Browsing by Subject "V(D)J recombination"

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Open Access
Mechanistic and Genetic Biases in Human Immunoglobulin Heavy Chain Development
(2008-04-23) Volpe, Joseph M
Broadly neutralizing antibodies against HIV are rare; most patients never develop them at detectable levels. The discovery of four such antibodies therefore warrants research into their origins and their presumed unique characteristics. Such studies, however, require baseline knowledge about commonalities and biases affecting human immunoglobulin development. Obtaining that knowledge requires large sets of gene sequence data and the appropriate statistical techniques and tools. The Genbank repository provides a free and easily accessible source for such data. Several large datasets cumulatively comprising over 10,000 human Ig heavy chain genes were identified, downloaded, and carefully filtered. We then developed a special software tool called SoDA, which employs a unique dynamic programming algorithm to provide a statistical reconstruction of the events that led to a given antigen receptor gene. Once developed, tested, and peer-reviewed, we used SoDA to provide initial data about each downloaded gene with respect to gene segment usage, n-nucleotide addition, CDR3 length, and mutation frequency, thereby establishing the most precise estimates currently available for human Ig heavy chain gene segment usage frequencies. We compared data from productive non-autoreactive Ig to non-productive Ig and found evidence for gene segment usage biases, D/J segment pairing preferences resulting from multiple sequential D-to-J recombination events, and biases in TdT action between the V-D and D-J. Further analysis of autoreactive Ig genes yielded evidence that n-nucleotide addition comes at a cost: the higher the ratio of n-nucleotides to germline-encoded nucleotides for a given CDR3 length, the greater the probability of autoreactivity. These results suggest that the germline gene segments have been selected for lack of autoreactivity. It has previously been shown that human Ig gene segments have evolved efficient evolvability under somatic hypermutation. We have now extended these results, showing that Ig gene sequences are "tuned" to preferentially produce consequential mutations in the antigen-binding domains, and synonymous mutations in the framework regions. Together, these analyses provide new insights into the genetic and mechanistic biases shaping the human Ig repertoire.
Open Access
The Role of Epigenetics in Regulating V(D)J Recombination and Allelic Exclusion
(2011) Kondilis-Mangum, Hrisavgi Demetrios
As members of the adaptive immune response, T- and B- cells express unique antigen receptors generated from antigen receptor loci. These loci encode multiple Variable (V), Diversity (D), and Joining (J) gene segments. Through a process known as V(D)J recombination, genomic rearrangements occur to generate a unique antigen receptor proteins. During each stage of lymphocyte development, antigen receptor loci are epigenetically regulated. The epigenetic regulation promotes and inhibits V(D)J recombination through different mechanisms. To generate an antigen receptor protein, the substrates for rearrangement (recombination signal sequences, RSSs) must be made accessible to the recombination machinery. Moreover, once an antigen receptor locus has rearranged and produced a successful in-frame protein, a mechanism known as allelic exclusion prevents further recombination.
The nucleosome can positively and negatively regulate V(D)J recombination. Therefore, we defined the in vivo nucleosome organization of accessible and inaccessible RSSs on the Tcr loci. We used Tcrb and Tcra alleles which lack various cis-elements (e.g. enhancers and promoters) and terminate transcription. By comparing nucleosome organization and histone octamer occupancy, we found that accessible alleles are characterized by lower histone octamer occupancy and in some cases movement of nucleosomes. Also, we found that some these changes are mediated by transcription through the RSS. We concluded that one mechanism by which cis-elements epigenetically regulate RSS accessibility is by histone octamer loss and nucleosome repositioning and that some of these changes are mediated by transcription.
In addition, we further investigated how allelic exclusion prevents Tcrb locus recombination in CD4, CD8 double positive (DP) thymocytes. A previous study had introduced the Tcra enhancer (Eα) into the middle of the Tcrb locus to test if allelic exclusion was mediated solely by RSS accessibility. That study found that Eα could force RSS accessibility in DP thymocytes, but Vβ RSS accessibility did not overcome additional mechanisms involved in allelic exclusion. One potential mechanism that has been suggested in the literature is changes in locus conformation. Thus, we tested if RSS accessibility and locus conformation together mediate allelic exclusion. We generated two alleles that overcome changes in RSS accessibility, due to the presence of Eα and that overcome changes in locus conformation, due to a decrease in distance between Vβ and DJβ RSSs. We found that both alleles are accessible in DP thymocytes and we detected Vβ to DJβ recombination in DP thymocytes. Therefore, the epigenetic mechanisms that regulate Tcrb allelic exclusion consists of changes in RSS accessibility and changes in locus conformation.
Open Access
The Role of Tcrb Subnuclear Positioning in V(D)J Recombination
(2014) Chan, Elizabeth Ann Wilcox
T cells and B cells each express unique antigen receptors used to identify, eliminate, and remember pathogens. These receptors are generated through a process known as V(D)J recombination, in which T cell receptor and B cell receptor gene loci undergo genomic recombination. Interestingly, recombination at certain genes is regulated so that a single in-frame rearrangement is present on only one allele per cell. This phenomenon, termed allelic exclusion, requires two steps. First, recombination can occur only on one allele at a time. In the second step, additional recombination must be prevented. Though the mechanism of the second step is well-understood, the first step remains poorly understood.
The first step of recombination necessitates that alleles rearrange one at a time. This could be achieved either through inefficient recombination or by halting further recombination in the presence of recombination. To separate these mechanisms, we analyzed recombination in nuclei unable to complete recombination. We found that rearrangement events accumulated at antigen receptor loci, suggesting that the presence of recombination does not stop additional rearrangements and asynchronous recombination likely results from inefficient recombination at both alleles.
Association with repressive subnuclear compartments has been proposed to reduce the recombination efficiency of allelically excluded antigen receptor loci. Of the alleleically excluded loci, Tcrb alleles are uniquely regulated during development. Other allelically excluded alleles are positioned at the transcriptionally-repressive nuclear periphery prior to recombination, and relocate to the nuclear interior at the stage in which they recombine. However Tcrb alleles remain highly associated with the nuclear periphery during rearrangement. Here we provide evidence that this peripheral subnuclear positioning of Tcrb alleles does suppress recombination. We go on to suggest that peripheral localization mediates the first step of allelic exclusion.
In search of the mechanism by which recombination is suppressed on peripheral Tcrb alleles, we investigated the subnuclear localization of a recombinase protein. Two recombinase proteins are required for recombination, one of which is recruited to actively transcribing (and more centrally located) DNA. Here we demonstrate that one recombinase protein is unable to localize to peripheral Tcrb alleles, potentially serving as the mechanism by which recombination is suppressed on peripheral alleles.