Abstract
Homologous recombination is essential for maintaining genomic integrity. A common repair mechanism, it uses a homologous or homeologous donor as a template for repair of a damaged target gene. Such repair must be regulated, both to identify appropriate donors for repair, and to avoid excess or inappropriate recombination. We show that modifications of donor chromatin structure can promote homology-directed repair. These experiments demonstrate that either the activator VP16 or the histone chaperone, HIRA, accelerated gene conversion approximately 10-fold when tethered within the donor array for Ig gene conversion in the chicken B cell line DT40. VP16 greatly increased levels of acetylated histones H3 and H4, while tethered HIRA did not affect histone acetylation, but caused an increase in local nucleosome density and levels of histone H3.3. Thus, epigenetic modification can stimulate genetic variation. The evidence that distinct activating modifications can promote similar functional outcomes suggests that a variety of chromatin changes may regulate homologous recombination, and that disregulation of epigenetic marks may have deleterious genetic consequences.
Highlights
Homologous recombination depends upon a DNA donor molecule to serve as a template for correction of a damaged recipient [1,2]
Tethered VP16 accelerates gene conversion To determine the effects of chromatin structure on gene conversion, we have taken advantage of the powerful physiological model provided by the chicken B cell line, DT40
In the DT40 cell line, which derives from a B cell lymphoma, the Ig heavy (IgH) and light (Igl) chain variable (V) regions constitutively diversify by gene conversion
Summary
Homologous recombination depends upon a DNA donor molecule to serve as a template for correction of a damaged recipient [1,2]. Homologous recombination is a critical pathway for error-free restoration of broken DNA, but it can lead to mutagenesis and chromosomal rearrangements. We recently showed that local repressive modifications at donor chromatin can diminish homologous recombination [12]. This raised the possibility that activating modifications might stimulate recombination. We have tested this, by asking if gene conversion can be promoted by local recruitment to the donors of factors associated with activation of chromatin. We assayed the effects of two distinct regulators, VP16 and HIRA. HIRA is a histone chaperone capable of nucleosome assembly and deposition outside of S-phase [17,18], with a role in deposition of the histone variant H3.3 [17,19]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
