Abstract
BackgroundFoxH1 is a forkhead transcription factor with conserved key functions in vertebrate mesoderm induction and left-right patterning downstream of the TGF-beta/Nodal signaling pathway. Binding of the forkhead domain (FHD) of FoxH1 to a highly conserved proximal sequence motif was shown to regulate target gene expression.ResultsWe identify the conserved microRNA-430 family (miR-430) as a novel target of FoxH1. miR-430 levels are increased in foxH1 mutants, resulting in a reduced expression of transcripts that are targeted by miR-430 for degradation. To determine the underlying mechanism of miR-430 repression, we performed chromatin immunoprecipitation studies and overexpression experiments with mutant as well as constitutive active and repressive forms of FoxH1. Our studies reveal a molecular interaction of FoxH1 with miR-430 loci independent of the FHD. Furthermore, we show that previously described mutant forms of FoxH1 that disrupt DNA binding or that lack the C-terminal Smad Interaction Domain (SID) dominantly interfere with miR-430 repression, but not with the regulation of previously described FoxH1 targets.ConclusionsWe were able to identify the distinct roles of protein domains of FoxH1 in the regulation process of miR-430. We provide evidence that the indirect repression of miR-430 loci depends on the connection to a distal repressive chromosome environment via a non-canonical mode. The widespread distribution of such non-canonical binding sites of FoxH1, found not only in our study, argues against a function restricted to regulating miR-430 and for a more global role of FoxH1 in chromatin folding.
Highlights
FoxH1 is a forkhead transcription factor with conserved key functions in vertebrate mesoderm induction and left-right patterning downstream of the TGF-beta/Nodal signaling pathway
We identified the microRNA-430 family (miR-430) loci as targets for NC interaction with FoxH1 and we show that this interaction is relevant for FoxH1 dependent miR-430 repression
Chromatin immunoprecipitation assay (ChIP)-seq and microarray revealed non-canonical FoxH1 regulation of miR-430 To gain insight into the function of FoxH1 during early embryonic development, we performed a combination of expression and ChIP-seq analysis on 6hpf epiboly-stage zebrafish embryos
Summary
FoxH1 is a forkhead transcription factor with conserved key functions in vertebrate mesoderm induction and left-right patterning downstream of the TGF-beta/Nodal signaling pathway. Binding of the forkhead domain (FHD) of FoxH1 to a highly conserved proximal sequence motif was shown to regulate target gene expression. Gastrulation and formation of the three germ layers endoderm, mesoderm, and ectoderm are a key step in development from single-cell to multicellular organism. Dose-dependent signaling by the TGF-beta factor Nodal is central for germ layer induction and patterning. Throughout the vertebrate phylogeny, the loss of Nodal signaling leads to loss of endodermal and mesodermal cell fate [1,2,3]. A central step in Nodal signaling is the ligand-induced phosphorylation and subsequent nuclear translocation of Smad. CAN binding sites critical for mediating Nodal signals are further characterized
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