Genomic imprinting defect in Zfp57 mutant iPS cell lines.

ZFP57 maintains genomic imprinting in mouse embryos and ES cells. To test its roles during iPS reprogramming,we derived iPS clones by utilizing retroviral infection to express reprogramming factors in mouse MEF cells. After analyzing four imprinted regions, we found that parentally derived DNA methylation imprint was largely maintained in the iPS clones with Zfp57 but missing in those without maternal or zygotic Zfp57. Intriguingly, DNA methylation imprint was lost at the Peg1 and Peg3 but retained at the Snrpn and Dlk1-Dio3 imprinted regions in the iPS clones without zygotic Zfp57. This finding will be pursued in future studies.

Mechanisms of triglyceride accumulation in activated macrophages.

LPS treatment of macrophages induces TG accumulation, which is accentuated by TG-rich lipoproteins or FFA. We defined pathways altered during macrophage activation that contribute to TG accumulation. Glucose uptake increased with activation, accompanied by increased GLUT1. Oxidation of glucose markedly decreased, whereas incorporation of glucose-derived carbon into FA and sterols increased. Macrophage activation also increased uptake of FFA, associated with an increase in CD36. Oxidation of FA was markedly reduced, whereas the incorporation of FA into TGs increased, associated with increased GPAT3 and DGAT2. Additionally, macrophage activation decreased TG lipolysis; however, expression of ATGL or HSL was not altered. Macrophage activation altered gene expression similarly when incubated with exogenous FA or AcLDL. Whereas activation with ligands of TLR2 (zymosan), TLR3 (poly I:C), or TLR4 (LPS) induced alterations in macrophage gene expression, leading to TG accumulation, treatment of macrophages with cytokines had minimal effects. Thus, activation of TLRs leads to accumulation of TG in macrophages by multiple pathways that may have beneficial effects in host defense but could contribute to the accelerated atherosclerosis in chronic infections and inflammatory diseases.

Zinc finger protein ZFP57 requires its co-factor to recruit DNA methyltransferases and maintains DNA methylation imprint in embryonic stem cells via its transcriptional repression domain.

Previously, we discovered that ZFP57 is a maternal-zygotic effect gene, and it maintains DNA methylation genomic imprint at multiple imprinted regions in mouse embryos. Despite these findings, it remains elusive how DNA methyltransferases are targeted to the imprinting control regions to initiate and maintain DNA methylation imprint. To gain insights into these essential processes in genomic imprinting, we examined how ZFP57 maintains genomic DNA methylation imprint in mouse embryonic stem (ES) cells. Here we demonstrate that the loss of ZFP57 in mouse ES cells led to a complete loss of genomic DNA methylation imprint at multiple imprinted regions, similar to its role in mouse embryos. However, reintroduction of ZFP57 into Zfp57-null ES cells did not result in reacquisition of DNA methylation imprint, suggesting that the memory for genomic imprinting had been lost or altered in Zfp57-null ES cells in culture. Interestingly, ZFP57 and DNA methyltransferases could form complexes in the presence of KAP1/TRIM28/TIF1ß when co-expressed in COS cells. We also found that the wild-type exogenous ZFP57 but not the mutant ZFP57 lacking the KRAB box that interacts with its co-factor KAP1/TRIM28/TIF1ß could substitute for the endogenous ZFP57 in maintaining the DNA methylation imprint in ES cells. These results suggest that ZFP57 may recruit DNA methyltransferases to its target regions to maintain DNA methylation imprint, and this interaction is likely facilitated by KAP1/TRIM28/TIF1ß.

ADRP/ADFP and Mal1 expression are increased in macrophages treated with TLR agonists.

Activation of macrophages by TLR agonists enhances foam cell formation, but the underlying mechanisms are not understood. We examined the effects of TLR agonists on ADRP/ADFP, a protein associated with forming lipid droplets, and Mal1 a fatty acid-binding protein, in two mouse macrophage cell lines and human monocytes. Low doses of LPS, a TLR4 agonist increased both mRNA and protein levels of ADRP/ADFP and Mal1 in RAW 264.7 macrophages. Following pretreatment with Intralipid, fatty acids, or acetyl-LDL to increase triglyceride or cholesterol ester storage, LPS treatment still increased ADRP/ADFP and Mal1 mRNA levels. LPS also induced ADRP/ADFP and Mal1 in J774 macrophages and ADRP/ADFP in human monocytes. Zymosan, a fungal product that activates TLR2, poly-I:C, a viral mimetic that activates TLR3, and imiquimod, a TLR7 agonist, also increased ADRP/ADFP. Zymosan, but not poly-I:C or imiquimod, induced Mal1. In contrast, neither gene was induced by TNFalpha, IL-1beta, IL-6, or interferon-gamma. Thus TLR agonists induce ADRP/ADFP and Mal1, which likely contributes to macrophage triglyceride and cholesterol ester storage leading to foam cell formation.

Interaction of Epstein-Barr virus BZLF1 C-terminal tail structure and core zipper is required for DNA replication but not for promoter transactivation.

The Epstein-Barr virus (EBV) protein BZLF1 contains a bZIP DNA binding domain in which C-terminal tail residues fold back against a zipper region that forms a coiled coil and mediates dimerization. Point mutagenesis in the zipper region reveals the importance of individual residues within the (208)SSENDRLR(215) sequence that is conserved in C/EBP for transactivation and EBV DNA replication. The restoration of BZLF1 DNA replication activity by the complementation of two deleterious mutations (S208E and D236K) indicates that the interaction of the C-terminal tail and the core zipper is required for DNA replication, identifying a functional role for this structural feature unique to BZLF1.

Adipocyte fatty acid-binding protein expression and lipid accumulation are increased during activation of murine macrophages by toll-like receptor agonists.

OBJECTIVE: Toll-like receptors (TLRs) recognize pathogens and mediate signaling pathways important for host defense. Recent studies implicate TLR polymorphisms in atherosclerosis risk in humans. Adipocyte fatty acid-binding protein (aP2) is present in macrophages and has an important role in atherosclerotic plaque development. We investigated aP2 expression in RAW 264.7 cells treated with lipopolysaccharide (LPS) and other TLR agonists and assessed lipid accumulation in these activated murine macrophages. METHODS AND RESULTS: Stimulation with LPS, a TLR4 ligand, resulted in a 56-fold increase in aP2 mRNA expression, and zymosan, a TLR2 ligand, induced an approximately 1500-fold increase. Polyinosine: polycytidylic acid (poly I:C), a TLR3 ligand, led to a 9-fold increase. Levels of aP2 protein were significantly increased in LPS or zymosan-treated macrophages compared with control or poly I:C-treated cells. In addition, the cholesteryl ester content of LPS or zymosan-treated macrophages was approximately 5-fold greater in the presence of low-density lipoprotein, and triglyceride content was approximately 2-fold greater in the absence of exogenous lipid than control or poly I:C-treated cells. CONCLUSIONS: Expression of macrophage aP2 is induced on TLR activation and parallels increases in cholesteryl ester and triglyceride levels. These results provide a molecular link between the known roles of TLR and aP2 in foam cell formation.