The regulatory role of histone deacetylase inhibitors in Fgf4 expression is dependent on the differentiation state of pluripotent stem cells.

The identity of embryonic stem cells (ESCs) is controlled by a set of pluripotency genes, including Oct4, Sox2, Nanog, and Fgf4. How their expression is repressed during differentiation and reactivated during reprogramming is largely unknown. Here, using mouse ESCs as well as F9 and P19 cells (mouse embryonal carcinoma cell lines, P19 being considered further differentiated than F9 cells) as models, we found that HDAC inhibitors elevated Fgf4 expression in P19 cells, but reduced it in F9 cells. We also observed that HDAC inhibitors enhanced the expression of Fgf4 and a subset of pluripotency genes in differentiated ESCs, but reduced their expression in undifferentiated and less differentiated ESCs. Mechanistically, we observed more HDAC1 recruitment and a weaker association of histone 4 lysine 5 acetylation at the Fgf4 enhancer in P19 cells compared to F9 cells. Additionally, we demonstrated the interaction between Sox2 and HDAC1 both in vitro and in vivo, implicating a possible role for Sox2 in the recruitment of HDAC1 to the Fgf4 enhancer. We also found that Nanog bound to the Fgf4 enhancer, and this binding was stronger in F9 cells, indicating the involvement of Nanog in the regulation of Fgf4 expression in undifferentiated and less differentiated pluripotent stem cells. This study uncovers an important role of HDAC1 and histone modifications in the repression of Fgf4 and perhaps other pluripotency genes during ESC differentiation. Our results also suggest that HDAC inhibitors may promote reprogramming partially through activating pluripotency genes at some intermediate stages.

Shi G ，Gao F ，Jin Y 《-》

Foxa1 contributes to the repression of Nanog expression by recruiting Grg3 during the differentiation of pluripotent P19 embryonal carcinoma cells.

Transcription factor Foxa1 plays a critical role during neural differentiation and is induced immediately after retinoic acid (RA)-initiated differentiation of pluripotent P19 embryonal carcinoma cells, correlated with the downregulated expression of pluripotency-related genes such as Nanog. To study whether Foxa1 participates in the repression of pluripotency factors, we expressed Foxa1 ectopically in P19 cells and identified that Nanog was repressed directly by Foxa1. We confirmed that Foxa1 was able to interact with Grg3, which is a transcriptional corepressor that expresses in P19 cells as well as during RA-induced P19 cell differentiation. Knockdown of Foxa1 or Grg3 delayed the downregulation of Nanog expression during RA-induced P19 cell differentiation. Furthermore, we found that Foxa1 recruited Grg3 to the Nanog promoter -2kb upstream region and switched the promoter to an inactive chromatin status represented by typical modifications in histone H3. Together, our results suggested a critical involvement of Foxa1 in the negative regulation of Nanog expression during the differentiation of pluripotent stem cells.

Chen T ，He S ，Zhang Z ，Gao W ，Yu L ，Tan Y ... -  《-》

Artd1/Parp1 regulates reprogramming by transcriptional regulation of Fgf4 via Sox2 ADP-ribosylation.

The recently established reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by Takahashi and Yamanaka represents a valuable tool for future therapeutic applications. To date, the mechanisms underlying this process are still largely unknown. In particular, the mechanisms how the Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc) directly drive reprogramming and which additional components are involved are still not yet understood. In this study, we aimed at analyzing the role of ADP-ribosyltransferase diphtheria toxin-like one (Artd1; formerly called poly(ADP-ribose) polymerase 1 [Parp1]) during reprogramming. We found that poly(ADP-ribosylation) (PARylation) of the reprogramming factor Sox2 by Artd1 plays an important role during the first days upon transduction with the reprogramming factors. A process that happens before Artd1 in conjunction with 10-11 translocation-2 (Tet2) mediates the histone modifications necessary for the establishment of an activated chromatin state at pluripotency loci (e.g., Nanog and Essrb) [Nature 2012;488:652-655]. Wild-type (WT) fibroblasts treated with an Artd1 inhibitor as well as fibroblasts deficient for Artd1 (Artd1-/-) show strongly decreased reprogramming capacity. Our data indicate that Artd1-mediated PARylation of Sox2 favors its binding to the fibroblast growth factor 4 (Fgf4) enhancer, thereby activating Fgf4 expression. The importance of Fgf4 during the first 4 days upon initiation of reprogramming was also highlighted by the observation that exogenous addition of Fgf4 was sufficient to restore the reprogramming capacity of Artd1-/- fibroblast to WT levels. In conclusion, our data clearly show that the interaction between Artd1 and Sox2 is crucial for the first steps of the reprogramming process and that early expression of Fgf4 (day 2 to day 4) is an essential component for the successful generation of iPSCs.

Weber FA ，Bartolomei G ，Hottiger MO ，Cinelli P ... -  《-》

PARP1 poly(ADP-ribosyl)ates Sox2 to control Sox2 protein levels and FGF4 expression during embryonic stem cell differentiation.

Gao F ，Kwon SW ，Zhao Y ，Jin Y ... -  《-》

BRG1 Governs Nanog Transcription in Early Mouse Embryos and Embryonic Stem Cells via Antagonism of Histone H3 Lysine 9/14 Acetylation.

During mouse preimplantation development, the generation of the inner cell mass (ICM) and trophoblast lineages comprises upregulation of Nanog expression in the ICM and its silencing in the trophoblast. However, the underlying epigenetic mechanisms that differentially regulate Nanog in the first cell lineages are poorly understood. Here, we report that BRG1 (Brahma-related gene 1) cooperates with histone deacetylase 1 (HDAC1) to regulate Nanog expression. BRG1 depletion in preimplantation embryos and Cdx2-inducible embryonic stem cells (ESCs) revealed that BRG1 is necessary for Nanog silencing in the trophoblast lineage. Conversely, in undifferentiated ESCs, loss of BRG1 augmented Nanog expression. Analysis of histone H3 within the Nanog proximal enhancer revealed that H3 lysine 9/14 (H3K9/14) acetylation increased in BRG1-depleted embryos and ESCs. Biochemical studies demonstrated that HDAC1 was present in BRG1-BAF155 complexes and BRG1-HDAC1 interactions were enriched in the trophoblast lineage. HDAC1 inhibition triggered an increase in H3K9/14 acetylation and a corresponding rise in Nanog mRNA and protein, phenocopying BRG1 knockdown embryos and ESCs. Lastly, nucleosome-mapping experiments revealed that BRG1 is indispensable for nucleosome remodeling at the Nanog enhancer during trophoblast development. In summary, our data suggest that BRG1 governs Nanog expression via a dual mechanism involving histone deacetylation and nucleosome remodeling.

Carey TS ，Cao Z ，Choi I ，Ganguly A ，Wilson CA ，Paul S ，Knott JG ... -  《-》