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Serine Threonine Kinase Receptor-Associated Protein Deficiency Impairs Mouse Embryonic Stem Cells Lineage Commitment Through CYP26A1-Mediated Retinoic Acid Homeostasis.
Jin L
,
Chang C
,
Pawlik KM
,
Datta A
,
Johnson LM
,
Vu T
,
Napoli JL
,
Datta PK
.
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Retinoic acid (RA) signaling is essential for the differentiation of embryonic stem cells (ESCs) and vertebrate development. RA biosynthesis and metabolism are controlled by a series of enzymes, but the molecular regulators of these enzymes remain largely obscure. In this study, we investigated the functional role of the WD-domain protein STRAP (serine threonine kinase receptor-associated protein) in the pluripotency and lineage commitment of murine ESCs. We generated Strap knockout (KO) mouse ESCs and subjected them to spontaneous differentiation. We observed that, despite the unchanged characteristics of ESCs, Strap KO ESCs exhibited defects for lineage differentiation. Signature gene expression analyses revealed that Strap deletion attenuated intracellular RA signaling in embryoid bodies (EBs), and exogenous RA significantly rescued this deficiency. Moreover, loss of Strap selectively induced Cyp26A1 expression in mouse EBs, suggesting a potential role of STRAP in RA signaling. Mechanistically, we identified putative Krüppel-like factor 9 (KLF9) binding motifs to be critical in the enhancement of non-canonical RA-induced transactivation of Cyp26A1. Increased KLF9 expression in the absence of STRAP is partially responsible for Cyp26A1 induction. Interestingly, STRAP knockdown in Xenopus embryos influenced anterior-posterior neural patterning and impaired the body axis and eye development during early Xenopus embryogenesis. Taken together, our study reveals an intrinsic role for STRAP in the regulation of RA signaling and provides new molecular insights for ESC fate determination. Stem Cells 2018;36:1368-1379.
Figure 1. Strap depletion has no effect on the morphology or viability of ESCs.  A. Schematic representation of the
strategy used to generate Strap heterozygous mice. The Strap locus (from exons 1 to 10) is shown. Exons are shown
as filled boxes and cutting positions of the two zinc finger nucleases (ZFNs) are indicated. Details of   targeted seâ
quences are shown in boxes. B. Left panel, phase contrast image showing the morphology of isolated E3.5 blastocysts
from Strap+/â intercrossings. Right panel, representative images of blastocystâderived Strap WT and KO ESCs cultured
on MEFs. Image magnification x 40; scale bar = 50 um. C. PCR genotyping of ESCs established from various blastoâ
cysts. D. Fluorescenceâactivated cell sorting (FACS) analysis of WT and KO ESC lines. The percentages of cell populaâ
tions at each cell cycle phase are displayed.  E. Growth curve of two independent WT and KO ESC lines. A total of
1X104
ESCs were seeded in 12âwell plates and cell numbers were counted every other day. F. Alkaline phosphatase
staining of WT and KO ESC cells. G. Western blots of pluripotent markers and STRAP in the indicated ESCs. βâActin was
used as a loading control.
Figure 2. Strapâdeficient EBs are unable to differentiate into primary germ layers. A. Phaseâcontrast images of EB
morphology during EB formation from Strap WT and KO ESCs at the indicated time points. Scale bar = 600 um. B. The
cell cycle distribution of D10 EBs from WT and KO ESC lines was analyzed by FACS. The percentages of cell populations
at each cell cycle phase are labeled. CâE. qPCR analyses of lineageâspecific geneâ expression at Day 0, Day 4, Day 8 and
Day 12 during EB formation of two independent WT and KO ESC lines. Error bars represent mean ±s.d. Each experiâ
ment was replicated at least three times.
Figure 3. Endogenous RA signaling is inactivated during differentiation of Strap KO ESCs. A. Gene set enrichment
analysis (GSEA) using gene expression profiles from Strap +/+ and Strap â/â MEFs. Representative gene expression patâ
terns predicted RA signaling components were enriched in Strapâ/â MEFs compared to Strap+/+ MEFs. NES, normalized
enrichment score.  B. RNA was extracted from EBs cultured without LIF for various time points, and the transcript
levels of RAâresponsive genes were assessed by RTâqPCR. Bars represent mean ±s.d., n=3, *P<0.05, **P<0.01,
***P<0.005, when compared with the corresponding control.  C. RTâqPCR analyses of RAâspecific gene expression at
Day 8 and Day 10 during EB formation with vitamin A (VA) or allâtrans retinoid acid (ATRA). DMSO was used as vehicle
control. No significance between the two groups was observed. Bars represent mean ±s.d., n=3. D. Freeâfloating EBs
formed after 4 days of suspension culture with removal of LIF. Individual EBs were seeded into 12âwell plates with
either RA (100 uM) or DMSO. 12âoldâday EBs were fixed and stained for antiâβâtubulin III (green) to illustrate neuronal
growth. Image magnification X 40.
Figure 4. Loss of STRAP results in increased CYP26A1 mRNA and protein levels during EB differentiation. A. RTâqPCR
analyses of RA metabolic gene expression at Day 10 during EB formation.  Bars represent mean ±s.d., n=3, **P<0.01,
when compared with the control.  B. RTâqPCR analyses of Cyp26A1 expression at Day 4, Day 6, Day 8 and Day 10 durâ
ing EB formation. Each point represents mean ±s.d., n=3.  C. Western blot analyses of the level of CYP26A1 in the inâ
dicated EBs at Day 10 and Day 12. βâActin was used as a loading control. D. Western blotting was used to determine
the expression of STRAP protein in the control and STRAP knockdown E14 polyclonal cells. E. Levels of CYP26A1 and
STRAP in EBs derived from shSTRAP and shCtrl clones (E14) were determined by western blotting using βâActin as a
loading control.  F. 8âdayâold EBs were treated with CYP26A1 inhibitor, liarozole (50 uM), for 48 hours and total RNA
from control and knockdown EBs was subjected to RTâqPCR analyses for detecting RAâtargeted genes. No significance
between the two groups was observed. Bars represent mean ±s.d., n=3.
Figure 5. Krüppelâlike factor 9 (KLF9) and Sp1 coordinately regulate Cyp26A1 transcription in STRAPâdepleted EBs.
A. Either E14 control or shSTRAP cells were transfected with the indicated Cyp26A1 luciferase reporters. Luciferase
activity was normalized to ï¢âGal activity and presented as mean ±s.d., n=3,  *P<0.05, when compared with the correâ
sponding control. B. Western blots of total cell lysates from indicated EBs with STRAP knockdown were analyzed for
KLF9 and Sp1 expression. C. E14 WT cells were coâtransfected with the indicated Cyp26A1 promoter reporter as well
as Sp1 or KLF9 expression plasmids.  Luciferase activity was normalized to ï¢âGal activity and presented as mean ±s.d.,
n=3, *P<0.05, **P<0.01.   D. The murine Cyp26A1 promoter showing the positions of potential KLF9 binding sites
(black boxes) and the transcription start site (TSS). Black lines indicate the amplified regions for ChIP primers.  E. ChIP
assays were performed from cells mentioned above using antiâKLF9 or antiâSp1 antibody.  PCR amplification was perâ
formed within the target region of the Cyp26A1 promoter. Results are expressed as percentages of immunoprecipiâ
tated DNA compared to total input DNA. *P<0.05, ** P<0.01, when compared with the corresponding control.
Figure 6. STRAP knockdown influences anteriorâ€posterior neural patterning and impairs the body axis and eye deâ€
velopment in early Xenopus embryogenesis.  A. Strap transcript was detected in the entire blastopore (a), early neuâ€
rulation (b), late neurula stage (c), and the tailbud and the tadpole stages (d and e, respectively). B. 50 ng Strapâ€MO
was injected into the animal regions of twoâ€cellâ€stage embryos and resulted in defects in the tadpole stage. C. Strap
RNA (0.20 ng) was injected alone or with 25 ng Strapâ€MO unilaterally into twoâ€cellâ€Â stage embryos. The embryos were
stained with the βâ€Gal substrate Redâ€Gal. D. 50 ng Strapâ€MO was injected unilaterally into twoâ€cellâ€Â stage embryos.
The embryos were examined at neurula stages 17â€18 by ISH for neural markers. E.  50 ng Strapâ€MO was injected into
the animal regions of twoâ€cellâ€stage embryos. Animal caps were dissected at blastula stage 9 and cultured until neuâ€
rula stages 19â€20. RNA was then extracted for RTâ€qPCR analysis of marker expression. Bars represent mean ±s.d., n=3.
* P<0.05,  **P<0.01, when compared with the control.
strap (serine/threonine kinase receptor associated protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 11, vegetal view.
strap (serine/threonine kinase receptor associated protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, dorsal view, anteriorleft.
strap (serine/threonine kinase receptor associated protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anteriorleft.
strap (serine/threonine kinase receptor associated protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anteriorleft, dorsal up.
strap (serine/threonine kinase receptor associated protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anteriorleft, dorsal up.
