Pathways of homologous recombination (HR) are critical for restoring or altering DNA sequence and structure after a variety of DNA damage events including nicks, and double strand breaks (DSBs). The process requires two DNA molecules, the recipient, which is the site of the DNA lesion, and the donor. HR can recreate the original DNA sequence at a DNA break or can promote a mutagenic outcome via recombination between non-allelic repeated sequences. Such mutagenic events can lead to genomic instability in the form of insertions, deletions or inversions, or, can generate loss of heterozygosity (LOH) by templating repair from a homolog rather than a sister chromatid.
Immunoglobulin (Ig) gene diversification in chicken B cells provides a powerful model for studying homologous recombination. The variable regions of chicken Ig genes, the V genes, undergo frequent and targeted sequence diversification by gene conversion (a type of HR), using an array of "homeologous" upstream pseudo-V (ψV) regions as donors. In addition to HR, chicken Ig genes can diversify by non-templated pathways involving misincorporation by error-prone DNA polymerases. We have previously demonstrated that the pseudogene donors in actively diversifying cells have features, such as histone acetylation, that are indicative of a permissive chromatin state. Using the chicken light chain gene as an experimental system, we have demonstrated that chromatin status can influence the pathway of choice for DNA repair (HR vs. nontemplated repair). For example, recruitment of the chromatin effector protein HP1 to the pseudo-V donors, by a LacI/LacO tethering system, reduced their use in HR. Instead, the initiating lesions of hypermutation resulted in nontemplated events, observed mostly as point mutations. Thus, HP1 elicits an inappropriate chromatin context for donor molecules, revealing that the regulation of chromatin is a major determinant of which DNA repair pathway is ultimately used.
Factors such as HP1, demonstrate how chromatin can serve as a barrier to HR, but we are also interested in how chromatin can activate HR since strategies for stimulating this form of repair may be potentially useful in gene therapy. We have observed that the histone H3.3 variant populates nucleosomes of the pseudogene donors. H3.3 is an activation-associated variant that is deposited in a replication-independent mechanism via the histone chaperone HIRA. Tethering HIRA to the pseudogene donors results in a more nucleosome-rich and ordered chromatin structure. Interestingly, this change is associated with greater IgM diversification, principally by virtue of an increase in short-tract gene conversion events (HR pathway repair). Similar to HIRA a tetherable version of the well-characterized transcriptional activator VP16, also stimulates Ig gene diversification creating a similar mutation spectrum. However, the two proteins differ in their effects on chromatin. HIRA leads to nucleosome enrichment, and VP16 principally acts to increase activation-associated histone modifications. Therefore, we have uncovered distinct paths of chromatin modification both that result in a stimulation of homologous recombination.
While our results are beginning to uncover the response to some specific factors, there is still much to learn about how the complex layers of chromatin regulation: histone post-translational modifications, composition of histone variants in nucleosomes, and nucleosome density at donor regions, affect homologous recombination. The LacI/LacO tethering system in the chicken Ig light chain gene has served as an exceptional experimental system allowing us to test in effects of several chromatin modifiers in a reversible way. Further research promises to unlock more of the epigenetic factors that regulate DNA repair pathways.
