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Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation
Published on: June 21, 2016
Role of SF-1 and DAX-1 during differentiation of P19 cells by retinoic acid
Bryan W Teets1, Kenneth J Soprano, Dianne Robert Soprano
1Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Abstract:
Retinoic acid (RA) is critical for embryonic development and cellular differentiation. Previous work in our laboratory has shown that blocking the RA-dependent increase in pre-β cell leukemia transcription factors (PBX) mRNA and protein levels in P19 cells prevents endodermal and neuronal differentiation. Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX-1) and steroidogenic factor (SF-1) were found by microarray analysis to be regulated by PBX in P19 cells. To determine the roles of DAX-1 and SF-1 during RA-dependent differentiation, P19 cells that inducibly express either FLAG-DAX-1 or FLAG-SF-1 were prepared. Unexpectedly, overexpression of DAX-1 had no effect on the RA-induced differentiation of P19 cells to either endodermal or neuronal cells. However, SF-1 overexpression prevented the RA-dependent loss of OCT-4, DAX-1 and the increase in COUP-TFI, COUP-TFII, and ETS-1 mRNA levels during the commitment stages of both endodermal and neuronal differentiation. Surprisingly, continued expression of SF-1 for 7 days caused the RA-independent loss of OCT-4 protein and RA-dependent loss of SSEA-1 expression. Despite the loss of well-characterized pluripotency markers, these cells did not terminally differentiate into either endodermal or neuronal cells. Instead, the cells gained the expression of many steroidogenic enzymes with a pattern consistent with adrenal cells. Finally, we found evidence for a feedback loop in which PBX reduces SF-1 mRNA levels while continued SF-1 expression blocks the RA-dependent increase in PBX levels. Taken together, these data demonstrate that SF-1 plays a dynamic role during the differentiation of P19 cells and potentially during early embryogenesis.
Insights
Steroidogenic factor 1 (SF-1) plays a key role in retinoic acid (RA)-induced cell differentiation. SF-1 overexpression in P19 cells blocked differentiation and led to adrenal cell characteristics, revealing a dynamic role in embryogenesis.
Area of Science:
- Developmental Biology
- Stem Cell Biology
- Endocrinology
Background:
- Retinoic acid (RA) is essential for embryonic development and cellular differentiation.
- Pre-β cell leukemia transcription factors (PBX) are involved in RA-dependent differentiation, regulating DAX-1 and SF-1.
- P19 cells are a valuable model for studying RA-induced differentiation into endodermal and neuronal lineages.
Purpose of the Study:
- To investigate the roles of DAX-1 and SF-1 in RA-dependent differentiation of P19 cells.
- To elucidate the molecular mechanisms by which SF-1 influences cell fate decisions during differentiation.
Main Methods:
- Generation of P19 cell lines with inducible expression of FLAG-DAX-1 or FLAG-SF-1.
- Analysis of gene expression (mRNA and protein levels) of key developmental and pluripotency markers (OCT-4, SSEA-1, DAX-1, SF-1, COUP-TFI/II, ETS-1, PBX).
- Assessment of cellular differentiation into endodermal, neuronal, and steroidogenic lineages.
Main Results:
- DAX-1 overexpression did not affect RA-induced differentiation.
- SF-1 overexpression blocked RA-dependent loss of pluripotency markers (OCT-4, DAX-1) and induction of differentiation markers.
- SF-1 overexpression led to RA-independent loss of OCT-4 and RA-dependent loss of SSEA-1, resulting in cells with steroidogenic enzyme expression, resembling adrenal cells.
- A feedback loop was identified where PBX reduces SF-1 mRNA, and SF-1 inhibits RA-induced PBX upregulation.
Conclusions:
- SF-1 plays a critical and dynamic role in regulating RA-dependent differentiation pathways.
- SF-1 can redirect P19 cell differentiation towards an adrenal cell fate, independent of terminal neuronal or endodermal differentiation.
- The interplay between PBX and SF-1 is crucial for controlling cell fate during early embryogenesis and differentiation.
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