contributed equally to this work Mammalian TIF1α and TIF1β (KAP-1/KRIP-1) are related transcriptional intermediary factors that possess intrinsic silencing activity. TIF1α is believed to be a euchromatic target for liganded nuclear receptors, while TIF1β may serve as a co-repressor for the large family of KRAB domain-containing zinc finger proteins. Here, we report an association of TIF1β with both heterochromatin and euchromatin in interphase nuclei. Co-immunoprecipitation of nuclear extracts shows that endogenous TIF1β, but not TIF1α, is associated with members of the heterochromatin protein 1 (HP1) family. However, in vitro, both TIF1α and TIF1β interact with and phosphorylate the HP1 proteins. This interaction involves a conserved amino acid motif, which is critical for the silencing activity of TIF1β but not TIF1α. We further show that trichostatin A, an inhibitor of histone deacetylases, can interfere with both TIF1 and HP1 silencing. The silencing activity of TIF1α appears to result chiefly from histone deacetylation, whereas that of TIF1β may be mediated via both HP1 binding and histone deacetylation.

The ric-3 gene is required for maturation of nicotinic acetylcholine receptors in Caenorhabditis elegans. The human homolog of RIC-3, hRIC-3, enhances expression of ␣7 nicotinic receptors in Xenopus laevis oocytes, whereas it totally abolishes expression of ␣4␤2 nicotinic and 5-HT 3 serotonergic receptors. Both the N-terminal region of hRIC-3, which contains two transmembrane segments, and the C-terminal region are needed for these differential effects. hRIC-3 inhibits receptor expression by hindering export of mature receptors to the cell membrane. By using chimeric proteins made of ␣7 and 5-HT 3 receptors, we have shown that the presence of an extracellular isoleucine close to the first transmembrane receptor fragment is responsible for the transport arrest induced by hRIC-3. Enhancement of ␣7 receptor expression occurs, at least, at two levels: by increasing the number of mature receptors and facilitating its transport to the membrane. Certain amino acids of a putative amphipathic helix present at the large cytoplasmic region of the ␣7 subunit are required for these actions. Therefore, hRIC-3 can act as a specific regulator of receptor expression at different levels.

The expression of several genes involved in intra-and extracellular lipid metabolism, notably those involved in peroxisomal and mitochondrial ␤-oxidation, is mediated by ligand-activated receptors, collectively referred to as peroxisome proliferator-activated receptors (PPARs). To gain more insight into the control of expression of carnitine palmitoyltransferase (CPT) genes, which are regulated by fatty acids, we have examined the transcriptional regulation of the human MCPT I gene. We have cloned by polymerase chain reaction the 5-flanking region of this gene and demonstrated its transcriptional activity by transfection experiments with the CAT gene as a reporter. We have also shown that this is a target gene for the action of PPARs, and we have localized a PPAR responsive element upstream of the first exon. These results show that PPAR regulates the entry of fatty acids into the mitochondria, which is a crucial step in their metabolism, especially in tissues like heart, skeletal muscle and brown adipose tissue in which fatty acids are a major source of energy.The incorporation of activated long-chain fatty acids into the mitochondria to be catabolized through ␤-oxidation is produced by the mitochondrial carnitine palmitoyltransferase (CPT) 1 enzyme system. CPT I, the outer membrane component of this system, is the main control point in the ␤-oxidation pathway. CPT I is thus a suitable site for pharmacological control of fatty acid oxidation in conditions such as diabetes or heart diseases.Two isoforms of CPT I have been described, which have been designated LCPT I and MCPT I since these isoforms are mainly expressed in liver and muscle respectively. The MCPT I gene is expressed not only in skeletal muscle but also in heart and brown and white adipose tissue (1-4). This expression pattern may be of great significance since fatty acids are a major source of energy for heart, skeletal muscle, and brown adipose tissue.The CPT I gene expression is regulated by fatty acids and peroxisome proliferators (5, 6). To gain more insight into the control of CPT I gene expression by fatty acids, we have examined the transcriptional regulation of CPT I genes. The expression of several genes involved in intra-and extracellular lipid metabolism, notably those involved in peroxisomal and mitochondrial ␤-oxidation, is mediated by ligand-activated receptors collectively referred to as peroxisome proliferator-activated receptors (PPARs); these receptors are members of the nuclear receptor superfamily. PPARs are activated by a wide array of peroxisome proliferators and also by natural and synthetic fatty acids (7,8), antidiabetic drugs (9, 10), prostaglandin J 2 (10), and leukotriene B 4 (11).We have amplified by polymerase chain reaction (PCR) the 5Ј region of the human heart and brown adipose tissue CPT I gene and demonstrate, first, the transcriptional activity of this fragment and, second, the presence of a PPRE in the 5Ј-flanking region of this gene. In CV1 cells, the activation of the CPT I gene by PPAR was dependent on the ad...

Members of the heterochromatin protein 1 (HP1) family are silencing nonhistone proteins. Here, we show that in P19 embryonal carcinoma (EC) nuclei, HP1 alpha, beta, and gamma form homo- and heteromers associated with nucleosomal core histones. In vitro, all three HP1s bind to tailed and tailless nucleosomes and specifically interact with the histone-fold of histone H3. Furthermore, HP1alpha interacts with the linker histone H1. HP1alpha binds to H3 and H1 through its chromodomain (CD) and hinge region, respectively. Interestingly, the Polycomb (Pc1/M33) CD also interacts with H3, and HP1alpha and Pc1/M33 binding to H3 is severely impaired by CD mutations known to abrogate HP1 and Polycomb silencing in Drosophila. These results define a novel function for the conserved CD and suggest that HP1 self-association and histone binding may play a crucial role in HP1-mediated heterochromatin assembly.

The Drosophila bonus (bon) gene encodes a homolog of the vertebrate TIF1 transcriptional cofactors. bon is required for male viability, molting, and numerous events in metamorphosis including leg elongation, bristle development, and pigmentation. Most of these processes are associated with genes that have been implicated in the ecdysone pathway, a nuclear hormone receptor pathway required throughout Drosophila development. Bon is associated with sites on the polytene chromosomes and can interact with numerous Drosophila nuclear receptor proteins. Bon binds via an LxxLL motif to the AF-2 activation domain present in the ligand binding domain of betaFTZ-F1 and behaves as a transcriptional inhibitor in vivo.

The purpose of this study was to estimate the prevalence of erectile dysfunction (ED) in Colombia, Ecuador, and Venezuela. A 49-item questionnaire was completed by 1946 men aged 40 years and older. The age-adjusted combined prevalence of minimal, moderate, and complete ED for all three countries was 53.4%, with 19.8% of all men reporting moderate to complete ED. Age was the variable most strongly linked to ED; the prevalence of complete ED increased markedly in men older than 79 y of age (31.9%) and 70 -79 y (17.2%) compared with men aged 40 -49 y (<3%). Several medical conditions, such as hypertension, benign prostatic hyperplasia, and diabetes, and the use of medications to treat these conditions were correlated with the prevalence of ED. This study corroborates earlier studies demonstrating that ED is very common, increases dramatically with age, and has multiple correlates, including some that are also risk factors for cardiovascular disease.

The isozymes CYP1A2, CYP2D6, and CYP3A4/5 are involved in the majority of all cytochrome P450-mediated drug biotransformations. In this study we investigated the inhibition profiles of CYP1A2 (substrate: caffeine) CYP2D6 (substrate: dextromethorphan), and CYP3A4/5 (substrate: dextrorphan) by cimetidine, ranitidine, and the novel H2-receptor antagonist ebrotidine in human liver microsomes. The inhibitory effect of the drugs on the enzymes activities were as follows: CYP1A2: cimetidine >> ranitidine = ebrotidine; CYP2D6: cimetidine >>> ranitidine = ebrotidine; CYP3A4/5: ebrotidine > cimetidine >>> ranitidine. The inhibition of CYP3A4/5 enzyme activity by ebrotidine was competitive. To test whether the inhibitory effect of ebrotidine in CYP3A activity was also found in vivo, we analyzed the biodisposition of midazolam in 8 healthy volunteers. Midazolam biodisposition was significantly reduced when administered together with cimetidine (P < .05), whereas no significant inhibition was observed with ebrotidine or ranitidine compared with placebo. Psychomotor performance analysis revealed no significant effect of the observed reduction on midazolam biodisposition. We concluded that patients who are receiving treatment with drugs metabolized through CYP3A may experience enhanced drug effects as a result of pharmacokinetic interaction when treated concomitantly with cimetidine. In contrast, the effect of ranitidine or ebrotidine on CYP3A activity in vivo seems to have little clinical significance.

The Krü ppel-associated box (KRAB) domain, originally identified as a 75-aa sequence present in numerous Krü ppel-type zinc-finger proteins, is a potent DNA-binding-dependent transcriptional repression domain that is believed to function through interaction with the transcriptional intermediary factor 1 (TIF1) ␤. On the basis of sequence comparison and phylogenetic analysis, we have recently defined three distinct subfamilies of KRAB domains. In the present study, individual members of each subfamily were tested for transcriptional repression and interaction with TIF1␤ and two other closely related family members (TIF1␣ and TIF1␥). All KRAB variants were shown, (i) to repress transcription when targeted to DNA through fusion to a heterologous DNA-binding domain in mammalian cells, and (ii) to interact specifically with TIF1␤, but not with TIF1␣ or TIF1␥. Taken together, these results implicate TIF1␤ as a common transcriptional corepressor for the three distinct subfamilies of KRAB zinc-finger proteins and suggest a high degree of conservation in the molecular mechanism underlying their transcriptional repression activity. The Krüppel-associated box (KRAB) domain is one of the most potent and widely distributed transcriptional repression domains yet identified in mammals (1, 2); it has been estimated that approximately one-third of the 300 to 700 human zinc-finger proteins (ZFPs) of the Krüppel Cys 2 His 2 -type contain a KRAB domain in their N termini (3). This regulatory domain consists of Ϸ75 amino acids and is composed of two contiguous modules, the KRAB-A box and the KRAB-B box (3), each encoded by separate exons (4, 5). When fused to a heterologous DNAbinding domain (DBD), the KRAB-A box silences both basal and activated transcription in transfected cells in a dosedependent manner and over large distances (1,2,6,7). This transcriptional silencing has recently been linked at a molecular level to chromatin remodeling through the demonstration of a physical association between several different KRAB domains and the transcriptional intermediary factor 1 (TIF1) ␤, a transcriptional corepressor involved in heterochromatin-mediated regulation (8-10). TIF1␤, also named KAP-1 (11) or KRIP-1 (12), was demonstrated to interact with numerous KRAB domains but not KRAB mutants deficient in repression, to enhance KRAB-mediated repression, and to silence transcription when directly tethered to DNA (11-13). This silencing activity requires histone deacetylation (9) and may result from the recruitment of a histone deacetylase complex, called N-CoR-1 (14), and͞or from an association of TIF1␤ with members of the heterochromatin protein 1 (HP1) family (9, 10), a class of nonhistone proteins with a well-established epigenetic gene silencing function (for review, see ref. 15).TIF1␤ is a member of a family of proteins (8) that also includes TIF1␣, a putative nuclear receptor cofactor (8,16,17), and TIF1␥, whose function is unknown (18). These proteins are defined by the presence of two conserved amino acid regions: an N-terminal R...