Epigenetic mechanisms, in particular histone acetylation and methylation, play critical roles in gene expression, cell differentiation and animal development. Histone acetylation generally correlates with gene activation. Histone methylation has been implicated in both gene activation and repression, depending on the specific lysine (K) residue that gets methylated. For example, methylation at K4 of histone H3 (H3K4) is associated with gene activation, whereas methylation at K27 of histone H3 (H3K27) is associated with gene repression. Histone lysine methylation is dynamically regulated by site-specific methyltransferases and demethylases.
PPARγ is a master regulator of adipogenesis. It cooperates with transcription factor C/EBPα to promote adipogenesis. PPARγ is a nuclear receptor and thus a ligand-activated transcription factor. Synthetic PPARγ ligands have been used to treat type 2 diabetes, but have undesirable side effects. Investigating how epigenetic mechanisms regulate ligand-induced nuclear receptor target gene expression may help the next generation of diabetes drugs.
Identification and characterization of histone methyltransferases and demethylases
In search of novel transcription cofactors for PPARγ, we identified the nuclear protein complex (i.e., the MLL3/MLL4 complex) that contains H3K4 methyltransferases MLL3 and MLL4, and the JmjC domain-containing protein UTX (JBC, 2007) [See Research Images Fig. 1.]. Further we show:
- The JmjC domain-containing proteins UTX and JMJD3 are H3K27-specific demethylases (PNAS, 2007). By adding an active epigenetic mark (methylation on H3K4) and removing a repressive one (methylation on H3K27), the MLL3/MLL4 complex may use two distinct histone modifying activities to activate target gene expression synergistically [See Research Images Fig. 2.]
- UTX protein, but not its demethylase activity, is required for embryonic stem cell differentiation and mouse development (PNAS, 2012). Interestingly, while the H3K27 demethylase activity of UTX is dispensable for normal muscle development, it is required for stem cell-mediated muscle regeneration (J Clin Invest, 2016).
- Enhancers play a central role in cell-type-specific gene expression. Primed enhancers are marked by H3K4me1/2. Active enhancers are further marked by H3K27ac. We have identified MLL3 and MLL4 as major H3K4me1/2 methyltransferases essential for enhancer activation and cell-type-specific gene expression during differentiation (eLife, 2013). MLL3/MLL4 are essential for adipogenesis, myogenesis, mammary gland differentiation, and heart development (eLife, 2013, Cell Rep, 2016, Development, 2016).
- Although enhancer priming by MLL3/MLL4 is dispensable for cell-identity maintenance, it controls cell fate transition by orchestrating H3K27 acetyltransferase p300-mediated enhancer activation (PNAS, 2016).
Epigenetic and transcriptional regulation of adipogenesis
We use adipogenesis as a model system to study epigenetic regulation of cell differentiation. Through our research, we show the following:
- H3K4 methyltransferases MLL3/MLL4 and associated PTIP protein directly control the induction of principal adipogenic transcription factors PPARγ and C/EBPα and are essential for adipogenesis (Cell Metab, 2009, eLife, 2013)
- H3K27 methyltransferase Ezh2 uses its enzymatic activity to constitutively represses Wnt genes to facilitate adipogenesis. H3K27ac and H3K27me3 appear to play opposing roles in regulating Wnt expression (PNAS, 2010).
- H3K9 methyltransferase G9a represses PPARγ expression and adipogenesis (EMBO J, 2013).
These results indicate that site-specific histone methylations control expression of both positive and negative master regulators of adipogenesis [Reviewed in BBA 2012 and Cell Biosci 2014; see Research Images Fig. 3.]
Adipogenesis is induced by treating confluent preadipocytes in culture with the adipogenic cocktail, which activates transcription factors (TFs) glucocorticoid receptor (GR) and CREB within minutes and increases expression of TFs C/EBPb, C/EBPd, KLF4 and Krox20 within hours. Using conditional knockout mice and derived preadipocytes, we show that GR accelerates, but is dispensable for, adipogenesis (MCB, 2016a). We also show that endogenous KLF4 and Krox20 are dispensable for adipogenesis in culture and in mice (MCB, 2016b). These unexpected results challenge the existing model on transcriptional regulation in the early phase of adipogenesis and highlight the need of studying adipogenesis in vivo.
Epigenetic regulation of nuclear receptor target gene expression
We report that the two pairs of histone acetyltransferases (HATs), GCN5/PCAF and CBP/p300, are specifically required for H3K9 acetylation (H3K9ac) and H3K18/27 acetylation (H3K18/27ac), respectively, in cells. Further, CBP/p300 and their HAT activities are essential, while GCN5/PCAF and associated H3K9ac are dispensable, for ligand-induced nuclear receptor target gene expression. These results highlight the substrate and site specificities of HATs in cells, and demonstrate the distinct roles of GCN5/PCAF- and CBP/p300-mediated histone acetylation in gene activation (EMBO J, 2011). We show that GCN5/PCAF-mediated H3K9ac correlates well with, but is surprisingly dispensable for, the expression of endogenous interferon-β (IFN-β) and the vast majority of active genes in fibroblasts. Instead, GCN5/PCAF repress IFN-β production and innate antiviral immunity in cells in a HAT-independent and non-transcriptional manner (EMBO Rep, 2014).
Our current research is focused on the following projects:
- Enhancer regulation by H3K4 methyltransferases MLL3/MLL4
- Epigenetic regulation of adipogenesis by histone methylation
- Epigenetic regulation of nuclear receptor target gene expression