The modulation of chromatin structure in the V(H) locus during lineage commitment and gene rearrangement.

摘要

The adaptive immune system protects its host from an almost infinite number of pathogens. To mediate this function, vertebrates have evolved a unique process of site-specific DNA recombination (V(D)J recombination) that generates a collection of highly diverse antigen receptors of surprisingly precise antigen specificity. While this process is crucial to the organism, it is also potentially dangerous because mistargeted double strand breaks can compromise the integrity of the genome.; My thesis work focuses on potential mechanisms by which VH-to-DJ H recombination is regulated during B cell development at the level of chromatin structure. We find B cell specific regulation of two histone modifications that have known roles in controlling RAG accessibility at the endogenous VH locus. First, we find that histone acetylation is dynamically regulated during B cell development at the endogenous VH locus, appearing in tight association with VH gene segments prior to recombination in pro B cells. Histone H4 acetylation accompanies preferential rearrangement of the DJH proximal VH gene segments in the fetal liver and is regulated in separate chromosomal domains in bone marrow pro B cells. Finally, decreased levels of histone acetylation accompany the inhibition of VH-to-DJH recombination during the process of allelic exclusion suggesting that histone acetylation is involved in both the promotion and inhibition of VH-to-DJ H during B cell ontogeny and development.; Secondly, we find that methylation of lysine 9 on histone H3 (H3K9), a mark of inactive chromatin, is found at the VH locus in non-B cells in which VH-to-DJH recombination is inhibited. The pattern of H3K9 methylation at the VH locus is different than histone acetylation and is not regulated during the process of allelic exclusion, revealing separate regulation processes for these two histone modifications. As such, we find that the B cell specific transcription factor Pax5 is both necessary and sufficient to remove H3K9 methylation from the VH locus through the mechanism of histone exchange, a mechanism that has not been previously described at an endogenous locus. Our data suggest that one function of Pax5 is to remove this inhibitory modification thus allowing B cell specific VH-to-DJH recombination and subsequent B cell development.