Epigenetic mechanisms, in particular histone acetylation and methylation, play critical roles in regulating 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 PTIP. Through our research, we show the following:
- In cells, PTIP and a novel protein PA1 are both subunits of a 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.]
- The JmjC domain-containing proteins UTX and JMJD3 are histone H3K27-specific demethylases (PNAS, 2007). By adding an active epigenetic marker (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 and are marked by H3K4me1/2. We identify MLL3 and MLL4 as major mammalian enhancer H3K4me1/2 methyltransferases. MLL3 and MLL4 are essential for enhancer activation and cell-type-specific gene expression during adipogenesis, myogenesis and heart development (eLife, 2013, Development, 2016).
Epigenetic regulation of adipogenesis by histone methylation
We use adipogenesis as a model system to study epigenetic regulation of cell differentiation. Through our research, we show the following:
- Histone H3K4 methylation regulator PTIP directly controls the induction of principal adipogenic transcription factors PPARγ and C/EBPα and is essential for adipogenesis (Cell Metab, 2009).
- Histone 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).
- Histone 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.]
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). Recently, 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