FOXC1 Downregulates Nanog Expression by Recruiting HDAC2 to Its Promoter in F9 Cells Treated by Retinoic Acid

FOXC1, a transcription factor involved in cell differentiation and embryogenesis, is demonstrated to be a negative regulator of Nanog in this study. FOXC1 is up-regulated in retinoic acid-induced differentiation of F9 Embryonal Carcinoma (EC) cells; furthermore, FOXC1 specifically inhibits the core pluripotency factor Nanog by binding to the proximal promoter. Overexpression of FOXC1 in F9 or knockdown in 3T3 results in the down-regulation or up-regulation of Nanog mRNA and proteins, respectively. In order to explain the mechanism by which FOXC1 inhibits Nanog expression, we identified the co-repressor HDAC2 from the FOXC1 interactome. FOXC1 recruits HDAC2 to Nanog promoter to decrease H3K27ac enrichment, resulting in transcription inhibition of Nanog. To the best of our knowledge, this is the first report that FOXC1 is involved in the epigenetic regulation of gene expression.

All-in-one rheology of multicellular aggregates

Tissues are generally subjected to external stresses, a potential stimulus for their differentiation or remodelling. While single-cell rheology has been extensively studied, mechanical tissue behavior under external stress is still poorly known because of a lack of adapted set-ups. Herein we introduce magnetic techniques designed both to form aggregates of controlled size, shape and content (magnetic molding) and to deform them under controlled applied stresses over a wide range of timescales and amplitudes (magnetic rheometer). We explore the rheology of multicellular aggregates (F9 cells) using both standard assays (creep and oscillatory response) and an innovative broad spectrum solicitation coupled with inference analysis. We find that multicellular aggregates exhibit a power-law response with non-linearities leading to tissue stiffening at high stress. Comparing magnetic measurements on aggregates to isolated F9 cells characterization by parallel-plates rheometry, we reveal the role of cell-cell adhesions in tissue mechanics. Thanks to its versatility, the magnetic rheometer thus stands as an essential tool to investigate model tissue rheology.

Frizzled gene expression and negative regulation of canonical WNT–β-catenin signaling in mouse F9 teratocarcinoma cells

Mouse F9 cells differentiate into primitive endoderm (PrE) following the activation of the canonical WNT–β-catenin pathway. The upregulation of Wnt6 and activation of β-catenin–TCF–LEF-dependent transcription is known to accompany differentiation, but the Frizzled (FZD) receptor responsible for transducing the WNT6 signal is not known. Eight of the 10 Fzd genes were found to be expressed in F9 cells, with Fzd7 being the most highly expressed, and chosen for further analysis. To alter steady-state Fzd7 levels and test the effect this has on differentiation, siRNA and overexpression approaches were used to knock-down and ectopically express the Fzd7 message, respectively. siRNA knock-down of Fzd7 resulted in reduced DAB2 levels, and the overexpression activated a TCF–LEF reporter, but neither approach affected differentiation. Our focus turned to how canonical WNT6 signaling was attenuated to allow PrE cells to form parietal endoderm (PE). Dkk1, encoding a WNT antagonist, was examined and results showed that its expression increased in F9 cells treated with retinoic acid (RA) or overexpressing Wnt6. F9 cells overexpressing human DKK1 or treated with DKK1-conditioned medium and then treated with RA failed to differentiate, indicating that a negative feedback loop involving WNT6 and DKK1 attenuates canonical WNT–β-catenin signaling, thereby allowing PE cells to differentiate.

Two appendages homologous between basal bodies and centrioles are formed using distinct Odf2 domains

Ciliogenesis is regulated by context-dependent cellular cues, including some transduced through appendage-like structures on ciliary basal bodies called transition fibers and basal feet. However, the molecular basis for this regulation is not fully understood. The Odf2 gene product, ODF2/cenexin, is essential for both ciliogenesis and the formation of the distal and subdistal appendages on centrioles, which become basal bodies. We examined the effects of Odf2 deletion constructs on ciliogenesis in Odf2-knockout F9 cells. Electron microscopy revealed that ciliogenesis and transition fiber formation required the ODF2/cenexin fragment containing amino acids (aa) 188–806, whereas basal foot formation required aa 1–59 and 188–806. These sequences also formed distal and subdistal appendages, respectively, indicating that the centriole appendages are molecularly analogous to those on basal bodies. We used the differential formation of appendages by Odf2 deletion constructs to study the incorporation and function of molecules associated with each appendage type. We found that transition fibers and distal appendages were required for ciliogenesis and subdistal appendages stabilized the centrosomal microtubules.