Biomedical subjects

Ferenc Livák

Publications and source records attributed to Ferenc Livák.

7 recordsLinked to original sources

PubMed2005

Identification of a ubiquitously active promoter of the murine activation-induced cytidine deaminase (AICDA) gene.

Somatic hypermutation and class switch recombination of immunoglobulin genes are dependent on the presence of the activation-induced cytidine deaminase (AICDA) enzyme. AICDA expression is restricted to activated B-lymphocytes in the germinal centers. It has been suggested that inappropriate expression of AICDA may lead to genome instability and aberrant affinity maturation of putative autoreactive antibodies. To better understand the molecular control of its tightly regulated expression we have identified the transcription initiation site and an upstream, conserved promoter region of the murine AICDA gene. The promoter lacks a consensus TATA box but contains an initiator (Inr) element and is active in several murine and human cell lines irrespective of endogenous AICDA expression. Mutagenesis analysis identified a functionally important Sp-binding site which binds both Sp1 and Sp3 in vitro in all cell types. Contrary to a recent report, no evidence was found for direct Pax5-binding at this DNA site. We discuss the role of ubiquitous and lymphoid-specific factors in the control of AICDA gene transcription.

Animals↗

PubMed2005

Beyond the 12/23 rule of VDJ recombination independent of the Rag proteins.

The combinatorial repertoire of AgRs is established through somatic recombination of V, D, and J gene segments during lymphocyte development. Incorporation of D segments into IgH, TCRbeta, and TCRdelta chains also contributes to junctional diversification by substantially extending the length of the third CDR. The V, D, and J gene segments are flanked by recombination signals (RS) of 12- or 23-mer spacer length that direct recombination according to the 12/23 rule. D genes in the TCRbeta and TCRdelta loci are flanked by a 12RS and 23RS, and their incorporation is controlled by mechanisms "beyond the 12/23 rule." In the TCRbeta locus, selective interactions between Rag proteins and the RS flanking the V-D and D-J genes, respectively, are sufficient to enforce D gene usage. In this article, we report that in the TCRdelta locus, the Rag proteins are not the major determinant of D gene incorporation. In developing mouse and human thymocytes, the two Ddelta genes rearrange predominantly to form D-D coding joints. In contrast, when tested in ex vivo transfection assays in a nonlymphoid cell line, the flanking RS mediate deletion, rather than incorporation, of the two D genes on both exogenous recombination substrates and the endogenous locus. These results suggest that selective Rag-RS interactions are not the sole regulators of D gene segment incorporation, and additional, perhaps lymphocyte-specific, mechanisms exist that allow proper shaping of the primary AgR repertoire.

Animals↗

PubMed2004

Recombination signal sequence variations and the mechanism of patterned T-cell receptor-beta locus rearrangement.

Rearrangement of antigen receptor genes is controlled at multiple levels. One important regulation is achieved through variation in the recombination signals (RS) that flank the rearranging variable (V), diversity (D), and joining (J) gene segments. Several functional and biochemical studies have confirmed the importance of RS variations in gene rearrangement but very few molecular analyses has been reported with known, endogenous RS motifs. We have shown previously that rearrangement of the murine T-cell receptor (TCR)-B, D, and J genes follows a stereotypical pattern determined largely by the BJ genes and their flanking RS. Therefore, we studied the mechanism by which the endogenous BJ RS determine patterned gene rearrangement. We have compared the activity of three representative RS in transfection and in vitro DNA cleavage assays. Mutagenesis studies identified residues in various components of the RS and flanking coding ends that cooperate to determine the ultimate efficiency of recombination. Surprisingly, we find that changes in one component of the RS can be compensated by other elements to restore functional activity. DNA electrophoretic mobility shift assays (EMSA) show that a combinatorial effect of various higher order protein/RS complexes can, in part, control the efficiency of recombination. We propose that evolution of a patterned primary antigen receptor repertoire reflects the delicate interplay between various components of the RS and flanking coding end motifs resulting from the highly flexible interactions of the recombinase with its target DNA.

Animals↗

PubMed2004

In vitro and in vivo studies on the generation of the primary T-cell receptor repertoire.

The primary T-cell receptor repertoire is generated by somatic rearrangement of discontinuous gene segments. The shape of the combinatorial repertoire is stereotypical and, in part, evolutionarily conserved among mammals. Rearrangement is initiated by specific interactions between the recombinase and the recombination signals (RSs) that flank the gene segments. Conserved sequence variations in the RS, which modulate its interactions with the recombinase, appear to be a major factor in shaping the primary repertoire. In vitro, biochemical studies have revealed distinct steps in these complex recombinase-RS interactions that may determine the final frequency of gene segment rearrangement. These studies offer a plausible model to explain gene segment selection, but new, more physiological approaches will have to be developed to verify and refine the mechanism by which the recombinase targets the RS in its endogenous chromosomal context in vivo.

Animals↗

PubMed2003

DNA-Rag protein interactions in the control of selective D gene utilization in the TCR beta locus.

Ordered assembly of Ag receptor genes by VDJ recombination is a key determinant of successful lymphocyte differentiation and function. Control of gene rearrangement has been traditionally viewed as a result of complex reorganization of the nucleochromatin mediated by several nuclear factors. Selective recombination of the variable (V) genes to the diversity (D), but not joining (J), gene segments within the TCRbeta locus has been shown to be controlled by recombination signal (RS) sequences that flank the gene segments. Through ex vivo and in vitro recombination assays, we demonstrate that the Rag proteins can discriminate between the RS of the D and J genes and enforce selective D gene incorporation into the TCRbeta variable domain in the absence of other nuclear factors or chromatin structure. DNA binding studies indicate that discrimination is not simply caused by higher affinity binding of the Rag proteins to the isolated 12RS of the D as opposed to the J genes. Furthermore, we also demonstrate that the 12RS within the TCRbeta locus is functionally inferior to the consensus 12RS. We propose that selective gene segment usage is controlled at the level of differential assembly and/or stability of synaptic RS complexes, and that evolutionary "deterioration" of the RS motifs may have been important to allow the VDJ recombinase to exert autonomous control over gene segment use during gene rearrangement.

Antibody Diversity↗

PubMed2003

Evolutionarily conserved pattern of gene segment usage within the mammalian TCRbeta locus.

Antigen receptor gene rearrangement is mediated by interactions between the VDJ recombinase and the recombination signal sequences that flank the antigen receptor gene segments. In this report I present phylogenetic analyses that suggest a remarkable evolutionary conservation of the recombination signal sequences flanking some of the orthologous T-cell receptor-beta locus gene segments between human and mouse. Comparison of published data on the usage of the same gene segments between human and mouse indicates similar conservation in the shape of the primary T-cell receptor-beta repertoire. I propose that interactions between the recombinase and its cognate recognition sequences play a hitherto underestimated role in the formation of the specific pattern of the primary, combinatorial antigen receptor repertoire and that this pattern appears to be conserved in diverse mammalian species. Generation of a conserved pattern of the primary T-cell receptor repertoire may be critical for efficient selection of immature T lymphocytes.

Animals↗

PubMed2002

Access roads for RAG-ged terrains: control of T cell receptor gene rearrangement at multiple levels.

Antigen-specific immune response requires the generation of a diverse antigen (Ag)-receptor repertoire. The primary repertoire is generated through somatic gene rearrangement and molded by subsequent cellular selection. Constraints during gene recombination influence the ultimate shape of the repertoire. One major control mechanism of gene rearrangement, investigated for many years, is exerted through regulated chromosomal accessibility of the recombinase to the antigen receptor loci. More recent studies began to explore the role of interactions between the recombinase and its cognate recognition DNA sequences. The emerging results suggest that formation of the primary repertoire is controlled by two, partially independent factors: chromosomal accessibility and direct recombinase-DNA interactions.

Animals↗