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Title: AID Induces Double-Strand Breaks at Immunoglobulin Switch Regions and c-MYC Causing Chromosomal Translocations in Yeast THO Mutants}
Article:
Abstract:
Transcription of the switch( S) regions of immunoglobulin genes in B cells generates stable R-loops that are targeted by Activation Induced Cytidine Deaminase( AID), triggering class switch recombination( CSR), as well as translocations with c-MYC responsible for Burkitt's lymphomas. In Saccharomyces cerevisiae, stable R-loops are formed co-transcriptionally in mutants of THO, a conserved nuclear complex involved in mRNP biogenesis. Such R-loops trigger genome instability and facilitate deamination by human AID. To understand the mechanisms that generate genome instability mediated by mRNP biogenesis impairment and by AID, we devised a yeast chromosomal system based on different segments of mammalian S regions and c-MYC for the analysis of chromosomal rearrangements in both wild-type and THO mutants. We demonstrate that AID acts in yeast at heterologous S and c-MYC transcribed sequences leading to double-strand breaks( DSBs) which in turn cause chromosomal translocations via Non-Homologous End Joining( NHEJ). AID–induced translocations were strongly enhanced in yeast THO null mutants, consistent with the idea that AID–mediated DSBs depend on R-loop formation. Our study not only provides new clues to understand the role of mRNP biogenesis in preventing genome rearrangements and the mechanism of AID-mediated genome instability, but also shows that, once uracil residues are produced by AID–mediated deamination, these are processed into DSBs and chromosomal rearrangements by the general and conserved DNA repair functions present from yeast to human cells.
Introduction:
Transcription is a source of genetic instability in eukaryotic cells( reviewed in[1]). High transcription rates can result in increased recombination and mutagenesis[2],[3], and a variety of factors have an influence on this relationship, including the collision between replication forks and the transcription machinery[4], and the occurrence of RNA–DNA hybrids( R-loops)[5],[6]. In mammalian B cells, class switch recombination( CSR) and somatic hypermutation( SHM) are strongly dependent on transcription. Transcription of the switch( S) regions generates DNA intermediates in which the C-rich template strand forms stable R-loops and the non-template G-rich strand can form secondary structures( reviewed in[7]). Activation Induced Deaminase( AID), an enzyme specifically expressed in mature B cells[8] that is essential in both CSR and SHM, appears to be associated with the transcription machinery[9]–[11], and AID-mediated mutations increase when RNAPII is stalled[12]. Current models of CSR suggest that AID triggers the process by deaminating cytosines in the non-template DNA strand displaced by the R-loop[13]–[15]. In addition, during transcription the DNA sequences upstream of the elongating RNA polymerase are negatively supercoiled, and this transient change in DNA topology may allow AID to access both DNA strands[16]. Cytosine residues deaminated by AID at S regions are converted into uracils, which are processed by base excision repair( BER) and/or mismatch repair( MMR) pathways into DSBs responsible for CSR[17]–[19]. However, the reason why in B cells uracils lead to DSBs instead of being repaired remains unclear, although a breakdown of the cellular protective error-free repair has been suggested[20]. An open question is whether B cells have specialized functions or DNA modifying enzymes not present in other eukaryotic cells to process uracils into DSBs. The same mechanism leading to CSR seems to be involved in the formation of chromosomal translocations( CTs) between the immunoglobulin S regions and c-MYC( Ig/myc) that are associated with B cell-derived Burkitt's lymphomas, also mediated by AID[21]–[23]. CTs are indeed a hallmark of cancer cells[24],[25], and are mediated by DSBs[26]. Repair of AID-mediated DSBs occurs via Non-Homologous End Joining( NHEJ)[27], but in the presence of sequence homology, CTs may also occur by homologous recombination[28]–[32]. R-loops have been shown to accumulate in yeast THO mutants. The THO complex is functionally involved in transcription elongation and mRNP biogenesis[33], and depletion of some of its components generates strong transcription impairment and hyper-recombination phenotypes, which are linked to the formation of R-loops[5]. These phenotypes depend on gene length, GC content and transcription levels[34],[35]. Importantly, THO depletion enhances AID-dependent mutations as a consequence of its ability to accumulate R-loops[36]. Taking advantage of this ability, we developed a yeast genetic system to study the mechanisms of AID-mediated chromosomal rearrangements. With this system we observed that transcription of S and c-MYC sequences in yeast generates AID-mediated DSBs that can be joined with an ectopic HO-induced DSB to yield a reciprocal CT. AID-induced CTs were strongly enhanced in yeast THO null mutants but not in hpr1-101 mutants, which do not form R-loops, consistent with the idea that AID-mediated DSBs depend on R-loop formation. CTs analyzed took place through NHEJ, confirming that the initial event was a DSB. This study not only provides new clues to understanding the role of mRNP biogenesis in preventing genome rearrangements and the mechanism of AID-mediated genome instability, but it shows that yeast cells, as mammalian B cells, have the factors and pathways required to channel AID-mediated DNA lesions into DSBs responsible for NHEJ-dependent genome rearrangements and CSR.
Discussion:
We show here that AID induces DSBs in the immunoglobulin S regions and in the c-MYC sequences in yeast THO mutants, which are impaired in mRNP biogenesis. Our data suggest that AID-mediated deamination of cytosines at S sequences in yeast are channeled by the Ung1 activity into DSBs as occurs in mammalian B cells, which are then repaired by NHEJ. The c-MYC oncogene, which shares features of S sequences, also acts as a hotspot of recombination in yeast THO mutants. Using a novel translocation assay we show that translocations involving S or c-MYC sequences occur efficiently in yeast THO mutants, but poorly in wild-type cells. This study demonstrates that the mechanisms of AID-mediated CSR and AID-dependent translocations are mediated by standard DNA repair functions existing in yeast and human cells. The only requirement for these events to occur is the formation of the appropriate substrates for AID action, as they are the R-loops known to be accumulated in THO null mutants. It is widely accepted that transcription of S regions plays a primary role in CSR in B cells by generating R-loops that are substrates for efficient AID action[7]. However, once cytosines at the single-stranded DNA sequence of the R-loop are converted into uracils by AID deamination, it is unclear whether B cells have specific DNA modification enzymes or pathways to channel such uracils into DSBs responsible for CSR or Ig/myc translocations instead of being efficiently repaired by either BER or MMR, although a breakdown of the protective accurate repair pathways during the process of B cell maturation has been suggested[20]. Our yeast assay for the detection of translocations generated by the NHEJ-mediated processing of DSBs, reveals that yeast cells also have the capability of processing AID-mediated lesions into DSBs. We found that transcription through mammalian Sµ regions in yeast generates AID-mediated DSBs that can lead to CTs, similarly to B cells. The breakpoints of these translocations are located at the 5' end of Sµ sequences, where most CSR junctions occur in vivo[45], and show short nucleotide microhomologies, consistent with data from mammalian B cells[46]. Notably, AID-dependent CTs in our assay occur at a low frequency in wild-type cells, but are synergistically enhanced in yeast THO null mutants, which are affected in mRNP biogenesis. This is in agreement with previous results showing the mutagenic and recombinogenic phenotype of yeast overexpressing AID[47], and that the transcription impairment in yeast THO null mutants enhances AID-dependent mutation of the non-template DNA strand, consistent with its exposure as a single stranded DNA during the transcription elongation in the form of R-loops[36]. These results, and the fact that AID-dependent CTs are poorly observed in hpr1-101 mutants, which do not form R-loops[41], suggest that the formation of a suboptimal structure of the ribonucleoparticle around the S region RNA fragment may be a major contributor to the formation and/or stabilization of a R-loop as an optimal substrate for AID action. Therefore, our data open the possibility that mRNP biogenesis may have an impact on the generation of genetic variability in mammalian B cells by controlling the formation of R-loops as an efficient substrate for the action of AID. It has been reported that CSR is orientation-dependent and that inversion of the S sequences decreases the efficiency of CSR[38],[45], as it happens with the formation of sequence-dependent secondary structures in DNA during transcription in vitro[39]. We found in our assays a lesser, although substantial, recombinogenic effect of the Sµ sequence in the reversal orientation, which suggests that in our yeast system the presence of G-rich non-transcribed sequences is not an unique requisite for recombinogenicity. The size of the S region seems important for the recombination hotspot activity in B cells, as longer S regions could generate more extensive R-loops[48]. A slight increase of the size of the Sµ sequence in our yeast assay did not enhance the translocation frequency, because it was still below a minimum size. Notably, the long GC-rich c-MYC oncogene, used in this study as a structural equivalent of the longer S region, was a hotspot for recombination between direct-repeats and NHEJ-mediated CTs in wild-type yeast in our assays. Genetic instability in c-MYC and other oncogenes has been correlated with the GC-richness[44],[49], and the identification of c-MYC as a target of AID[23] also suggests that c-MYC shares structural features with S regions that make them highly recombinogenic during transcription in B cells. Interestingly, the recombinogenic potential generated by the transcription of the c-MYC sequence in our assays in hpr1Δ cells was also observed in the absence of AID expression. This is explained by the direct effect of hpr1Δ mutation observed in stability of long and GC-rich sequences such as c-MYC, as previously shown for bacterial DNA sequences[40], and by the lower density of AID-recognition motifs along this sequence. Altogether, our results confirm that c-MYC behaves as a natural hotspot of recombination, but this is more pronounced in THO depleted cells. The results suggest that the lower efficiency of mRNP biogenesis caused by THO mutation predispose c-MYC to breakage, leading to translocations. The similarity of phenotypes observed for mutations in the THSC/TREX-2 complex[50], which is located at the nuclear periphery in close contact with the nuclear pore complex and also causes transcription and RNA-dependent hyper-recombination, suggests that AID action would enhance DSB formation at S and c-MYC sequences provoking NHEJ-mediated translocations also in other mRNP biogenesis mutants, THO mutants being a paradigmatic example. In summary, our work points to the idea that the structure of the nascent mRNA molecule may be a major determinant in the formation of R-loops at the S regions in B cells in vivo. Accordingly, previous data demonstrate that the RNA transcript formed during transcription of S sequences in vitro can thread back onto the template DNA strand, leading to an R-loop[51],[52]. Our model yeast system provides a validated assay to study the molecular mechanisms mediated by AID leading to DSBs at S regions and the c-MYC oncogene, such as those occurring in B cells responsible for class switching or the Ig/myc translocations that are associated with Burkitt's lymphomas, thus opening new perspectives in understanding AID-mediated rearrangements.
Mammalian B cells have developed complex processes to create genetic diversity from DNA double-strand breaks ( DSBs ) . These must be tightly controlled to avoid harmful chromosomal translocations . Here we report an experimental yeast assay to analyze how the B-cell specific Activation Induced Deaminase ( AID ) induces transcription-dependent DSBs in mammalian DNA sequences . Our data suggest that in yeast AID is able to mediate deamination of cytosines in transcribed DNA that are then channeled into DSBs as it occurs in mammalian B cells , leading finally to reciprocal chromosomal translocations . These events are strongly enhanced in THO yeast mutants , which indicates that the impairment of the mRNP biogenesis may generate the appropriate substrates for AID action . Our study demonstrates that the AID–dependent genomic instability mechanisms are mediated by standard DNA repair functions existing from yeast to human cells . The only requirement for these events to occur is the formation of the appropriate substrates for AID action , as they are the transcription-mediated RNA–DNA hybrids known to be accumulated in THO null mutants . This experimental model provides a useful tool for the study of the sequences and the mechanisms leading to genomic instability that are primarily caused by chromosomal rearrangements .