Eric Kalkhoven

A signature motif in transcriptional co-activators mediates binding to nuclear receptors.

The binding of lipophilic hormones, retinoids and vitamins to members of the nuclear-receptor superfamily modifies the DNA-binding and transcriptional properties of these receptors, resulting in the activation or repression of target genes,. Ligand binding induces conformational changes in nuclear receptors and promotes their association with a diverse group of nuclear proteins, including SRC-1/p160 (refs 3-5), TIF-2/GRIP-1 (refs 6, 7) and CBP/p300 (refs 4, 5, 8, 9) which function as co-activators of transcription, and RIP-140 (ref. 10), TIF-1 (ref. 11) and TRIP-1/SUG-1 (refs 12, 13) whose functions are unclear. Here we report that a short sequence motif LXXLL (where L is leucine and X is any amino acid) present in RIP-140, SRC-1 and CBP is necessary and sufficient to mediate the binding of these proteins to liganded nuclear receptors. We show that the ability of SRC-1 to bind the oestrogen receptor and enhance its transcriptional activity is dependent upon the integrity of the LXXLL motifs and on key hydrophobic residues in a conserved helix (helix 12) of the oestrogen receptor that are required for its ligand-induced activation function. We propose that the LXXLL motif is a signature sequence that facilitates the interaction of different proteins with nuclear receptors, and is thus a defining feature of a new family of nuclear proteins.

Regulation of Treg functionality by acetylation-mediated Foxp3 protein stabilization

Regulatory T cells (Tregs) are a specific subset of lymphocytes that are critical for the maintenance of self-tolerance. Expression levels of the transcription factor Foxp3 have been causally associated with Treg differentiation and function. Recent studies show that Foxp3 can also be transiently expressed in effector T cells; however, stable Foxp3 expression is required for development of a functional Treg suppressor phenotype. Here, we demonstrate that Foxp3 is acetylated, and this can be reciprocally regulated by the histone acetyltransferase p300 and the histone deacetylase SIRT1. Hyperacetylation of Foxp3 prevented polyubiquitination and proteasomal degradation, therefore dramatically increasing stable Foxp3 protein levels. Moreover, using mouse splenocytes, human peripheral blood mononuclear cells, T cell clones, and skin-derived T cells, we demonstrate that treatment with histone deacetylase inhibitors resulted in significantly increased numbers of functional Treg cells. Taken together, our data demonstrate that modulation of the acetylation state of Foxp3 provides a novel molecular mechanism for assuring rapid temporal control of Foxp3 levels in T cells, thereby regulating Treg numbers and functionality. Manipulating Foxp3 acetylation levels could therefore provide a new therapeutic strategy to control inappropriate (auto)immune responses.

AF-2 activity and recruitment of steroid receptor coactivator 1 to the estrogen receptor depend on a lysine residue conserved in nuclear receptors.

Hormone-dependent transcriptional activation by nuclear receptors depends on the presence of a conserved C-terminal amphipathic alpha-helix (helix 12) in the ligand-binding domain. Here we show that a lysine residue, which is conserved in most nuclear receptors in the predicted helix 3, is also required for estrogen-dependent transactivation. The replacement of lysine 366 with alanine appreciably reduced activation function 2 (AF-2) activity without affecting steroid- or DNA-binding activity in the mouse estrogen receptor. The mutation dramatically reduced the ability of the receptor to bind steroid receptor coactivator 1 (SRC-1) but had no effect on receptor-interacting protein 140 (RIP-140) binding, indicating that while their sites of interaction overlap, they are not entirely consistent and in keeping with the proposal that the recruitment of coactivators, such as SRC-1, is required for AF-2 activity. Although the function of RIP-140 remains to be established, RIP-140 appears to be capable of recruiting the basal transcription machinery, since overexpression of the protein markedly increased the transcriptional activity of the mutant receptor. Since the lysine residue is conserved, we propose that it is required, together with residues in helix 12, to form the surface by which members of the nuclear receptor family interact with coactivators.

Adipose tissue-resident immune cells: key players in immunometabolism

Adipose tissue (AT) plays a pivotal role in whole-body lipid and glucose homeostasis. AT exerts metabolic control through various immunological mechanisms that instigated a new research field termed immunometabolism. Here, we review AT-resident immune cells and their role as key players in immunometabolism. In lean subjects, AT-resident immune cells have housekeeping functions ranging from apoptotic cell clearance to extracellular matrix remodeling and angiogenesis. However, obesity provides bacterial and metabolic danger signals that mimic bacterial infection, and drives a shift in immune-cell phenotypes and numbers, classified as a prototypic T helper 1 (Th1) inflammatory response. The resulting AT inflammation and insulin resistance link obesity to its metabolic sequel, and suggests that targeted immunomodulatory interventions may be beneficial for obese patients.

Natural killer T cells in adipose tissue prevent insulin resistance

Lipid overload and adipocyte dysfunction are key to the development of insulin resistance and can be induced by a high-fat diet. CD1d-restricted invariant natural killer T (iNKT) cells have been proposed as mediators between lipid overload and insulin resistance, but recent studies found decreased iNKT cell numbers and marginal effects of iNKT cell depletion on insulin resistance under high-fat diet conditions. Here, we focused on the role of iNKT cells under normal conditions. We showed that iNKT cell-deficient mice on a low-fat diet, considered a normal diet for mice, displayed a distinctive insulin resistance phenotype without overt adipose tissue inflammation. Insulin resistance was characterized by adipocyte dysfunction, including adipocyte hypertrophy, increased leptin, and decreased adiponectin levels. The lack of liver abnormalities in CD1d-null mice together with the enrichment of CD1d-restricted iNKT cells in both mouse and human adipose tissue indicated a specific role for adipose tissue-resident iNKT cells in the development of insulin resistance. Strikingly, iNKT cell function was directly modulated by adipocytes, which acted as lipid antigen-presenting cells in a CD1d-mediated fashion. Based on these findings, we propose that, especially under low-fat diet conditions, adipose tissue-resident iNKT cells maintain healthy adipose tissue through direct interplay with adipocytes and prevent insulin resistance.

Posttranslational Modifications of PPAR‐γ: Fine‐tuning the Metabolic Master Regulator

Peroxisome proliferator–activated receptors (PPARs) are members of the nuclear receptor family of ligand-dependent transcription factors, which comprise a subgroup of three closely homologous genes, PPAR-α (NR1C1), PPAR-β/δ (NR1C2), and PPAR-γ (NR1C3) (refs. 1,2). All PPARs bind to PPAR responsive elements in the promoter regions of target genes as obligate heterodimers with retinoic acid X receptors. Similar to other nuclear receptors, PPARs consist of distinct functional domains including an N-terminal transactivation domain (AF1), a highly conserved DNA-binding domain (DBD), and a C-terminal ligand-binding domain (LBD) that contains a ligand-dependent transactivation function (AF2; Figure 1a). Ligand binding stabilizes the active conformation of the LBD, thereby serving as a “molecular switch” between activation and repression functions of the receptor (3). On the promoters of some target genes, unliganded PPARs recruit corepressors such as N-CoR and SMRT, which are part of multiprotein complexes containing histone deacetylase activity that repress gene transcription (4). Upon ligand binding, these corepressor complexes are exchanged for activating co-activator complexes, including the SRC1/CBP and TRAP/DRIP/ARC complexes (5). Besides ligand-mediated regulation, the transcriptional activity of nuclear receptors can also be regulated via the ligand-independent AF1 domain. In common with other NRs (6), the AF1 domains of the different PPAR isotypes probably lack a stable secondary structure in aqueous solution, only to adopt a stable conformation upon interaction with other proteins. Although all PPAR nuclear receptors play a part in lipid homeostasis and energy metabolism, the different PPAR proteins exhibit different physiological roles due to (i) distinct expression patterns, (ii) specific activation by different ligands, and (iii) intrinsic functional differences between the different isotype proteins. Alternative splicing and differential promoter usage results in two PPAR-γ isoforms, PPAR-γ1 and PPAR-γ2, with the latter harboring a 30-residue extension at its N-terminus (1,2). PPAR-γ1 is expressed in several tissues, including the lower intestine, macrophages, and white adipose tissue (WAT), whereas PPAR-γ2 expression is almost exclusively restricted to WAT. The endogenous ligands for PPAR-γ are not firmly established, although some natural compounds, such as polyunsaturated fatty acids, prostaglandin J2 derivatives (15-deoxy-δ12,14-PGJ2), and nitro-oleic acid, have been shown to be able to activate PPAR-γ (7,8). Synthetic PPAR-γ agonists include the thiazolidinediones (TZDs), which ameliorate insulin resistance and lower blood glucose levels in patients with type 2 diabetes (9). A huge amount of data indicates that PPAR-γ is one of the key players in the differentiation of fibroblast-like mesenchymal stem cells into adipocytes, a process known as adipogenesis. For example, PPAR-γ+/– mice lack most adipose tissue (10–12), whereas in vitro differentiation of fibroblasts into mature adipocytes can be induced by the introduction of PPAR-γ (13). In addition, PPAR-γ is also essential for the maintenance of adipose tissue, because conditional knock-out of the Pparg gene resulted in reduced in vivo survival of mature adipocytes (14). Besides its role in adipocyte differentiation and maintenance, PPAR-γ directly regulates the expression of a number of genes involved in net lipid partitioning into mature adipocytes, underscoring the importance of PPAR-γ in glucose and lipid homeostasis. Compelling genetic evidence for this view comes from human familial partial lipodystrophy subtype 3 (MIM 604367) patients, harboring heterozygous mutations in the PPARG gene, as they are characterized by aberrant fat distribution and metabolic disturbances, including insulin resistance and dyslipidemia (15). Interestingly, PPAR-γ was recently shown to be involved in the inhibition of osteoblast differentiation and osteoclastogenesis (16), indicating that novel biological functions of this transcription factor remain to be identified beyond its role in lipid and glucose homeostasis. Although the three PPAR isotypes display a high degree in primary amino acid Posttranslational Modifications of PPAR-γ: Fine-tuning the Metabolic Master Regulator

Peroxisome Proliferator-Activated Receptor β/δ (PPARβ/δ) but Not PPARα Serves as a Plasma Free Fatty Acid Sensor in Liver

ABSTRACT Peroxisome proliferator-activated receptor α (PPARα) is an important transcription factor in liver that can be activated physiologically by fasting or pharmacologically by using high-affinity synthetic agonists. Here we initially set out to elucidate the similarities in gene induction between Wy14643 and fasting. Numerous genes were commonly regulated in liver between the two treatments, including many classical PPARα target genes, such as Aldh3a2 and Cpt2. Remarkably, several genes induced by Wy14643 were upregulated by fasting independently of PPARα, including Lpin2 and St3gal5, suggesting involvement of another transcription factor. Using chromatin immunoprecipitation, Lpin2 and St3gal5 were shown to be direct targets of PPARβ/δ during fasting, whereas Aldh3a2 and Cpt2 were exclusive targets of PPARα. Binding of PPARβ/δ to the Lpin2 and St3gal5 genes followed the plasma free fatty acid (FFA) concentration, consistent with activation of PPARβ/δ by plasma FFAs. Subsequent experiments using transgenic and knockout mice for Angptl4, a potent stimulant of adipose tissue lipolysis, confirmed the stimulatory effect of plasma FFAs on Lpin2 and St3gal5 expression levels via PPARβ/δ. In contrast, the data did not support activation of PPARα by plasma FFAs. The results identify Lpin2 and St3gal5 as novel PPARβ/δ target genes and show that upregulation of gene expression by PPARβ/δ is sensitive to plasma FFA levels. In contrast, this is not the case for PPARα, revealing a novel mechanism for functional differentiation between PPARs.

Effect of Synthetic Dietary Triglycerides: A Novel Research Paradigm for Nutrigenomics

Background The effect of dietary fats on human health and disease are likely mediated by changes in gene expression. Several transcription factors have been shown to respond to fatty acids, including SREBP-1c, NF-κB, RXRs, LXRs, FXR, HNF4α, and PPARs. However, it is unclear to what extent these transcription factors play a role in gene regulation by dietary fatty acids in vivo. Methodology/Principal Findings Here, we take advantage of a unique experimental design using synthetic triglycerides composed of one single fatty acid in combination with gene expression profiling to examine the effects of various individual dietary fatty acids on hepatic gene expression in mice. We observed that the number of significantly changed genes and the fold-induction of genes increased with increasing fatty acid chain length and degree of unsaturation. Importantly, almost every single gene regulated by dietary unsaturated fatty acids remained unaltered in mice lacking PPARα. In addition, the majority of genes regulated by unsaturated fatty acids, especially docosahexaenoic acid, were also regulated by the specific PPARα agonist WY14643. Excellent agreement was found between the effects of unsaturated fatty acids on mouse liver versus cultured rat hepatoma cells. Interestingly, using Nuclear Receptor PamChip® Arrays, fatty acid- and WY14643-induced interactions between PPARα and coregulators were found to be highly similar, although several PPARα-coactivator interactions specific for WY14643 were identified. Conclusions/Significance We conclude that the effects of dietary unsaturated fatty acids on hepatic gene expression are almost entirely mediated by PPARα and mimic those of synthetic PPARα agonists in terms of regulation of target genes and molecular mechanism. Use of synthetic dietary triglycerides may provide a novel paradigm for nutrigenomics research.

Paneth cell extrusion and release of antimicrobial products is directly controlled by immune cell–derived IFN-γ

IFN-γ directly triggers degranulation and death of Paneth cells, which do not respond to microbial stimulation in primary culture.

Scaffold/matrix attachment region elements interact with a p300-scaffold attachment factor A complex and are bound by acetylated nucleosomes.

ABSTRACT The transcriptional coactivator p300 regulates transcription by binding to proteins involved in transcription and by acetylating histones and other proteins. These transcriptional effects are mainly at promoter and enhancer elements. Regulation of transcription also occurs through scaffold/matrix attachment regions (S/MARs), the chromatin regions that bind the nuclear matrix. Here we show that p300 binds to the S/MAR binding protein scaffold attachment factor A (SAF-A), a major constituent of the nuclear matrix. Using chromatin immunoprecipitations, we established that both p300 and SAF-A bind to S/MAR elements in the transiently silent topoisomerase I gene prior to its activation at G1 during cell cycle. This binding is accompanied by local acetylation of nucleosomes, suggesting that p300-SAF-A interactions at S/MAR elements of nontranscribed genes might poise these genes for transcription.