Figures and data in A stochastic epigenetic switch controls the dynamics of T-cell lineage commitment

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Kenneth KH Ng

Mary A Yui

Arnav Mehta

Sharmayne Siu

Blythe Irwin

Shirley Pease

Satoshi Hirose

Michael B Elowitz

Ellen V Rothenberg

Hao Yuan Kueh

(2018)
A stochastic epigenetic switch controls the dynamics of T-cell lineage commitment

eLife 7:e37851.

https://doi.org/10.7554/eLife.37851
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Figures
Videos
Tables
Additional files

8 figures, 1 video, 7 tables and 2 additional files

Figures

Figure 1 with 1 supplement

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Dual-color *Bcl11b* reporter strategy can reveal epigenetic mechanisms controlling T-cell lineage commitment.

(A) Overview of early T-cell development. *Bcl11b* turns on to silence alternate fate potentials and drive T-cell fate commitment. ETP – early thymic progenitor; DN2 – CD4^- CD8^-double negative-2A …
https://doi.org/10.7554/eLife.37851.002

Figure 1—figure supplement 1

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Experimental strategy for generating different *Bcl11b* reporter mouse strains.

The two *Bcl11b* loci were targeted in embryonic stem (ES) cells using homologous recombination, followed by drug selection using the indicated drug-resistance markers (vertical arrows). ES cells were …
https://doi.org/10.7554/eLife.37851.003

Figure 2 with 1 supplement

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Two copies of *Bcl11b* switch on independently and stochastically in the same cell in single lineages of T-cell progenitors.

(A) *Bcl11b*-negative DN2 cells derived from bone-marrow progenitors were isolated by flow cytometry, cultured within microwells, and followed for 5 days using fluorescence imaging. Cells were then …
https://doi.org/10.7554/eLife.37851.004

Figure 2—source data 1 Differential Bcl11b allelic expression states over time for a cohort of ~200 starting cells. File contains a table of the fractions of mono-, bi-allelic, and non-expressing Bcl11b cells sampled at various time points over 105hrs. This data is plotted in Figure 2E.: https://doi.org/10.7554/eLife.37851.006
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Figure 2—figure supplement 1

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*Bcl11b* shows heterogeneity in locus activation within clonal progenitor lineages.

Bone-marrow-derived Bcl11b-YFP^-mCherry^- DN2 progenitors were sorted, seeded on OP9-DL1 monolayers within PDMS micro-well arrays, and continuously observed using long-term timelapse imaging. …
https://doi.org/10.7554/eLife.37851.005

Figure 3 with 4 supplements

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A distal enhancer region controls *Bcl11b* activation probability.

(A) Schematic of normal and enhancer-deleted two-color *Bcl11b* reporter strains (left). Genome browser plots (right), showing +850 kb enhancer of *Bcl11b,* showing distributions of histone marks …
https://doi.org/10.7554/eLife.37851.008

Figure 3—source data 1 Comparison of Bcl11b allelic expression between wildtype and mutant dual reporter mice in early thymic populations. Data gives the population percentages of mono- and bi-allelic expressing cells for early thymic populations analyzed using flow cytometry. 4 biological replicates of each strain (wildtype and enhancer deleted) are presented. Bar graphs in Figure 3C are generated from this data.: https://doi.org/10.7554/eLife.37851.016
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Figure 3—figure supplement 1

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Levels of mono-allelic *Bcl11b* expression in thymus subsets: mono-allelic expression can persist throughout thymic development.

(A) Representative flow cytometry plots showing gating strategies for thymic subsets and two-color Bcl11b expression in these populations from Bcl11b^YFP/mCh(neo) (wildtype) or Bcl11b^{YFPΔEnh/mCh(neo)} …
https://doi.org/10.7554/eLife.37851.009

Figure 3—figure supplement 1—source data 1 Percentages of mono- and bi-allelic expressing cells in specific thymic populations analyzed for wildtype (Bcl11b^YFP/mCh(neo)) and mutant (Bcl11b^{YFPΔEnh/mCh(neo)}) dual reporter mice. Thymic populations were analyzed using flow cytometry according to the representative plots shown in Figure 3—figure supplement 1A, and percentages of cells with mono- and bi-allelic expression are shown. 4-6 biological replicates of each strain are presented. Plots in Figure 3—figure supplement 1B are generated from percentages of mono-expressing cells only.: https://doi.org/10.7554/eLife.37851.010
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Figure 3—figure supplement 2

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Mono-allelic *Bcl11b* expression persists in peripheral splenic T-cell subsets and is cell autonomous.

(A) Representative flow cytometry plots showing gating strategies for splenic subsets and two-color Bcl11b expression in these populations from Bcl11b^YFP/mCh(neo) (wildtype) or Bcl11b^{YFPΔEnh/mCh(neo)}…
https://doi.org/10.7554/eLife.37851.011

Figure 3—figure supplement 2—source data 1 Percentages of mono- and bi-allelic expressing cells in specific spleen populations analyzed for wildtype (Bcl11b^YFP/mCh(neo)) and mutant (Bcl11b^{YFPΔEnh/mCh(neo)}) dual reporter mice. Figure 3—figure supplement 1—source data 1 shows data comparing Bcl11b expressing cells between wildtype and mutant dual reporter mice. T cell subsets in the spleen were analyzed using flow cytometry according to representative plots shown in Figure 3—figure supplement 3A. Data represents 2-8 animals of each strain and shows percentages of mono- and bi-allelic expressing cells. Plots in Figure 3—figure supplement 2B are generated from percentages of mono-expressing cells only.: https://doi.org/10.7554/eLife.37851.012
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Figure 3—figure supplement 3

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Cell autonomy of Bcl11b expression control in hematopoietic chimeric mice.

B6.Cd45.1 mice were irradiated with 1000 rads and injected retro-orbitally with 10⁶ fetal liver cells from Bcl11b^YFP/mCh(neo) (wt) and Bcl11b^{YFPΔEnh/mCh(neo)} (YFPΔenh) (*Cd45.2+*) mice (F₀ …
https://doi.org/10.7554/eLife.37851.013

Figure 3—figure supplement 3—source data 1 Percentages of mono- and bi-allelic expressing cells in thymic and splenic populations analyzed for wildtype (Bcl11b^YFP/mCh(neo)) and mutant (Bcl11b^{YFPΔEnh/mCh(neo)}) chimeric mice. Figure 3—figure supplement 3—source data 1 shows data comparing Bcl11b expression in hematopoietic chimeric mice. Thymic and splenic T cell populations were analyzed using flow cytometry according to the representative plots shown in Figure 3—figure supplements 1A,2A,3A. 2 chimeric animals of each strain were analyzed for Figure 3—figure supplement 3—source data 1. Plots in Figure 3—figure supplement 2B are generated from percentages of mono-expressing cells only.: https://doi.org/10.7554/eLife.37851.014
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Figure 3—figure supplement 4

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Thymocytes from homozygous mutant enhancer Bcl11b^{YFPΔEnh/YFPΔEnh} mice are able to generate T-cell subsets expressing Bcl11b at normal levels relative to wild-type enhancer Bcl11b YFP/YFP mice.

Representative FACS plots showing gates used for CD4 and CD8 double negative (DN), double positive (DP) and single positive (CD4 and CD8) populations (left plots) and the relative levels of …
https://doi.org/10.7554/eLife.37851.015

Figure 4 with 2 supplements

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A *trans*-acting step, occurring in parallel with the *cis*-acting step, provides an additional input for *Bcl11b* activation.

(A) Candidate models for *Bcl11b* activation from the DN2 stage, involving a single *cis*-acting switch (top left), sequential *trans*-, then *cis*-acting switches (bottom left), and parallel, independent *tr…*
https://doi.org/10.7554/eLife.37851.017

Figure 4—source data 1 Quantitative analysis of timelapse imaging data used to test three minimal models. Figure 4—source data 1B shows the mean population fractions and 95% confidence intervals of mono- and bi-allelic expressing cells as observed by timelapse imaging. Figure 4B was plotted from these data points. Figure 4—source data 1C gives the reduced chi-squared values calculated for each model and is represented in Figure 4C. Figure 4—source data 1E, plotted as pie charts in Figure 4E, shows expected fractions of each class of Bcl11b activation state from 30,000 Monte-Carlo simulations for both sequential and parallel trans-cis models. Figure 4—source data 1F shows number of single cell lineages scored for each class of Bcl11b activation state in each observed experiment (3 independent experiments). Both sequential and parallel trans-cis models predict different frequencies of activation states.: https://doi.org/10.7554/eLife.37851.020
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Figure 4—figure supplement 1

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Least-squares fitting of 2D histograms of Bcl11b expression levels.

Two-dimensional plots show experimental (right) and best-fit (left) heat maps of Bcl11b levels at the indicated time points. Data are taken from 5 hr time windows centered on the indicated time …
https://doi.org/10.7554/eLife.37851.018

Figure 4—figure supplement 2

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Clones show mono-allelic expression from a single predominant allele during *Bcl11b* activation.

(A) Table shows observed numbers of clones with indicated allelic activation patterns, showing data from three independent experiments. Simulations of clonal lineages from sequential or parallel *tran…*
https://doi.org/10.7554/eLife.37851.019

Figure 5 with 1 supplement

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Notch signaling controls a parallel *trans*-acting step for *Bcl11b* activation.

BM-derived DN2 progenitors with different *Bcl11b* allelic activation states were sorted, cultured on either OP9-Control (-Notch) or OP9-DL1 (+Notch) monolayers for four days, and analyzed using flow …
https://doi.org/10.7554/eLife.37851.021

Figure 5—source data 1 Flow Cytometry Analysis of BM-derived DN2 progenitors cultured in the presence or absence of Notch. File shows percentages of mono- and bi-allelic state cells analyzed after 4 days culture from each group of starting progenitors. Data was used to generate Figure 5B.: https://doi.org/10.7554/eLife.37851.023
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Figure 5—figure supplement 1

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Notch controls a parallel *trans*-acting step for *Bcl11b* activation.

The parallel and sequential *trans-cis Bcl11b* activation models (upper and lower panels, respectively). Effects of perturbation of *cis* (blue) or *trans* (green) acting steps in both models are shown …
https://doi.org/10.7554/eLife.37851.022

Figure 6

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Probabilistic *Bcl11b* activation occurs within a limited developmental time window.

Cells expressing only one *Bcl11b* allele at the indicated stages were sorted from thymocytes, cultured for 4d on OP9-DL1 monolayers, and analyzed for activation of the initially inactive *Bcl11b* …
https://doi.org/10.7554/eLife.37851.024

Figure 7

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Model of *Bcl11b* regulation by parallel *cis* and *trans-*limiting steps.

*Bcl11b* activation requires two rate-limiting steps: a switch of the *Bcl11b* locus from an inactive to active epigenetic state, and the activation of a *trans* factor is necessary for transcription of *Bc…*
https://doi.org/10.7554/eLife.37851.026

Author response image 1

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A slower rate of bi-allelic activation does not significantly improve model fit in the parallel *trans-cis* model.

A) Diagram showing parallel *trans-cis* model shown in main text (left), and variant parallel *trans-cis* model with a slower rate of activation of the second *Bcl11b* allele (right). B) Best fits of …
https://doi.org/10.7554/eLife.37851.036

Videos

Video 1

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posterframe for video — Timelapse movie of a single clonal DN2 progenitor lineage.

Bcl11b-YFP^-mCh^- DN2 progenitors were cultured on OP9-DL1 monolayers with 5 ng/mL IL-7 and Flt3-L within individual PDMS micro-wells, and continuously imaged for 100 hr. Images show superposition of …
https://doi.org/10.7554/eLife.37851.007

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Recombinant DNA reagent	pTarget Bcl11b IRES-H2Bm Cherry-neo/3pUTR	This paper	N/A	Gene targeting vector with IRES-H2B-mCherry- loxP-neo-loxP cassette knocked into 3' UTR of Bcl11b
Recombinant DNA reagent	pTarget Bcl11b dEnh-hygro	This paper	N/A	Gene targeting vector with Enhancer replaced by hygromycin cassette
Recombinant DNA reagent	FRT-PGK-gb2- hygromycin-FRT cassette	Genebridges	Cat# A010
Recombinant DNA reagent	MSCV IRES H2B-mCerulean	Kueh et al., 2013	N/A
Recombinant DNA reagent	pCL-Eco	Imgenex	Cat# NBP2-29540
Strain, strain background (mouse)	Bcl11b^{YFP(neo)/mCh(neo)}	This paper	N/A	Two color reporter mice generated from breeding animals homozygous for either Bcl11b YFP(neo) or Bcl11b mCh(neo). See Materials and methods for details.
Strain, strain background (mouse)	Bcl11b^{mCh(neo)/mCh(neo)}	This paper	N/A	Homozygous Bcl11b mCh(neo) reporter mice used to generate two color reporter mice. Derived from Bcl11b YFP/mCh(neo) F0 chimeric mice. See Materials and methods for details.
Strain, strain background (mouse)	Bcl11b^YFP/mCh(neo)	This paper	N/A	Control mice for comparing the effects of the enhancer on Bcl11b expression. Generated by targeting Bcl11b mCherry gene targeting vector to V6.5 mouse embryonic stem (ES) cells with single modified Bcl11b mCitrine dneo allele. See Materials and methods for details.
Strain, strain background (mouse)	Bcl11b^{YFPdEnh/mCh(neo)}	This paper	N/A	Two color reporter mouse with Bcl11b enhancer deleted. Generated by targeting dEnh gene target vector to V6.5 mouse ES cells with genotype Bcl11b YFP/mCh(neo). See Materials and methods for details.
Strain, strain background (mouse)	Bcl11b^YFPdEnh/dEnh	This paper	N/A	Homozygous deleted enhancer mice generated from Bcl11b YFP dEnh/mCh(neo) mice. See Materials and methods for details.
Strain, strain background (mouse)	CD45.1 C57BL/6: B6.SJL-Ptprc^a Pepc^b/BoyJ	Jackson Laboratory	Stock No# 002014
Cell line (mouse)	OP9-DL1-GFP	Schmitt and Zúñiga-Pflücker, 2002	N/A
Cell line (mouse)	OP9-Mig	Schmitt and Zúñiga-Pflücker, 2002	N/A
Cell line (mouse)	OP9-DL1-hCD8	Kueh et al., 2016	N/A
Cell line (human)	Human Phoenix-ECO	ATCC	Cat# CRL-3214
Antibody	Anti-mouse CD8a Biotin (clone 53–6.7)	eBioscience	Cat# 13-0081-86; RRID:AB_466348	(1:100)
Antibody	Anti-mouse TCRb Biotin (clone H57-597)	eBioscience	Cat# 13-5961-85; RRID:AB_466820	(1:100)
Antibody	Anti-mouse TCRgd Biotin (clone GL3)	eBioscience	Cat# 13-5711-85; RRID:AB_466669	(1:100)
Antibody	Anti-mouse Ter119 Biotin (clone TER-119)	eBioscience	Cat# 13-5921-85; RRID:AB_466798	(1:100)
Antibody	Anti-mouse NK1.1 Biotin (clone PK136)	eBioscience	Cat# 13-5941-85; RRID:AB_466805	(1:100)
Antibody	Anti-mouse Gr-1 Biotin (clone RB6-8C5)	eBioscience	Cat# 13-5931-86; RRID:AB_466802	(1:100)
Antibody	Anti-mouse CD11c Biotin (clone N418)	eBioscience	Cat# 13-0114-85; RRID:AB_466364	(1:100)
Antibody	Anti-mouse CD11b Biotin (clone M1/70)	eBioscience	Cat# 13-0112-86; RRID:AB_466361	(1:100)
Antibody	Anti-mouse CD19 Biotin (clone 1D3/6D5)	eBioscience	Cat# 13-0193-85; RRID:AB_657658	(1:100)
Antibody	Anti-mouse CD3e Biotin (clone 145–2 C11)	eBioscience	Cat# 13-0031-85; RRID:AB_466320	(1:100)
Antibody	Anti-human/mouse B220 Biotin (clone RA3-6B2)	eBioscience	Cat# 13-0452-85; RRID:AB_466450	(1:100)
Antibody	Anti-mouse F4/80 Biotin (clone BM8)	eBioscience	Cat# 13-4801-85; RRID:AB_466658	(1:100)
Antibody	Anti-mouse CD4 Biotin (clone GK1.5)	eBioscience	Cat# 13-0041-85; RRID:AB_466326	(1:100)
Antibody	Anti-human/mouse CD44 eFluor 450 (clone IM7)	eBioscience	Cat# 48-0441-82; RRID:AB_1272246	(1:300)
Antibody	Anti-mouse CD25 Brilliant Violet 510 (clone PC61)	Biolegend	Cat# 102041; RRID:AB_2562269	(1:300)
Antibody	Anti-mouse CD117 (cKit) APC-eFluor 780 (clone 2B8)	eBioscience	Cat# 47-1171-82; RRID:AB_1272177	(1:300)
Antibody	Anti-mouse HSA eFluor 450 (clone M1/69)	eBioscience	Cat# 48-0242-82; RRID:AB_1311169	(1:300)
Antibody	Anti-mouse CD4 Brilliant Violet 510 (clone GK1.5)	Biolegend	Cat# 100449; RRID:AB_2564587	(1:300)
Antibody	Anti-mouse CD8a APC (clone 53–6.7)	eBioscience	Cat# 17-0081-82; RRID:AB_469335	(1:300)
Antibody	Anti-mouse TCRb APC-eFluor 780 (clone H57-597)	eBioscience	Cat# 47-5961-82; RRID:AB_1272173	(1:300)
Antibody	Anti-mouse CD25 APC-eFluor 780 (clone PC61.5)	eBioscience	Cat# 47-0251-82; RRID:AB_1272179	(1:300)
Antibody	Anti-mouse CD19 eFluor 450 (clone 1D3/6D5)	eBioscience	Cat# 48-0193-82; RRID:AB_2734905	(1:300)
Antibody	Anti-mouse CD117 (cKit) APC (clone 2B8)	eBioscience	Cat# 17-1171-82; RRID:AB_469430	(1:300)
Antibody	Anti-mouse CD45 APC-eFluor 780 (clone 30-F11)	eBioscience	Cat# 47-0451-82; RRID:AB_1548781	(1:300)
Antibody	Anti-mouse CD25 APC (clone PC61.5)	eBioscience	Cat# 17-0251-82; RRID:AB_469366	(1:300)
Antibody	Anti-mouse CD4 APC-eFluor 780 (clone GK1.5)	eBioscience	Cat# 47-0041-82; RRID:AB_11218896	(1:300)
Antibody	Anti-mouse CD8a APC-eFluor 780 (clone 53–6.7)	eBioscience	Cat# 47-0081-82; RRID:AB_1272185	(1:300)
Antibody	Anti-mouse CD45 APC (clone 30-F11)	eBioscience	Cat# 17-0451-82; RRID:AB_469392	(1:300)
Antibody	Anti-mouse CD5 eFluor 450 (clone 53–7.3)	eBioscience	Cat# 48-0051-82; RRID:AB_1603250	(1:300)
Antibody	Anti-mouse TCRgd APC (clone GL3)	eBioscience	Cat# 17-5711-82; RRID:AB_842756	(1:300)
Antibody	Anti-mouse CD49b eFluor 450 (clone DX5)	eBioscience	Cat# 48-5971-82; RRID:AB_10671541	(1:300)
Antibody	Anti-mouse NK1.1 APC (clone PK136)	eBioscience	Cat# 17-5941-82; RRID:AB_469479	(1:300)
Antibody	Anti-mouse CD3e APC-eFluor 780 (clone 145–2 C11)	eBioscience	Cat# 47-0031-82; RRID:AB_11149861	(1:300)
Antibody	Anti-mouse TCRb eFluor 450 (clone H57-597)	eBioscience	Cat# 48-5961-82; RRID:AB_11039532	(1:300)
Antibody	Anti-mouse CD49b Biotin (clone DX5)	eBioscience	Cat# 13-5971-82; RRID:AB_466825	(1:300)
Antibody	Anti-mouse CD62L APC (clone MEL-14)	eBioscience	Cat# 17-0621-82; RRID:AB_469410	(1:300)
Antibody	Anti-mouse CD45.2 Brilliant Violet 510 (clone 104)	Biolegend	Cat# 109837; RRID:AB_2561393	(1:300)
Antibody	Anti-mouse CD4 eFluor 450 (clone GK1.5)	eBioscience	Cat# 48-0041-82; RRID:AB_10718983	(1:300)
Antibody	Anti-mouse CD45 eFluor 450 (clone 30-F11)	eBioscience	Cat# 48-0451-82; RRID:AB_1518806	(1:300)
Antibody	Streptavidin PerCP-Cyanine5.5	Biolegend	Cat# 405214; RRID:AB_2716577	(1:300)
Antibody	Streptavidin Brilliant Violet 510	Biolegend	Cat# 405234	(1:300)
Peptide, recombinant protein	Recombinant Human Flt3-Ligand	PeproTech	Cat# 300–19
Peptide, recombinant protein	Recombinant Human IL-7	PeproTech	Cat# 200–07
Peptide, recombinant protein	Recombinant Human Stem Cell Factor (SCF)	PeproTech	Cat# 300–07
Peptide, recombinant protein	Recombinant Mouse IL-6	eBioscience	Cat# 14-8061-62
Peptide, recombinant protein	Recombinant Mouse Stem Cell Factor (SCF)	eBioscience	Cat# 34-8341-82
Peptide, recombinant protein	Recombinant Mouse IL-3	eBioscience	Cat# 14-8031-62
Peptide, recombinant protein	Retronectin	Takara	Cat# T100B
Peptide, recombinant protein	DL1-extIgG Protein	Varnum-Finney et al., 2000	N/A
Software, algorithm	FlowJo (v10.0.8)	Tree Star	N/A
Software, algorithm	MATLAB (R2016a)	MathWorks	N/A
Other	FuGENE 6 Transfection Reagent	Promega	Cat# E2691
Other	MACS Streptavidin Microbeads	Miltenyi Biotec	Cat# 130-048-101
Other	LS Columns	Miltenyi Biotec	Cat# 130-042-401
Other	250mm-diameter PDMS circular micromesh arrays	Microsurfaces Pty Ltd	Cat# MMA-0250-100-08-01

Appendix 1—table 1

Best fit parameters of the sequential trans-cis activation model to data, with $95 %$ confidence intervals.

https://doi.org/10.7554/eLife.37851.029

Parameter	Units	Best-fit	Lower bound	Upper bound
$k_{c}$	1/hr	$3.5 \times 10^{- 2}$	$3.3 \times 10^{- 2}$	$3.6 \times 10^{- 2}$
$k_{t}$	1/hr	$3.3 \times 10^{- 2}$	$3.1 \times 10^{- 2}$	$3.6 \times 10^{- 2}$
$m_{0}$	I	$0.81$	$0.78$	$0.83$

Appendix 1—table 2

Best fit parameters of the parallel trans-cis activation model to data, with $95 %$ confidence intervals.

https://doi.org/10.7554/eLife.37851.030

Parameter	Units	Best-fit	Lower bound	Upper bound
$k_{c}$	1/hr	$5.2 \times 10^{- 3}$	$4.3 \times 10^{- 3}$	$6.2 \times 10^{- 3}$
$k_{t}$	1/hr	$2.7 \times 10^{- 2}$	$2.6 \times 10^{- 2}$	$2.8 \times 10^{- 2}$
$m_{0}$	(fraction)	$0.21$	$0.14$	$0.33$
$m_{r}$ , $m_{y}$	(fraction)	$0.29$	$0.27$	$0.31$
$m_{r y}$	(fraction)	$0.21$	$0.20$	$0.23$

Appendix 1—table 3

Perturbing the cis-acting step in the sequential activation model.

https://doi.org/10.7554/eLife.37851.031

Parameter	Description
$k_{f}^{0} = k_{C} (1 - d)$	cis-activation rate, from non-expressing to mono-allelic state
$k_{f}^{1} = k_{C} (1 - f \cdot d)$	cis-activation rate, from mono-allelic to bi-allelic state
$k_{r}^{0} = k_{C} \cdot d$	back cis-activation rate, from mono-allelic to non-expressing state
$k_{r}^{1} = k_{C} \cdot f \cdot d$	back cis-activation rate, from bi-allelic to mono-allelic state
$k_{T}$	trans-activation rate
$d$	0 to 0.35
$f$	0.4

Appendix 1—table 4

Perturbing the trans-acting step in the sequential activation model.

https://doi.org/10.7554/eLife.37851.032

Parameter	Description
$k_{C}$	cis-activation rate
$k_{f}^{0} = k_{T} (1 - d)$	trans-activation rate
$k_{r}^{0} = k_{T} \cdot d$	back trans-activation rate
$d$	0 to 0.35

Appendix 1—table 5

Perturbing the cis-acting step in the parallel activation model.

https://doi.org/10.7554/eLife.37851.033

Parameter	Description
$k_{f}^{0} = k_{C} (1 - d)$	cis-activation rate, from non-expressing to mono-allelic state
$k_{f}^{1} = k_{C} (1 - f \cdot d)$	cis-activation rate, from mono-allelic to bi-allelic state
$k_{r}^{0} = k_{C} \cdot d$	back cis-activation rate, from mono-allelic to non-expressing state
$k_{r}^{1} = k_{C} \cdot f \cdot d$	back cis-activation rate, from bi-allelic to mono-allelic state
$k_{T}$	trans-activation rate
$d$	0 to 0.35
$f$	0.4

Appendix 1—table 6

Perturbing the trans-acting step in the parallel activation model.

https://doi.org/10.7554/eLife.37851.034

Parameter	Description
$k_{c}$	cis-activation rate
$k_{f}^{0} = k_{T} (1 - d)$	trans-activation rate, from non-expressing/mono-allelic state
$k_{f}^{1} = k_{T} (1 - f \cdot d)$	trans-activation rate, from bi-allelic state
$k_{r}^{0} = k_{T} \cdot d$	back trans-activation rate, from non-expressing/mono-allelic state
$k_{r}^{1} = k_{T} \cdot f \cdot d$	back trans-activation rate, from bi-allelic state
$d$	0 to 0.65
$f$	0.2

Additional files

Supplementary file 1 List of antibodies used for magnetic bead protocols, flow cytometry analysis, and sorting. Each antibody specifies the cell populations targeted and their corresponding reference figures.: https://doi.org/10.7554/eLife.37851.025
Download elife-37851-supp1-v1.docx
Transparent reporting form: https://doi.org/10.7554/eLife.37851.027
Download elife-37851-transrepform-v1.docx

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Dual-color Bcl11b reporter strategy can reveal epigenetic mechanisms controlling T-cell lineage commitment.

Experimental strategy for generating different Bcl11b reporter mouse strains.

Two copies of Bcl11b switch on independently and stochastically in the same cell in single lineages of T-cell progenitors.

Figure 2—source data 1

Bcl11b shows heterogeneity in locus activation within clonal progenitor lineages.

A distal enhancer region controls Bcl11b activation probability.

Figure 3—source data 1

Levels of mono-allelic Bcl11b expression in thymus subsets: mono-allelic expression can persist throughout thymic development.

Figure 3—figure supplement 1—source data 1

Mono-allelic Bcl11b expression persists in peripheral splenic T-cell subsets and is cell autonomous.

Figure 3—figure supplement 2—source data 1

Cell autonomy of Bcl11b expression control in hematopoietic chimeric mice.

Figure 3—figure supplement 3—source data 1

Thymocytes from homozygous mutant enhancer Bcl11bYFPΔEnh/YFPΔEnh mice are able to generate T-cell subsets expressing Bcl11b at normal levels relative to wild-type enhancer Bcl11b YFP/YFP mice.

A trans-acting step, occurring in parallel with the cis-acting step, provides an additional input for Bcl11b activation.

Figure 4—source data 1

Least-squares fitting of 2D histograms of Bcl11b expression levels.

Clones show mono-allelic expression from a single predominant allele during Bcl11b activation.

Notch signaling controls a parallel trans-acting step for Bcl11b activation.

Figure 5—source data 1

Notch controls a parallel trans-acting step for Bcl11b activation.

Probabilistic Bcl11b activation occurs within a limited developmental time window.

Model of Bcl11b regulation by parallel cis and trans-limiting steps.

A slower rate of bi-allelic activation does not significantly improve model fit in the parallel trans-cis model.

Timelapse movie of a single clonal DN2 progenitor lineage.

Best fit parameters of the sequential trans-cis activation model to data, with 95%<math id="inf23"><mrow><mn>95</mn><mi>%</mi></mrow></math> confidence intervals.

Best fit parameters of the parallel trans-cis activation model to data, with 95%<math id="inf40"><mrow><mn>95</mn><mi>%</mi></mrow></math> confidence intervals.

Perturbing the cis-acting step in the sequential activation model.

Perturbing the trans-acting step in the sequential activation model.

Perturbing the cis-acting step in the parallel activation model.

Perturbing the trans-acting step in the parallel activation model.

Supplementary file 1

Transparent reporting form

Thymocytes from homozygous mutant enhancer Bcl11b^{YFPΔEnh/YFPΔEnh} mice are able to generate T-cell subsets expressing Bcl11b at normal levels relative to wild-type enhancer Bcl11b YFP/YFP mice.

Best fit parameters of the sequential trans-cis activation model to data, with $95 %$ confidence intervals.

Best fit parameters of the parallel trans-cis activation model to data, with $95 %$ confidence intervals.