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2006

A screen for cohesion mutants uncovers Ssl3, the fission yeast counterpart of the cohesin loading factor Scc4.

Author(s) : Bernard P, Drogat J, Maure J, Dheur S, Vaur S, Genier S, Javerzat J,
Journal : Curr Biol
2006
Sister-chromatid cohesion is mediated by cohesin, a ring-shape complex made of four core subunits called Scc1, Scc3, Smc1, and Smc3 in Saccharomyces cerevisiae(Rad21, Psc3, Psm1, and Psm3 in Schizosaccharomyces pombe). How cohesin ensures cohesion is unknown, although its ring shape suggests that it may tether sister DNA strands by encircling them . Cohesion establishment is a two-step process. Cohesin is loaded on chromosomes before replication and cohesion is subsequentlyestablished during S phase. In S. cerevisiae, cohesin loading requires a separate complex containing the Scc2 and Scc4 proteins. Cohesin rings fail to associate with chromatin and cohesion can not establish when Scc2 is impaired . The mechanism of loading is unknown, although some data suggest that hydrolysis of ATP bound to Smc1/3 is required . Scc2 homologs exist in fission yeast (Mis4), Drosophila, Xenopus, and human . By contrast, no homolog of Scc4 has been identified so far. We report here on the identification of fission yeast Ssl3 asa Scc4-like factor. Ssl3 is in complex with Mis4 and, as a bona fide loading factor, Ssl3 is required in G1 for cohesin binding to chromosomes but dispensable in G2 when cohesion is established. The discovery of a functional homolog of Scc4 indicates that the machinery of cohesin loading is conserved among eukaryotes.

An exon skipping-associated nonsense mutation in the dystrophin gene uncovers a complex interplay between multiple antagonistic splicing elements.

Author(s) : Disset A, Bourgeois C, Benmalek N, Claustres M, Stevenin J, Tuffery-Giraud S,
Journal : Hum Mol Genet
2006
A nonsense mutation c.4250T>A (p.Leu1417X) in the dystrophin gene of a patient with an intermediate phenotype of muscular dystrophy induces partial in-frame skipping of exon 31. On the basis of UV cross-linking assays and pull-down analysis, we present evidence that the skipping of this exon is because of the creation of an exonic splicing silencer, which acts as a highly specific bindingsite (UAGACA) for a known repressor protein, hnRNP A1. Recombinant hnRNP A1 represses exon inclusion both in vitro and in vivo upon transient transfection of C2C12 cells with Duchenne muscular dystrophy (DMD) minigenes carrying the c.4250T>A mutation. Furthermore, we identified a downstream splicing enhancer inthe central region of exon 31. This region functions as a Tra2beta-dependent exonic splicing enhancer (ESE) in vitro when inserted into a heterologous splicing reporter, and deletion of the ESE showed that incorporation of exon 31 depends on the Tra2beta-dependent enhancer both in the wild-type and mutant context. We conclude that dystrophin exon 31 contains juxtaposed sequence motifsthat collaborate to regulate exon usage. This is the first elucidation of the molecular mechanism leading to exon skipping in the dystrophin gene and allowingthe occurrence of a milder phenotype than the expected DMD phenotype. The knowledge of which cis-acting sequence within an exon is important for its definition will be essential for the alternative gene therapy approaches based on modulation of splicing to bypass DMD-causing mutations in the endogenous dystrophin gene.

Broadening of DNA replication origin usage during metazoan cell differentiation.

Author(s) : Dazy S, Gandrillon O, Hyrien O, Prioleau M,
Journal : EMBO Rep
2006
We have examined whether replication of the chicken beta-globin locus changes during differentiation of primary erythroid progenitors into erythrocytes. In undifferentiated progenitors, four principal initiation sites and a replication fork pausing region (RFP) were observed. Forty-eight hours after induction of differentiation, the principal sites were maintained, even in the activated beta(A)-globin gene, some minor sites were enhanced, three new sites appeared and the RFP disappeared. One of the activated origins showed increased histone H3 K9K14 diacetylation, but the others did not. These results demonstrate a broadening of DNA replication origin usage during differentiation of untransformed metazoan cells and indicate that histone H3 diacetylation, other histone modifications so far reported and transcription are not crucial determinants of origin selection in this system.

Characterization of the Nurr1 ligand-binding domain co-activator interaction surface.

Author(s) : Volakakis N, Malewicz M, Kadkhodai B, Perlmann T, Benoit G,
Journal : J Mol Endocrinol
2006
The recently solved crystal structure of the orphan nuclear receptor (NR) Nurr1 ligand-binding domain (LBD) showed that Nurr1 lacks a cavity for ligand binding and a canonical NR co-activator-binding site. Computer modeling of the Nurr1 LBDstructure identified a hydrophobic region on the surface of the Nurr1 LBD that was positioned on the opposite side from the classical co-activator-binding site. Site-directed mutagenesis demonstrated that this region is critical for the activity of the Nurr1 LBD. Most mutations introduced in this region reduced or abolished transcriptional activity of the Nurr1 LBD, but mutation at lysine (K577) resulted in a drastically increased activity. Moreover, the activity of the Nurr1 LBD was shown to correlate with a propensity for proteasome-dependent degradation revealing a close association between activity and Nurr1 protein turnover. These data provide novel insights into the mechanisms of transcriptionvia the Nurr1 LBD and identify an alternative co-activator-binding surface that is unique to the NR4A family of NRs.

Characterization of the phosphorylation sites of Mycobacterium tuberculosis serine/threonine protein kinases, PknA, PknD, PknE, and PknH by mass spectrometry.

Author(s) : Molle V, Zanella-Cleon I, Robin J, Mallejac S, Cozzone A, Becchi M,
Journal : Proteomics
2006
In Mycobacterium tuberculosis (Mtb), regulatory phosphorylation of proteins at serine and/or threonine residues by serine/threonine protein kinases (STPKs) is an emerging theme connected with the involvement of these enzymes in virulence mechanisms. The identification of phosphorylation sites in proteins provides a powerful tool to study signal transduction pathways and to identify the corresponding interaction networks. Detection of phosphorylated proteins as wellas assignment of the phosphorylated sites in STPKs is a major challenge in proteomics since some of these enzymes might be interesting therapeutical targets. Using different strategies to identify phosphorylated residues, we report, in the present work, MS studies of the entire intracellular regions of recombinant protein kinases PknA, PknD, PknE, and PknH from Mtb. The on-target dephosphorylation/MALDI-TOF for identification of phosphorylated peptides was used in combination with LC-ESI/MS/MS for localization of phosphorylation sites.By doing so, seven and nine phosphorylated serine and/or threonine residues wereidentified as phosphorylation sites in the recombinant intracellular regions of PknA and PknH, respectively. The same technique led also to the identification of seven phosphorylation sites in each of the two recombinant kinases, PknD and PknE.

Cytochrome c-d regulates developmental apoptosis in the Drosophila retina.

Author(s) : Mendes C, Arama E, Brown S, Scherr H, Srivastava M, Bergmann A, Steller H, Mollereau B,
Journal : EMBO Rep
2006
The role of cytochrome c (Cyt c) in caspase activation has largely been established from mammalian cell-culture studies, but much remains to be learned about its physiological relevance in situ. The role of Cyt c in invertebrates has been subject to considerable controversy. The Drosophila genome contains distinct cyt c genes: cyt c-p and cyt c-d. Loss of cyt c-p function causes embryonic lethality owing to a requirement of the gene for mitochondrial respiration. By contrast, cyt c-d mutants are viable but male sterile. Here, we show that cyt c-d regulates developmental apoptosis in the pupal eye. cyt c-d mutant retinas show a profound delay in the apoptosis of superfluous interommatidial cells and perimeter ommatidial cells. Furthermore, there is no apoptosis in mutant retinaltissues for the Drosophila homologues of apoptotic protease-activating factor 1 (Ark) and caspase 9 (Dronc). In addition, we found that cyt c-d--as with ark anddronc-regulates scutellar bristle number, which is known to depend on caspase activity. Collectively, our results indicate a role of Cyt c in caspase regulation of Drosophila somatic cells.

International Union of Pharmacology. LXVI. Orphan nuclear receptors.

Author(s) : Benoit G, Cooney A, Giguere V, Ingraham H, Lazar M, Muscat G, Perlmann T, Renaud J, Schwabe J, Sladek F, Tsai M, Laudet V,
Journal : Pharmacol Rev
2006
Half of the members of the nuclear receptors superfamily are so-called "orphan" receptors because the identity of their ligand, if any, is unknown. Because of their important biological roles, the study of orphan receptors has attracted much attention recently and has resulted in rapid advances that have helped in the discovery of novel signaling pathways. In this review we present the main features of orphan receptors, discuss the structure of their ligand-binding domains and their biological functions. The paradoxical existence of a pharmacology of orphan receptors, a rapidly growing and innovative field, is highlighted.

Modulation of HIV-1 Rev protein abundance and activity by polyubiquitination with unconventional Lys-33 branching.

Author(s) : Vitte A, Buchsbaum S, Jalinot P,
Journal : FEBS Lett
2006
The HIV-1 Rev protein plays a key role in virus replication by allowing export to the cytoplasm of unspliced or singly-spliced RNAs. In this report, we investigated whether Rev is modified by ubiquitination or sumoylation. Whereas no evidence of sumoylation was obtained, transient expression experiments showed that ubiquitin conjugates to Rev as high molecular weight polyubiquitin chains. Mutation of the three lysine residues of Rev showed that the site of ubiquitin conjugation is Lys-115. Experiments with ubiquitin mutants including a single lysine at every seven possible position indicated that branching of the polyubiquitin chains mainly involves Lys-33. Mutation of Rev Lys-115 to argininereduces markedly the steady state amount of the protein, but does not impair itsability to export RNA via the Rev response element. These observations support the notion that polyubiquitination of Rev stabilizes the viral protein but hinders its activity.

Optimal estimates of free energies from multistate nonequilibrium work data.

Author(s) : Maragakis P, Spichty M, Karplus M,
Journal : Phys Rev Lett
2006
We derive the optimal estimates of the free energies of an arbitrary number of thermodynamic states from nonequilibrium work measurements; the work data are collected from forward and reverse switching processes and obey a fluctuation theorem. The maximum likelihood formulation properly reweights all pathways contributing to a free energy difference and is directly applicable to simulations and experiments. We demonstrate dramatic gains in efficiency by combining the analysis with parallel tempering simulations for alchemical mutations of model amino acids.

Recruitment of TFIIH to the HIV LTR is a rate-limiting step in the emergence of HIV from latency.

Author(s) : Kim Y, Bourgeois C, Pearson R, Tyagi M, West M, Wong J, Wu S, Chiang C, Karn J,
Journal : EMBO J
2006
Latently infected cells rapidly initiate HIV transcription after exposure to signals that induce NF-kappaB. To investigate the role of TFIIH during HIV reactivation in vivo, we developed a population of Jurkat cells containing integrated, but transcriptionally silent, HIV proviruses. Surprisingly, the HIV promoter in unactivated Jurkat T cells is partially occupied and carries Mediator containing the CDK8 repressive module, TFIID and RNAP II that is hypophosphorylated and confined to the promoter region. Significantly, the promoter is devoid of TFIIH. Upon stimulation of the cells by TNF-alpha, NF-kappaB and TFIIH are rapidly recruited to the promoter together with additional Mediator and RNAP II, but CDK8 is lost. Detailed time courses show that the levels of TFIIH at the promoter fluctuate in parallel with NF-kappaB recruitment to the promoter. Similarly, recombinant p65 activates HIV transcription in vitro and stimulates phosphorylation of the RNAP II CTD by the CDK7 kinase module of TFIIH. We conclude that the recruitment and activation of TFIIH represents a rate-limiting step for the emergence of HIV from latency.

The C. elegans HP1 homologue HPL-2 and the LIN-13 zinc finger protein form a complex implicated in vulval development.

Author(s) : Coustham V, Bedet C, Monier K, Schott S, Karali M, Palladino F,
Journal : Dev Biol
2006
HP1 proteins are essential components of heterochromatin and contribute to the transcriptional repression of euchromatic genes via the recruitment to specific promoters by corepressor proteins including TIF1 and Rb. The Caenorhabditis elegans HP1 homologue HPL-2 acts in the "synMuv" (synthetic multivulval) pathway, which defines redundant negative regulators of a Ras signaling cascade required for vulval induction. Several synMuv genes encode for chromatin-associated proteins involved in transcriptional regulation, including Rb and components of the Mi-2/NuRD and TIP60/NuA4 chromatin remodeling complexes. Here, we show that HPL-2 physically interacts in vitro and in vivo with the multiple zinc finger protein LIN-13, another member of the synMuv pathway. A variant of the conservedPXVXL motif found in many HP1-interacting proteins mediates LIN-13 binding to the CSD of HPL-2. We further show by in vivo localization studies that LIN-13 is required for HPL-2 recruitment in nuclear foci. Our data suggest that the LIN-13/HPL-2 complex may physically link a subset of the Rb related synMuv proteins to chromatin.

Unique and redundant functions of C. elegans HP1 proteins in post-embryonic development.

Author(s) : Schott S, Coustham V, Simonet T, Bedet C, Palladino F,
Journal : Dev Biol
2006
HP1 proteins are essential components of heterochromatin and contribute to the transcriptional repression of euchromatic genes. Although most species contain more than one HP1 family member which differ in their chromosomal distribution, it is not known to what extent the activity of these different family members isredundant or specific in a developmental context. C. elegans has two HP1 homologues, HPL-1 and HPL-2. While HPL-2 functions in vulval and germline development, no function has so far been attributed to HPL-1. Here we report thecharacterization of an hpl-1 null allele. We show that while the absence of hpl-1 alone results in no obvious phenotype, hpl-1;hpl-2 double mutants show synthetic, temperature sensitive phenotypes including larval lethality and severe defects in the development of the somatic gonad. Furthermore, we find that hpl-1 has an unexpected role in vulval development by acting redundantly with hpl-2, but not other genes previously implicated in vulval development. Localization studies show that like HPL-2, HPL-1 is a ubiquitously expressed nuclear protein. However, HPL-1 and HPL-2 localization does not completely overlap. Our results show that HPL-1 and HPL-2 play both unique and redundant functions in post-embryonic development.