1. Stem Cells and Regenerative Medicine
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Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

  1. Lucy LeBlanc
  2. Bum-Kyu Lee
  3. Andy C Yu
  4. Mijeong Kim
  5. Aparna V Kambhampati
  6. Shannon M Dupont
  7. Davide Seruggia
  8. Byoung U Ryu
  9. Stuart H Orkin
  10. Jonghwan Kim  Is a corresponding author
  1. The University of Texas at Austin, United States
  2. Boston Children’s Hospital, United States
  3. Harvard Medical School, United States
  4. Dana-Farber Cancer Institute (DFCI), United States
  5. Howard Hughes Medical Institute, United States
Research Article
Cite this article as: eLife 2018;7:e40167 doi: 10.7554/eLife.40167
8 figures, 1 table and 3 additional files

Figures

Figure 1 with 1 supplement
Loss of Yap1 substantially increases apoptosis during ESC differentiation.

(A) Lactate dehydrogenase (LDH) assay of WT and Yap1 KO ESCs in ±LIF. Cells were treated with either Z-VAD-FMK (Z-VAD), necrostatin-1, DMSO, or no treatment. Values were normalized to wells that had been lysed completely. (B) LDH assay measuring cell death after Yap1 KO in three different ESC lines during differentiation (72 hr) or self-renewal. (C) LDH assay measuring cell death in Yap1 KO, WT, and three different stable FLAG-Bio (FB) Yap1 overexpression cell lines during differentiation (72 hr). (D) Representative brightfield and fluorescence microscopy images of WT and Yap1 KO ESCs incubated with NucView 488 Casp3 substrate at the indicated times after LIF withdrawal. (E) Representative flow cytometry density plots of WT and Yap1 KO ESCs detecting fluorescent signal from annexin-V (conjugated to CF594) and NucView 488 reagent during differentiation (60 hr). (F) Fold enrichment of annexin-V and active Casp3-positive Yap1 KO vs. WT ESCs according to flow cytometry. (G) Immunoblot of Casp9, Casp8, Casp3, cleaved Casp3, and cleaved Parp1 in WT and Yap1 KO cells during differentiation. β-actin was used as a loading control. (H) Luminescent assay of caspase activity in Yap1 KO vs. WT ESCs in ±LIF media. (I) LDH assay of WT and Yap1 KO cells ± KD of Casp9 during differentiation (72 hr). All data are expressed as mean ±standard deviation (n = 4 independent samples for LDH assays and n = 3 for other experiments). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

https://doi.org/10.7554/eLife.40167.002
Figure 1—figure supplement 1
Yap1 expression in KO/KD/OE cell lines, STS sensitivity, and caspase expression during ES cell differentiation.

(A) Immunoblot of Yap1 to verify knockout in Yap1 KO cells. β-actin was used as a loading control. (B) Representative brightfield microscopy images of WT and Yap1 KO ES cells in ±LIF. Scale bar, 200 μm. (C) Immunoblot of Yap1 to verify knockout of Yap1 in three different ESC lines (J1, E14, and CJ7). β-actin was used as a loading control. J1 clone #5 was used as a positive control for knockout. (D) RT-qPCR measuring the expression of Yap1 after lentiviral shRNA-mediated Yap1 KD in differentiating WT ESCs (-LIF 72 hr). (E) LDH assay measuring cell death of Yap1 KD vs. control KD cells during differentiation (-LIF 72 hr). (F) Immunoblot of Yap1 to verify stable overexpression (OE) of FLAG-Bio-Yap1 in three different clones compared to WT ESCs. β-actin was used as a loading control. (G) Immunoblot of cleaved Casp3 and cleaved Parp1 in WT and Yap1 KO cells that had been treated with 1 μM STS for the indicated number of hours during differentiation (treatment started 43–48 hr after withdrawal of LIF depending on the length of STS treatment). (H) RT-qPCR measuring the expression of Casp9 upon shRNA-mediated lentiviral KD in WT and Yap1 KO cells during differentiation (72 hr) relative to empty vector KD. (I) RT-qPCR measuring the expression of Casp2, Casp3, Casp6, Casp7, Casp8, and Casp9 in Yap1 KO cells compared to WT cells in ±LIF. All data are expressed as mean ±standard deviation (n = 3 independent samples). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

https://doi.org/10.7554/eLife.40167.003
Figure 2 with 1 supplement
Loss of Yap1 augments apoptosis in several differentiation conditions, but its role is largely restricted to the exit from self-renewal.

(A) Schematic of 3 differentiation protocols (ectoderm, endoderm, and epiblast) used in Figures 2, 3 and 5. (B) LDH assay of WT and Yap1 KO ESCs in N2B27 with or without 2i and Z-VAD. (C) LDH assay of WT and Yap1 KO ESCs in low serum DMEM supplemented with IDE1 ±Z VAD (48 hr). (D) LDH assay of ESC towards EpiLC conversion in WT and Yap1 KO ESCs (72 hr). (E) Schematic of verteporfin (vert) treatment timings during late and early differentiation in WT ESCs in -LIF. (F) Timecourse LDH assay of verteporfin-treated dESCs at the indicated timepoints along with positive controls (treatment with verteporfin just after -LIF as well as untreated Yap1 KO ESCs, the latter of which are n = 8). All data are expressed as mean ±standard deviation (n = 4 independent samples unless otherwise stated). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

https://doi.org/10.7554/eLife.40167.004
Figure 2—figure supplement 1
Lineage marker expression during various differentiation methods.

(A) RT-qPCR measuring the expression of neural ectoderm markers Nes, Otx2, and Gbx2, as well as pluripotency marker Nanog, in cells that had been differentiating in N2B27 neural differentiation media for 48 hr. Expression was normalized to ESCs grown in 2i. (B) RT-qPCR measuring the expression of endoderm markers Gata4, Gata6, and Sox17, as well as pluripotency marker Nanog, in cells that had been differentiating in low serum DMEM supplemented with 5 μM IDE1 for 48 hr. Expression was normalized to ESCs grown in 2i. (C) RT-qPCR measuring the expression of epiblast markers Fgf5, Wnt3, and Dnmt3b, as well as pluripotency marker Nanog, in cells that had been differentiating in EpiLC conditions for 48 hr. Expression was normalized to ESCs grown in 2i. All data are expressed as mean ±standard deviation (n = 3 independent samples).

https://doi.org/10.7554/eLife.40167.005
Figure 3 with 1 supplement
Loss of Yap1 leads to abnormal expression of apoptosis-related genes.

(A) Immunoblot of Bcl-2, Bcl-xL, and Mcl-1 in WT and Yap1 KO cells in -LIF after 72 hr of differentiation. (B) RT-qPCR measuring the expression of anti-apoptotic (blue) and pro-apoptotic (red) genes in WT ESCs cultured in the indicated differentiation conditions (all at 48 hr) normalized to their respective self-renewal conditions. (C) RT-qPCR measuring the expression of anti- and pro-apoptotic genes in Yap1 KO vs. WT cells (log2) in various differentiation conditions (all at 48 hr). (D) RT-qPCR measuring the expression of Bcl2, Bcl2l1, and Mcl1 in Yap1 KO cells vs. WT cells during differentiation (timecourse). (E) RT-qPCR measuring the expression of Bcl2 in WT and Yap1 KO cells during differentiation (timecourse) relative to +LIF. All data are expressed as mean ±standard deviation (n = 3 independent samples unless otherwise stated). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

https://doi.org/10.7554/eLife.40167.006
Figure 3—figure supplement 1
Depletion or loss of Yap1 leads to dysregulation of apoptosis-related genes.

(A) Representative confocal images (63X oil objective) of immunocytochemistry of WT and Yap1 KO ESCs in -LIF (72 hr). Blue represents the nucleus, red represents Bcl-2 (top 6) or Mcl-1 (bottom 6), and yellow represents mitochondria. White squares indicate location of zoom images. Scale bar, 20 μm. (B) Quantification of fluorescence corresponding to Bcl-2, Mcl-1, and mitochondria using ImageJ, normalized to the number of nuclei in each view as stained by NucBlue. (C) RT-qPCR of WT ESCs with transient OE of Yap1 (48 hr) in -LIF (72 hr) normalized to empty vector. Blue indicates anti-apoptotic genes, red indicates pro-apoptotic genes. (D) Boxplots of expression of pro-apoptotic genes and anti-apoptotic genes in Yap1 OE cells versus BirA cells (log2) in +LIF. Plus symbols represent the average and middle red bands represent the median. Outliers are represented by hollow circles. Significance stars indicate p-values from paired t-test. (E) Boxplots of expression of pro- and anti-apoptotic genes in Yap1 KD and empty KD in -LIF relative to +LIF (log2). Plus symbols represent the average and middle red bands represent the median. Outliers are represented by hollow circles. Significance stars indicate p-values from paired t-test. All data are expressed as mean ±standard deviation (n = 3 independent samples unless otherwise stated). Two sample two-tailed t-test (unless otherwise specified) compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

https://doi.org/10.7554/eLife.40167.007
Figure 4 with 1 supplement
Yap1 directly regulates target apoptotic genes during differentiation.

(A) RNA-seq heatmap (Yap1 KD/empty vector KD, in both undifferentiated and differentiating ESCs) and line graph depicting Yap1 peak score, normalized to BirA, calculated using a moving window average (window = 150). Color bar indicates extent of upregulation (red) or downregulation (green) upon Yap1 KD. (B) ChIP-seq peak heatmaps using coordinates centered on the top Yap1 peaks (p-value cutoff, 1e-5) in dESCs (-LIF), which are shown in the second heatmap from the left. The other heatmaps represent occupancy of Yap1 in ESCs (first) or p300 in ESCs (third) or dESCs (fourth) corresponding to Yap1 dESC peak centers ± 3 kb (bin size = 100). (C) Signal tracks of Yap1 (red) and p300 (blue) occupancy on apoptosis-related genes in dESCs and EpiLCs, respectively. (E and F) Dual luciferase assay of Yap1-occupied cis-regulatory elements from anti- and pro-apoptotic genes in (E) Yap1 KO and WT cells ± LIF (48 hr) or (F) WT cells with Yap1 or empty OE (in -LIF, 48 hr), relative to pGL3-promoter, 24 hr after transfection. (G) Dual luciferase assay of Bcl-2 and Mcl-1 regulatory elements in Yap1 KO cells after transfection of empty vector or vectors containing FLAG-Bio Yap1 with or without a Ser79Ala mutation. (H) Dual luciferase assay of Mcl-1 with a deletion of its Tead binding motif (GGAAT on the reverse strand) in WT ESCs ± Yap1 OE. All data are expressed as mean ±standard deviation (n = 3 independent samples unless otherwise stated). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

https://doi.org/10.7554/eLife.40167.009
Figure 4—figure supplement 1
Yap1 binds to distal regulatory elements, primarily through Tead factors.

(A) Gene feature analysis of Yap1 ChIP-seq quantifying the proportion of Yap1 peaks in promoter, intergenic, upstream, intron, or exon regions. (B) Co-IP followed by immunoblot of Tead4 and p300 after pull-down using magnetic streptavidin beads in Yap1 FB cells during differentiation (72 hr). (C) Known motif analysis of Yap1 ChIP-seq peaks using BirA ES cells as a background control. Top five motifs with the lowest p-values corresponding to known factors are presented. (D) Peak-centered histogram of Yap1 ChIP-seq peaks indicating the presence of the motifs of Tead4, Zic3, and AP-1 complex members JunB and Fra1 (Fosl1). Esrrb is presented as a negative control, as known motif analysis in (C) showed no significant enrichment of the Esrrb motif. Input represents 0.03% of total protein lysate. (E) Peak-centered histogram of Yap1 ChIP-seq peaks indicating p300 occupancy and H3K27ac presence in ESCs maintained in 2i and EpiLCs, which represent dESCs. (F) Gene ontology (GO) analysis of genes bound by Yap1 that are also upregulated (white) or downregulated (black) after Yap1 KD in -LIF (96 hr). (G) Schematic of dual luciferase essay using putative Yap1-responsive cis-regulatory elements. (H) Schematic of tandem Bcl-2 enhancer creation showing the location of both Yap1-occupied elements that were combined into the same construct. (I) Correlation heatmap of YAP1 occupancy on apoptosis-related genes in SF268 glioblastoma cells, NCI-H2052 lung mesothelioma cells, IMR90 lung fibroblasts, and MDA-MB-231 triple negative breast cancer cells. Genes that were not occupied by any factor were removed from the analysis to reduce noise. (J) Signal tracks of YAP1 occupancy on MCL1, BCL2, and BCL2L1 (BCL-XL) in the cell types described in (I). (K) Correlation heatmap of occupancy of Yap1 in mouse dES cells (from this study), Tead1 in pre-B progenitor cells, Tead2 in Py2T breast cancer cells, and Tead4 in hemogenic epithelium, all on apoptosis-related genes. Genes that were not occupied by any factor were removed from the analysis to reduce noise. (L) Signal tracks of Yap1 (red), Tead1, Tead2, and Tead4 (all in blue) on Mcl1, Bcl2, and Bcl2l1 (Bcl-xL) in the cell types described in (I).

https://doi.org/10.7554/eLife.40167.010
Figure 5 with 1 supplement
Yap1 regulates mitochondrial priming and addiction to anti-apoptotic proteins.

(A) JC-10 mitochondrial membrane potential assay in WT and Yap1 KO cells during various forms of differentiation (72 hr for Pan and EpiLC, 48 hr for Neural and Endo) and self-renewal (maintained for an equal amount of time). Values (525/570 nm ratio, n = 6) corresponding to loss in ∆ψ (mitochondrial membrane potential) in Yap1 KO cells were normalized to WT cells. (A) JC-10 assay in WT and Yap1 KO cells in ±LIF after 12 hr of treatment with BH3 mimetics ABT-737, Venetoclax, A-1210477, and A1155463 (total differentiation time: 36 hr). Values (525/570 nm ratio) corresponding to loss in ∆ψ were normalized to DMSO as a control. (C) LDH assays of BH3 mimetic dose response curves after 24 hr of treatment in WT and Yap1 KO cells in ±LIF (48 hr differentiation). (D) LDH assay of WT and Yap1 KO cells after KD of Bmf or Puma in -LIF conditions (72 hr). (E) LDH assay of inducible Bmf and Puma OE (±Dox, 48 hr, 500 ng/mL) in WT and Yap1 KO cells in ±LIF (48 hr differentiation). (F) Immunoblot of cleaved Casp3, cleaved Parp1, and Mcl-1 in WT and Yap1 KO dESCs (28 hr) after 4 hr of treatment with BH3 mimetics A-1210477 (Mcl-1 inhibitor) and ABT-737 (inhibitor of Bcl-2, Bcl-xL, and Bcl-w). β-actin was used as a loading control. All data are expressed as mean ±standard deviation (n = 4 independent samples unless otherwise stated). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

https://doi.org/10.7554/eLife.40167.012
Figure 5—figure supplement 1
Verification of KD and OE of pro-apoptotic factors Bmf and Puma.

(A) RT-qPCR measuring the expression of Bmf and Puma in Yap1 KO cells during differentiation (48 hr) relative to empty vector KD (n = 3). (B) Immunoblot of Bmf and Puma after KD in Yap1 KO cells during differentiation (48 hr) relative to empty vector KD. β-actin was used as a loading control. (C) RT-qPCR measuring the expression of Bmf and Puma in WT and Yap1 KO ESCs ± Dox (24 hr) in +LIF (n = 2). (D) Immunoblot of Bmf and Puma after OE in WT ESCs in +LIF. β-actin was used as a loading control.

https://doi.org/10.7554/eLife.40167.013
Figure 6 with 1 supplement
Overexpression of Taz or individual anti-apoptotic proteins fully rescues the survival defect in the absence of Yap1.

(A) LDH assay of WT, Yap1 KO, and Yap1 KO constitutively overexpressing Bcl-xL or Yap1 in -LIF (72 hr). (B) LDH assay of inducible Bcl-2 (±Dox, 48 hr, 500 ng/mL) in WT and Yap1 KO cells -LIF (72 hr). (C) LDH assay of inducible Taz (±Dox, 48 hr, 500 ng/mL) in WT and Yap1 KO cells ± LIF (72 hr differentiation). (D) Immunoblot of cleaved Parp1, cleaved Casp3, Bcl-xL, and Mcl-1 in Yap1 KO cells inducibly overexpressing Taz (±Dox, 48 hr, 500 ng/mL) in -LIF (72 hr). (E) LDH assay of WT ESCs during differentiation (72 hr) after 48 hr KD of Bcl-xL or Mcl-1. (F) LDH assay of WT ESCs ± LIF (72 hr)±KD of Bcl-2. (G) RT-qPCR measuring the expression of lineage markers (trophectoderm: Cdx2 and Gata3, ectoderm: Nes and Otx2, endoderm: Gata4, mesoderm: Gsc and T) in WT and Yap1 KO cells in -LIF (72 hr, n = 3). Expression is indicated as a fold change in +Dox samples relative to -Dox. (H) Model proposing roles for Yap1 specific to the exit from self-renewal. In complex with Tead factors like Tead4, Yap1 co-activates anti-apoptotic genes and mildly co-represses pro-apoptotic genes to dampen mitochondrial priming, which thus prevents hyperactivation of the apoptotic cascade through Casp9. All data are expressed as mean ±standard deviation (n = 4 independent samples unless otherwise stated). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

https://doi.org/10.7554/eLife.40167.014
Figure 6—figure supplement 1
Modulation of the expression of individual anti- or pro-apoptotic genes influences cell death during differentiation.

(A) Immunoblot of Yap1 and Bcl-xL after OE in Yap1 KO cells relative to WT or empty vector Yap1 KO in +LIF. β-actin was used as a loading control. (B) Representative brightfield and fluorescent microscopy images of Yap1 KO cells showing ZsGreen fluorescence ±Dox. Scale bar, 400 μm. (C) RT-qPCR measuring the expression of Bcl-2 in WT and Yap1 KO cells during differentiation (72 hr)±Dox (48 hr, 500 ng/mL). (D) Immunoblot of Bcl-2 in Yap1 KO cells ± Dox (48 hr, 500 ng/mL) in +LIF. (E) Immunoblot of Taz in WT and Yap1 KO cells ± Dox (48 hr, 500 ng/mL) in +LIF. (F) Immunoblot of Bcl-xL and Mcl-1 after siRNA KD in WT cells in -LIF (48 hr). (G) Immunoblot of Bcl-2 after shRNA KD in -LIF (72 hr). (H) Quantification of fold increase in cell death from Figure 6E and F observed upon KD of Bcl-xL, Mcl-1, or Bcl-2, relative to control KD, in -LIF (72 hr). (I) RT-qPCR measuring the expression of lineage markers in WT cells transfected with siRNA against Bcl-xL or Mcl-1 in -LIF (72 hr). Expression is indicated as a fold change compared to control siRNA. (J) RT-qPCR measuring the induction of lineage markers in WT cells transduced with shRNA against Bcl-2 in -LIF relative to +LIF (72 hr). All data are expressed as mean ±standard deviation (n = 3 independent samples). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

https://doi.org/10.7554/eLife.40167.015
Author response image 1
dESCs were treated with 1 μM STS for the indicated hours and expression of various anti- and pro-apoptotic genes was measured at each treatment timepoint (n = 2).

Expression values were normalized to Gapdh and to 0 hr (DMSO-treated) dESCs

Author response image 2
(A) Signal tracks indicating Yap1 (red) and p300 (blue) ChIP-seq performed in ESCs (+LIF for Yap1, 2i for p300) and dESCs (-LIF for Yap1, EpiLC for p300).

Grey bars indicate regions during primer design for ChIP-qPCR. (B) ChIP-qPCR of regulatory elements associated with apoptosis-related genes in +LIF (black) and -LIF (grey) FLAG-Bio-Yap1 ESCs (n = 2). Stars indicate p-values of 0.0015 and 0.0066, respectively (two-tailed t-test).

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
AntibodyMouse anti
-β-actin
AbgentCat#AM1829b
RRID:AB_10664137
1:20,000
in 5% BSA
AntibodyMouse
anti-Yap1
Santa Cruz
Biotechnology
Cat#sc-101199
RRID:AB_1131430
1:1000
in 5% milk
AntibodyRabbit anti-
cleaved
caspase-3
Cell Signaling
Technology
Cat#9661S
RRID:AB_2341188
1:1000
in 5% BSA
AntibodyMouse anti-
cleaved Parp1
Cell Signaling
Technology
Cat#9548S
RRID:AB_2160592
1:1000
in 5% BSA
AntibodyMouse anti-
caspase-9
Cell Signaling
Technology
Cat#9508S
RRID:AB_10695598
1:1000
in 5% BSA
AntibodyRabbit anti-
Bcl-2
Cell Signaling
Technology
Cat#3498S
RRID:AB_1903907
1:1000
in 5% BSA
(WB), 1:200 in
4% BSA,
1% NGS (ICC)
AntibodyRabbit anti-Bcl-xLCell Signaling
Technology
Cat#2764S
RRID:AB_10695729
1:1000 in 5% BSA
AntibodyRabbit anti-Mcl-1Cell Signaling
Technology
Cat#94296S
RRID:AB_2722740
1:1000 in 5% BSA,
1:800 in 4% BSA,
1% NGS (ICC)
AntibodyMouse anti-PumaSanta Cruz
Biotechnology
Cat#sc-374223
RRID:AB_10987708
1:500 in 5% BSA
AntibodyRabbit anti-BmfBiossCat#bs-7587R RRID:
AB_2722741
1:1000 in 5% BSA
AntibodyHorse anti-mouse
secondary, HRP-
conjugated
Cell Signaling T
echnology
Cat#7076P2 RRID:
AB_330924
1:10,000 in TBST
AntibodyGoat anti-rabbit
secondary, HRP-
conjugated
Cell Signaling
Technology
Cat#7074S
RRID:AB_2099233
1:10,000 in TBST
AntibodyGoat anti-rabbit
IgG Alexa Fluor 594
Thermo
Scientific
Cat#R37117
RRID:AB_2556545
1:1000 in 4% BSA,
1% NGS (ICC)
AntibodyDynabeads MyOne Streptavidin
T1
Thermo
Scientific
Cat#656011:2000 in 5% BSA
AntibodyRabbit anti-
Taz (Wwtr1)
Sigma AldrichHPA007415
RRID:AB_1080602
1:500 in 5% milk
Chemical
compound,
drug
Z-VAD-FMKApexBioCat#A1902
Chemical
compound,
drug
Necrostatin-1Selleck
Chemicals
Cat#S8037
Chemical
compound,
drug
CHIR99021Selleck
Chemicals
Cat#S2924
Chemical
compound,
drug
PD184352Selleck
Chemicals
Cat#S1020
Chemical
compound,
drug
IDE1Cayman
Chemical
Company
Cat#13816
Chemical
compound,
drug
StaurosporineCell Signaling
Technology
Cat#9953S
Chemical
compound,
drug
PolybreneMilliporeCat#TR‐
1003‐G
Chemical
compound,
drug
PuromycinThermo
Scientific
Cat#A111
3803-02
Chemical
compound,
drug
Geneticin
/G418
Thermo
Scientific
Cat#10131027
Chemical
compound,
drug
ABT-737SelleckchemCat#S1002
Chemical
compound,
drug
Venetoclax
/ABT-199
SelleckchemCat#S8048
Chemical
compound,
drug
A-1210477SelleckchemCat#S7790
Chemical
compound,
drug
A-1155463SelleckchemCat#S7800
Chemical
compound,
drug
Lipofectamine
3000
Life TechnologiesCat#L3000008
Chemical
compound,
drug
INTERFERinPolyplus
Transfection
Cat#409–10
Chemical
compound,
drug
VerteporfinSelleck
Chemicals
S1786
Chemical
compound,
drug
Recombinant
Human
/Mouse/Rat
Activin A Protein
R and D Systems338-AC-010
Chemical
compound,
drug
Gibco FGF Basic
Recombinant
Mouse
Protein
Thermo Fisher
Scientific
PMG0034
Chemical
compound,
drug
KnockOut
Serum
Replacement
Thermo Fisher
Scientific
10828028
Commercial
assay or kit
Pierce LDH
Cytotoxicity Assay
Kit
Thermo
Scientific
Cat#88954
Commercial
assay or kit
RNeasy Plus
Mini Kit
QiagenCat#74136
Commercial
assay or kit
qScript cDNA
SuperMix
QuantaBio
/VWR
Cat#101414–108
Commercial
assay or kit
PerfeCTa SYBR
Green FastMix
VWRCat#95072–012
Commercial
assay or kit
Caspase-Glo 3/7PromegaCat#G8090
Commercial
assay or kit
Caspase-Glo 8PromegaCat#G8200
Commercial
assay or kit
Caspase-Glo 9PromegaCat#G8210
Commercial
assay or kit
Cell Meter JC-10
Mitochondrion
Membrane
Potential Assay Kit
AAT BioquestCat#22800
Commercial
assay or kit
NEBNext Ultra II
DNA Library Prep Kit
for Illumina
New England
Biolabs
Cat#E7645S
Commercial
assay or kit
Dual-Glo
Luciferase
Assay System
PromegaCat#E2920
Recombinant
DNA reagent
pLKO-puroMillipore
Sigma
See table S3
Recombinant
DNA reagent
pLVX-TRE3G
-ZsGreen1
ClontechCat#631164
Recombinant
DNA reagent
pCMV-Tet3GClontechCat#631164
Recombinant
DNA reagent
pCMV3-
Bcl2l1/Bcl-xL
Sino BiologicalCat#MG50012-UT
Recombinant
DNA reagent
pGL3-promoterPromegaCat#E1761

Recombinant
DNA reagent
pRL-TKPromegaCat#E2231
Recombinant
DNA reagent
3149 pSFFV-neo
Bcl-2 cDNA
AddGeneCat#8750
Recombinant
DNA reagent
Mus musculus
BCL2
binding
component 3
(Bbc3), mRNA.
NM_
133234.2
GenScriptCat#OMu19350D
Recombinant
DNA reagent
pcDNA3.1/
HisC-mTAZ
AddGeneCat#31793
Cell line
(Mus musculus,
male)
J1 Embryonic
Stem Cells
ATCCATCC SCRC
-1010
Cell line
(M. musculus,
male)
CJ7 Embryonic
Stem Cells
ENCODERRID:CVCL_C316
Cell line
(M. musculus,
male)
ES-E14TG2a
Embryonic
Stem Cells
ATCCATCC CRL-1821
Cell line
(Homo sapiens)
HEK293T cellsATCCATCC CRL-3216
Software,
algorithm
FlowJoTreestar
Software,
algorithm
BoxPlotRhttp://shiny.chemgrid.org/boxplotr/
Software,
algorithm
Java TreeViewhttp://jtreeview.sourceforge.net/
Software,
algorithm
AmiGO 2http://amigo.geneontology.org
Software,
algorithm
Primer3http://primer3.ut.ee/
Software,
algorithm
HOMERhttp://homer.ucsd.edu/homer/
Software,
algorithm
GOrillahttp://cbl-gorilla.cs.technion.ac.il/
Software,
algorithm
Cistromehttp://cistrome.org/db/#/
Software,
algorithm
Galaxyhttp://cistrome.org/ap/
Software,
algorithm
SRA Toolkithttps://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=toolkit_doc
Software,
algorithm
Bowtie 2http://bowtie-bio.sourceforge.net/bowtie2/index.shtml
Software,
algorithm
MACS2https://github.com/taoliu/MACS
Software,
algorithm
Vassar Statshttp://vassarstats.net/matrix2.html
Software,
algorithm
Integrated
Genome Viewer
http://software.broadinstitute.org/software/igv/
Software,
algorithm
ZEN Microscope
Software
https://www.zeiss.com/microscopy/int/downloads/zen.html
Software,
algorithm
ImageJhttps://imagej.nih.gov/ij/index.html
Software,
algorithm
STARhttps://github.com/alexdobin/STAR

Additional files

Source code 1

Code used to analyze raw sequencing files using the programs STAR, Bowtie2, MACS, and Homer.

https://doi.org/10.7554/eLife.40167.016
Supplementary file 1

Supplementary Table S1.

Table of RT-qPCR primers used for qPCR gene expression assays in this study. Primers were designed using Primer3 and verified by melt curve analysis. Supplementary Table S2. Table of cloning primers used for dual luciferase assay including chromosome coordinates (using mm9) and regulatory element length. Supplementary Table S3. Table of shRNA and siRNA used in KD experiments including target, ID, and sequence or target position.

https://doi.org/10.7554/eLife.40167.017
Transparent reporting form
https://doi.org/10.7554/eLife.40167.018

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