Abstract
The bHLH transcription factor Hand1 is essential for placentation and cardiac morphogenesis in the developing embryo. Here we implicate Hand1 as a molecular switch that determines whether a trophoblast stem cell continues to proliferate or commits to differentiation. We identify a novel interaction of Hand1 with a protein that contains an I-mfa (inhibitor of myogenic factor) domain that anchors Hand1 in the nucleolus where it negatively regulates Hand1 activity. In the trophoblast stem-cell line Rcho-1, nucleolar sequestration of Hand1 accompanies sustained cell proliferation and renewal, whereas release of Hand1 into the nucleus leads to its activation, thus committing cells to a differentiated giant-cell fate. Site-specific phosphorylation is required for nucleolar release of Hand1, for its dimerization and biological function, and this is mediated by the non-canonical polo-like kinase Plk4 (Sak). Sak is co-expressed in Rcho-1 cells, localizes to the nucleolus during G2 and phosphorylates Hand1 as a requirement for trophoblast stem-cell commitment to a giant-cell fate. This study defines a novel cellular mechanism for regulating Hand1 that is a crucial step in the stem-cell differentiation pathway.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Animals
-
Basic Helix-Loop-Helix Transcription Factors / genetics
-
Basic Helix-Loop-Helix Transcription Factors / metabolism*
-
Basic Helix-Loop-Helix Transcription Factors / physiology
-
Blotting, Northern
-
Blotting, Western
-
Cell Differentiation*
-
Cell Nucleolus / metabolism*
-
Cell Proliferation
-
Gene Expression Regulation, Developmental
-
Giant Cells / cytology
-
Giant Cells / metabolism
-
Green Fluorescent Proteins / genetics
-
Green Fluorescent Proteins / metabolism
-
Immunoprecipitation
-
Mice
-
Myogenic Regulatory Factors / metabolism
-
NIH 3T3 Cells
-
Phosphorylation
-
Protein Binding
-
Protein Phosphatase 2
-
Protein Serine-Threonine Kinases / metabolism
-
Recombinant Fusion Proteins / genetics
-
Recombinant Fusion Proteins / metabolism
-
Reverse Transcriptase Polymerase Chain Reaction
-
Stem Cells / cytology
-
Stem Cells / metabolism*
-
Trophoblasts / cytology
-
Trophoblasts / metabolism
-
Two-Hybrid System Techniques
Substances
-
Basic Helix-Loop-Helix Transcription Factors
-
Hand1 protein, mouse
-
Mdfi protein, mouse
-
Myogenic Regulatory Factors
-
Recombinant Fusion Proteins
-
Green Fluorescent Proteins
-
Plk4 protein, mouse
-
Protein Serine-Threonine Kinases
-
Ppp2r5d protein, mouse
-
Protein Phosphatase 2