Impact of AHR Ligand TCDD on Human Embryonic Stem Cells and Early Differentiation
Abstract
:1. Introduction
2. Results
2.1. AHR Is Downregulated during Non-Directed Differentiation into Embryoid Bodies (EBs)
2.2. AHR Expression Shows Distinct Patterns during Directed Differentiation into Three Lineages
2.3. TCDD Does Not Affect Pluripotency- or Differentiation-Specific Marker Gene Expression
2.4. Impact of TCDD on hESCs and Early Differentiation
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Embryoid Body Formation
4.3. Directed Differentiation of hESCs
4.4. RNA Isolation and Measurement of mRNA Levels
4.5. Western Blotting
4.6. Flow Cytometry
4.7. RNA-seq
4.8. ChIP-seq
4.9. ATAC-seq
4.10. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
AHR | Aryl hydrocarbon receptor |
AIP | Aryl hydrocarbon receptor interacting protein |
ARNT | Aryl hydrocarbon receptor nuclear translocator |
bHLH/PAS | Basic helix-loop-helix/PER-ARNT-SIM |
DMSO | Dimethyl sulfoxide |
EB | Embryoid body |
EXOC2 | Exocyst complex component 2 |
hESC | Human embryonic stem cell |
HSP90 | Heat shock protein 90 |
LRAT | Lecithin retinol acyltransferase |
MAP2 | Microtubule-associated protein 2 |
RORA | Retinoic acid receptor-related orphan receptor alpha |
TCDD | 2,3,7,8-tetrachlorodibenzo-p-dioxin |
TH | Tyrosine hydroxylase |
TIPARP | TCDD-inducible poly(ADP-ribose) polymerase |
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RNA-seq | ChIP-seq | ||||
---|---|---|---|---|---|
Gene | Fold Change | p-Value | AHR Binding | Distance Relative to TSS | |
DMSO | TCDD | ||||
AHRR | 2.26 | 3.50 × 10−3 | + | +69,749 | |
+ | +95,863 | ||||
APOL3 | 3.6 | 2.40 × 10−2 | + | +15,455 | |
CASQ1 | 1.94 | 3.50 × 10−2 | + | −18,394 | |
CCKBR | 1.64 | 1.00 × 10−2 | + | +44,133 | |
CTIF | 1.41 | 2.60 × 10e−2 | + | −539 | |
CUZD1 | 2.54 | 1.70 × 10e−6 | + | + | −25,162 |
CYP1A1 | 10 | 8.40 × 10−11 | + | −702 | |
CYP1B1 | 2.73 | 2.00 × 10−3 | + | −779 | |
EXOC2 | 1.38 | 1.10 × 10−3 | + | + | +9040 |
GALNT5 | 4.04 | 2.10 × 10−3 | + | −11,039 | |
LHX1 | 2.1 | 4.00 × 10−2 | + | + | +129,491 |
LHX4 | 3.64 | 3.20 × 10−5 | + | −168 | |
+ | +912 | ||||
LINC00886 | 4.21 | 4.5 × 10−6 | + | + | 0 |
LRAT | 8.57 | 7.70 × 10−7 | + | −2640 | |
+ | + | −1296 | |||
+ | −212 | ||||
LRP4 | 1.3 | 2.80 × 10−2 | + | +14,565 | |
LRRTM3 | 2.58 | 3.00 × 10−2 | + | −32,899 | |
MYADML2 | 5.4 | 3.70 × 10−6 | + | +5254 | |
PCDH8 | 3.02 | 5.50× 10−3 | + | −3158 | |
PDP1 | 1.66 | 1.90 × 10−3 | + | −70,169 | |
+ | −448 | ||||
PKNOX2 | 1.52 | 3.30 × 10−2 | + | +98,739 | |
PXDNL | 4.5 | 1.4 × 10−4 | + | +191,524 | |
RORA | 1.9 | 2.48 × 10−5 | + | + | −177,729 |
SIX3 | 2.46 | 6.10 × 10−3 | + | −11,295 | |
SIX6 | 9.43 | 1.50 × 10−10 | + | +4316 | |
SLC16A12 | 2.41 | 6.00 × 10−3 | + | +1255 | |
SLC27A2 | 1.6 | 2.10 × 10−2 | + | +47 | |
TIPARP | 4.82 | 1.34 × 10−34 | + | + | +142,217 |
TESC | 1.66 | 3.60 × 10−3 | + | +46,627 |
RNA-seq | ChIP-seq | ||||
---|---|---|---|---|---|
Gene | Fold Change | p-Value | AHR Binding (hESC) | Distance Relative to TSS | |
DMSO | TCDD | ||||
CDCA7L | 1.39 | 2.9 × 10−2 | + | + | −120,620 |
CUZD1 | −10.16 | 4.2 × 10−2 | + | + | −25,162 |
MYADML2 | −28.61 | 7.4 × 10−4 | + | +5254 | |
S1PR1 | 2.55 | 3.0 × 10−2 | + | +73,148 |
RNA-seq | ChIP-seq | ||||||||
---|---|---|---|---|---|---|---|---|---|
Gene | Fold Change | p-Value | AHR Binding | Distance Relative to TSS | |||||
hESC | Endo | Neur | |||||||
DMSO | TCDD | DMSO | TCDD | DMSO | TCDD | ||||
ADM | −2.59 | 4.9 × 10−2 | + | +46,975 | |||||
AK4 | 1.75 | 2.9 × 10−2 | + | +131,446 | |||||
AOAH | −3.9 | 3.2 × 10−2 | + | +65,017 | |||||
CBX4 | −4.6 | 2.8 × 10−5 | + | −110,655 | |||||
CCNG1 | 1.32 | 3.6 × 10−2 | + | +387 | |||||
CRABP1 | 3.1 | 3.8 × 10−2 | + | +3175 | |||||
CYP1A1 | −9.07 | 4.1 × 10−2 | + | + | −702 | ||||
EPC2 | −1.43 | 1.3 × 10−2 | + | + | + | + | + | +450 | |
FGFR3 | 1.71 | 1.8 × 10−2 | + | −15,013 | |||||
GADD45G | −4.29 | 2.0 × 10−3 | + | +72,997 | |||||
GSE1 | −1.70 | 6.9 × 10−3 | + | + | −29,402 | ||||
HSPB8 | −5.34 | 1.5 × 10−3 | +124,278 | ||||||
LRAT | 3.79 | 1.6 × 10−3 | + | −2640 | |||||
+ | + | + | −1296 | ||||||
+ | −212 | ||||||||
MAP3K8 | 1.51 | 2.2 × 10−2 | + | + | +2436 | ||||
SLC7A5 | −1.91 | 4.4 × 10−2 | + | −1151 | |||||
SLC25A25 | −1.46 | 3.2 × 10−2 | + | + | + | −896 |
RNA-seq | ChIP-seq | ||||||||
---|---|---|---|---|---|---|---|---|---|
Gene | Fold Change | p-Value | AHR Binding | Distance Relative to TSS | |||||
hESC | Endo | Neur | |||||||
DMSO | TCDD | DMSO | TCDD | DMSO | TCDD | ||||
AHRR | 3.95 | 2.2 × 10−6 | + | + | +69,749 | ||||
+ | + | +95,863 | |||||||
C3orf80 | −9.2 | 4.7 × 10−3 | + | −186,661 | |||||
CCDC60 | 1.84 | 2.7 × 10−2 | + | −31,792 | |||||
CCKBR | 2.45 | 2.3 × 10−6 | + | + | +44,133 | ||||
CRYBB1 | −2.84 | 4.8 × 10−2 | + | + | + | −30,267 | |||
CYP1A1 | 34.78 | 1.4 × 10−30 | + | + | −702 | ||||
CYP1B1 | 7.6 | 1.2 × 10−9 | + | −20,263 | |||||
+ | + | −779 | |||||||
CYP27A1 | 1.65 | 2.2 × 10−2 | + | +24,031 | |||||
GRB7 | 2.84 | 2.0 × 10−5 | + | +62,663 | |||||
HS3ST5 | 3.07 | 2.6 × 10−6 | + | + | −20,093 | ||||
IKZF3 | 7.62 | 1.0 × 10−3 | + | +62,753 | |||||
LINC00886 | 2.02 | 7.5 × 10−3 | + | + | + | + | + | 0 | |
LRAT | 3.85 | 1.3 × 10−3 | + | −2640 | |||||
+ | + | + | −1296 | ||||||
+ | −212 | ||||||||
PDP1 | 1.49 | 1.5 × 10−2 | + | + | −70,169 | ||||
+ | −448 | ||||||||
TPRA1 | 1.44 | 2.4 × 10−3 | + | + | + | + | + | −376 | |
RUNX1T1 | 3.38 | 1.3 × 10−2 | + | + | + | −8068 | |||
TIPARP | 2.27 | 6.96 × 10−9 | + | + | + | + | + | +142,217 | |
TSC22D1 | 1.34 | 4.0 × 10−2 | + | +179,610 |
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Teino, I.; Matvere, A.; Pook, M.; Varik, I.; Pajusaar, L.; Uudeküll, K.; Vaher, H.; Trei, A.; Kristjuhan, A.; Org, T.; et al. Impact of AHR Ligand TCDD on Human Embryonic Stem Cells and Early Differentiation. Int. J. Mol. Sci. 2020, 21, 9052. https://doi.org/10.3390/ijms21239052
Teino I, Matvere A, Pook M, Varik I, Pajusaar L, Uudeküll K, Vaher H, Trei A, Kristjuhan A, Org T, et al. Impact of AHR Ligand TCDD on Human Embryonic Stem Cells and Early Differentiation. International Journal of Molecular Sciences. 2020; 21(23):9052. https://doi.org/10.3390/ijms21239052
Chicago/Turabian StyleTeino, Indrek, Antti Matvere, Martin Pook, Inge Varik, Laura Pajusaar, Keyt Uudeküll, Helen Vaher, Annika Trei, Arnold Kristjuhan, Tõnis Org, and et al. 2020. "Impact of AHR Ligand TCDD on Human Embryonic Stem Cells and Early Differentiation" International Journal of Molecular Sciences 21, no. 23: 9052. https://doi.org/10.3390/ijms21239052