Effect of natural genetic variation on enhancer selection and function.

The mechanisms by which genetic variation affects transcription regulation and phenotypes at the nucleotide level are incompletely understood. Here we use natural genetic variation as an in vivo mutagenesis screen to assess the genome-wide effects of sequence variation on lineage-determining and signal-specific transcription factor binding, epigenomics and transcriptional outcomes in primary macrophages from different mouse strains. We find substantial genetic evidence to support the concept that lineage-determining transcription factors define epigenetic and transcriptomic states by selecting enhancer-like regions in the genome in a collaborative fashion and facilitating binding of signal-dependent factors. This hierarchical model of transcription factor function suggests that limited sets of genomic data for lineage-determining transcription factors and informative histone modifications can be used for the prioritization of disease-associated regulatory variants.

Use of mouse models to evaluate roles of nuclear receptors and their ligands in the pathogenesis and treatment of atherosclerosis.

Nuclear receptors form a large family of ligand-dependent transcription factors that regulate diverse aspects of development and homeostasis. Several of these receptors have been demonstrated to play important roles in controlling biological processes that influence the development and clinical consequences of atherosclerosis. Because nuclear receptors are regulated by small molecules, they are potential targets for anti-atherogenic drugs. In this chapter, we review the use of mouse models to evaluate roles of nuclear receptors and their ligands in the pathogenesis and treatment of atherosclerosis.

Differential recruitment of the coactivator proteins CREB-binding protein and steroid receptor coactivator-1 to peroxisome proliferator-activated receptor gamma/9-cis-retinoic acid receptor heterodimers by ligands present in oxidized low-density lipoprotein.

Peroxisome proliferator-activated receptor gamma (PPARgamma) colocalizes with oxidized low-density lipoprotein (LDL) in foam cells in atherosclerotic lesions. We have explored a potential role of oxidized fatty acids in LDL as PPARgamma activators. LDL from patients suffering from intermittent claudication due to atherosclerosis was analyzed using HPLC and gas chromatography/mass spectrophotometry and found to contain 9-hydroxy and 13-hydroxyoctadecadienoic acid (9- and 13-HODE), as well as 5-hydroxy-, 12-hydroxy- and 15-hydroxyeicosatetraenoic acid (5-, 12- and 15-HETE respectively). PPARgamma was potently activated by 13(S)-HODE and 15(S)-HETE, as judged by transient transfection assays in macrophages or CV-1 cells. 5(S)- and 12(S)-HETE as well as 15-deoxy-Delta(12,14)-prostaglandin J(2) also activated PPARgamma but were less potent. Interestingly, the effect of the lipoxygenase products 13(S)-HODE and 15(S)-HETE as well as of the drug rosiglitazone were preferentially enhanced by the coactivator CREB-binding protein, whereas the effect of the cyclooxygenase product 15-deoxy-Delta(12,14)-prostaglandin J(2) was preferentially enhanced by steroid receptor coactivator-1. We interpret these results, which may have relevance to the pathogenesis of atherosclerosis, to indicate that the lipoxygenase products on the one hand and the cyclooxygenase product on the other exert specific effects on the transcription of target genes through differential coactivator recruitment by PPARgamma/9-cis retinoic acid receptor heterodimer complexes.

Defect of histone acetyltransferase activity of the nuclear transcriptional coactivator CBP in Rubinstein-Taybi syndrome.

CREB-binding protein (CBP) is a transcriptional coactivator that has intrinsic histone acetyltransferase (HAT) activity. CBP is the causative gene of Rubinstein-Taybi syndrome (RTS). To investigate the relationships between CBP HAT activity and RTS, we analyzed 16 RTS patients. A microdeletion was identified in one patient by fluorescent in situ hybridization analysis. Heteroallelic mutations were identified in five patients by reverse transcriptase-polymerase chain reaction-single-strand conformation polymorphism analysis and sequencing. These included a 2 bp deletion between nucleotides 4319 and 4320, an 11 bp deletion between nucleotides 4898 and 4908, a 14 bp insertion (CCTCGGTCCTGCAC) between nucleotides 5212 and 5213, a 2 bp deletion between nucleotides 5222 and 5223, and a missense mutation from guanine (G) to cytosine (C) at nucleotide 4951 that changed codon 1378 from CGG (arginine) to CCG (proline). The identical missense mutation was introduced into the recombinant mouse CBP. It abolished the HAT activity of CBP and the ability of CBP to transactivate cyclic AMP-response element binding protein (CREB), in HAT assays and in microinjection experiments, respectively. These results suggest that the loss of the HAT activity of CBP may cause RTS, as the first example of a defect of HAT activity in a human disease. Our findings raise the possibility that treatment of RTS patients with histone deacetylase inhibitors might have beneficial effects.

Cyclopentenone prostaglandins: new insights on biological activities and cellular targets.

The cyclopentenone prostaglandins PGA2, PGA1, and PGJ2 are formed by dehydration within the cyclopentane ring of PGE2, PGE1, and PGD2. PGJ2 is metabolized further to yield Delta(12)-PGJ(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)). Various compounds within the cyclopentenone prostaglandin family possess potent anti-inflammatory, anti-neoplastic, and anti-viral activity. Most actions of the cyclopentenone prostaglandins do not appear to be mediated by binding to G-protein coupled prostanoid receptors. Rather, the bioactivity of these compounds results from their interaction with other cellular target proteins. 15-deoxy-Delta(12,14)-PGJ(2) is a high affinity ligand for the nuclear receptor PPARgamma and modulates gene transcription by binding to this receptor. Other activities of the cyclopentenone prostaglandins are mediated by the reactive alpha,beta-unsaturated carbonyl group located in the cyclopentenone ring. The transcription factor NF-kappaB and its activating kinase are key targets for the anti-inflammatory activity of 15d-PGJ2, which inhibits NF-kappaB-mediated transcriptional activation by PPARgamma-dependent and independent molecular mechanisms. Other cyclopentenone prostaglandins, such as Delta(7)-PGA1 and Delta(12)-PGJ2, have strong anti-tumor activity. These compounds induce cell cycle arrest or apoptosis of tumor cells depending on the cell type and treatment conditions. We review here recent progress in understanding the mechanisms of action of the cyclopentenone prostaglandins and their possible use as therapeutic agents.

Regulation of somatic growth by the p160 coactivator p/CIP.

A family of p160 coactivators was initially identified based on ligand-dependent interactions with nuclear receptors and thought to function, in part, by recruiting CREB-binding protein/p300 to several classes of transcription factors. One of the p160 factors, p/CIP/AIB1, often amplified and overexpressed in breast cancer, also exhibits particularly strong interaction with CREB-binding protein/p300. In this manuscript, we report that p/CIP, which exhibits regulated transfer from cytoplasm to nucleus, is required for normal somatic growth from embryonic day 13.5 through maturity. Our data suggest that a short stature phenotype of p/CIP gene-deleted mice reflect both altered regulation of insulin-like growth factor-1 (IGF-1) gene expression in specific tissues and a cell-autonomous defect of response to IGF-1, including ineffective transcriptional activities by several classes of regulated transcription factors under specific conditions. The actions of p/CIP are therefore required for full expression of a subset of genes critical for regulating physiological patterns of somatic growth in mammals.

Mildly oxidized low density lipoprotein activates multiple apoptotic signaling pathways in human coronary cells.

Apoptosis of arterial cells induced by oxidized low density lipoproteins (OxLDL) is thought to contribute to the progression of atherosclerosis. However, most data on apoptotic effects and mechanisms of OxLDL were obtained with extensively oxidized LDL unlikely to occur in early stages of atherosclerotic lesions. We now demonstrate that mildly oxidized LDL generated by incubation with oxygen radical-producing xanthine/xanthine oxidase (X/XO) induces apoptosis in primary cultures of human coronary endothelial and SMC, as determined by TUNEL technique, DNA laddering, and FACS analysis. Apoptosis was markedly reduced when X/XO-LDL was generated in the presence of different oxygen radical scavengers. Apoptotic signals were mediated by intramembrane domains of both Fas and tumor necrosis factor (TNF) receptors I and II. Blocking of Fas ligand (FasL) reduced apoptosis by 50% and simultaneous blocking of FasL and TNF receptors by 70%. Activation of apoptotic receptors was accompanied by an increase of proapoptotic and a decrease in antiapoptotic proteins of the Bcl-2 family and resulted in marked activation of class I and II caspases. Mildly oxidized LDL also activated MAP and Jun kinases and increased p53 and other transcription factors (ATF-2, ELK-1, CREB, AP-1). Inhibitors of Map and Jun kinase significantly reduced apoptosis. Our results provide the first evidence that OxLDL-induced apoptosis involves TNF receptors and Jun activation. More important, they demonstrate that even mildly oxidized LDL formed in atherosclerotic lesions may activate a broad cascade of oxygen radical-sensitive signaling pathways affecting apoptosis and other processes influencing the evolution of plaques. Thus, we suggest that extensive oxidative modifications of LDL are not necessary to influence signal transduction and transcription in vivo.

Combinatorial roles of the nuclear receptor corepressor in transcription and development.

Transcriptional repression plays crucial roles in diverse aspects of metazoan development, implying critical regulatory roles for corepressors such as N-CoR and SMRT. Altered patterns of transcription in tissues and cells derived from N-CoR gene-deleted mice and the resulting block at specific points in CNS, erythrocyte, and thymocyte development indicated that N-CoR was a required component of short-term active repression by nuclear receptors and MAD and of a subset of long-term repression events mediated by REST/NRSF. Unexpectedly, N-CoR and a specific deacetylase were also required for transcriptional activation of one class of retinoic acid response element. Together, these findings suggest that specific combinations of corepressors and histone deacetylases mediate the gene-specific actions of DNA-bound repressors in development of multiple organ systems.

Oxidized LDL reduces monocyte CCR2 expression through pathways involving peroxisome proliferator-activated receptor gamma.

The CCR2-mediated recruitment of monocytes into the vessel wall plays an important role in all stages of atherosclerosis. In recent studies, we have shown that lipoproteins can modulate CCR2 expression and have identified native LDL as a positive regulator. In contrast, oxidized LDL (OxLDL), which is mainly formed in the aortic intima, reduces CCR2 expression, promotes monocyte retention, and may cause pathological accumulation of monocytes in the vessel wall. We now provide evidence that OxLDL reduces monocyte CCR2 expression by activating intracellular signaling pathways that may involve peroxisome proliferator-activated receptor gamma (PPARgamma). Receptor-mediated uptake of the lipoprotein particle was required and allows for delivery of the exogenous ligand to the nuclear receptor. The suppression of CCR2 expression by OxLDL was mediated by lipid components of OxLDL, such as the oxidized linoleic acid metabolites 9-HODE and 13-HODE, known activators of PPARgamma. Modified apoB had no such effect. Consistent with a participation of the PPARgamma signaling pathway, BRL49653 reduced CCR2 expression in freshly isolated human monocytes ex vivo and in circulating mouse monocytes in vivo. These results implicate PPARgamma in the inhibition of CCR2 gene expression by oxidized lipids, which may help retain monocytes at sites of inflammation, such as the atherosclerotic lesion.

Peroxisome proliferator-activated receptor gamma ligands inhibit development of atherosclerosis in LDL receptor-deficient mice.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that regulates fat-cell development and glucose homeostasis and is the molecular target of a class of insulin-sensitizing agents used for the management of type 2 diabetes mellitus. PPARgamma is highly expressed in macrophage foam cells of atherosclerotic lesions and has been demonstrated in cultured macrophages to both positively and negatively regulate genes implicated in the development of atherosclerosis. We report here that the PPARgamma-specific agonists rosiglitazone and GW7845 strongly inhibited the development of atherosclerosis in LDL receptor-deficient male mice, despite increased expression of the CD36 scavenger receptor in the arterial wall. The antiatherogenic effect in male mice was correlated with improved insulin sensitivity and decreased tissue expression of TNF-alpha and gelatinase B, indicating both systemic and local actions of PPARgamma. These findings suggest that PPARgamma agonists may exert antiatherogenic effects in diabetic patients and provide impetus for efforts to develop PPARgamma ligands that separate proatherogenic activities from antidiabetic and antiatherogenic activities.

Polymerase chain reaction-based method for quantifying recruitment of monocytes to mouse atherosclerotic lesions in vivo: enhancement by tumor necrosis factor-alpha and interleukin-1 beta.

The critical role of monocyte recruitment in atherogenesis has been appreciated for some time. However, until recently, there have been no sufficiently sensitive methods for measuring rates of monocyte recruitment to the arterial wall in vivo. We have developed a novel highly sensitive method, based on the polymerase chain reaction, for quantitatively tracking DNA-marked monocytes and have adapted it for use in mice. We use the uniquely male gene, SRY:, on the Y chromosome as a gene marker. We transfuse monocytes from a male donor into a congenic female mouse, euthanize the mouse after 24 to 48 hours, and then quantify the arterial uptake of monocytes by quantitative polymerase chain reaction. This study describes the techniques used and their sensitivity and reproducibility and demonstrates the approach by assessing the effects of cytokines. In control low density lipoprotein receptor-negative mice, monocyte recruitment decreased slightly but significantly as lesions progressed. Intraperitoneal injection of a combination of tumor necrosis factor-alpha and interleukin-1 beta more than doubled the rate of monocyte recruitment into developing lesions. However, the response to the cytokines was much greater in younger mice with less advanced lesions than in older animals with more advanced lesions.

Peroxisome proliferator-activated receptor gamma-dependent repression of the inducible nitric oxide synthase gene.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily that activates target gene transcription in a ligand-dependent manner. In addition, liganded PPARgamma can inhibit transcription of genes induced by gamma interferon (IFN-gamma) and/or lipopolysaccharides (LPSs), including the inducible nitric oxide synthase (iNOS) gene. Inhibition of the iNOS promoter is achieved partially through antagonizing the activities of NF-kappaB, AP-1, and STAT1, which are known to mediate effects of LPS and IFN-gamma. Previous studies have suggested that transrepression of these factors by nuclear receptors involves competition for limiting amounts of the general coactivators CREB-binding protein (CBP) and p300. CBP and p300 are thought to be recruited to nuclear receptors through bridging factors that include SRC-1, although CBP also interacts directly with PPARgamma through its amino terminus. These observations have raised questions concerning the involvement of SRC-1-like factors in CBP recruitment and transrepression. We here provide evidence that PPARgamma's ability to repress iNOS transcription requires the ligand-dependent charge clamp that mediates interactions with CBP and SRC-1. Single amino acid mutations in PPARgamma that abolished ligand-dependent interactions with SRC-1 and CBP not only resulted in complete loss of transactivation activity but also abolished transrepression. Conversely, a CBP deletion mutant containing the SRC-1 interaction domain but lacking the N-terminal PPARgamma interaction domain was inactive as a PPARgamma coactivator and failed to rescue transrepression. Together, these findings are consistent with a model in which transrepression by PPARgamma is achieved by targeting CBP through direct interaction with its N-terminal domain and via SRC-1-like bridge factors.

The histone deacetylase-3 complex contains nuclear receptor corepressors.

Acetylation and deacetylation of nucleosomal histones have profound effects on gene transcription in all eukaryotes. In humans, three highly homologous class I and four class II histone deacetylase (HDAC) enzymes have been identified to date. The class I deacetylases HDAC1 and HDAC2 are components of multisubunit complexes, one of which could associate with the nuclear hormone receptor corepressor, N-CoR. N-CoR also interacts with class II deacetylases HDAC4, HDAC5, and HDAC7. In comparison with HDAC1 and HDAC2, HDAC3 remains relatively uncharacterized, and very few proteins have been shown to interact with HDAC3. Using an affinity purification approach, we isolated an enzymatically active HDAC3 complex that contained members of the nuclear receptor corepressor family. Deletion analysis of N-CoR revealed that HDAC3 binds multiple N-CoR regions in vitro and that all of these regions are required for maximal binding in vivo. The N-CoR domains that interact with HDAC3 are distinct from those that bind other HDACs. Transient overexpression of HDAC3 and microinjection of Abs against HDAC3 showed that a component of transcriptional repression mediated by N-CoR depends on HDAC3. Interestingly, data suggest that interaction with a region of N-CoR augments the deacetylase activity of HDAC3. These results provide a possible molecular mechanism for HDAC3 regulation and argue that N-CoR is a platform in which distinct domains can interact with most of the known HDACs.

Ligand-dependent interactions of coactivators steroid receptor coactivator-1 and peroxisome proliferator-activated receptor binding protein with nuclear hormone receptors can be imaged in live cells and are required for transcription.

Members of the nuclear receptor superfamily are thought to activate transcription by recruitment of one or more recently identified coactivator complexes. Here we demonstrate that both peroxisome proliferator-activated receptor binding protein (PBP) and steroid receptor coactivator-1 (SRC-1) are required for ligand-dependent transcription of transiently transfected and chromosomally integrated reporter genes by the estrogen receptor (ER) and retinoic acid receptor (RAR). To examine ligand-dependent interactions between nuclear receptors and specific coactivators in living cells, these proteins were tagged with cyan (CFP) and yellow (YFP) mutants of the green fluorescent protein. Fluorescence resonance energy transfer (FRET) from the CFP to the YFP indicated interaction between the receptor and coactivator. CFP fusions to RAR or its ligand-binding domain exhibited rapid ligand-dependent FRET to YFP-tagged nuclear receptor interaction domains of the coactivators SRC-1 and PBP. The ER-ligand-binding domain, unlike RAR, also exhibited some basal interaction with coactivators in unstimulated cells that was abolished by the receptor antagonists tamoxifen or ICI182,780. Inhibition of FRET by tamoxifen but not ICI182,780 could be reversed by estradiol, whereas estradiol-enhanced FRET could not be inhibited by either antagonist, indicating that ligand effects can show varying degrees of hysteresis. These findings suggest that ligand-dependent transcriptional activities of the RAR and ER require concurrent or sequential recruitment of SRC-1 and PBP-containing coactivator complexes.

15-deoxy-delta 12,14-prostaglandin J2 inhibits multiple steps in the NF-kappa B signaling pathway.

Prostaglandin J(2) (PGJ(2)) and its metabolites Delta(12)-PGJ(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) are naturally occurring derivatives of prostaglandin D(2) that have been suggested to exert antiinflammatory effects in vivo. 15d-PGJ(2) is a high-affinity ligand for the peroxisome proliferator-activated receptor gamma (PPARgamma) and has been demonstrated to inhibit the induction of inflammatory response genes, including inducible NO synthase and tumor necrosis factor alpha, in a PPARgamma-dependent manner. We report here that 15d-PGJ(2) potently inhibits NF-kappaB-dependent transcription by two additional PPARgamma-independent mechanisms. Several lines of evidence suggest that 15d-PGJ(2) directly inhibits NF-kappaB-dependent gene expression through covalent modifications of critical cysteine residues in IkappaB kinase and the DNA-binding domains of NF-kappaB subunits. These mechanisms act in combination to inhibit transactivation of the NF-kappaB target gene cyclooxygenase 2. Direct inhibition of NF-kappaB signaling by 15d-PGJ(2) may contribute to negative regulation of prostaglandin biosynthesis and inflammation, suggesting additional approaches to the development of antiinflammatory drugs.