Peroxisome Proliferator-Activated Receptor-γ (PPARγ) is considered the master regulator of adipogenesis. It is a member of the nuclear receptor superfamily of ligand-activated transcription factors and is both necessary and sufficient for adipogenesis (Farmer, 2006
; Rosen et al., 2002
). PPARγ has two isoforms, PPARγ1 and PPARγ2, generated by usage of two distinct promoters and alternative splicing (Zhu et al., 1995
). PPARγ1 is ubiquitously expressed while PPARγ2 expression is restricted to adipose tissues. However, both isoforms are strongly induced during preadipocyte differentiation in vitro
and both are highly expressed in adipose tissues in animals. PPARγ1 is induced earlier than PPARγ2 and is maintained at a level higher than PPARγ2 during preadipocyte differentiation (Morrison and Farmer, 1999
). Data from PPARγ2 isoform-specific knockout mice indicate that PPARγ2 is not absolutely required for adipogenesis in vivo
(Rosen and MacDougald, 2006
), suggesting that PPARγ1 may be critical for adipogenesis. Although the functional difference between endogenous PPARγ1 and PPARγ2 remains unclear, ectopic expression of either PPARγ1 or PPARγ2 is sufficient to stimulate immortalized, non-adipogenic MEFs to differentiate into adipocytes (Mueller et al., 2002
C/EBPα (CCAAT/enhancer binding protein α) is another principal adipogenic transcription factor and is strongly induced in the early phase of preadipocyte differentiation. Ectopic expression of C/EBPα stimulates non-adipogenic MEFs to undergo adipogenesis while deletion of C/EBPα in mice results in almost complete absence of white adipose tissue (WAT) (Farmer, 2006
). PPARγ and C/EBPα positively regulate each other's expression and cooperate to control preadipocyte differentiation (adipogenesis) (Farmer, 2006
; Rosen and MacDougald, 2006
). However, the factors and the underlying mechanisms that regulate the induction of PPARγ
expression during adipogenesis remain unclear.
PTIP (Pax transactivation domain-interacting protein) is a ubiquitously expressed nuclear protein that associates with active chromatin. Knockout of PTIP in mice leads to lethality by embryonic day 9.5 (Patel et al., 2007
). PTIP carries six tandem BRCT domains that are predominantly found in proteins involved in DNA damage response. Indeed, ectopically expressed PTIP has been implicated in cellular response to DNA damage (Manke et al., 2003
). However, the physiological function of endogenous PTIP has remained largely unclear.
Histone lysine methylation has been implicated in both gene activation and repression, depending on the specific lysine residue that gets methylated. For example, methylation on histone H3 lysine 4 (H3K4) associates with gene activation while methylation on histone H3 lysine 27 (H3K27) associates with gene repression (Li et al., 2007
). We and others recently show that endogenous PTIP is a component of a histone methyltransferase (HMT) complex that contains histone H3K4 methyltransferases MLL3 and MLL4 (also known as ALR), and the JmjC domain-containing protein UTX (Cho et al., 2007
; Issaeva et al., 2007
; Patel et al., 2007
). Further, we and others demonstrate UTX is a histone H3K27-specific demethylase (Hong et al., 2007
; Swigut and Wysocka, 2007
). Thus, endogenous PTIP associates with two H3K4 methyltransferases and one H3K27 demethylase. Since both methylation of H3K4 and demethylation of H3K27 presumably associate with gene activation, these data strongly suggest a role of PTIP in gene activation.
In this report, we identify PTIP as a factor that regulates PPARγ and C/EBPα expression in MEFs as well as during preadipocyte differentiation. In MEFs, PTIP deletion leads to over 10 fold decrease of PPARγ expression and over 5 fold decrease of PPARγ-stimulated C/EBPα expression. In preadipocytes, while PTIP is dispensable for the basal level expression of PPARγ and C/EBPα before differentiation, it is essential for the robust induction of PPARγ and C/EBPα but not C/EBPβ during differentiation. Further, we show PTIP is required for the enrichment of methyltransferase MLL4, H3K4 trimethylation (H3K4me3) and RNA polymerase II (Pol II) on PPARγ and C/EBPα promoters. Accordingly, PTIP-deficient MEFs and white and brown preadipocytes all show striking defects in adipogenesis. Finally, tissue-specific deletion of PTIP significantly reduces brown adipose tissue weight and leads to cold intolerance in mice.