Developmental regulation of Hand1 via nucleolar sequestration.

The bHLH transcription factor Hand1 is essential for placentation and cardiac morphogenesis in the developing embryo. However, how the activity of Hand1 is regulated in either lineage remains largely unknown. Here we demonstrate that Hand1 is anchored in the nucleolus and negatively-regulated by the murine orthologue of the human I-mfa domain-containing protein (HIC). Nucleolar sequestration controls Hand1 activity during the differentiation of rat choriocarcinoma-1 (Rcho-1) trophoblast cells. Hand1 is sequestered in the nucleoli of Rcho-1 stem cells but is released into the nucleoplasm at the onset of their differentiation into trophoblast giant cells. Site-specific phosphorylation of Hand1 was previously shown to modulate the affinity of Hand1 for its nucleoplasm E-factor binding partners. We demonstrate that Hand1 phosphorylation is required for its nucleolar release, as a pre-requisite for dimerisation and biological function. Moreover, the polo-like kinase Plk4 (Sak) is responsible for this phosphorylation event. Plk4 localises to the nucleolus of Rcho-1 stem cells at phase G2 and interacts with Hand1 in vitro and in vivo to promote mitotic cell cycle exit and entry into the endocycle. We also demonstrate that the B568 subunit of the PP2A phosphatase, shown previously to target Hand1 for dephosphorylation, is exported from the nucleus during Rcho-1 differentiation. In this thesis we present findings that describe a novel mode of Hand1 regulation that is a crucial step in trophoblast stem cell differentiation and placentation and support previous studies that implicate the nucleolus as a molecular 'sink'. We suggest that nucleolar sequestration is an important mode of protein regulation and this may impact on a broad range of transcription factors