Feedback on hypothalamic TRH transcription is dependent on thyroid hormone receptor N terminus.

The beta thyroid hormone receptor (TRbeta), but not TRalpha1, plays a specific role in mediating T(3)-dependent repression of hypothalamic TRH transcription. To investigate the structural basis of isoform specificity, we compared the transcriptional regulation and DNA binding obtained with chimeric and N-terminally deleted TRs. Using in vivo transfection assays to follow hypothalamic TRH transcription in the mouse brain, we found that TRbeta1 and chimeras with the TRbeta1 N terminus did not affect either transcriptional activation or repression from the rat TRH promoter, whereas N-terminally deleted TRbeta1 impaired T(3)-dependent repression. TRalpha1 or chimeras with the TRalpha1 N terminus reduced T(3)-independent transcriptional activation and blocked T(3)-dependent repression of transcription. Full deletion of the TRalpha1 N terminus restored ligand-independent activation of transcription. No TR isoform specificity was seen after transcription from a positive thyroid hormone response element. Gel mobility assays showed that all TRs tested bound specifically to the main negative thyroid hormone response element in the TRH promoter (site 4). Addition of neither steroid receptor coactivator 1 nor nuclear extracts from the hypothalamic paraventricular nuclei revealed any TR isoform specificity in binding to site 4. Thus N-terminal sequences specify TR T(3)-dependent repression of TRH transcription but not DNA recognition, emphasizing as yet unknown neuron-specific contributions to protein-promoter interactions in vivo.

Drosophila Myc interacts with host cell factor (dHCF) to activate transcription and control growth.

The Myc proto-oncoproteins are transcription factors that recognize numerous target genes through hexameric DNA sequences called E-boxes. The mechanism by which they then activate the expression of these targets is still under debate. Here, we use an RNAi screen in Drosophila S2 cells to identify Drosophila host cell factor (dHCF) as a novel co-factor for Myc that is functionally required for the activation of a Myc-dependent reporter construct. dHCF is also essential for the full activation of endogenous Myc target genes in S2 cells, and for the ability of Myc to promote growth in vivo. Myc and dHCF physically interact, and they colocalize on common target genes. Furthermore, down-regulation of dHCF-associated histone acetyltransferase and histone methyltransferase complexes in vivo interferes with the Myc biological activities. We therefore propose that dHCF recruits such chromatin-modifying complexes and thereby contributes to the expression of Myc targets and hence to the execution of Myc biological activities.

Genetic and epigenetic analysis of SSAT gene dysregulation in suicidal behavior.

It has recently been proposed that the SSAT gene plays a role in the predisposition to suicidal behavior. SSAT expression was found to be down-regulated in the brain of suicide completers. In addition, a single nucleotide polymorphism (SNP) rs6526342 was associated both with variation in SSAT expression and with suicidal behavior. In this study, we aimed to characterize the relationship between SSAT dysregulation and suicide behavior. To this end, we measured SSAT expression levels in the ventral prefrontal cortex (VPFC) of suicide completers (n = 20) and controls (n = 20) and found them to be significantly down-regulated in suicide victims (P = 0.007). To identify the basis of the regulation of SSAT expression, we performed an association analysis of 309 SNPs with SSAT transcript levels in 53 lymphoblastoid cell lines from the CEPH collection. We then examined the methylation status of the SSAT promoter region in males and females suicide completers and control subjects whose SSAT brain expression had been measured. We found no evidence to support a role for SNPs in controlling the level of SSAT expression. SSAT promoter methylation levels were not different between suicide completers and controls and did not correlate with SSAT expression levels. In addition, we found no indication of a genetic association between suicidal behavior and SNPs located within the SSAT gene. Our study provides new results which show that dysregulation of SSAT expression does play a role in suicide behavior. However, our data do not support any association between rs6526342 and variation in SSAT expression or suicidal behavior.

Identification of estrogen-responsive DNA sequences by transient expression experiments in a human breast cancer cell line.

The expression of a hybrid gene formed by the promoter region of the Xenopus laevis vitellogenin gene B1 and the CAT coding region is regulated by estrogen when the gene is transfected into hormone-responsive MCF-7 cells. Furthermore, the 5' flanking region of the gene B1 alone can confer inducibility to heterologous promoters, although to a varying extent depending on the promoter used. Deletion mapping of he vitellogenin hormone-responsive sequences revealed that a 13 bp element 5'-AGTCACTGTGACC-3' at position -334 is essential for estrogen inducibility. We have shown previously that this 13 bp element is present upstream of several liver-specific estrogen-inducible genes.

Linking epigenetics to lipid metabolism: focus on histone deacetylases.

A number of recent studies revealed that epigenetic modifications play a central role in the regulation of lipid and of other metabolic pathways such as cholesterol homeostasis, bile acid synthesis, glucose and energy metabolism. Epigenetics refers to aspects of genome functions regulated in a DNA sequence-independent fashion. Chromatin structure is controlled by epigenetic mechanisms through DNA methylation and histone modifications. The main modifications are histone acetylation and deacetylation on specific lysine residues operated by two different classes of enzymes: Histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively. The interaction between these enzymes and histones can activate or repress gene transcription: Histone acetylation opens and activates chromatin, while deacetylation of histones and DNA methylation compact chromatin making it transcriptionally silent. The new evidences on the importance of HDACs in the regulation of lipid and other metabolic pathways will open new perspectives in the comprehension of the pathophysiology of metabolic disorders.

Estrogenic activity assessment of environmental chemicals using in vitro assays: identification of two new estrogenic compounds.

Environmental chemicals with estrogenic activities have been suggested to be associated with deleterious effects in animals and humans. To characterize estrogenic chemicals and their mechanisms of action, we established in vitro and cell culture assays that detect human estrogen receptor [alpha] (hER[alpha])-mediated estrogenicity. First, we assayed chemicals to determine their ability to modulate direct interaction between the hER[alpha] and the steroid receptor coactivator-1 (SRC-1) and in a competition binding assay to displace 17ss-estradiol (E(2)). Second, we tested the chemicals for estrogen-associated transcriptional activity in the yeast estrogen screen and in the estrogen-responsive MCF-7 human breast cancer cell line. The chemicals investigated in this study were o,p'-DDT (racemic mixture and enantiomers), nonylphenol mixture (NPm), and two poorly analyzed compounds in the environment, namely, tris-4-(chlorophenyl)methane (Tris-H) and tris-4-(chlorophenyl)methanol (Tris-OH). In both yeast and MCF-7 cells, we determined estrogenic activity via the estrogen receptor (ER) for o,p'-DDT, NPm, and for the very first time, Tris-H and Tris-OH. However, unlike estrogens, none of these xenobiotics seemed to be able to induce ER/SRC-1 interactions, most likely because the conformation of the activated receptor would not allow direct contacts with this coactivator. However, these compounds were able to inhibit [(3)H]-E(2) binding to hER, which reveals a direct interaction with the receptor. In conclusion, the test compounds are estrogen mimics, but their molecular mechanism of action appears to be different from that of the natural hormone as revealed by the receptor/coactivator interaction analysis.

Epigenetics in sepsis: targeting histone deacetylases.

Severe sepsis and septic shock are lethal complications of infection, characterised by dysregulated inflammatory and immune responses. Our understanding of the pathogenesis of sepsis has improved markedly in recent years, but unfortunately has not been translated into efficient treatment strategies. Epigenetic mechanisms such as covalent modification of histones by acetylation are master regulators of gene expression under physiological and pathological conditions, and strongly impact on inflammatory and host defence responses. Histone acetylation is controlled by histone acetyltransferases and histone deacetylases (HDACs), which affect gene expression also by targeting non-histone transcriptional regulators. Numerous HDAC inhibitors (HDACi) are being tested in clinical trials, primarily for the treatment of cancer. We performed the first comprehensive study of the impact of HDACi on innate immune responses in vitro and in vivo. We showed that HDACi act essentially as negative regulators of the expression of critical immune receptors and antimicrobial pathways in innate immune cells. In agreement, HDACi impaired phagocytosis and killing of bacteria by macrophages, and increased susceptibility to non-severe bacterial and fungal infections. Strikingly, proof-of-principle studies demonstrated that HDACi protect from lethal toxic shock and septic shock. Overall, our observations argue for a close monitoring of the immunological and infection status of patients treated with HDACi, especially immunocompromised cancer patients. They also support the concept of pharmacological inhibitors of HDACs as promising drugs to treat inflammatory diseases, including sepsis.

Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors controlling the expression of genes involved in lipid homeostasis. PPARs activate gene transcription in response to a variety of compounds including hypolipidemic drugs as well as natural fatty acids. From the plethora of PPAR activators, Scatchard analysis of receptor-ligand interactions has thus far identified only four ligands. These are the chemotactic agent leukotriene B4 and the hypolipidemic drug Wy 14,643 for the alpha-subtype and a prostaglandin J2 metabolite and synthetic antidiabetic thiazolidinediones for the gamma-subtype. Based on the hypothesis that ligand binding to PPAR would induce interactions of the receptor with transcriptional coactivators, we have developed a novel ligand sensor assay, termed coactivator-dependent receptor ligand assay (CARLA). With CARLA we have screened several natural and synthetic candidate ligands and have identified naturally occurring fatty acids and metabolites as well as hypolipidemic drugs as bona fide ligands of the three PPAR subtypes from Xenopus laevis. Our results suggest that PPARs, by their ability to interact with a number of structurally diverse compounds, have acquired unique ligand-binding properties among the superfamily of nuclear receptors that are compatible with their biological activity.

PPARs: nuclear receptors for fatty acids, eicosanoids, and xenobiotics.

The peroxisome proliferator-activated receptors have enjoyed the spotlight for many reasons. These transcription factors are ligand-inducible nuclear receptors that modulate gene expression in response to a broad spectrum of compounds. The recognition that PPARs are indeed nuclear receptors for polyunsaturated fatty acids, some eicosanoids and also lipid-lowering and antidiabetic drugs, has opened many exciting avenues of research and drug discovery. Recent studies on the PPAR function have extended the role of these transcription factors beyond energy homeostasis to master gene in adipogenesis and also determinants in inflammation control. While rapid advances have been made, it is clear that we are far from a global understanding of the mechanisms and functions of PPARs.

Functional analysis of the 5' flanking sequence of a vaccinia virus late gene.

A series of mutations, including 5' and 3' deletions, as well as insertions were introduced into the 5' flanking nucleotide sequence of a vaccinia virus late gene. This DNA has been shown previously to contain all the necessary elements for correct regulation of the gene most probably transcribed by the viral RNA polymerase. To facilitate the assays, the mutated DNA was fused to the chloramphenicol acetyltransferase gene and inserted into the genome of live vaccinia virus. The effects of the mutations on expression of the chimeric gene were studied by both enzyme assays and nuclease S1 analysis. The results showed that 5' deletions up to about 15 bp from the putative initiation site of transcription still yielded high levels of gene expression. All mutations, however, that deleted the authentic late mRNA start site, abolished promoter activity.

In Vivo Persistence of Codominant Human CD8+ T Cell Clonotypes Is Not Limited by Replicative Senescence or Functional Alteration.

T cell responses to viral epitopes are often composed of a small number of codominant clonotypes. In this study, we show that tumor Ag-specific T cells can behave similarly. In a melanoma patient with a long lasting HLA-A2/NY-ESO-1-specific T cell response, reaching 10% of circulating CD8 T cells, we identified nine codominant clonotypes characterized by individual TCRs. These clonotypes made up almost the entire pool of highly differentiated effector cells, but only a fraction of the small pool of less differentiated "memory" cells, suggesting that the latter serve to maintain effector cells. The different clonotypes displayed full effector function and expressed TCRs with similar functional avidity. Nevertheless, some clonotypes increased, whereas others declined in numbers over the observation period of 6 years. One clonotype disappeared from circulating blood, but without preceding critical telomere shortening. In turn, clonotypes with increasing frequency had accelerated telomere shortening, correlating with strong in vivo proliferation. Interestingly, the final prevalence of the different T cell clonotypes in circulation was anticipated in a metastatic lymph node withdrawn 2 years earlier, suggesting in vivo clonotype selection driven by metastases. Together, these data provide novel insight in long term in vivo persistence of T cell clonotypes associated with continued cell turnover but not replicative senescence or functional alteration.

Histone deacetylase inhibitors impair innate immune responses to Toll-like receptor agonists and to infection.

Regulated by histone acetyltransferases and deacetylases (HDACs), histone acetylation is a key epigenetic mechanism controlling chromatin structure, DNA accessibility, and gene expression. HDAC inhibitors induce growth arrest, differentiation, and apoptosis of tumor cells and are used as anticancer agents. Here we describe the effects of HDAC inhibitors on microbial sensing by macrophages and dendritic cells in vitro and host defenses against infection in vivo. HDAC inhibitors down-regulated the expression of numerous host defense genes, including pattern recognition receptors, kinases, transcription regulators, cytokines, chemokines, growth factors, and costimulatory molecules as assessed by genome-wide microarray analyses or innate immune responses of macrophages and dendritic cells stimulated with Toll-like receptor agonists. HDAC inhibitors induced the expression of Mi-2β and enhanced the DNA-binding activity of the Mi-2/NuRD complex that acts as a transcriptional repressor of macrophage cytokine production. In vivo, HDAC inhibitors increased the susceptibility to bacterial and fungal infections but conferred protection against toxic and septic shock. Thus, these data identify an essential role for HDAC inhibitors in the regulation of the expression of innate immune genes and host defenses against microbial pathogens.

Epigenetic regulation of histone H3 serine 10 phosphorylation status by HCF-1 proteins in C. elegans and mammalian cells.

BACKGROUND: The human herpes simplex virus (HSV) host cell factor HCF-1 is a transcriptional coregulator that associates with both histone methyl- and acetyltransferases, and a histone deacetylase and regulates cell proliferation and division. In HSV-infected cells, HCF-1 associates with the viral protein VP16 to promote formation of a multiprotein-DNA transcriptional activator complex. The ability of HCF proteins to stabilize this VP16-induced complex has been conserved in diverse animal species including Drosophila melanogaster and Caenorhabditis elegans suggesting that VP16 targets a conserved cellular function of HCF-1. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the role of HCF proteins in animal development, we have characterized the effects of loss of the HCF-1 homolog in C. elegans, called Ce HCF-1. Two large hcf-1 deletion mutants (pk924 and ok559) are viable but display reduced fertility. Loss of Ce HCF-1 protein at reduced temperatures (e.g., 12 degrees C), however, leads to a high incidence of embryonic lethality and early embryonic mitotic and cytokinetic defects reminiscent of mammalian cell-division defects upon loss of HCF-1 function. Even when viable, however, at normal temperature, mutant embryos display reduced levels of phospho-histone H3 serine 10 (H3S10P), a modification implicated in both transcriptional and mitotic regulation. Mammalian cells with defective HCF-1 also display defects in mitotic H3S10P status. CONCLUSIONS/SIGNIFICANCE: These results suggest that HCF-1 proteins possess conserved roles in the regulation of cell division and mitotic histone phosphorylation.

Detection of CBP rearrangements in acute myelogenous leukemia with t(8;16).

The CREB-binding protein (CBP) is a large nuclear protein that regulates many signal transduction pathways and is involved in chromatin-mediated transcription. The translocation t(8;16)(p11;p13.3) consistently disrupts two genes: the CBP gene on chromosome band 16p13.3 and the MOZ gene on chromosome band 8p11. Although a fusion of these two genes as a result of the translocation is expected, attempts at detecting the fusion transcript by reverse transcriptase polymerase chain reaction (RT-PCR) have proven difficult; to date, only one in-frame CBP/MOZ fusion transcript has been reported. We therefore sought other reliable means of detecting CBP rearrangements. We applied fluorescence in situ hybridization (FISH) and Southern blot analyses to a series of AML patients with a t(8;16) and detected DNA rearrangements of both the CBP and the MOZ loci in all cases tested. All six cases examined for CBP rearrangements have breakpoints within a 13 kb breakpoint cluster region at the 5' end of the CBP gene. Additionally, we used a MOZ cDNA probe to construct a surrounding cosmid contig and detect DNA rearrangements in three t(8;16) cases, all of which display rearrangements within a 6 kb genomic fragment of the MOZ gene. We have thus developed a series of cosmid probes that consistently detect the disruption of the CBP gene in t(8;16) patients. These clones could potentially be used to screen other cancer-associated or congenital translocations involving chromosome band 16p13.3 as well.

The estrogen-responsive element as an inducible enhancer: DNA sequence requirements and conversion to a glucocorticoid-responsive element.

The estrogen-responsive element (ERE) present in the 5'-flanking region of the Xenopus laevis vitellogenin (vit) gene B1 has been characterized by transient expression analysis of chimeric vit-tk-CAT (chloramphenicol acetyltransferase) gene constructs transfected into the human estrogen-responsive MCF-7 cell line. The vit B1 ERE behaves like an inducible enhancer, since it is able to confer estrogen inducibility to the heterologous HSV thymidine kinase (tk) promoter in a relative position- and orientation-independent manner. In this assay, the minimal B1 ERE is 33 bp long and consists of two 13 bp imperfect palindromic elements both of which are required for the enhancer activity. A third imperfect palindromic element is present further upstream within the 5'-flanking region of the gene but is unable to confer hormone responsiveness by itself. Similarly, neither element forming the B1 ERE can alone confer estrogen inducibility to the tk promoter. However, in combinations of two, all three imperfect palindromes can act cooperatively to form a functional ERE. In contrast a single 13 bp perfect palindromic element, GGTCACTGTGACC, such as the one found upstream of the vit gene A2, is itself sufficient to act as a fully active ERE. Single point mutations within this element abolish estrogen inducibility, while a defined combination of two mutations converts this ERE into a glucocorticoid-responsive element.