Biomedical subjects

Alexandru Olaru

Publications and source records attributed to Alexandru Olaru.

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

Analysis of transcription factor expression during discrete stages of postnatal thymocyte differentiation.

Postnatal T lymphocyte differentiation in the thymus is a multistage process involving serial waves of lineage specification, proliferative expansion, and survival/cell death decisions. Although these are believed to originate from signals derived from various thymic stromal cells, the ultimate consequence of these signals is to induce the transcriptional changes that are definitive of each step. To help to characterize this process, high density microarrays were used to analyze transcription factor gene expression in RNA derived from progenitors at each stage of T lymphopoietic differentiation, and the results were validated by a number of appropriate methods. We find a large number of transcription factors to be expressed in developing T lymphocytes, including many with known roles in the control of differentiation, proliferation, or cell survival/death decisions in other cell types. Some of these are expressed throughout the developmental process, whereas others change substantially at specific developmental transitions. The latter are particularly interesting, because stage-specific changes make it increasingly likely that the corresponding transcription factors may be involved in stage-specific processes. Overall, the data presented here represent a large resource for gene discovery and for confirmation of results obtained through other methods.

Animals↗

PubMed2004

53BP1 is required for class switch recombination.

53BP1 participates early in the DNA damage response and is involved in cell cycle checkpoint control. Moreover, the phenotype of mice and cells deficient in 53BP1 suggests a defect in DNA repair (Ward et al., 2003b). Therefore, we asked whether or not 53BP1 would be required for the efficient repair of DNA double strand breaks. Our data indicate that homologous recombination by gene conversion does not depend on 53BP1. Moreover, 53BP1-deficient mice support normal V(D)J recombination, indicating that 53BP1 is not required for "classic" nonhomologous end joining. However, class switch recombination is severely impaired in the absence of 53BP1, suggesting that 53BP1 facilitates DNA end joining in a way that is not required or redundant for the efficient closing of RAG-induced strand breaks. These findings are similar to those observed in mice or cells deficient in the tumor suppressors ATM and H2AX, further suggesting that the functions of ATM, H2AX, and 53BP1 are closely linked.

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↗

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↗

PubMed2002

Genomic instability in mice lacking histone H2AX.

Higher order chromatin structure presents a barrier to the recognition and repair of DNA damage. Double-strand breaks (DSBs) induce histone H2AX phosphorylation, which is associated with the recruitment of repair factors to damaged DNA. To help clarify the physiological role of H2AX, we targeted H2AX in mice. Although H2AX is not essential for irradiation-induced cell-cycle checkpoints, H2AX-/- mice were radiation sensitive, growth retarded, and immune deficient, and mutant males were infertile. These pleiotropic phenotypes were associated with chromosomal instability, repair defects, and impaired recruitment of Nbs1, 53bp1, and Brca1, but not Rad51, to irradiation-induced foci. Thus, H2AX is critical for facilitating the assembly of specific DNA-repair complexes on damaged DNA.

Amino Acid Sequence↗