isabelle villey - Academia.edu (original) (raw)

Papers by isabelle villey

Pharmacology & Therapeutics

European Journal of Immunology, Dec 1, 1999

Immunol Lett, 1997

We have generated two in vivo mouse models to study the regulation of DNA accessibility to the V(... more We have generated two in vivo mouse models to study the regulation of DNA accessibility to the V(D)J recombinase machinery in the T cell receptor (TCR)-J alpha locus. In recombination activating gene (RAG)-deficient mice, both injection of a TCR-beta chain transgene (RTB mice) or anti-CD3-epsilon treatment in vivo (RT3 mice) lead to the same phenotype with homogeneous thymocyte populations blocked at the CD4+ CD8+ double positive (DP) stage. At this developmental stage, the TCR-alpha rearrangements are about to start, and the TCR-J alpha locus is frozen in an accessible but yet unrearranged configuration in these mice. We show high level of TCR-alpha germ-line transcription in thymocytes from RTB and RT3 mice. Transcripts are skewed towards the 5' end of the TCR-J alpha locus, and the T early alpha (TEA) sterile transcript is predominant and therefore provides a useful marker for the TCR-J alpha locus opening. Analysis of the DNA methylation status reveals a global surmethylation of the TCR-J alpha locus in the thymus in comparison with non-lymphoid cells in these mice. We propose that hypermethylation of the locus could precede a progressive demethylation, providing a specific protective and regulatory role in the rearrangement events.

International Journal of Neuropsychopharmacology, 2015

Background: Most currently available active antidepressant drugs are selective serotonin/noradren... more Background: Most currently available active antidepressant drugs are selective serotonin/noradrenaline reuptake inhibitors. However, as their clinical efficacy is not immediate, long-term administration is often accompanied by substantial side effects, and numerous patients remain non-or partial responders. We have recently found that the synthetic neurosteroid derivative 3β-methoxypregnenolone, which binds to the microtubule-associated protein-2, can provide a novel therapeutic approach in experimental model of depressive disorders in rats. To further validate the antidepressant-like efficacy of 3β-methoxypregnenolone, we investigated effects of a longer treatment (4-week oral administration; 50 mg/kg/d) in a nonrodent species, the tree shrew, exposed to psychosocial stress that elicits close-to-human alterations observed in patients with depressive disorders. Methods: During the experimental period, physiological parameters were registered, including core body temperature and electroencephalogram, while animals were videotaped to analyze their avoidance behavior. Morning urine samples were collected for measurements of cortisol and noradrenaline levels. Results: We found that treatment with 3β-methoxypregnenolone abolished stress-triggered avoidance behavior and prevented hormone hypersecretion, hypothermia, and sleep disturbances, further suggesting its antidepressant-like efficacy. Comparative treatment with fluoxetine also prevented some of the physiological alterations, while the hypersecretion of cortisol and sleep disturbances were not or partially restored by fluoxetine, suggesting a better efficacy of 3β-methoxypregnenolone. Alpha-tubulin isoforms were measured in hippocampi: we found that 3β-methoxypregnenolone reversed the specific decrease in acetylation of α-tubulin induced by psychosocial stress, while it did not modify the psychosocial stress-elicited reduction of tyrosinated α-tubulin. Conclusions: Taken together, these data strongly suggest a potent antidepressant-like effect of 3β-methoxypregnenolone on translational parameters.

The V(D)J recombination/DNA repair factor Artemis belongs to the metallo- � -lactamase ( � -Lact)... more The V(D)J recombination/DNA repair factor Artemis belongs to the metallo- � -lactamase ( � -Lact) superfamily of enzymes. Three regions can be defined within the Artemis protein sequence: (a) the � -Lact homology domain, to which is appended (b) the � -CASP region, specific of members of the � -Lact superfamily acting on nucleic acids, and (c) the COOH-terminal domain.

European journal of immunology, 2003

Enhancer alpha-dependent histone acetylation has been proposed as a molecular mechanism underlyin... more Enhancer alpha-dependent histone acetylation has been proposed as a molecular mechanism underlying the control of accessibility of recombination signal sequences along the TCRalpha locus. Here we show that chromatin acetylation along the first Jalpha segments is under the dependence of the T early alpha element (TEA), located upstream of TCRJalpha locus. The targeted deletion of TEA leads to an absence of histones H3 and H4 tail acetylation, while maintaining histone acetylation in the region spanning downstream Jalpha segments. During thymocyte maturation, TEA-dependent histone acetylation appears at immature single-positive stage, known to represent the stage of ValphaJalpha initiation. TEA-dependent histone acetylation of the most upstream Jalpha segments leads to enhanced DNA accessibility thus optimizing TCRJalpha usage and increasing Ag receptor diversity potential.

Molecular cell, 2000

During V(D)J recombination, the RAG1 and RAG2 proteins cooperate to catalyze a series of DNA bond... more During V(D)J recombination, the RAG1 and RAG2 proteins cooperate to catalyze a series of DNA bond breakage and strand transfer reactions. The structure, location, and number of active sites involved in RAG-mediated catalysis have as yet not been determined. Using protein secondary structure prediction algorithms, we have identified a region of RAG1 with possible structural similarities to the active site regions of transposases and retroviral integrases. Based on this information, we have identified two aspartic acid residues in RAG1 (D600 and D708) that function specifically in catalysis. The results support a model in which RAG1 contains a single, divalent metal ion binding active site structurally related to the active sites of transposases/integrases and responsible for all catalytic functions of the RAG protein complex.

Molecular and cellular biology, 1999

V(D)J recombination is initiated by double-strand cleavage at recombination signal sequences (RSS... more V(D)J recombination is initiated by double-strand cleavage at recombination signal sequences (RSSs). DNA cleavage is mediated by the RAG1 and RAG2 proteins. Recent experiments describing RAG protein-RSS complexes, while defining the interaction of RAG1 with the nonamer, have not assigned contacts immediately adjacent to the site of DNA cleavage to either RAG polypeptide. Here we use UV cross-linking to define sequence- and site-specific interactions between RAG1 protein and both the heptamer element of the RSS and the coding flank DNA. Hence, RAG1-DNA contacts span the site of cleavage. We also detect cross-linking of RAG2 protein to some of the same nucleotides that cross-link to RAG1, indicating that, in the binding complex, both RAG proteins are in close proximity to the site of cleavage. These results suggest how the heptamer element, the recognition surface essential for DNA cleavage, is recognized by the RAG proteins and have implications for the stoichiometry and active site ...

Nucleic Acids Research, 1999

RAG1 and RAG2 are the two lymphoid-specific proteins required for the cleavage of DNA sequences k... more RAG1 and RAG2 are the two lymphoid-specific proteins required for the cleavage of DNA sequences known as the recombination signal sequences (RSSs) flanking V, D or J regions of the antigen-binding genes. Previous studies have shown that RAG1 alone is capable of binding to the RSS, whereas RAG2 only binds as a RAG1/RAG2 complex. We have expressed recombinant core RAG1 (amino acids 384-1008) in Escherichia coli and demonstrated catalytic activity when combined with RAG2. This protein was then used to determine its oligomeric forms and the dissociation constant of binding to the RSS. Electrophoretic mobility shift assays show that up to three oligomeric complexes of core RAG1 form with a single RSS. Core RAG1 was found to exist as a dimer both when free in solution and as the minimal species bound to the RSS. Competition assays show that RAG1 recognizes both the conserved nonamer and heptamer sequences of the RSS. Zinc analysis shows the core to contain two zinc ions. The purified RAG1 protein overexpressed in E.coli exhibited the expected cleavage activity when combined with RAG2 purified from transfected 293T cells. The high mobility group protein HMG2 is stably incorporated into the recombinant RAG1/RSS complex and can increase the affinity of RAG1 for the RSS in the absence of RAG2.

The Journal of Immunology, 2003

Journal of Experimental Medicine, 2004

The V(D)J recombination/DNA repair factor Artemis belongs to the metallo-β-lactamase (β-Lact) sup... more The V(D)J recombination/DNA repair factor Artemis belongs to the metallo-β-lactamase (β-Lact) superfamily of enzymes. Three regions can be defined within the Artemis protein sequence: (a) the β-Lact homology domain, to which is appended (b) the β-CASP region, specific of members of the β-Lact superfamily acting on nucleic acids, and (c) the COOH-terminal domain. Using in vitro mutagenesis, here we show that the association of the β-Lact and the β-CASP regions suffices for in vivo V(D)J recombination of chromosome-integrated substrates. Single amino acid mutants point to critical catalytic residues for V(D)J recombination activity. The results presented here define the β-Lact/β-CASP domain of Artemis as the minimal core catalytic domain needed for V(D)J recombination and suggest that Artemis uses one or two Zn(II) ions to exert its catalytic activity, like bacterial class B β-Lact enzymes hydrolyzing β-lactam compounds.

Immunity, 1996

To address the role of the TEA germline transcription, which initiates upstream of the TCR–Jαs, i... more To address the role of the TEA germline transcription, which initiates upstream of the TCR–Jαs, in the regulation of TCR–Jα locus accessibility, we created a mouse in which this region has been removed by homologous recombination. Normal development of Tαβ cells and the expression of other TCR α germline transcripts in TEA−/− mice ruled out an exclusive role for TEA in the overall accessibility of the Jα cluster. However, the rearrangement of the most 5′ Jα (Jα61 to Jα53) was severely impaired, indicating that TEA may control the DNA accessibility of a particular Jα window. Moreover, the relative usage of every Jα segment was affected. These results are consistent with TEA acting as a “rearrangement-focusing” element, targeting the primary waves of Vα–Jα recombination to the most 5′ Jαs in an ongoing TCR–Jα rearrangement model.

European Journal of Immunology, 2003

Enhancer §-dependent histone acetylation has been proposed as a molecular mechanism underlying th... more Enhancer §-dependent histone acetylation has been proposed as a molecular mechanism underlying the control of accessibility of recombination signal sequences along the TCR § locus. Here we show that chromatin acetylation along the first J § segments is under the dependence of the T early § element (TEA), located upstream of TCRJ § locus. The targeted deletion of TEA leads to an absence of histones H3 and H4 tail acetylation, while maintaining histone acetylation in the region spanning downstream J § segments. During thymocyte maturation, TEA-dependent histone acetylation appears at immature single-positive stage, known to represent the stage of V § J § initiation. TEA-dependent histone acetylation of the most upstream J § segments leads to enhanced DNA accessibility thus optimizing TCRJ § usage and increasing Ag receptor diversity potential.

European Journal of Immunology, 1999

European Journal of Immunology, 2001

Both TCRA alleles are rearranged in mature T lymphocytes, as a result of a lack of allelic exclus... more Both TCRA alleles are rearranged in mature T lymphocytes, as a result of a lack of allelic exclusion at the TRCA locus. We show in a series of T cell clones that the two TCRJA segments are not randomly, but rather coincidentally, rearranged in a given T cell. The TCRJA coincidence relies, in part, on the presence of "T early alpha" (TEA), a cis-regulatory genetic element located upstream of the TCRJA cluster. TEA promotes specific recombinational accessibility that targets primary TCRVAJA rearrangements on the 5' side of the TCRA locus. In a model of multiple waves of TCRVAJA recombination, this cis-regulatory effect of TEA allows for the scanning of the entire TCRJA cluster, thereby increasing the TCR § / g diversity potential.