Bonnet 2009 JI Eb169 SupFig

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.


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bHLH ETS*

KLF*

HumanMouseDogRabbitCowOpossum

GATA KLF bHLH* ETS*

EE1/2 EE3 EE4




RUNX* ETS*

EE6 EE7


RUNX*

EE4 EE5

HumanMouseDogRabbit

CowOpossum

bHLH


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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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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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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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


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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


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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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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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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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

Bonnet 2009 JI Eb169 SupFig

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