Supplementary Materials for
Distinct use of super-enhancer elements controls cell type–specific CD25
transcription and function
Rosanne Spolski
et al.
Corresponding author: Warren J. Leonard, wjl@helix.nih.gov
Sci. Immunol.
8
, eadi8217 (2023)
DOI: 10.1126/sciimmunol.adi8217
The PDF file includes:
Materials and Methods
Figs. S1 to S9
Tables S1, S2, and S5
Legends for tables S3, S4, and S6
Other Supplementary Material for this manuscript includes the following:
Tables S3, S4, and S6
Data file S1
MDAR Reproducibility Checklist
Supplementary Materials
Supplementary Methods
Cell Culture
For DN2 and DN3 cell culture, bone marrow progenitor cells from
8-12 week-old mice
with a C57BL/6J background were first obtained by depleting mature cells expressing CD3
ɛ
(clone 145-2C11), CD19 (clone 1D3), B220 (clone RA3-6B2), NK1.1 (clone PK136), CD11b
(clone M1/70), CD11c (clone N418), Ly6G/C (clone RB6-8C5), and Ter119 (clone TER-119)
using MACS LS magnetic columns (Miltenyi Biotec).
To generate DN cells from bone marrow
-derived progenitors in Artificial Thymic
Organoid (ATO) cultures (
fig. S1, E to G
), the protocol described by (
28
) was followed, using
stromal cells kindly supplied by A. Montel-Hagen and G. Crooks (UCLA). For differentiation of
DN cells in OP9-DLL1 cocultures (
fig. S1, H to L
;
fig. S2
), enriched progenitor cells were co
-
cultured with OP9-DLL1 cells with IL-7 (Peprotech) and FLT3L (Peprotech) (10 ng/ml each) for
7 days in OP9 medium (α-MEM, 20% FBS, 2 mM glutamine, 100 IU/ml penicillin, 100 ug/ml
streptomycin, and 50 μM β-ME). To generate DN2 cells for ChIP-Seq with and without IL-7
stimulation (
fig. S2A
), precursors from conventional C57BL/6J mice were differentiated in OP9
-
DLL1 culture. For IL-7 stimulation, FACS-sorted DN2 (cKit
+
CD25
+
) cells that were deprived of
IL-7 during the ~6 hour purification period were incubated with 20 ng/ml of IL
-7 for 2 hours and
compared with controls incubated without IL
-
7. All in vitro pro
-
T cell cultures were in a 37 °C,
7% CO
2
environment.
Acute deletion of STAT5 coding genes in developing pro
-
T cells
Generation of
Stat5a;Stat5b
double knockout (DKO) was carried out in cells from
Cas9;
Bcl2
transgenic (tg) animals, as described
(
31
)
, or alternatively in cells from
Cas9
;
Bcl2
-
tg;
Bcl11b
mCh/mCh
or
Cas9
;
Bcl2
-
tg;
Bcl11b
mCitrine/mCitrine
mice in order to differentiate pre
-
commitment of
BCL11B
-
nonexpressing cells from post
-
commitment
BCL11B
+
cells. The
Bcl11b
mCh
and
Bcl11b
mCitrine
alleles respectively encode intact BCL11b together with an internal
ribosome entry site plus an mCherry or mCitrine fluorescent reporter. Retroviral vectors
encoding guide RNAs (sgRNA) against the
Stat5a
and/or
Stat5b
genes, or control sgRNAs (see
Table S6
), together with an hNGFR or mTurquoise reporter were generated as described
(
30,
31
)
, and were introduced into the cells at the time points indicated in
fig. S1
, prior to or during T
cell differentiation culture in vitro. Data shown in
fig. S1M
are from
(
30
)
, where
Cas9
;
Bcl2
-
tg;
Bcl11b
mCh/mCh
progenitor cells were transduced with guide RNAs after different times of culture
and
then harvested five days later. For RNA
-
Seq analysis (
fig. S1, K and L and Table S3
), cells
were collected and sorted to purify DN2
BCL11B
-
and DN2
BCL11B
+
subsets generated from
both control and
Stat5a;Stat5b
DKO cells with a
Bcl11b
mCh
transgene.
To determine cell surface protein expression by flow cytometry, cells were first incubated
in 2.4G2 hybridoma cell supernatant to block Fc receptors. Cell surface staining was performed
by incubating cells in a biotin
-
conjugated lineage cocktail (TCR
β,
TCR
γδ,
CD19, NK1.1,
CD11b, CD11c, and Ly6G/C) followed by secondary surface staining with fluorescently
conjugated streptavidin, CD45, cKit, CD44, CD25, and hNGFR (for sgRNA expressing vector).
To exclude dead cells, a viability dye (Life Technologies, Aqua) or 7AAD (eBioscience) was
used. To determine STAT5 phosphorylation, cells were stimulated with IL
-
7 for 2 hr, fixed with
4% paraformaldehyde for 15
min at room temperature, and then permeabil
ized with 100%
methanol at
-
20°C for 30 min prior to intracellular staining at room temperature for 45 min.
Samples were acquired using a CytoFlex cytometer (Beckman Coulter), and data were analyzed
with FlowJo version 10.8.1 (BD Biosciences). The followin
g antibodies were used for flow
cytometry analysis:
Anti
-
mouse pSTAT5 (pY694) Alexa Fluor 647 (clone
47/Stat5)
BD Biosciences
Cat#612599
Anti
-
human/mouse CD44 PE (clone IM7)
eBioscience
Cat#12
-
0441
-
83
Anti
-
mouse CD117 (cKit) APC (clone 2B8)
eBioscience
Cat#17
-
1171
-
82
Anti
-
mouse CD117 (cKit) APCe780 (clone 2B8)
eBioscience
Cat#47
-
1171
-
82
Anti
-
mouse CD25 APCe780 (clone PC61.5)
eBioscience
Cat#47
-
0251
-
82
Anti
-
mouse CD25 eFluor450 (clone PC61.5)
eBioscience
Cat#48
-
0251
-
82
Anti
-
mouse CD45 PECy7 (clone 30
-
F11)
eBioscience
Cat#25
-
0451
-
82
anti
-
human NGFR APC
(clone ME20.4)
eBioscience
Cat#17
-
9400
-
42
anti
-
human NGFR Super Bright 436 (clone ME20.4)
eBioscience
Cat# 62
-
9400
-
42
Anti
-
mouse TCRβ Biotin (clone H57
-
597)
eBioscience
Cat#13
-
5961
-
85
Anti
-
mouse TCRγδ Biotin (clone GL
-
3)
eBioscience
Cat#13
-
5711
-
85
Anti
-
mouse CD3
ɛ
Biotin (clone 145
-
2C11)
eBioscience
Cat#13
-
0031
-
82
Anti
-
mouse CD8α Biotin (clone 53
-
6.7)
eBioscience
Cat#13
-
0081
-
86
Anti
-
mouse CD19 Biotin (clone 1D3)
eBioscience
Cat#13
-
0193
-
85
Anti
-
mouse B220 Biotin (RA3
-
6B2)
eBioscience
Cat#13
-
0452
-
85
Anti
-
mouse Ly6G/C (clone RB6
-
8C5)
eBioscience
Cat#
13
-
5931
-
86
Anti
-
mouse NK1.1 Biotin (clone PK136)
eBioscience
Cat#13
-
5941
-
85
Anti
-
mouse CD11b Biotin (clone M1/70)
eBioscience
Cat#13
-
0112
-
86
Anti
-
mouse CD11c Biotin
(clone N418)
eBioscience
Cat#13
-
0114
-
85
Analysis of skin
Skin was analyzed as previously described
(
73
)
. Briefly, subcutaneous tissue was
removed with forceps, skin was then minced with scissors in 5 ml RPMI containing 0.25 mg/ml
Liberase T
-
Flex Research grade (Roche) and 1 mg/ml DNase I (Sigma) in 6 well plates and then
incubated at 37 C for 2 hrs.
1
mL of 0.25%Trypsin
-
1 mM EDTA (Gibco) was added for the last
10 min of incubation. After the addition of 5% FBS in PBS, tissue was further dissociated with a
12 ml syringe and then filtered through 100
μ
m Falcon Cell Strainer (Celltreat). Cells were
washed with 5% FBS/PBS and filtered through a 40
μ
m Cell Strainer (Celltreat) into a new 50
ml conical tube. Cells were then stained for flow cytometry using the antibodies listed below in
the table which could distinguish specific lymphoid populations. Cells were acquired on a
Symphony A3 flow cytometer
(BD) and analyzed
with Flowjo Software (Version 10.8.1, FlowJo,
LLC). Skin samples were harvested and fixed in 10% formalin at room temperature and were
sent to Histoserv (Germantown, MD) for immunohistochemical staining (Germantown, MD)
with anti
-
CD45 (Abcam #10558), anti
-
CD4 (Abcam #183685), anti
-
CD8 (Cell Signaling
#98941), and anti
-
FOXP3 (Abcam #215206).
Anti
-
mouse CD3 FITC (clone 145
-
2C11)
Biolegend
Cat#100306
Anti
-
mouse CD11c PerCPCy5.5 (clone N418)
Biolegend
Cat#117328
Anti
-
mouse CD11b PerCPCy5.5 (clone M1/70)
Biolegend
Cat#101228
Anti
-
mouse CD19 PerCPCy5.5 (clone 6D5)
Biolegend
Cat#115534
Anti
-
mouse FceR1a PerCPCy5.5 (clone MAR
-
1)
Biolegend
Cat#134320
Anti
-
mouse CD5 PerCPCy5.5 (clone 53
-
7.3)
Biolegend
Cat#100624
Anti
-
mouse NK1.1 PerCPCy5.5 (clone
PK136)
Biolegend
Cat#108728
Anti
-
mouse CD8a BV421 (clone 53
-
6.7)
Biolegend
Cat#100738
Anti
-
mouse CD45 BV650 (clone 30
-
F11)
Biolegend
Cat#103151
Anti
-
mouse TCRb BV711 (clone H57
-
597)
Biolegend
Cat#109243
Anti
-
mouse CD25 APC (clone 7D4)
BD
Biosciences
Cat#563598
Anti
-
mouse Thy1 BUV395 (clone 53
-
2.1)
BD Biosciences
Cat#565257
Anti
-
mouse FoxP3 PE (clone 150D)
Biolegend
Cat#320008
Anti
-
mouse CD4 PE
-
CF594 (clone RM4
-
5)
BD Biosciences
Cat#562285
Anti
-
mouse TCRgD (clone GL3)
Biolegend
Cat#
118124
Anti
-
mouse CD16/32 (Fc Block, clone 93)
Biolegend
Cat#101320
Zombie aqua fixable viability kit
Biolegend
Cat#423102
The flow cytometric gating strategy is
shown immediately
below.
HiChIP Protocol
HiChIP
assays for H3K27Ac modification were performed using pre
-
activated CD8
+
T
cells from either wild
-
type or mutant mice, as described previously
(
80
)
. Briefly, cells were
crosslinked with 1% formaldehyde and flash
-
frozen in liquid nitrogen. Fixed cells were lysed to
obtain the nuclear fraction, and then chromatin was digested using intact nuclei and 200 U of the
4
-
base cutter Mbo I (New England Biolabs
), and restricted ends religated as described
(
81
)
.
Pelleted nuclei were dissolved in 130
μ
l of nuclear lysis buffer (50 mM Tris
-
HCl, pH 7.5, 10 mM
EDTA, and 1% SDS) and sonicated using a Covaris S220 for 4 min. Sonicated chromatin was
diluted ten times in ChIP Dilution Buffer and immunoprecipitation was
done overnight at 4 °C
by incubating 2.5
μ
l (7
μ
g) of anti
-
H3K27ac antibody (C15410196; Diagenode) precoated on 25
-
μ
l protein A
-
coated magnetic beads (Thermo Fisher Scientific). Immunocomplexes were
captured and washed three times for 5 min each with RIPA
buffer, high
-
salt buffer, LiCl buffer
and low
-
salt buffer (10 mM Tris
-
HCl, pH 8, 1 mM EDTA, and 50 mM NaCl). Beads were
resuspended in TE (10 mM Tris
-
HCl, pH 8, 1 mM EDTA), transferred to fresh tubes, captured,
and then resuspended in 200
μ
l of elution buffer (50 mM NaHCO
3
and 1% SDS). Samples were
treated with 10
μ
g of RNase A (Thermo Fisher Scientific) for 30 min at 37 °C, then with 2
μ
l of
20 mg ml
−
1
proteinase K (Thermo Fisher Scientific) for 1 h at 55° C, and then incubated
overnight at 65° C. DNA was purified using affinity columns (Zymo Research) and eluted in
20 μ
l of DNA elution buffer (10 mM Tris
-
HCl). Adapters were ligated to DNA using NEBNext
Ultra DNA Library Prep Kit (New England Biolabs) according t
o the manufacturer’s protocol.
Streptavidin C
-
1 beads were used to capture biotinylated DNA according to the manufacturer’s
protocol and resuspended in 20
μ
l of DNA elution buffer. To generate the sequencing library,
PCR amplifications of the DNA were perf
ormed while the DNA was still bound to the beads.
Purified HiChIP libraries were size
-
selected to 300
–
800 bp using AMPure XP beads (Beckman
Coulter Life Sciences) according to the manufacturer’s protocol and subjected to 2 × 50
-
bp
paired
-
end sequencing on
an HiSeq 2500 or NovaSeq6000.
HiChIP Analysis
Reads were mapped as follows: For individual samples in different cell types, we applied
the HiC
-
Pro pipeline on the respective paired
-
end reads. Each was mapped independently to the
mouse mm10 reference genome, using the aligner bowtie2. Aligned reads wer
e then paired, and
paired reads involving two different Mbo I restriction sites were retained. FitHiChIP
(
82
)
was
applied on the input set of valid HiChIP reads, and 1 kb binning was applied. Since the point of
junction does not create a new Mbo I site, custom indexing is not required.
FitHiChIP
(
82
)
uses
the valid read pairs generated by HiC
-
Pro
(
83
)
and a set of reference ChIP
–
Seq or HiChIP peaks
corresponding to the target protein or histone modifications of interest (here H3K27ac) to derive
statistically significant interactions. FitHiChIP applies fixed
-
size binning (here 5 kb) on the input
set of valid HiChIP reads and attributes a bin as peak
-
bin if that bin overlaps with a ChIP
–
Seq or
HiChIP
-
inferred peak in the reference peaks file, subject to a 1
-
bp minimum overlap without any
slack. Otherwise, the bin is labeled as a non
-
peak
-
bin. The b
ackground (set of locus pairs used to
infer the null model) as well as the foreground (that is, set of locus pairs that were assigned a
significance estimate) of FitHiChIP can be either peak
-
to
-
peak (that is, interactions between two
peak bins) or peak
-
to
-
all (that is, interactions involving peak bins in at least one end). We used
the default mode (peak
-
to
-
all pairs) for the foreground, which is the setting employed in this
study. Note that FitHiChIP estimates the background contact probability and employs
the
binomial distribution on the generated contact probabilities to estimate P values, which are then
corrected for multiple testing. Interactions having false discovery rate (FDR) < 0.01 are
considered significant and reported as loop calls. These statist
ical analyses support the validity of
interactions identified by FitHiChIP. Lack of loop counts (
Il2ra
gene promoter to regions close to
UP1
-
6) in FitHiChIP
-
L (that detect peak
-
to
-
all interactions) in
D
UP1
-
6 KO cells also suggest that
these are not from closely associated open chromatin regions. Loops calls were visualized using
the Washington University Epigenome Browser [http://epigenomegateway.wustl.edu/browser/].
Cell types
Stimulation
Top enriched motifs derived from the
most significant 500
ChIP
-
Seq peaks
GAS%
E
-
value
CD8
+
T
IL
-
2
88.2%
8.9e
-
505
iTreg
IL
-
2
89.2%
2.6e
-
520
NK
IL
-
15
88.6%
7.4e
-
658
DC
GM
-
CSF
79.6%
6.5e
-
380
Mast cell
IgE
100%
1e
-
634
DN2
IL
-
7
91.4%
1.3e
-
726
MEME (no SSC) 29.06.22 16:30
0
1
2
bits
1
A
C
G
T
2
T
3
C
A
T
4
C
5
A
C
T
6
A
T
G
7
T
A
G
8
A
G
9
G
T
A
10
G
A
11
C
G
A
MEME (no SSC) 29.06.22 16:30
0
1
2
bits
1
C
G
T
A
2
C
A
G
T
3
T
4
C
A
T
5
C
6
G
T
C
7
T
A
C
8
T
G
A
9
G
10
T
A
11
G
A
MEME (no SSC) 29.06.22 16:30
0
1
2
bits
1
G
A
C
T
2
C
T
3
C
G
A
T
4
C
5
A
C
T
6
T
C
G
A
7
G
A
8
A
G
9
A
10
A
11
C
A
MEME (no SSC) 29.06.22 16:30
0
1
2
bits
1
C
G
T
A
2
C
A
G
T
3
T
4
A
T
5
T
C
6
G
C
T
7
A
T
C
8
T
G
A
9
T
G
10
T
A
11
G
A
MEME (no SSC) 29.06.22 16:30
0
1
2
bits
1
C
T
2
C
A
T
3
C
4
A
C
T
5
T
A
G
6
T
A
G
7
A
G
8
T
G
A
9
A
10
T
C
G
A
11
G
C
T
MEME (no SSC) 29.06.22 16:30
0
1
2
bits
1
G
T
A
2
A
G
C
T
3
C
T
4
C
A
T
5
C
6
A
T
C
7
C
A
8
C
G
A
9
G
10
G
T
A
11
A
Ta b l e S 1 . D e n o v o m o t i f a n a l y s i s o f S TAT 5
ChIP
-
Seq data in various cell types.
Table S1 legend: De novo motif analysis of STAT5
ChIP
-
Seq data in various cell types. For each STAT5
ChIP
-
Seq library
in the indicated cell types, we identified binding sites (peaks) by comparing to the IgG control. We then selected the top
500 peaks with lowest
FDR
values, extracted 100 bp of DNA sequence centered on the peak summits, and performed
de
novo
motif analysis using MEME
to characterize the STAT5 consensus binding motifs in CD8
+
T (IL
-
2),
iTreg
(IL
-
2), NK
(IL
-
15), DC (GM
-
CSF), mast cells (
IgE
) and DN2 (IL
-
7). The most significant motifs are shown, as well as motif
frequencies (GAS%) and the corresponding consensus E
-
values.
STAT5 peaks
chrom
start
end
Peak Score
-
10log(
pval
)
GAS motif
E
-
value
Motif DNA
sequence
UP1
chr2
11617088
11617096
961.8
GASn
7.45E
-
05
TTCGAGGGA
UP2
chr2
11620444
11620452
2017.56
GASc
0.00002
TTCAATGAA
UP3
chr2
11622823
11622831
475.59
GASc
1.63E
-
05
TTCAGAGAA
UP4
chr2
11626094
11626102
775.34
GASc
1.79E
-
06
TTCTAAGAA
UP5
chr2
11629859
11629867
2032.24
GASc
4.63E
-
06
TTCTGAGAA
UP6
chr2
11630596
11630604
301.23
GASn
1.16E
-
04
TCCACAGAA
UP7/PRR5
chr2
11636314
11636322
570.14
GASc
5.47E
-
06
TTCACAGAA
PRR3
chr2
11641489
11641497
700.8
GASc
2.36E
-
06
TTCTGAGAA
Promoter
chr2
11642554
11642562
533.34
GASn
3.86E
-
05
TGCCAAGAA
PRR4
chr2
11645576
11645584
340.29
GASc
6.64E
-
06
TTCTAGGAA
IN1a
chr2
11651616
11651624
1053.78
GASc
7.68E
-
07
TTCAAAGAA
IN1m
chr2
11653492
11653500
172.97
GASn
4.04E
-
05
TTCCAGGGA
IN1b
chr2
11655221
11655229
2802.08
GASc
3.04E
-
05
TTCTCAGAA
GASc
: GAS motif, canonical,
TTCnnnGAA
GASn
: GAS motif, non
-
canonical, with 1 bp mismatch compared to
GASc
UP7/PRR5: PRR5 is ~1kb away from UP7. PRR5 is not a STAT5 binding site
Ta b l e S 2 . S TAT 5 b i n d i n g G A S m o t i f a t e a c h
Il2ra
enhancer element.
Table S2 legend: For each STAT5
binding site (peak) at the
Il2ra
locus, canonical GAS or GAS
-
like motifs with only single
mismatches are shown with their chromosomal positions, E
-
value and motif DNA sequence. MAST (Motif Alignment and
Search Tool) was used to scan the GAS motif at each enhancer element.
Supplemental
legends for Tables S3, S4, and S6 (excel
files)
Table S3.
STAT5 responsive genes in BCL11b
-
and BCL11b
+
DN2 cells upon acute deletion of
Stat5a
and
Stat5b
using CRISPR/Cas9.
Legend and methodological features of the analysis are
given on the “Readme” page.
Table S4
: Single cell
RNA
-
Sequencing from skin infiltrating CD45
+
immune cells. Marker
genes for sorted CD45+ immune cells and differentially expressed genes in indicated
comparisons are shown.
Table S6.
Sequences for gStat5a and gStat5b.
Three
gRNA sequences targeting
Stat5a
or
Stat5b
are shown with mouse genomic coordinates.