Disruption of aryl hydrocarbon receptor homeostatic levels during embryonic stem cell differentiation alters expression of homeobox transcription factors that control cardiomyogenesis.
BACKGROUND
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates the expression of xenobiotic detoxification genes and is a critical mediator of gene-environment interactions. Many AHR target genes identified by genome-wide gene expression profiling have morphogenetic functions, suggesting that AHR may play a role in embryonic development.


OBJECTIVE
To characterize the developmental functions of the AHR, we studied the consequences of AHR activation by the agonist 2,3,7,8-tetrachlorodibenzo-p-doxin (TCDD), and the result of its repression by the antagonists 6,2,4-trimethoxyflavone and CH 223191 or by short-hairpin RNA (shRNA)-mediated Ahr knockdown during spontaneous differentiation of embryonic stem (ES) cells into cardiomyocytes.


METHODS
We generated an AHR-positive cardiomyocyte lineage differentiated from mouse ES cells that expresses puromycin resistance and enhanced green fluorescent protein (eGFP) under the control of the Cyp1a1 (cytochrome P450 1a1) promoter. We used RNA sequencing (RNA.Seq) to analyze temporal trajectories of TCDD-dependent global gene expression in these cells during differentiation.


RESULTS
Activation, inhibition, and knockdown of Ahr significantly inhibited the formation of contractile cardiomyocyte nodes. Global expression analysis of AHR-positive cells showed that activation of the AHR/TCDD axis disrupted the concerted expression of genes that regulate multiple signaling pathways involved in cardiac and neural morphogenesis and differentiation, including dozens of genes encoding homeobox transcription factors and Polycomb and trithorax group proteins.


CONCLUSIONS
Disruption of AHR expression levels resulted in gene expression changes that perturbed cardiomyocyte differentiation. The main function of the AHR during development appears to be the coordination of a complex regulatory network responsible for attainment and maintenance of cardiovascular homeostasis.
Qin
Q
Wang
Jing
J
Chen
Chia-I
CI
Ko
Yunxia
Y
Fan
Vinicius
V
Carreira
Yinglei
Y
Chen
Ying
Y
Xia
Mario
M
Medvedovic
Alvaro
A
Puga
D000818:Animals; D001391:Azo Compounds / Q000494:pharmacology; D002454:Cell Differentiation / Q000502:physiology*; D019070:Cell Lineage; D004351:Drug Resistance / Q000502:physiology; D053595:Embryonic Stem Cells / Q000378:metabolism* / Q000502:physiology; D047309:Flavones / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000502:physiology*; D055785:Gene Knockdown Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006321:Heart / Q000196:embryology*; D018398:Homeodomain Proteins / Q000378:metabolism; D006706:Homeostasis / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D024510:Muscle Development / Q000502:physiology*; D009200:Myocardial Contraction / Q000502:physiology; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D011691:Puromycin; D011720:Pyrazoles / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D018336:Receptors, Aryl Hydrocarbon / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA; D014157:Transcription Factors / Q000378:metabolism
C511621:2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazophenyl)amide; C546607:6,2',4'-trimethoxyflavone; D001391:Azo Compounds; D047309:Flavones; D018398:Homeodomain Proteins; D000072317:Polychlorinated Dibenzodioxins; D011720:Pyrazoles; D034741:RNA, Small Interfering; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011691:Puromycin