Twist1 is involved in trophoblast syncytialization by regulating GCM1

• Twist1 is required for cytotrophoblast cell syncytialization.
• GCM1 and Twist exhibit correlated expression patterns during syncytialization.
• Twist1 binds to the E-box-enriched region in intron 2 of GCM1.

IntroductionThe multinucleated syncytiotrophoblast (STB) is maintained and regenerated by the fusion of underlying cytotrophoblast cells (CTBs) and is responsible for a number of functions in the human placenta. Deficiencies in this structure may result in pregnancy-associated diseases. However, the detailed mechanisms underlying trophoblast syncytialization await further investigation.MethodsThe location of the transcription factor Twist1 in human placental tissues was identified by immunohistochemistry. The expression of Twist1 and glial cells missing-1 (GCM1) was evaluated by qPCR or western blotting in two cell-fusion models including forskolin-induced fusion of BeWo cells and spontaneous syncytialization of CTBs. The key role of Twist1 in trophoblast differentiation was identified using BeWo cells transfected with Twist1-specific siRNA. We investigated the effect of hypoxia on the expression of Twist1 and GCM1 in primary CTBs cultured with 2% oxygen. The Twist1 binding region in the GCM1 gene was detected by chromatin-immunoprecipitation.ResultsTwist1 was expressed in human placental tissues, and the expression of Twist1 and GCM1 increased in a time-dependent manner during spontaneous syncytialization of primary CTBs and forskolin-induced fusion of BeWo cells. A reduction in Twist1 and GCM1 expression was observed under hypoxic conditions and was accompanied by inhibition of trophoblast syncytialization. Moreover, siRNA-mediated silencing of Twist1 resulted in inhibition of BeWo cells fusion and down-regulation of GCM1 expression. Furthermore, Twist1 was found to bind to the E-box-enriched region in intron 2 of the GCM1 gene during forskolin-induced fusion of BeWo cells.DiscussionThe above results suggest that Twist1 is required during trophoblast syncytialization. Twist1 may promote trophoblast syncytialization by regulating the expression of GCM1.