Abstract
The coordinated and sequential actions of lineage-specific transcription factors and epigenetic regulators are essential for the initiation and maintenance of cellular differentiation. We here report KDM4D histone demethylase as a key regulator of adipogenesis in C3H10T1/2 mesenchymal stem cells. The depletion of KDM4D results in impaired differentiation, which can be rescued by exogenous KDM4D, PPARγ, and C/EBPα, but not by C/EBPβ. In addition, KDM4D interacts physically and functionally with both NFIB and MLL1 complex to regulate C/EBPα and PPARγ expression upon adipogenic hormonal induction. Although KDM4D is dispensable for the binding of both NFIB and MLL1 complex to the target promoters, the demethylation of tri-methylated H3K9 by KDM4D is required for NFIB and MLL1 complex to deposit tri-methylated H3K4 and activate PPARγ and C/EBPα expression. Taken together, our data provide a molecular framework for lineage-specific transcription factor and histone modifiers to cooperate in adipogenic differentiation, in which KDM4D removes repressive histone marks at genes with a bivalent chromatin domain and allows NFIB and MLL1 complex to promote the expression of key adipogenic regulators.
Highlights
The differentiation of mesenchymal stem cells (MSCs) to adipocytes is initiated and maintained by the coordinated actions of signal transduction pathways and a series of transcription programs operating at different stages of differentiation[1,2,3]
To investigate whether KDM4D is required for adipogenesis, two stable cell lines, in which endogenous KDM4D is decreased by shRNAs (Supplementary Fig. S1c), and their ability to differentiate into adipocytes upon MDI induction was tested
Whereas significant increases in the level of key adipogenic markers such as Peroxisome proliferator activator receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), and aP2 were observed in control cells during differentiation, activation of PPARγ2, C/EBPα, and aP2 were significantly inhibited and activation of PPARγ1 was markedly decreased in both shKDM4D-1 and shKDM4D-2 cells after induction of adipogenic differentiation (Fig. 1b,c)
Summary
The differentiation of mesenchymal stem cells (MSCs) to adipocytes is initiated and maintained by the coordinated actions of signal transduction pathways and a series of transcription programs operating at different stages of differentiation[1,2,3]. A number of pro-/anti-adipogenic transcription factors have been identified, which include CCAAT/enhancer binding proteins (C/EBPs), early B-cell factors (EBFs), Krüppel-like factors (KLFs), sterol regulatory element-binding protein-1 (SREBP-1), and nuclear factor I family of transcription factors (NFI)[6,7,8] These factors have been shown to regulate the expression or function of PPARγ either directly or indirectly. KMTs and KDMs are often found in the same complex, and it is believed that they cooperate to regulate gene expression[30,31] Both MLL3/MLL4 and an H3K27 specific histone demethylase, UTX, are associated with the Pax transactivation domain-interacting protein (PTIP), and they cooperate to regulate the expression of PPARγ and C/EBPα in adipogenic differentiation[32]. We demonstrate coordinated actions of KDM4D, NFIB, and MLL1 complex in the expression of PPARγ and C/EBPα, in which KDM4D functions as a ‘gate-opener’ by removing the repressive H3K9me[3] and allows NFIB and MLL1 complex to mediate the tri-methylation of H3K4 and activate the transcription of PPARγ and C/EBPα
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