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Published on: March 17, 2023
Functional analysis of Hes-1 in preadipocytes
David A Ross1, Sridhar Hannenhalli, John W Tobias
1Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Abstract:
Notch signaling blocks differentiation of 3T3-L1 preadipocytes, and this can be mimicked by constitutive expression of the Notch target gene Hes-1. Although considered initially to function only as a repressor, recent evidence indicates that Hes-1 can also activate transcription. We show here that the domains of Hes-1 needed to block adipogenesis coincide with those necessary for transcriptional repression. HRT1, another basic-helix-loop-helix protein and potential Hes-1 partner, was also induced by Notch in 3T3-L1 cells but did not block adipogenesis, suggesting that Hes-1 functions primarily as a homodimer or possibly as a heterodimer with an unknown partner. Purification of Hes-1 identified the Groucho/transducin-like enhancer of split family of corepressors as the only significant Hes-1 interacting proteins in vivo. An evaluation of global gene expression in preadipocytes identified approximately 200 Hes-1-responsive genes comprising roughly equal numbers of up-regulated and down-regulated genes. However, promoter analyses indicated that the down-regulated genes were significantly more likely to contain Hes-1 binding sites, indicating that Hes-1 is more likely to repress transcription of its direct targets. We conclude that Notch most likely blocks adipogenesis through the induction of Hes-1 homodimers, which repress transcription of key target genes.
Insights
Notch signaling inhibits fat cell development by inducing Hes-1. This study shows Hes-1 primarily represses gene targets, likely as a homodimer, to block adipogenesis.
Area of Science:
- Cell biology
- Molecular biology
- Gene regulation
Background:
- Notch signaling plays a role in blocking preadipocyte differentiation.
- Hes-1, a Notch target gene, mimics this inhibitory effect.
- Hes-1's role in transcription, both repression and activation, is complex.
Purpose of the Study:
- To elucidate the mechanism by which Hes-1 blocks adipogenesis.
- To identify Hes-1's interacting partners and transcriptional targets.
- To determine if Hes-1 acts as a repressor or activator in adipogenesis.
Main Methods:
- Analysis of Hes-1 domains required for blocking adipogenesis.
- Investigating Hes-1 interactions with corepressors using protein purification.
- Global gene expression analysis to identify Hes-1-responsive genes.
- Promoter analysis to determine Hes-1 binding site enrichment in target genes.
Main Results:
- Domains for blocking adipogenesis in Hes-1 overlap with those for transcriptional repression.
- Groucho/transducin-like enhancer of split corepressors were identified as Hes-1 interacting proteins.
- Approximately 200 Hes-1-responsive genes were identified, with down-regulated genes enriched for Hes-1 binding sites.
- Hes-1 is more likely to repress transcription of its direct targets.
Conclusions:
- Notch signaling likely blocks adipogenesis via Hes-1 induction.
- Hes-1 functions primarily as a transcriptional repressor, likely through homodimers.
- Hes-1 represses key target genes, thereby inhibiting adipocyte differentiation.
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