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8/4/2019 Bonnet 2009 JI Eb169 SupFig
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Bonnet et al., Supplementary Fig. S1
EE3EE4EE5EE6
EE7
KLF
Naked
WTTc
ETS
EE2/1
EE3
EE4
Naked
EE169/1
69 Tc
Hypersensitive nucleotides
Protected nucleotides
EE1/2
Fig. S1: In vivo DMS genomic footprinting profiles throughout the EE region in WT and
EE169/169 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 EEwt and EE169 sequences, the WT and mutant
samples were analyzed in separate acrylamide gels. The dotted line indicate the
corresponding regions in the EEwt and EE169 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 EE169/169 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 receptorE gene
core enhancer of V(D)J recombination by transgenic mapping. Mol. Cell Biol. 20: 42-53.
8/4/2019 Bonnet 2009 JI Eb169 SupFig
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bHLH ETS*
KLF*
HumanMouseDogRabbitCowOpossum
GATA KLF bHLH* ETS*
EE1/2 EE3 EE4
HumanMouseDogRabbitCowOpossum
HumanMouseDogRabbitCowOpossum
HumanMouseDogRabbitCowOpossum
RUNX* ETS*
EE6 EE7
HumanMouseDogRabbitCowOpossum
RUNX*
EE4 EE5
HumanMouseDogRabbit
CowOpossum
bHLH
Bonnet et al., Supplementary Fig. S2
Fig. S2: Phylogenetic conservations within EE-overlapping sequences from the indicated species. Toretrieve E orthologues, human C and V20S1 (Genbank ID: U66061) as well as mouse C andV14S1 (Genbank ID: AE000665) nucleotide sequences were blasted against the dog, rabbit, cow andopossum Ensembl databases (http://www.ensembl.org). Orthologous loci were defined as the regionsshowing the highest scoring alignments. The E region was defined by aligning the reference mouse Esequence (Genbank ID: X07177) to these regions. All conserved regions were investigated using the
JASPAR database for human and mouse TFs. The EE169 insert is underlined in yellow. Highlyconserved motifs matching known TFBS are shown. TFBS overlapping with the footprinted sequencesidentified in vivo are marked by an asterisk (*).
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Bonnet et al., Supplementary Fig. S3
WT EE
169n
eo/W
T
EE169n
eo/16
9neo
3kb
4kb
TCRE locusB HN N E Bg
3kb
probeCE2 VE14
B E
TK Neo
EE
EE169
BN E Bg
4kb
probeCE2 VE14NeoEE169
N
N
BN E Bg
probeCE2 VE14EE169
Homologous
recombination
Cre-mediated
deletion
pEE169neoconstruct
EE169neoallele
EE169 alleleWTEE1
69/16
9
EE169/W
T
0.5 kb
1kbWT allele
Mutant allele
Fig. S3: The diagram depicts the experimental strategy used for the replacement of the EE enhancer
by the truncated EE169 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 pGEb169neo (upper middle row)
generated the EE169neo modified allele. The EE169 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-kbHindIII-
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, EEWT/169neo and EE169neo/169neo
mice (top panel) and WT, EEWT/169, EE169/169 mice (bottom panel). DNA fragments were gel-
fractionated, southern-blotted and hybridized with the probe specific for the EE169 insert. Fragments
of the expected sizes are indicated.
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Bonnet et al., Supplementary Fig. S4
B220 TCRE CD169
WT
EE-/-EE169/169
EE169/169;TCRG-/-
Fig. S4: Immuno-histological analysis of splenic T-cell areas in mice homozygous for the EE169
mutation. Spleens from WT, EE169/169, EE169/169;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, EE169/169 and E169/169;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. ONE3:e3467.
100 m
100 m 100 m
100 m
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Bonnet et al., Supplementary Fig. S5
10.2 12.3
51.126.4
2.4 12.4
84.21
610.1
64.419.5
2.1 12.7
841.237.337.6 37.337.6
14.9
37.337.6
10.2
EE-/-WT EE169/169 EE169/169;
TCRG-/- EE-/-;
TCRG-/-
CD25
CD44
%Cells
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, EE169/169, EE169/169;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, EE169/169 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 fromEE169/169;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.
EE169/169;TCRG-/-
RAG2-/-
1.73 36
61.80.54
3.87 44.8
50.90.38
93.2
81.9
14.7 6.68
2.6776
25.7 1.45
0.4372.4
A
B
%Cells
Thy1.2
CD44
CD25
CD27
icTCRE
8/4/2019 Bonnet 2009 JI Eb169 SupFig
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WT
E169/169
Mm = 300 65
Mm = 140 5
CVm = 134 43
CVm = 414 89
IgG
TCRE
A
D0 2d week 5th week
TCRhi sorted cellsUnsorted cells
TCRlo sorted cells
B
Bonnet et al., Supplementary Fig. S6
E169/169
WT
TCRE
%Cells
TCRE
%Cells
Fig. S6 : Flow cytometric analysis of EE169/169 splenic T-cell derived hybridomas. (A)
TCRE expression at the cell surface of WT (top panels) vs. EE169/169 (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
(TCRElo and TCREhi) from WT (top panels) and EE169/169 (bottom panels) T-cell
hybridomas were sorted and cultured for 5 weeks. Levels of TCRE expression of unsorted
(grey histograms), TCRElo (blue lines) and TCREhi (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 EE169/169 T-cell outliers (either TCRElo or TCREhi) 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
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Bonnet et al., Supplementary Fig. S7
DN Tcs
+
WT
- +- - + -+-1/15625
H2O
1/125
1/625
1/3
125
VE5.1-CEVE
6
-CE
VE8.1-CE
VE14-CE
VE11-CEVE4-CE
CEDE2-CEE-Actin
EE169/169 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,
EE169/169 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 32P-labelled specific probes. RT-PCR forE-
actin was used to control for the amount of the investigated cDNAs.
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Bonnet et al., Fig. S8
Fig. S8: Comparative analysis in the efficiency of TCRE gene expression and V(D)J recombination
at the E169 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, E169/169, SJL/WT, SJL and SJL/E169 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 byPCR 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 32P-labelled specific probes.
A
B
V
expression(%)
0
5
10
15
20
25
30
VE3 VE5VE8 VE11
WT
E169/169SJL/WT
SJL
SJL/E169
VE14
DE2-JE2
C
EE169/169
SJL/EE
169
WT SJL/WT
SJL
KdH 2O
VE3-JE2
VE14-JE2
VE11-JE2
VE5.1.2-JE2
VE8.1.2-JE2
CD3H
4
145.2
10
8.1
12 9 6 73
11TG TG
8.3
5.1
8.213
16 1
15
19
20 18
DJC1 DJC2pD1
V2
E
EE169/169
SJL/EE
169
WT SJL/WT
SJL
KdH 2O
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Bonnet et al., Supplementary Fig. S9
R-/-EEWT
R-/-EEc/c
R-/-EE-/-
VE8.1 VE13 VE12 VE11 VE9 VE6 VE15
R-/-EEc/c
R-/-EE-/-
VE19 VE20 VE3 VE VE18
R-/-EEWT
R-/-EEc/c
R-/-EE-/-
VE5.2
VE8.3VE5.1
VE8.2MoxD2
VE2T1
VE4
VE16VE10
VE1
R-/-EEc/c
R-/-EE-/-
CE1 CE2 EE VE14DE1JE1 DEJE2 Ephb6T20
A
Chr6:
Chr6:
Chr6:
Chr6:
R-/-EEWT
R-/-EEWT
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Bonnet et al., Supplementary Fig. S9
CD3JCD3G
CD3H
R-/-EEWT
R-/-EE169/169
R-/-EE-/-
B
R-/-EEWTR-/-EE169/169R-/-EE-/-
C
Chr6:
Fig. S9: ChIP-on-chip analysis of enhancer-dependent H3K4me2 enrichment at the TCR locus. (A)
H3K4me2 enrichments (Log2 IP/input) at the TCR locus were analyzed using cell suspensions from
thymus of Rag2-deficient (R-/-
EWT
) mice, and of R-/-
E169/169
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-/-EWT cells were set to 1.
NormalizedH3K4me2enrichment
1,4
1,2
1
0,8
0,6
0,4
0,2
0
All 3V
Es
TCRED
J1
TCRED
J2
TCREC
1
TCREC
2
TCREV
14S1
pEphb6
CD3clu
ster
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Table SI. Absolute numbers of LN T cells and thymocytes in WT, EE169/WT, EE169/169, EE169TCR
EE-/-
;TCRG-/-
micea
LN T cellsb
CD4+
CD8+
Totalthymocytes
DN DP SP CD4+
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
EE169/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
EE169/169
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
EE169/169
;TCRG-/- 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
EE-/- 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
EE-/-
;TCRG-/- 0.01 0.01 0.01 3.0 0.7 3 0.02 0.01 0.01
aAverage cell numbers (x 106) in each individual cell subset were calculated for 8 mice of each stra
were prepared from the inguinal, axillary and mesenteric LNs and the number of T cells determin
Thy1.2 cell staining. In the EE-/-;TCRG-/- mice, the numbers and percentages of LN T cells, and of DP
were generally very low and therefore not determined precisely.
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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
CE2 F TGT GGC AGG CTC TAA TTA AAT DNA-PCR
CE2 R GCT ATA ATT GCT CTC CTT GAT GGC CTG DNA-PCR
CE2 Probe ATT CAC CCA CCA GCT CAG CTC CAC GTG DNA-PCR
DE1 F TGG TTT CTT CCA GCC CTC AAG DNA-PCR
JE1.6 R GCA GAC AGA GCT CTA TGT ATC AAA DNA-PCR
JE1.6 Probe CTC TAC TTT GCG GCA GGC ACC DNA-PCR
DE2 F GTA GGC ACC TGT GGG GAA GAA ACT DNA-PCR
JE2.6 R TGAGAGCTGTCTCCTACTATCGAT DNA-PCR
JE2.6 Probe TTT CCC TCC CGG AGA TTC CCT AA DNA-PCR
VE3 F GTC ATT CAG CTC CAA GAT ATC TG DNA-PCR
VE5 F CCC AGC AGA TTC TCA GTC CAA CAG DNA-PCR
VE8 F TAT ATG TAC TGG TAT CGG CAG GAC A DNA-PCR
VE11 F CCT GGA GTT CCT GAC TTA CTT TCG DNA-PCR
VE14 F GTG CTC AGA CTA TCC ATC A DNA-PCR
E Actine F CTC TTT GAT GTC ACG CAC GAT TTC RT-PCR
E Actine R GTG GGC CGC TCT AGG CAC CAA RT-PCR
E Actine Probe CTG GGT CAT CTT TTC ACG GT RT-PCR
DE1F TGC AGC CTG CTA GGC CAA GAT RT-PCR
CE1 GL R AGA CAA GAC CCC TTG TTG ATA G RT-PCR
CE1 R Probe GCC TCT GCA CTG ATG TTC TGT RT-PCR
DE2 F TCC GTT CCC AAG CCA AAA G RT-PCR
CE2 R TTC TTG ACC ATG GCC ATC AGC ACC RT-PCR
CE2 GL R TTC TTG ACC ATG GCC ATC AGC ACC RT-PCR
VE4 F CTG GTG GCA GGT CCA GTC G RT-PCR
VE5 F CTC CTG GGA ACA AGT TCA GCA A RT-PCR
VE5 GL R GAT TAA GTT ACA GAA AGC CAG TAG C RT-PCR
VE5 Probe CCC AGC AGA TTC TCA GTC CAA CAG RT-PCR
VE6 F CCT TAC TGT AGA GAC CAC ACA TGG RT-PCR
VE8 F TAT ATG TAC TGG TAT CGG CAG GAC A RT-PCR
VE8 GL R GAT TAT CAA GAG TGA TCA TGA CCT TCA RT-PCR
VE8 Probe TCATATGTCGCTGACAGCACGGAGA RT-PCR
VE11 F GCT TCT TGA GAG CAG AAC CAA CA RT-PCR
VE11 GL R GGA AGC GTA TGG TTT CTG CCT CAG RT-PCR
VE11 Probe TGC TGG TGT CAT CCA AAC ACC TAG RT-PCR
VE14 F CAT GTT CTT GGG TGT TAG TGC TCA RT-PCR
VE14 GL R TTG CAC AGA TGT CTG CCC CA RT-PCR
VE14 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
pDE1 F TTT CAA TGA CAC CCA GCG CCA A qPCR
pDE1 R TTG TGC AAG GTG GTG GTA AGA TGC qPCR
EE F GGA AGG GGT GGA AGC ATC TC qPCR
EE R TGT AGG ACC TGG TAA ATG TCA AAC qPCR