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Title: Chromatin Structure Regulates Gene Conversion}
Article:
Abstract:
Homology-directed repair is a powerful mechanism for maintaining and altering genomic structure. We asked how chromatin structure contributes to the use of homologous sequences as donors for repair using the chicken B cell line DT40 as a model. In DT40, immunoglobulin genes undergo regulated sequence diversification by gene conversion templated by pseudogene donors. We found that the immunoglobulin Vλ pseudogene array is characterized by histone modifications associated with active chromatin. We directly demonstrated the importance of chromatin structure for gene conversion, using a regulatable experimental system in which the heterochromatin protein HP1( Drosophila melanogaster Su[var]205), expressed as a fusion to Escherichia coli lactose repressor, is tethered to polymerized lactose operators integrated within the pseudo-Vλ donor array. Tethered HP1 diminished histone acetylation within the pseudo-Vλ array, and altered the outcome of Vλ diversification, so that nontemplated mutations rather than templated mutations predominated. Thus, chromatin structure regulates homology-directed repair. These results suggest that histone modifications may contribute to maintaining genomic stability by preventing recombination between repetitive sequences.
Introduction:
Homologous recombination provides a pathway for restoring or altering DNA sequence and structure[1–7]. Homologous recombination can recreate the original DNA sequence at a DNA break, and predominates in S/G2 phases of cell cycle, when sister chromatids can serve as donors for faithful repair[8, 9]. Homologous recombination can also have a mutagenic outcome by promoting recombination between nonallelic repeated sequences, leading to genomic instability, or by templating repair from a homolog rather than a sister chromatid, leading to loss of heterozygosity( LOH). In a living cell, multiple pathways compete to repair the same kinds of damage. For example, double-strand breaks( DSBs) can be repaired by nonhomologous end-joining, which may be accompanied by sequence loss or translocation[5, 10, 11]. Nicks can be efficiently repaired in situ, or by short- or long-patch repair pathways that use the complementary strand as a template[12, 13]. Chromatin structure plays an important role in repair at the site of DNA damage. A critical signal for DSB repair is C-terminal phosphorylation of the variant histone H2AX by ATM and ATR, to generate γ-H2AX[14–16]. γ-H2AX is recruited to the break and extends over a large region surrounding the break site, creating a boundary of modified chromatin, and recruits the cohesin complex to sites of damage, to promote DSB repair using the sister chromatid as a template[17, 18]. γ-H2AX alerts DNA damage checkpoints, and is recognized by histone acetyltransferases and chromatin remodeling complexes[19–22]. Changes in chromatin structure also facilitate synapsis of severed DNA ends for nonhomologous end-joining[23]. Homologous recombination involves two DNA molecules, the recipient, which is the site of the DNA lesion, and the donor. Two lines of evidence suggest that donor chromatin structure may contribute to homologous recombination. At the yeast mating type locus, changes in histone acetylation occur at the donor locus that are distinct from those at or near the DNA break[24]. In human cells, transcription of a donor promotes its use in gene conversion[25]. However, the role of donor chromatin structure in regulating recombination has not been directly tested in vertebrate cells. Immunoglobulin( Ig) gene diversification in chicken B cells provides a powerful model for studying homologous recombination. Chickens have a limited number of functional heavy and light chain variable( V) regions, which undergo V( D) J recombination early in B cell development[26, 27]. The rearranged V genes then undergo sequence diversification by gene conversion, using an array of homeologous upstream pseudo-V( ψV) regions as donors( Figure 1A). The ψV regions are nonfunctional, as they lack promoters and cannot be transcribed. The mechanism of Ig gene conversion is readily studied in the DT40 cell line, which derives from a bursal lymphoma and constitutively diversifies its Ig heavy and Ig light chain( Igλ) genes by gene conversion[28–30]. DT40 also supports very high levels of homologous gene targeting, thought to reflect elevated expression or activity of factors that promote recombinational repair[31–33]. Ig gene conversion in DT40 depends upon ubiquitous and conserved factors. The substrate for repair is a nick produced by successive action of three factors. The B-cell-specific enzyme activation-induced deaminase( AID)[34–37] deaminates cytosine to uracil in transcribed and targeted genes[38–41]; uracil DNA glycosylase excises the uracil produced by AID to generate an abasic site[42–46]; and the MRE11 abasic lyase, functioning within the MRE11/RAD50/NBS1 complex, nicks at the abasic site[47, 48]. Strand transfer and new DNA synthesis are carried out by ubiquitous DNA repair factors including the RAD51 paralogs, BRCA2, FANCC, FANCD2, and polη[49–55]. Deficiencies in some of these factors, particularly the RAD51 paralogs, or targeted deletion of some or all of the ψV donors[56], impair gene conversion and can contribute to a shift in the processing of AID-initiated breaks so that templated repair is accompanied or even supplanted by nontemplated mutagenesis. ψV regions preferentially used as donors are in opposite orientation to the functional V region, suggesting that local chromosomal architecture may guide templated repair[57]. However, nothing is known about how epigenetic features of the donors affect recombination. To understand how donor chromatin structure affects gene conversion in particular, and homologous recombination more generally, we characterized and experimentally manipulated chromatin structure at the Igλ locus in the chicken B cell line DT40. We found that the ψVλ donors contain acetylated histones, consistent with an open chromatin structure. To test whether this reflects requirements of gene conversion, we tethered HP1( Drosophila melanogaster Su[var]205) to the ψVλ array in a DT40 derivative in which polymerized lactose operator( PolyLacO) has been inserted into that chromosomal region. HP1 is known to promote heterochromatic gene silencing[58–60]. Tethered HP1 caused a local transition of the donor sequences from an open to a nonpermissive state, and a switch from templated to nontemplated diversification, evident as point mutations. These observations demonstrate that permissive chromatin structure at the donor is a key regulator of gene conversion, and that nonpermissive chromatin structure can prevent homologous recombination and result in point mutagenesis. These results have implications for our understanding of homologous recombination and of the mechanisms that promote LOH, leading to tumorigenesis and nonallelic recombination between repeats. These results should also inform design of donor constructs for targeted gene therapy.
Discussion:
Gene conversion at Vλ results from priming of new DNA synthesis at the 3′ end of a break using a ψVλ region as template. Gene conversion requires synapsis between the donor and recipient DNA, as well as access to the donor by factors that carry out homology-directed repair. The elevated levels of H3 and H4 acetylation characteristic of the ψVλ array in wild-type DT40 are evidence of a relaxed chromatin structure, which would increase the accessibility of the ψVλ genes to trans-acting factors and also create a three-dimensional architecture that is favorable for sequence synapsis. HP1 tethered within the ψVλ donor array impaired gene conversion at the rearranged VλR, without affecting Vλ gene expression. Chromatin changes caused by tethered HP1 may impair gene conversion by impeding access of repair factors and the invading strand to the donor template. Tethered HP1 may also contribute to larger chromosomal architecture that affects the mechanics of DNA repair pathways, such as looping necessary to juxtapose donor and recipient sequences. The point mutations that accumulated in LacI-HP1 transfectants are typical of thwarted recombinational repair, and are characteristic of cells lacking either trans-acting factors essential for recombination[49–55] or some or all of the ψV donor array[56]. HP1 regulates chromatin structure and heterochromatic gene silencing in two ways, by partnering with a histone methyltransferase[65] and by recruiting histone deacetylases[60]. Tethered HP1 caused modification changes characteristic of a nonpermissive chromatin structure within ψVλ. Histone acetylation has been documented at actively transcribed mammalian Ig genes undergoing somatic hypermutation and class switch recombination, but whether hyperacetylation contributes to targeting of diversification has yet to be resolved[72–76]. A connection between histone acetylation and gene conversion was suggested by experiments showing that treatment of DT40 cells with the histone deacetylase inhibitor trichostatin A promotes genome-wide histone deacetylation accompanied by increased gene conversion at VλR[77]. However, the interpretation of those results is complicated by the fact that the effects of trichostatin A are genome-wide, and not specific. The DT40 PolyLacO-λR cell line permits local manipulation of chromatin structure, avoiding that complication. Moreover, we were able to demonstrate that the effects of tethering a LacI-HP1 fusion protein were largely reversed upon culture with IPTG, so an active mechanism must determine chromatin modification at ψVλ. For studies of homologous recombination, the DT40 PolyLacO-λR B cell line has the further advantage that Ig gene conversion is a physiological process within an endogenous locus, avoiding the need for a transgene reporter. The importance of chromatin structure to the outcome of homologous recombination has implications for understanding the mechanisms that normally maintain genomic stability. There are vast numbers of repetitive elements distributed throughout the vertebrate genome, and recombination between these elements can lead to genomic instability[78]. In the human genome, there are approximately one million Alu elements, and recombination between Alu elements can cause duplications leading to tumorigenesis and genetic disease[79, 80]. Histones carrying repressive modifications are enriched at repetitive elements[81]. These modifications undoubtedly maintain transcriptional repression; our results suggest they may also contribute to suppression of recombination. LOH occurs as a result of unequal mitotic recombination between homologs at allelic sites. The mechanism of LOH is of particular interest, because it contributes to loss of tumor suppressor gene function, leading to tumorigenesis[82]. Recent experiments have demonstrated an age-dependent increase in LOH in Saccharomyces cerevisiae[83] and in reporter genes in Drosophila germ cells[84], and an increase in homologous recombination in mouse pancreatic cells[85]. Mechanisms proposed to explain age-associated LOH include elevated rates of DNA damage, changes in the cell cycle distribution, and inactivation of homology-independent repair pathways with aging. Our results suggest another possibility, that relaxation of chromatin structure may accompany aging and promote a genome-wide increase in homologous recombination in aging cells. This possibility is supported by recent analysis of Drosophila[86], as well as by recent evidence that the mutant form lamin A polypeptide( product of the human LMNA gene) responsible for Hutchinson-Gilford progeria syndrome leads to a genome-wide loss of H3 methylation[87]. The finding that chromatin structure regulates homologous recombination also has practical ramifications. Considerable current effort is directed toward developing strategies that harness a cell's capacity for homology-dependent repair to promote gene therapy, by providing an intact donor gene to replace a deficient target gene[88]. Our results suggest that permissive structure at the donor will be an important design parameter in developing donor genes for therapeutic applications.
Homologous recombination promotes genetic exchange between regions containing identical or highly related sequences . This is useful in repairing damaged DNA , or in reassorting genes in meiosis , but uncontrolled homologous recombination can create genomic instability . Chromosomes are made up of a complex of DNA and protein , called chromatin . DNA within chromatin is packed tightly in order to fit the entire genome inside a cell; but chromatin structure may become relaxed to allow access to enzymes that regulate gene expression , transcribe genes into mesenger RNA , or carry out gene replication . We asked if chromatin packing regulates homologous recombination . To do this , we tethered a factor associated with compact chromatin , called HP1 , adjacent to an immunoglobulin gene locus at which homologous recombination occurs constitutively , in order to produce a diverse repertoire of antibodies . We found that the compact , repressive chromatin structure produced by HP1 prevents homologous recombination . This finding suggests that regulated changes in chromatin structure may contribute to maintaining genomic stability by preventing recombination between repetitive sequences .