Detected footprints at EE4 and EE6
match previously identified footprinted regions [1]. New footprinted sequences and
associated putative TF binding sites discussed in the text of the Results section are shown.
Note that, in the EE
169/169
nuclei, the 5 KLF-binding nucleotides displayed patterns of strong
protection or hypersensitivity to DMS treatment suggestive of full occupancy by cognate TF,
whereas the downstream EE4 site showed no obvious protection indicating poor TF loading.
KLF
1. Tripathi, R. K., N. Mathieu, S. Spicuglia, D. Payet, C. Verthuy, G. Bouvier, D. Depetris,
M. G. Mattei, W. M. Hempel, and P. Ferrier. 2000. Definition of a T-cell receptor E gene
core enhancer of V(D)J recombination by transgenic mapping. Mol. Cell Biol. 20: 42-53.
bHLH ETS*
KLF*
Human
Mouse
Dog
Rabbit
Cow
Opossum
GATA KLF bHLH* ETS*
EE1/2 EE3 EE4
Human
Mouse
Dog
Rabbit
Cow
Opossum
Human
Mouse
Dog
Rabbit
Cow
Opossum
Human
Mouse
Dog
Rabbit
Cow
Opossum
RUNX* ETS*
EE6 EE7
Human
Mouse
Dog
Rabbit
Cow
Opossum
RUNX*
EE4 EE5
Human
Mouse
Dog
Rabbit
Cow
Opossum
bHLH
Bonnet et al., Supplementary Fig. S2
Fig. S2: Phylogenetic conservations within EE-overlapping sequences from the indicated species. To
retrieve E orthologues, human C and V20S1 (Genbank ID: U66061) as well as mouse C and
V14S1 (Genbank ID: AE000665) nucleotide sequences were blasted against the dog, rabbit, cow and
opossum Ensembl databases (http://www.ensembl.org). Orthologous loci were defined as the regions
showing the highest scoring alignments. The E region was defined by aligning the reference mouse E
sequence (Genbank ID: X07177) to these regions. All conserved regions were investigated using the
JASPAR database for human and mouse TFs. The EE
169
insert is underlined in yellow. Highly
conserved motifs matching known TFBS are shown. TFBS overlapping with the footprinted sequences
identified in vivo are marked by an asterisk (*).
Bonnet et al., Supplementary Fig. S3
W
T
E
E
1
6
9
n
e
o
/
W
T
E
E
1
6
9
n
e
o
/
1
6
9
n
e
o
3kb
4kb
TCRE locus
B H N N E Bg
3kb
probe
CE2 VE14
B E
TK Neo
EE
EE169
B N E Bg
4kb
probe
CE2 VE14 Neo EE169
N
N
B N E Bg
probe
CE2 VE14 EE169
Homologous
recombination
Cre-mediated
deletion
pEE
169neo
construct
EE
169neo
allele
EE
169
allele
W
T
E
E
1
6
9
/
1
6
9
E
E
1
6
9
/
W
T
0.5 kb
1kb
WT allele
Mutant allele
Fig. S3: The diagram depicts the experimental strategy used for the replacement of the EE enhancer
by the truncated EE
169
insert in mouse ES cells and southern blot analysis of the resulting knockin
mice. Homologous recombination between the endogenous TCRE gene (top panel; a partial structure
of the TCRE locus 3 region is shown) and the targeting vector pGEb
169neo
(upper middle row)
generated the EE
169neo
modified allele. The EE
169
mutated allele was obtained following Cre-mediated
loxP recombination. Restriction sites are indicated as follows: B, BamHI; Bg, BglI; E, EcoRI; H,
HpaI; N, NcoI. The TCRE regions used in the targeting vector for homologous recombination are
shown; neo: neomicyn-phosphotransferase resistance gene flanked by loxP target sequences of the
Cre recombinase; tk, thymidine kinase gene (flanked by the HSV promoter). The arrows indicate the
transcriptional orientation of the neo and tk cassettes. The location of the 3 probe (a 1.7-kb HindIII-
BglI fragment) is shown. The panels on the right show Southern blot analysis for the identification of
enhancer-associated genotypes in tail DNA from, respectively, WT, EE
WT/169neo
and EE
169neo/169neo
mice (top panel) and WT, EE
WT/169
, EE
169/169
mice (bottom panel). DNA fragments were gel-
fractionated, southern-blotted and hybridized with the probe specific for the EE
169
insert. Fragments
of the expected sizes are indicated.
Bonnet et al., Supplementary Fig. S4
B220 TCRE CD169
WT
EE
-/-
EE
169/169
EE
169/169
;
TCRG
-/-
Fig. S4: Immuno-histological analysis of splenic T-cell areas in mice homozygous for the EE
169
mutation. Spleens from WT, EE
169/169
, EE
169/169
;TCRG
-/-
and EE
-/-
mice were fixed in 4%
paraformaldehyde and embedded on dry ice in OCT (optimal cutting temperature) compound
(Miles Inc. Elkhart, IN, USA). Sections (12 mm) of splenic pulp specimens were processed for
staining as described previously [1], using the following antibodies: TCRE-PE for T-cells (in red),
B220-APC for B-cells (in blue) and CD169-FITC for marginal metallophilic macrophages (in
green). Pictures were acquired by confocal microscopy at a 16X enlargement. Scale bars [in
micrometers (m)] are shown. The results demonstrate the presence of genuine TCRE
+
areas in
spleens from WT, EE
169/169
and E
169/169
;TCRG
-/-
, but not in those from EE
-/-
mice.
1. Mugnier, B., B. Nal, C. Verthuy, C. Boyer, D. Lam, L. Chasson, V. Nieoullon, G. Chazal, X. J.
Guo, H. T. He, D. Rueff-Juy, A. Alcover, and P. Ferrier. 2008. Coronin-1A links cytoskeleton
dynamics to TCR alpha beta-induced cell signaling. PLoS. ONE 3:e3467.
100 m
100 m 100 m
100 m
Bonnet et al., Supplementary Fig. S5
10.2 12.3
51.1 26.4
2.4 12.4
84.2 1
6 10.1
64.4 19.5
2.1 12.7
84 1.2 37.3 37.6 37.3 37.6
14.9
37.3 37.6
10.2
EE
-/-
WT EE
169/169
EE
169/169
;
TCRG
-/-
EE
-/-
;
TCRG
-/-
CD25
C
D
4
4


%

C
e
l
l
s
TCRG
Fig. S5: Flow cytometric analysis of CD4
-
CD8
-
DN Tcs. (A) Top histograms: cell surface
expression of CD44 vs. CD25 analyzed in single cell suspension of thymic cells, gated on
CD4
-
CD8
-
Lin
-
cells (see Material and Methods) from WT, EE
169/169
, EE
169/169
;TCRG
-/-
, EE
-/-
and EE
-/-
;TCRG
-/-
four week old mice. Bottom histograms: analysis of TCRG chain
expression in DN Tcs revealed expression in cells from WT, EE
169/169
and EE
-/-
mice that
was more abundant in the latter two strains, consistent with an accumulation of JG T cells.
(B) Cell surface expression of CD44 vs. CD25 analyzed in CD4
-
CD8
-
Lin
-
cells from
EE
169/169
;TCRG
-/-
and RAG2
-/-
four week old mice (left histograms). The middle histograms
show the expression of the T-cell marker Thy1.2 within the DN4 (CD44
-
CD25
-
) thymic cell
population. The histograms on the right depict intra-cytoplasmic CD27 vs. TCRE chain
labeling of CD44
-
CD25
-
DN Tcs. Percentages of each subpopulation are indicated in the
corresponding quadrant. All experiments were performed three times with consistent results.
EE
169/169
;
TCRG
-/-
RAG2
-/-
1.73 36
61.8 0.54
3.87 44.8
50.9 0.38
93.2
81.9
14.7 6.68
2.67 76
25.7 1.45
0.43 72.4
A
B
%

C
e
l
l
s
Thy1.2
C
D
4
4
CD25
C
D
2
7
icTCRE
WT
E
169/169
Mm = 300 s 65
Mm = 140 s 5
CVm = 134 s 43
CVm = 414 s 89
IgG
TCRE
A
D0 2d week 5th week
TCR
hi
sorted cells
Unsorted cells
TCR
lo
sorted cells
B
Bonnet et al., Supplementary Fig. S6
E
169/169
WT
TCRE
%

C
e
l
l
s
TCRE
%

C
e
l
l
s
Fig. S6 : Flow cytometric analysis of EE
169/169
splenic T-cell derived hybridomas. (A)
TCRE expression at the cell surface of WT (top panels) vs. EE
169/169
(bottom panels) T-
cells. Three hybridomas of each genotype are shown; consistent results were obtained in
similar analyses of 79 additional hybridomas. Median (M) and coefficients of variation
(CV) values are shown above each panel; averaged values (Mm and CVm) from analyses
of the additional hybridomas are indicated on the right. An anti-IgG2O1 isotype antibody
was used as a negative control (grey areas). (B) Low and high TCRE expressing cells
(TCRE
lo
and TCRE
hi
) from WT (top panels) and EE
169/169
(bottom panels) T-cell
hybridomas were sorted and cultured for 5 weeks. Levels of TCRE expression of unsorted
(grey histograms), TCRE
lo
(blue lines) and TCRE
hi
(black lines) cells were analyzed by
FACS after 2 and 5 weeks of culture, as indicated. The results show that, during a 5-
weeks culture period, the EE
169/169
T-cell outliers (either TCRE
lo
or TCRE
hi
) re-adjusted
their TCRE expression levels to the parental profile less proficiently compared to their
WT counterparts. These experiment were performed three times with consistent results.
M = 382
Cv = 103
M = 255
Cv = 143
M = 234
Cv = 169
M = 140
Cv = 303
M = 135
Cv = 418
M = 146
Cv = 521
Bonnet et al., Supplementary Fig. S7
DN Tcs
+
WT
-
+
- -
+
-
+
-
1
/
1
5
6
2
5
H
2
O
1
/
1
2
5
1
/
6
2
5
1
/
3
1
2
5
VE
5.1
-CE
VE
6
-CE
VE
8.1
-CE
VE
14
-CE
VE
11
-CE
VE
4
-CE
CE
DE2-CE
E-Actin
EE
169/169
EE
-/-
Fig. S7: RT-PCR assays to detect rearranged transcripts within the indicated
regions of the TCRE locus using total RNA isolated from purified DN Tcs of WT,
EE
169/169
and EE
-/-
mice. (-) and (+) reverse transcriptions are indicated. cDNA from
the WT samples was subjected to serial 5-fold dilutions starting from the 1/125
dilution. cDNAs were PCR amplified for 43 rounds. The resulting amplicons were
analyzed by Southern blotting using
32
P-labelled specific probes. RT-PCR for E-
actin was used to control for the amount of the investigated cDNAs.
Bonnet et al., Fig. S8
Fig. S8: Comparative analysis in the efficiency of TCRE gene expression and V(D)J recombination
at the E
169
vs. EE (SJL) alleles. (A) Structural organization of the TCRE locus in the SJL mouse
depicting the deletion in this strain of a 80 kb region spanning from V5.2 to V9 (dashed area).
(B) Percentages of expression of the indicated TCR VE chains on the surface of Thy1.2
+
LN cells
from WT, E
169/169
, SJL/WT, SJL and SJL/E
169
mice. Standard error bars are from five separate
FACS analyses. (C) D2-J2 and V-DJ2 CJ products of V(D)J recombination were analyzed by
PCR amplification of genomic DNA from sorted LN T-cells. SJL/WT DNA was subjected to a
serial 3-fold dilution prior to amplification. DNA from WT kidney (Kd) was used as a control for
amplification of germline DNA. The amount of each DNA sample was monitored by amplification
of genomic DNA fragment within the CD3H gene. Amplicons were detected by southern blotting
using
32
P-labelled specific probes.
A
B
V
E
e
x
p
r
e
s
s
i
o
n

(
%
)

0
5
10
15
20
25
30
VE
3
VE
5
VE
8
VE
11
WT
E
169/169
SJL/WT
SJL
SJL/E
169
VE
14
DE2-JE2
C
E
E
1
6
9
/
1
6
9
S
J
L
/
E
E
1
6
9
W
T SJL/WT
S
J
L
K
d
H
2
O
VE
3
-JE2
VE
14
-JE2
VE
11
-JE2
VE
5.1.2
-JE2
VE
8.1.2
-JE2
CD3H
4
1 4
5.2
10
8.1
12 9 6 7 3
11
TG TG
8.3
5.1
8.2
13
16 1
15
19
20 18
DJC1 DJC2
pD1
V
2
E
E
E
1
6
9
/
1
6
9
S
J
L
/
E
E
1
6
9
W
T SJL/WT
S
J
L
K
d
H
2
O
Bonnet et al., Supplementary Fig. S9
R
-/-
EE
WT
R
-/-
EE
c/c
R
-/-
EE
-/-
VE
8.1
VE
13
VE
12 VE
11
VE
9
VE
6
VE
15
R
-/-
EE
c/c
R
-/-
EE
-/-
VE
19
VE
20
VE
3
VE

VE
18
R
-/-
EE
WT
R
-/-
EE
c/c
R
-/-
EE
-/-
VE
5.2
VE
8.3
VE
5.1
VE
8.2
MoxD2
VE
2
T1
VE
4
VE
16
VE
10
VE
1
R
-/-
EE
c/c
R
-/-
EE
-/-
CE1 CE2 EE VE
14
DE1JE1 DEJE2 Ephb6 T20
A
Chr6:
Chr6:
Chr6:
Chr6:
R
-/-
EE
WT
R
-/-
EE
WT
Bonnet et al., Supplementary Fig. S9
CD3J
CD3G
CD3H
R
-/-
EE
WT
R
-/-
EE
169/169
R
-/-
EE
-/-
B
R
-/-
EE
WT
R
-/-
EE
169/169
R
-/-
EE
-/-
C
Chr6:
Fig. S9: ChIP-on-chip analysis of enhancer-dependent H3K4me2 enrichment at the TCR locus. (A)
H3K4me2 enrichments (Log
2
IP/input) at the TCR locus were analyzed using cell suspensions from
thymus of Rag2-deficient (R
-/-
E
WT
) mice, and of R
-/-
E
169/169
or R
-/-
E
-/-
compound mice.
Chromosomal locations are shown at the top of each panel. The position of the various coding
sequences and enhancer sequences within or adjacent to the TCR locus are indicated. (B) H3K4me2
enrichments at the CD3 genomic cluster. (C) Relative H3K4me2 enrichments were calculated by
averaging the values of the probes that overlap the corresponding genomic region, following by
normalization using the total signal in the microarray (excluding the TCR locus). The levels
registered for the R
-/-
E
WT
cells were set to 1.
N
o
r
m
a
l
i
z
e
d

H
3
K
4
m
e
2

e
n
r
i
c
h
m
e
n
t
1,4
1,2
1
0,8
0,6
0,4
0,2
0
A
l
l
3

V
E
s
T
C
R
E
D
J
1
T
C
R
E
D
J
2
T
C
R
E
C
1
T
C
R
E
C
2
T
C
R
E
V
1
4
S
1
p
E
p
h
b
6
C
D
3
c
l
u
s
t
e
r
Table SI. Absolute numbers of LN T cells and thymocytes in WT, E|
169/WT
, E|
169/169
, E|
169/169
;TCRo
-/-
, E|
-/-
and
E|
-/-
;TCRo
-/-
mice
a
LN T cells
b
CD4
+
CD8
+

Total
thymocytes
DN DP SP CD4
+
SP CD8
+

WT 21.5 5.8 13.1 2.6 8.0 1.0 208.0 67.3 7.1 2.5 168.5 18.9 20.5 8.1 5.2 3.3
E|
169/WT
23.4 6.5 15.8 1.8 7.2 0.7 126.5 19.3 8.5 4.5 89.4 10.6 16.7 2.9 4.7 1.4
E|
169/169
3.2 0.9 2,0 0,9 1,5 0,5 11,3 6,4 5,4 1,9 4,2 2,1 0,4 0,2 0,6 0,4
E|
169/169
;TCRo
-/-
1.6 0.4 1.0 0.7 1.3 1.0 4.8 1.3 4.5 0.4 0.2 0.1 0.13 0.1 0.12 0.1
E|
-/-
2.4 1.3 0.5 0.4 0.7 0.3 7.9 3.7 6.2 0.9 1.2 0.6 0.1 0.1 0.1 0.1
E|
-/-
;TCRo
-/-
0.01 0.01 0.01 3.0 0.7 3 0.02 0.01 0.01 0.01
a
Average cell numbers (x 10
6
) in each individual cell subset were calculated for 8 mice of each strain.
b
LN cells
were prepared from the inguinal, axillary and mesenteric LNs and the number of T cells determined following
Thy1.2 cell staining. In the E|
-/-
;TCRo
-/-
mice, the numbers and percentages of LN T cells, and of DP and SP Tcs
were generally very low and therefore not determined precisely.

Table SII. Primers and probes used for DNA rearrangements, transcription and
ChIP analyses
Name Sequence Usage
CD3 F TGC CAT AGT AGG ATG AAG G DNA-PCR
CD3 R CAA ACA TTT CCA AGT GAC G DNA-PCR
C|2 F TGT GGC AGG CTC TAA TTA AAT DNA-PCR
C|2 R GCT ATA ATT GCT CTC CTT GAT GGC CTG DNA-PCR
C|2 Probe ATT CAC CCA CCA GCT CAG CTC CAC GTG DNA-PCR
D|1 F TGG TTT CTT CCA GCC CTC AAG DNA-PCR
J|1.6 R GCA GAC AGA GCT CTA TGT ATC AAA DNA-PCR
J|1.6 Probe CTC TAC TTT GCG GCA GGC ACC DNA-PCR
D|2 F GTA GGC ACC TGT GGG GAA GAA ACT DNA-PCR
J|2.6 R TGAGAGCTGTCTCCTACTATCGAT DNA-PCR
J|2.6 Probe TTT CCC TCC CGG AGA TTC CCT AA DNA-PCR
V|3 F GTC ATT CAG CTC CAA GAT ATC TG DNA-PCR
V|5 F CCC AGC AGA TTC TCA GTC CAA CAG DNA-PCR
V|8 F TAT ATG TAC TGG TAT CGG CAG GAC A DNA-PCR
V|11 F CCT GGA GTT CCT GAC TTA CTT TCG DNA-PCR
V|14 F GTG CTC AGA CTA TCC ATC A DNA-PCR
| Actine F CTC TTT GAT GTC ACG CAC GAT TTC RT-PCR
| Actine R GTG GGC CGC TCT AGG CAC CAA RT-PCR
| Actine Probe CTG GGT CAT CTT TTC ACG GT RT-PCR
D|1F TGC AGC CTG CTA GGC CAA GAT RT-PCR
C|1 GL R AGA CAA GAC CCC TTG TTG ATA G RT-PCR
C|1 R Probe GCC TCT GCA CTG ATG TTC TGT RT-PCR
D|2 F TCC GTT CCC AAG CCA AAA G RT-PCR
C|2 R TTC TTG ACC ATG GCC ATC AGC ACC RT-PCR
C|2 GL R TTC TTG ACC ATG GCC ATC AGC ACC RT-PCR
V|4 F CTG GTG GCA GGT CCA GTC G RT-PCR
V|5 F CTC CTG GGA ACA AGT TCA GCA A RT-PCR
V|5 GL R GAT TAA GTT ACA GAA AGC CAG TAG C RT-PCR
V|5 Probe CCC AGC AGA TTC TCA GTC CAA CAG RT-PCR
V|6 F CCT TAC TGT AGA GAC CAC ACA TGG RT-PCR
V|8 F TAT ATG TAC TGG TAT CGG CAG GAC A RT-PCR
V|8 GL R GAT TAT CAA GAG TGA TCA TGA CCT TCA RT-PCR
V|8 Probe TCATATGTCGCTGACAGCACGGAGA RT-PCR
V|11 F GCT TCT TGA GAG CAG AAC CAA CA RT-PCR
V|11 GL R GGA AGC GTA TGG TTT CTG CCT CAG RT-PCR
V|11 Probe TGC TGG TGT CAT CCA AAC ACC TAG RT-PCR
V|14 F CAT GTT CTT GGG TGT TAG TGC TCA RT-PCR
V|14 GL R TTG CAC AGA TGT CTG CCC CA RT-PCR
V|14 Probe GTG CTC AGA CTA TCC ATC A RT-PCR
CD3 F TGC CAT AGT AGG ATG AAG G qPCR
CD3 R CAA ACA TTT CCA AGT GAC G qPCR
pD|1 F TTT CAA TGA CAC CCA GCG CCA A qPCR
pD|1 R TTG TGC AAG GTG GTG GTA AGA TGC qPCR
E| F GGA AGG GGT GGA AGC ATC TC qPCR
E| R TGT AGG ACC TGG TAA ATG TCA AAC qPCR