Er occupancy. [A] The image depicts the results of ChIP assays using chromatin from HepG2 cells infected with GFP, HNF4a and/or lipin 1b. Chromatin was immunoprecipitated with antibodies directed against HNF4a, the HA tag of lipin 1b or IgG control. Input represents 0.2 of the total chromatin used in the IP reactions. PCR primers were designed to flank 
the HNF4a response elements in the Apoc3 or Ppara gene MedChemExpress SC-1 promoters. Control primers were designed to amplify the 36B4 gene. The graph depicts results of real-time PCR (SYBR GREEN) to quantify immunoprecipitated chromatin. The results are the mean of 3 independent experiments done in duplicate. *p,0.05 versus pCDNA control. **p,0.05 versus HNF4a alone. [B] Graphs depict results of luciferase assays using lysates from HepG2 cells transfected with UAS.TKLuc and cotransfected with Gal4-HNF4a or Gal4-DNA binding domain (DBD) control and/or lipin 1expression constructs as indicated. The results are the mean of 3 independent experiments done in triplicate. *p,0.05 versus pCDNA control. doi:10.1371/journal.pone.0051320.gDiscussionHNF4a is a nuclear receptor transcription factor that is a critical regulator of hepatic gene expression. Previous work has demonstrated important roles for HNF4a in regulating the expression of enzymes involved in VLDL metabolism [16,31,32,33], fatty acid oxidation [18], and a broad profile of genes that define liver development [34]. In this work, we show that the expression of Lpin1 is also under the control of HNF4a in HepG2 cells and I-BRD9 site hepatocytes and that this occurs via a direct transcriptional mechanism involving a promoter in the first intron(Figure 4B). These data suggest that lipin 1 modulates HNF4a activity to selectively induce fatty acid catabolism whilst suppressing expression of genes encoding apoproteins.Lipin 1 and HNFof the Lpin1 gene. There have been hints in previous studies using `omic’ approaches that lipin 1 may be a target gene of HNF4a. Lpin1 was down-regulated by siRNA against HNF4a and identified in HNF4a ChIP-seq experiments by Bolotin and collegues [35]. In that work, the interaction of HNF4a was generally localized to 39 to the transcriptional start site of the Lpin1 gene, which coincides with our findings using promoter luciferase reporter constructs and targeted ChIP approaches. We have also shown that PGC-1a is a critical regulator of lipin 1 expression [10]. HNF4a is also an important partner of PGC-1a for mediating many aspects of the hepatic fasting response; a physiologic condition associated with increased lipin 1 expression [10]. In cardiac myocytes, we have recently shown that PGC-1a coactivates member of the ERR family through these same response elements to induce lipin 1 expression [13]. This suggests that the nuclear receptor partner coactivated by PGC-1a varies depending upon the cell type and expression level of the partners. 15755315 HNF4a is enriched in hepatocytes, but few other tissues [31]. ERRa and ERRc expression levels were at or below 
the edge of detection in HepG2 cells (unpublished observation), but these nuclear receptors are well expressed in muscle cells [27,36]. Collectively, these data strongly support the idea that lipin 1 is a direct HNF4a target gene in liver cells that is induced in physiologic conditions wherein PGC-1a is activated to coactivate HNF4a. We have previously shown that lipin 1 and HNF4a physically interact [10], but the physiologic consequences of the interaction and the induction of lipin 1 by HNF4a wa.Er occupancy. [A] The image depicts the results of ChIP assays using chromatin from HepG2 cells infected with GFP, HNF4a and/or lipin 1b. Chromatin was immunoprecipitated with antibodies directed against HNF4a, the HA tag of lipin 1b or IgG control. Input represents 0.2 of the total chromatin used in the IP reactions. PCR primers were designed to flank the HNF4a response elements in the Apoc3 or Ppara gene promoters. Control primers were designed to amplify the 36B4 gene. The graph depicts results of real-time PCR (SYBR GREEN) to quantify immunoprecipitated chromatin. The results are the mean of 3 independent experiments done in duplicate. *p,0.05 versus pCDNA control. **p,0.05 versus HNF4a alone. [B] Graphs depict results of luciferase assays using lysates from HepG2 cells transfected with UAS.TKLuc and cotransfected with Gal4-HNF4a or Gal4-DNA binding domain (DBD) control and/or lipin 1expression constructs as indicated. The results are the mean of 3 independent experiments done in triplicate. *p,0.05 versus pCDNA control. doi:10.1371/journal.pone.0051320.gDiscussionHNF4a is a nuclear receptor transcription factor that is a critical regulator of hepatic gene expression. Previous work has demonstrated important roles for HNF4a in regulating the expression of enzymes involved in VLDL metabolism [16,31,32,33], fatty acid oxidation [18], and a broad profile of genes that define liver development [34]. In this work, we show that the expression of Lpin1 is also under the control of HNF4a in HepG2 cells and hepatocytes and that this occurs via a direct transcriptional mechanism involving a promoter in the first intron(Figure 4B). These data suggest that lipin 1 modulates HNF4a activity to selectively induce fatty acid catabolism whilst suppressing expression of genes encoding apoproteins.Lipin 1 and HNFof the Lpin1 gene. There have been hints in previous studies using `omic’ approaches that lipin 1 may be a target gene of HNF4a. Lpin1 was down-regulated by siRNA against HNF4a and identified in HNF4a ChIP-seq experiments by Bolotin and collegues [35]. In that work, the interaction of HNF4a was generally localized to 39 to the transcriptional start site of the Lpin1 gene, which coincides with our findings using promoter luciferase reporter constructs and targeted ChIP approaches. We have also shown that PGC-1a is a critical regulator of lipin 1 expression [10]. HNF4a is also an important partner of PGC-1a for mediating many aspects of the hepatic fasting response; a physiologic condition associated with increased lipin 1 expression [10]. In cardiac myocytes, we have recently shown that PGC-1a coactivates member of the ERR family through these same response elements to induce lipin 1 expression [13]. This suggests that the nuclear receptor partner coactivated by PGC-1a varies depending upon the cell type and expression level of the partners. 15755315 HNF4a is enriched in hepatocytes, but few other tissues [31]. ERRa and ERRc expression levels were at or below the edge of detection in HepG2 cells (unpublished observation), but these nuclear receptors are well expressed in muscle cells [27,36]. Collectively, these data strongly support the idea that lipin 1 is a direct HNF4a target gene in liver cells that is induced in physiologic conditions wherein PGC-1a is activated to coactivate HNF4a. We have previously shown that lipin 1 and HNF4a physically interact [10], but the physiologic consequences of the interaction and the induction of lipin 1 by HNF4a wa.