Abstract
BackgroundThe Hedgehog (HH) signalling pathway regulates cardiomyogenesis in vivo and in differentiating P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (mES) cell model. To further assess the transcriptional role of HH signalling during cardiomyogenesis in stem cells, we studied the effects of overexpressing GLI2, a primary transducer of the HH signalling pathway, in mES cells.ResultsStable GLI2 overexpression resulted in an enhancement of cardiac progenitor-enriched genes, Mef2c, Nkx2-5, and Tbx5 during mES cell differentiation. In contrast, pharmacological blockade of the HH pathway in mES cells resulted in lower expression of these genes. Mass spectrometric analysis identified the chromatin remodelling factor BRG1 as a protein which co-immunoprecipitates with GLI2 in differentiating mES cells. We then determined that BRG1 is recruited to a GLI2-specific Mef2c gene element in a HH signalling-dependent manner during cardiomyogenesis in P19 EC cells, a mES cell model.ConclusionsThus, we propose a mechanism where HH/GLI2 regulates the expression of Mef2c by recruiting BRG1 to the Mef2c gene, most probably via chromatin remodelling, to ultimately regulate in vitro cardiomyogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-016-0127-8) contains supplementary material, which is available to authorized users.
Highlights
The Hedgehog (HH) signalling pathway regulates cardiomyogenesis in vivo and in differentiating P19 embryonal carcinoma (EC) cells, a mouse embryonic stem cell model
Overexpression of FLAG-GLI2S662A results in increased levels of GLI2 protein during mouse embryonic stem (mES) cell differentiation Since GLI2 protein is prone to degradation when phosphorylated [60], we chose to stably transfect mES cells with a Flag-Gli2S662A, a vector used to express a stabilized version of GLI2 [43]. quantitative PCR (qPCR) showed that over the course of differentiation, transgene expression declines in all four clones analyzed (Additional file 1: Figure S1), possibly reflecting post-transcriptional down-regulation of the Gli2 mRNA or transcript
Mef2c site C is of significant interest as it is located proximally to the ISL-1-dependent secondary heart field (SHF) enhancer region [82] (Fig. 5b, SHF1), it is upstream of the start site of a transcript expressed in the developing heart [83], and it is the closest GLI2-binding site to a region previously shown to associate with BRG1 [33, 84] (Fig. 5b, light blue circle)
Summary
The Hedgehog (HH) signalling pathway regulates cardiomyogenesis in vivo and in differentiating P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (mES) cell model. In the cardiac crescent a transcriptional network regulates cardiac progenitor differentiation in a spatiotemporal manner through a crosstalk of inductive signals from surrounding tissues, including hedgehog (HH) [4, 5]. These signals lead to the expression of cardiac progenitorenriched transcription factors, including NK2 homeobox 5 protein (NKX2-5), myocyte-specific enhancer factor 2C (MEF2C), T-box protein 5 (TBX5), and GATA-binding protein 4 (GATA-4), which are essential for efficient heart looping and morphogenesis [5,6,7,8,9,10]. While phosphorylation of GLI3 mostly yields a partially cleaved GLI3 transcriptional repressor (GLI3R), which represses the expression of HH
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