Identification and characterization of cvHsp. A novel human small stress protein selectively expressed in cardiovascular and insulin-sensitive tissues.

Starting with computational tools that search for tissue-selective expression of assembled expressed sequenced tags, we have identified by focusing on heart libraries a novel small stress protein of 170 amino acids that we named cvHsp. cvHsp was found as being computationally selectively and highly (0.3% of the total RNA) expressed in human heart. The cvHsp gene mapped to 1p36.23-p34.3 between markers D1S434 and D1S507. The expression of cvHsp was analyzed with RNA dot, Northern blots, or reverse transcription-polymerase chain reaction: expression was high in heart, medium in skeletal muscle, and low in aorta or adipose tissues. In the heart of rat models of cardiac pathologies, cvHsp mRNA expression was either unchanged (spontaneous hypertension), up-regulated (right ventricular hypertrophy induced by monocrotaline treatment), or down-regulated (left ventricular hypertrophy following aortic banding). In obese Zucker rats, cvHsp mRNA was increased in skeletal muscle, brown, and white adipose tissues but remained unchanged in the heart. Western blot analysis using antipeptide polyclonal antibodies revealed two specific bands at 23 and 25 kDa for cvHsp in human heart. cvHsp interacted in both yeast two-hybrid and immunoprecipitation experiments with alpha-filamin or actin-binding protein 280. Within cvHsp, amino acid residues 56-119 were shown to be important for its specific interaction with the C-terminal tail of alpha-filamin.

The Saccharomyces cerevisiae protein YJR043C (Pol32) interacts with the catalytic subunit of DNA polymerase alpha and is required for cell cycle progression in G2/M.

We have analysed the YJR043c gene of Saccharomyces cerevisiae, previously identified by systematic sequencing. The deletion mutant (yjr043cdelta) shows slow growth at low temperature (15 degrees C), while at 30 degrees C and 37 degrees C the growth rate of mutant cells is only moderately affected. At permissive and nonpermissive temperatures, mutant cells were larger and showed a high proportion of large-budded cells with a single duplicated nucleus at or beyond the bud neck and a short spindle. This phenotype was even more striking at low temperature, the mutant cells becoming dumbbell shaped. All these phenotypes suggest a role for YJR043C in cell cycle progression in G2/M phase. In two-hybrid assays, the YJR043c gene product specifically interacted with Pol1, the catalytic subunit of DNA polymerase alpha. The pol1-1 /yjr043cdelta double mutant showed a more severe growth defect than the pol1-1 single mutant at permissive temperature. Centromeric plasmid loss rate elevated in yjr043cdelta. Analysis of the sequence upstream of the YJR043c ORF revealed the presence of an MluI motif (ACGCGT), a sequence associated with many genes involved in DNA replication in budding yeast. The cell cycle phenotype of the yjr043cdelta mutant, the evidence for genetic interaction with Pol1, the presence of an MluI motif upstream and the elevated rate of CEN plasmid loss in mutants all support a function for YJR043C in DNA replication.

TIF1alpha: a possible link between KRAB zinc finger proteins and nuclear receptors.

Ligand-induced gene activation by nuclear receptors (NRs) is thought to be mediated by transcriptional intermediary factors (TIFs), that interact with their ligand-dependent AF-2 activating domain. Included in the group of the putative AF-2 TIFs identified so far is TIF1alpha, a member of a new family of proteins which contains an N-terminal RBCC (RING finger-B boxes-coiled coil) motif and a C-terminal bromodomain preceded by a PHD finger. In addition to these conserved domains present in a number of transcriptional regulatory proteins, TIF1alpha was found to contain several protein-protein interaction sites. Of these, one specifically interacts with NRs bound to their agonistic ligand and not with NR mutants that are defective in the AF-2 activity. Immediately adjacent to this 'NR box', TIF1alpha contains an interaction site for members of the chromatin organization modifier (chromo) family, HP1alpha and MOD1, which both are heterochromatinic proteins. Finally, TIF1alpha also has a binding site for KRAB silencing domains of C2H2 zinc finger proteins. TIF1beta, another member of the TIF1 gene family, has some interacting partners in common with TIF1alpha. TIF1beta can interact with HP1alpha, MOD1 and KRAB domains, but apparently not with NRs. Both TIF1alpha and TIF1beta repress transcription when fused to a DNA binding domain in transiently transfected mammalian cells. A model discussing the potential function(s) of TIF1s in the control of transcription at the level of the chromatin template will be presented.

The putative cofactor TIF1alpha is a protein kinase that is hyperphosphorylated upon interaction with liganded nuclear receptors.

Ligand-induced gene activation by nuclear receptors (NRs) is a complex process requiring dissociation of corepressors and recruitment of coactivators. The putative transcriptional intermediary factor TIF1alpha has been previously characterized as a nuclear protein that interacts directly with the AF-2 ligand-dependent activating domain present in the ligand-binding domain of numerous steroid and nonsteroid receptors, including the estrogen (ERalpha) and retinoid X (RXRalpha) receptors. We report here that TIF1alpha is both a phosphoprotein and a protein kinase. TIF1alpha coexpressed in COS-1 cells with RXRalpha or ERalpha is phosphorylated and becomes hyperphosphorylated upon ligand treatment. This hyperphosphorylation requires the binding of TIF1alpha to transcriptionally active NRs since it is prevented by mutations either in the core (alpha-helix 12 of the ligand-binding domain) of the AF-2 activating domains of RXRalpha and ERalpha or in the NR box of TIF1alpha that are known to prevent TIF1alpha-NR interactions. Thus, TIF1alpha is a phosphoprotein that undergoes ligand-dependent hyperphosphorylation as a consequence of nuclear receptor binding. We further show that purified recombinant TIF1alpha possesses intrinsic kinase activity and that, in addition to autophosphorylation, TIF1alpha selectively phosphorylates the transcription factors TFIIEalpha, TAFII28, and TAFII55 in vitro. These latter results raise the possibility that TIF1alpha may act, at least in part, by phosphorylating and modifying the activity of components of the transcriptional machinery.

A possible involvement of TIF1 alpha and TIF1 beta in the epigenetic control of transcription by nuclear receptors.

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.

Transcriptional repression by RING finger protein TIF1 beta that interacts with the KRAB repressor domain of KOX1.

Many of the vertebrate zinc finger factors of the Kruppel type (C2H2 zinc fingers) contain in their N-terminus a conserved sequence referred to as the KRAB (Kruppel-associated box) domain that, when tethered to DNA, efficiently represses transcription. Using the yeast two-hybrid system, we have isolated an 835 amino acid RING finger (C3HC4 zinc finger) protein, TIF1 beta (also named KAP-1), that specifically interacts with the KRAB domain of the human zinc finger factor KOX1/ZNF10. TIF1 beta, TIF1 alpha, PML and efp belong to a characteristic subgroup of RING finger proteins that contain one or two other Cys/His-rich clusters (B boxes) and a putative coiled-coil in addition to the classical C3HC4 RING finger motif (RBCC configuration). Like TIF1 alpha, TIF1 beta also contains an additional Cys/His cluster (PHD finger) and a bromo-related domain. When tethered to DNA, TIF1 beta can repress transcription in transiently transfected mammalian cells both from promoter-proximal and remote (enhancer) positions, similarly to the KRAB domain itself. We propose that TIF1 beta is a mediator of the transcriptional repression exerted by the KRAB domain.

Ligand-dependent interaction of nuclear receptors with potential transcriptional intermediary factors (mediators).

The activity of the ligand-inducible activation function 2 (AF-2) contained in the ligand binding domain (LBD) of nuclear receptors (NRs) is thought to be mediated by transcriptional intermediary factors (TIFs). We have recently reported the isolation and characterization of two novel mouse proteins, designated TIF1 and mSUG1, that interact in a ligand-dependent fashion with the LBD (region E) of several NRs in vivo as well as in vitro. Remarkably, these interactions require the conserved core motif of the AF-2 activating domain (AF-2 AD) and can be blocked by AF-2 antagonists. TIF1 and mSUG1 might therefore represent TIFs/mediators for the ligand-dependent AF-2 of NRs. By comparing the interaction properties of these two putative TIFs with different NRs including the oestrogen (ER), thyroid hormone (TR), vitamin D3 (VDR), retinoic acid (RAR alpha) and retinoid X (RXR) receptors, we demonstrate that: (i) RXR alpha efficiently interacts with TIF1, but not with mSUG1, whereas TR alpha interacts much more efficiently with mSUG1 than with TIF1, and RAR alpha, VDR and ER efficiently interact with both TIF1 and mSUG1; (ii) the amphipathic alpha helix core of AF-2 AD is differentially involved in the interactions of RAR alpha with TIF1 and mSUG1; and (iii) the AF-2 AD cores of RAR alpha and ER are similarly involved in their interaction with TIF1, but not with mSUG1. Thus the interaction interfaces between the various NRs and either TIF1 or mSUG1 may vary depending on the nature of both the receptor and the putative mediator of its AF-2 function. We discuss the possible roles of TIF1 and mSUG1 as mediators of the transcriptional activity of the AF-2 of NRs.

Differential ligand-dependent interactions between the AF-2 activating domain of nuclear receptors and the putative transcriptional intermediary factors mSUG1 and TIF1.

Using a yeast two-hybrid system we report the isolation of a novel mouse protein, mSUG1, that interacts with retinoic acid receptor alpha (RAR alpha) both in yeast cells and in vitro in a ligand- and AF-2 activating domain (AF-2 AD)-dependent manner and show that it is a structural and functional homologue of the essential yeast protein SUG1. mSUG1 also efficiently interacts with other nuclear receptors, including oestrogen (ER), thyroid hormone (TR), Vitamin D3 (VDR) and retinoid X (RXR) receptors. By comparing the interaction properties of these receptors with mSUG1 and TIF1, we demonstrate that: (i) RXR alpha efficiently interacts with TIF1, but not with mSUG1, whereas TR alpha interacts much more efficiently with mSUG1 than with TIF1, and RAR alpha, VDR and ER efficiently interact with mSUG1 and TIF1; (ii) the amphipathic alpha-helix core of the AF-2 AD is differentially involved in interactions of RAR alpha with mSUG1 and TIF1; (iii) the AF-2 AD cores of RAR alpha and ER are similarly involved in their interaction with TIF1, but not with mSUG1. Thus, the interaction interfaces between the different receptors and either mSUG1 or TIF1 may vary depending on the nature of the receptor and the putative mediator of its AF-2 function. We discuss the possibility that mSUG1 and TIF1 may mediate the transcriptional activity of the AF-2 of nuclear receptors through different mechanisms.

The N-terminal part of TIF1, a putative mediator of the ligand-dependent activation function (AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein T18.

Nuclear receptors (NRs) bound to response elements mediate the effects of cognate ligands on gene expression. Their ligand-dependent activation function, AF-2, presumably acts on the basal transcription machinery through intermediary proteins/mediators. We have isolated a mouse nuclear protein, TIF1, which enhances RXR and RAR AF-2 in yeast and interacts in a ligand-dependent manner with several NRs in yeast and mammalian cells, as well as in vitro. Remarkably, these interactions require the amino acids constituting the AF-2 activating domain conserved in all active NRs. Moreover, the oestrogen receptor (ER) AF-2 antagonist hydroxytamoxifen cannot promote ER-TIF1 interaction. We propose that TIF1, which contains several conserved domains found in transcriptional regulatory proteins, is a mediator of ligand-dependent AF-2. Interestingly, the TIF1 N-terminal moiety is fused to B-raf in the mouse oncoprotein T18.

High-level production of a peroxisomal enzyme: Aspergillus flavus uricase accumulates intracellularly and is active in Saccharomyces cerevisiae.

Strains of Saccharomyces cerevisiae producing Aspergillus flavus uricase (Uox) have been constructed. An artificial promoter which combined the upstream and downstream sequences of the GAL7 and ADH2 promoters, respectively, was found to be efficient in directing the synthesis of uaZ mRNAs encoding Uox. A good proportionality between the copy number of the uaZ expression cassette and the level of Uox production was found in the range of 1-10 copies. Transformants accumulated active and soluble Uox to a level exceeding 13% of total protein, as deduced from enzymatic assays. This relative level could be improved two- to threefold by using a recipient strain in which the wild-type GAL4 gene had been deleted and which expressed a GAL4 construct placed under the control of the ADH2 promoter.