No abstract

Nuclear receptors (NRs) are ligand‐inducible transcription factors that mediate complex effects on development, differentiation and homeostasis. They regulate the transcription of their target genes through binding to cognate DNA sequences as homodimers or heterodimers. The molecular mechanisms underlying transcriptional activation by NRs are still poorly understood, although intermediary factors (mediators) appear to be involved in mediating the transactivation functions of NRs. TIF1 has been identified previously as a protein that interacts specifically with the ligand binding domain of several nuclear receptors, both in yeast and in vitro. The characteristics of these interactions have led us to suggest that TIF1 might be a mediator of the NR ligand‐inducible activation function AF‐2. Using a two‐hybrid screening in yeast, we have now identified two TIF1‐binding proteins, mHP1 alpha and mMOD1, that are mouse homologues of the Drosophila heterochromatinic protein 1. Using mHP1 alpha as a bait in a second two‐hybrid screening, we have isolated cDNAs encoding proteins that are also very likely to be involved in chromatin structure and function, as well as a protein structurally and functionally related to TIF1 (renamed TIF1 alpha), which was named TIF1 beta. Here we discuss how the function of members of the TIF1 family in the control of transcription could be exerted at the level of the structure of the chromatin template.

The free-draining properties of DNA normally make it impossible to separate nucleic acids by free-flow electrophoresis. However, little is known, either theoretically or experimentally, about the diffusion coefficient of DNA molecules during free-flow electrophoresis. In fact, many authors simply assume that the Nernst-Einstein relation between the mobility and the diffusion coefficient still holds under such conditions. In this paper, we present an experimental study of the diffusion coefficient of both ssDNA and dsDNA molecules during free-flow electrophoresis. Our results unequivocally show that a simplistic use of Nernst-Einstein's relation fails, and that the electric field actually has no effect on the thermal diffusion process. Finally, we compare the dependence of the diffusion coefficient upon DNA molecular size to results obtained previously by other groups and to Zimm's theory.

The "specific reactivity" of B-galactosidase from Escherichia coli for different substrates has been studied a t the optimal pH value. The pH dependence of the enzyme activity has been reinvestigated in highly controlled conditions with respect to Mg2+ and Na+ concentrations and ionic strength. The different kinetic parameters kcat, Km and the kcat/Km ratio have been determined with o-nitrophenyl-B-D-galactoside and o-nitrophenyl-,8-D-fucoside, a t different pH values between 5.16 and 10, for both Mg2+-enzyme and Mg2+-free enzyme, since a residual activity has been found in the absence of Mg2+.The activity of both types of enzyme is controlled by a protonated group which ionizes in the alkaline range and by a t least one unprotonated group which ionizes in the acidic range. This latter group has a pK smaller than 6 in both types of enzyme; it is plausible to assume that this group is a carboxylate.I n the alkaline range, the p K of the involved group shifts from about 6.5 in Mg2+-free enzyme to 8.4 in the Mg2+ enzyme. Either the substrate or the Mg binding induces this shift in the ionization of the group.The B-galactosidase catalyzed reactions proceed via two intermediary complexes. For o-nitrophenyl-B-D-galactoside substrate the limiting process is not the same for the Mg2+ enzyme and for the Mg2+-free enzyme.j3-Galactosidase from Escherichia coli can be conveniently obtained in the crystalline form and is characterized by its high molecular weight and single polypeptide chain of 135000 associated in tetramers [l-71. Different studies of the enzyme activity as a function of pH have been reported. Kuby and Lardy [8] have shown that the enzyme is stable between pH 6 and pH 8. G. Cohen-Bazire and Monod [9] have observed a competition between alkaline ions and protons on some sites of the protein.A bell-shaped pH activity curve has already been observed but under conditions of variable ionic strength and salt concentrations and in the presence of Tris buffer [8, 10-131, all factors known to modify enzyme activity [8,11-141. I n an attempt to bring new information on the mechanism of catalysis, the pH dependence of the enzymic activity has been reinvestigated under more controlled conditions than previously. The investigations have been carried out in a large pH range, in the presence and in the absence of Mg2+ ions, since a residual activity has been found in the absence of this cation THEORETICAL SECTIONIt has been previously established that @-galactoside hydrolysis by B-galactosidase proceeds by a pathway involving a t least two intermediates [16]. Since the pH profile of the enzymic activity could be described by a bell-shaped curve, different ionization states of the enzyme could classically be written [17,18]: Kb'l l Kkll ES'E assuming a "bottle neck" mechanism, in which only EH and the corresponding complexes EHS and EHS' are the active forms. This scheme is the simplest that accounts for the experimental data ; some preliminary results indicate that k, and k3 pH variations are the same.

Thirty newly detected mutations in the PHEX gene are reported, and pooled with all the previously published mutations. The spectrum of mutations displayed 16% deletions, 8% insertions, 34% missense, 27% nonsense, and 15% splice site mutations, with two peaks in exon 15, and 17. Since 32.8% of PHEX amino acids were conserved in the endopeptidases family, the number of missense mutations detected at non-conserved residues was smaller than expected, whereas the number of nonsense mutations observed at non-conserved residues was very close to the expected number. Compared with conserved amino acids, the changes in nonconserved amino acids may result in benign polymorphisms or possibly mild disease that may go undiagnosed.

The low density lipoprotein receptor-related protein 1 (LRP1) is a ubiquitously expressed cell surface receptor that protects from intracellular cholesterol accumulation. However, the underlying mechanisms are unknown. Here we show that the extracellular (␣) chain of LRP1 mediates TGF␤-induced enhancement of Wnt5a, which limits intracellular cholesterol accumulation by inhibiting cholesterol biosynthesis and by promoting cholesterol export. Moreover, we demonstrate that the cytoplasmic (␤) chain of LRP1 suffices to limit cholesterol accumulation in LRP1؊/؊ cells. Cholesterol is a major component of mammalian cell membranes that accumulates in the vascular wall during atherosclerosis, the leading cause of death in industrialized societies (1, 2). The low density lipoprotein receptor-related protein 1 (LRP1), 3 a cell surface receptor that belongs to the LDL receptor family, endocytoses multiple ligands (3). It consists of an 85-kDa membrane-bound carboxyl fragment (␤ chain) and a non-covalently attached 515-kDa (␣ chain) amino-terminal fragment (4). We previously demonstrated that LRP1 limits cholesterol accumulation in the arterial wall. Mice deficient for LRP1 in vascular smooth muscle cells (vSMCs) (smLRP1 mice) develop vSMCs proliferation, cholesterol accumulation (5), and massive foam cell formation when fed a cholesterol-rich diet (6 -10). Whereas LRP1 integrates the platelet-derived growth factor (PDGF-BB) (8, 9) and transforming growth factor-␤ (TGF-␤) at the plasma membrane, two pathways known to regulate vSMCs proliferation (7), the physiological importance and function of LRP1 in regulating intracellular cholesterol homeostasis is still poorly understood. Several mechanisms have been proposed. LRP1 has been shown to promote cholesterol export in vSMCs through induction of ATP binding cassette transporter A1 (ABCA1) levels (5) and to induce a Wnt5a/␤-catenin pathway to limit cholesterol overload in mouse embryonic fibroblasts (11). Moreover, smLRP1 mice express very low levels of Wnt5a in vSMCs (12). TGF-␤ also stimulates a non-canonical Wnt5a pathway in airway smooth muscle cells (13). These data strongly suggest that a TGF-␤/LRP1/Wnt5a pathway limits intracellular cholesterol accumulation.How Wnt5a interferes with cholesterol homeostasis is unknown. It might increase cholesterol export and/or block cholesterol synthesis. ABCA1 and the ATP binding cassette transporter G1 (ABCG1) are two proteins that promote cholesterol efflux. Cholesterol synthesis is tightly regulated by a feedback system that senses the level of cholesterol and modulates the transcription of genes encoding enzymes of cholesterol biosynthesis and uptake (14, 15). For instance, when cholesterol levels rise in cells, the membrane-embedded protein of the endoplasmic reticulum (ER), Scap, senses the increase and binds to Insigs, proteins located to the ER. Insigs then limit cleavage and nuclear translocation of sterol regulatory element-binding proteins (SREBPs), in particular SREBP-2, an activator of cholesterol synthesis in liver and a...

The human general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). In eukaryotic cells, TFIID is thought to nucleate RNA polymerase II (Pol II) preinitiation complex formation on all protein coding gene promoters and thus, be crucial for Pol II transcription. In a child with intellectual disability, mild microcephaly, corpus callosum agenesis and poor growth, we identified a homozygous splice-site mutation in TAF8 (NM_138572.2: c.781–1G > A). Our data indicate that the patient’s mutation generates a frame shift and an unstable TAF8 mutant protein with an unrelated C-terminus. The mutant TAF8 protein could not be detected in extracts from the patient’s fibroblasts, indicating a loss of TAF8 function and that the mutation is most likely causative. Moreover, our immunoprecipitation and proteomic analyses show that in patient cells only partial TAF complexes exist and that the formation of the canonical TFIID is impaired. In contrast, loss of TAF8 in mouse embryonic stem cells and blastocysts leads to cell death and to a global decrease in Pol II transcription. Astonishingly however, in human TAF8 patient cells, we could not detect any cellular phenotype, significant changes in genome-wide Pol II occupancy and pre-mRNA transcription. Thus, the disorganization of the essential holo-TFIID complex did not affect global Pol II transcription in the patient’s fibroblasts. Our observations further suggest that partial TAF complexes, and/or an altered TFIID containing a mutated TAF8, could support human development and thus, the absence of holo-TFIID is less deleterious for transcription than originally predicted.

The whole exome analysis of a consanguineous Turkish family revealed MAGEB4 as a possible new X-linked cause of inherited male infertility. This study provides the first clue to the physiological function of a MAGE protein.