Albert G. Tsai

Human Chromosomal Translocations at CpG Sites and a Theoretical Basis for Their Lineage and Stage Specificity

We have assembled, annotated, and analyzed a database of over 1700 breakpoints from the most common chromosomal rearrangements in human leukemias and lymphomas. Using this database, we show that although the CpG dinucleotide constitutes only 1% of the human genome, it accounts for 40%-70% of breakpoints at pro-B/pre-B stage translocation regions-specifically, those near the bcl-2, bcl-1, and E2A genes. We do not observe CpG hotspots in rearrangements involving lymphoid-myeloid progenitors, mature B cells, or T cells. The stage specificity, lineage specificity, CpG targeting, and unique breakpoint distributions at these cluster regions may be explained by a lesion-specific double-strand breakage mechanism involving the RAG complex acting at AID-deaminated methyl-CpGs.

Single-stranded DNA ligation and XLF-stimulated incompatible DNA end ligation by the XRCC4-DNA ligase IV complex: influence of terminal DNA sequence

The double-strand DNA break repair pathway, non-homologous DNA end joining (NHEJ), is distinctive for the flexibility of its nuclease, polymerase and ligase activities. Here we find that the joining of ends by XRCC4-ligase IV is markedly influenced by the terminal sequence, and a steric hindrance model can account for this. XLF (Cernunnos) stimulates the joining of both incompatible DNA ends and compatible DNA ends at physiologic concentrations of Mg2+, but only of incompatible DNA ends at higher concentrations of Mg2+, suggesting charge neutralization between the two DNA ends within the ligase complex. XRCC4-DNA ligase IV has the distinctive ability to ligate poly-dT single-stranded DNA and long dT overhangs in a Ku- and XLF-independent manner, but not other homopolymeric DNA. The dT preference of the ligase is interesting given the sequence bias of the NHEJ polymerase. These distinctive properties of the XRCC4-DNA ligase IV complex explain important aspects of its in vivo roles.

H3K4me3 Stimulates the V(D)J RAG Complex for Both Nicking and Hairpinning in Trans in Addition to Tethering in Cis: Implications for Translocations

The PHD finger of the RAG2 polypeptide of the RAG1/RAG2 complex binds to the histone H3 modification, trimethylated lysine 4 (H3K4me3), and in some manner increases V(D)J recombination. In the absence of biochemical studies of H3K4me3 on purified RAG enzyme activity, the precise role of H3K4me3 remains unclear. Here, we find that H3K4me3 stimulates purified RAG enzymatic activity at both the nicking (2- to 5-fold) and hairpinning (3- to 11-fold) steps of V(D)J recombination. Remarkably, this stimulation can be achieved with free H3K4me3 peptide (in trans), indicating that H3K4me3 functions via two distinct mechanisms. It not only tethers the RAG enzyme complex to a region of DNA, but it also induces a substantial increase in the catalytic turnover number (k(cat)) of the RAG complex. The H3K4me3 catalytic stimulation applies to suboptimal cryptic RSS sites located at H3K4me3 peaks that are critical in the inception of human T cell acute lymphoblastic lymphomas.

Nonhomologous DNA End Joining (NHEJ) and Chromosomal Translocations in Humans

Double-strand breaks (DSBs) arise in dividing cells about ten times per cell per day. Causes include replication across a nick, free radicals of oxidative metabolism, ionizing radiation, and inadvertent action by enzymes of DNA metabolism (such as failures of type II topoisomerases or cleavage by recombinases at off-target sites). There are two major double-strand break repair pathways. Homologous recombination (HR) can repair double-strand breaks, but only during S phase and typically only if there are hundreds of base pairs of homology. The more commonly used pathway is nonhomologous DNA end joining, abbreviated NHEJ. NHEJ can repair a DSB at any time during the cell cycle and does not require any homology, although a few nucleotides of terminal microhomology are often utilized by the NHEJ enzymes, if present. The proteins and enzymes of NHEJ include Ku, DNA-PKcs, Artemis, DNA polymerase mu (Pol micro), DNA polymerase lambda (Pol lambda), XLF (also called Cernunnos), XRCC4, and DNA ligase IV. These enzymes constitute what some call the classical NHEJ pathway, and in wild type cells, the vast majority of joining events appear to proceed using these components. NHEJ is present in many prokaryotes, as well as all eukaryotes, and very similar mechanistic flexibility evolved both convergently and divergently. When two double-strand breaks occur on different chromosomes, then the rejoining is almost always done by NHEJ. The causes of DSBs in lymphomas most often involve the RAG or AID enzymes that function in the specialized processes of antigen receptor gene rearrangement.

Mechanisms of chromosomal rearrangement in the human genome.

Many human cancers are associated with characteristic chromosomal rearrangements, especially hematopoietic cancers such as leukemias and lymphomas. The first and most critical step in the rearrangement process is the induction of two DNA double-strand breaks (DSB). In all cases, at least one of the two DSBs is generated by a pathologic process, such as (1) randomly-positioned breaks due to ionizing radiation, free radical oxidative damage, or spontaneous hydrolysis; (2) breaks associated with topoisomerase inhibitor treatment; or (3) breaks at direct or inverted repeat sequences, mediated by unidentified strand breakage mechanisms. In lymphoid cells, one of the two requisite DSBs is often physiologic, the result of V(D)J recombination or class switch recombination (CSR) at the lymphoid antigen receptor loci. The RAG complex, which causes the DSBs in V(D)J recombination, can cause (4) sequence-specific, pathologic DSBs at sites that fit the consensus of their normal V(D)J recombination signal targets; or (5) structure-specific, pathologic DSBs at regions of single- to double-strand transition. CSR occurs specifically in the B-cell lineage, and requires (6) activation-induced cytidine deaminase (AID) action at sites of single-stranded DNA, which may occur pathologically outside of the normal target loci of class switch recombination regions and somatic hypermutation (SHM) zones. Recent work proposes a seventh mechanism: the sequential action of AID and the RAG complex at CpG sites provides a coherent model for the pathologic DSBs at some of the most common sites of translocation in human lymphoma – the bcl-2 gene in follicular lymphoma and diffuse large B-cell lymphoma, and the bcl-1 gene in mantle cell lymphoma.

IgH partner breakpoint sequences provide evidence that AID initiates t(11;14) and t(8;14) chromosomal breaks in mantle cell and Burkitt lymphomas

Previous studies have implicated activation-induced cytidine deaminase (AID) in B-cell translocations but have failed to identify any association between their chromosomal breakpoints and known AID target sequences. Analysis of 56 unclustered IgH-CCND1 translocations in mantle cell lymphoma across the ~ 344-kb bcl-1 breakpoint locus demonstrates that half of the CCND1 breaks are near CpG dinucleotides. Most of these CpG breaks are at CGC motifs, and half of the remaining breaks are near WGCW, both known AID targets. These findings provide the strongest evidence to date that AID initiates chromosomal breaks in translocations that occur in human bone marrow B-cell progenitors. We also identify WGCW breaks at the MYC locus in Burkitt lymphoma translocations and murine IgH-MYC translocations, both of which arise in mature germinal center B cells. Finally, we propose a developmental model to explain the transition from CpG breaks in early human B-cell progenitors to WGCW breaks in later stage B cells.

Conformational Variants of Duplex DNA Correlated with Cytosine-rich Chromosomal Fragile Sites

We found that several major chromosomal fragile sites in human lymphomas, including the bcl-2 major breakpoint region, bcl-1 major translocation cluster, and c-Myc exon 1-intron 1 boundary, contain distinctive sequences of consecutive cytosines exhibiting a high degree of reactivity with the structure-specific chemical probe bisulfite. To assess the inherent structural variability of duplex DNA in these regions and to determine the range of structures reactive to bisulfite, we have performed bisulfite probing on genomic DNA in vitro and in situ; on duplex DNA in supercoiled and linearized plasmids; and on oligonucleotide DNA/DNA and DNA/2′-O-methyl RNA duplexes. Bisulfite is significantly more reactive at the frayed ends of DNA duplexes, which is expected given that bisulfite is an established probe of single-stranded DNA. We observed that bisulfite also distinguishes between more subtle sequence/structural differences in duplex DNA. Supercoiled plasmids are more reactive than linear DNA; and sequences containing consecutive cytosines, namely GGGCCC, are more reactive than those with alternating guanine and cytosine, namely GCGCGC. Circular dichroism and x-ray crystallography show that the GGGCCC sequence forms an intermediate B/A structure. Molecular dynamics simulations also predict an intermediate B/A structure for this sequence, and probe calculations suggest greater bisulfite accessibility of cytosine bases in the intermediate B/A structure over canonical B- or A-form DNA. Electrostatic calculations reveal that consecutive cytosine bases create electropositive patches in the major groove, predicting enhanced localization of the bisulfite anion at homo-C tracts over alternating G/C sequences. These characteristics of homo-C tracts in duplex DNA may be associated with DNA-protein interactions in vivo that predispose certain genomic regions to chromosomal fragility.

Unexpected complexity at breakpoint junctions in phenotypically normal individuals and mechanisms involved in generating balanced translocations t(1;22)(p36;q13)

Approximately one in 500 individuals carries a reciprocal translocation. Balanced translocations are usually associated with a normal phenotype unless the translocation breakpoints disrupt a gene(s) or cause a position effect. We investigated breakpoint junctions at the sequence level in phenotypically normal balanced translocation carriers. Eight breakpoint junctions derived from four nonrelated subjects with apparently balanced translocation t(1;22)(p36;q13) were examined. Additions of nucleotides, deletions, duplications, and a triplication identified at the breakpoints demonstrate high complexity at the breakpoint junctions and indicate involvement of multiple mechanisms in the DNA breakage and repair process during translocation formation. Possible detailed nonhomologous end-joining scenarios for t(1;22) cases are presented. We propose that cryptic imbalances in phenotypically normal, balanced translocation carriers may be more common than currently appreciated.

Both CpG Methylation and Activation-Induced Deaminase Are Required for the Fragility of the Human bcl-2 Major Breakpoint Region: Implications for the Timing of the Breaks in the t(14;18) Translocation

ABSTRACT The t(14;18) chromosomal translocation typically involves breakage at the bcl-2 major breakpoint region (MBR) to cause human follicular lymphoma. A theory to explain the striking propensity of the MBR breaks at three CpG clusters within the 175-bp MBR region invoked activation-induced deaminase (AID). In a test of that theory, we used here minichromosomal substrates in human pre-B cell lines. Consistent with the essential elements of the theory, we found that the MBR breakage process is indeed highly dependent on DNA methylation at the CpG sites and highly dependent on the AID enzyme to create lesions at peak locations within the MBR. Interestingly, breakage of the phosphodiester bonds at the AID-initiated MBR lesions is RAG dependent, but, unexpectedly, most are also dependent on Artemis. We found that Artemis is capable of nicking small heteroduplex structures and is even able to nick single-base mismatches. This raises the possibility that activated Artemis, derived from the unjoined D to JH DNA ends at the IgH locus on chromosome 14, nicks AID-generated TG mismatches at methyl CpG sites, and this would explain why the breaks at the chromosome 18 MBR occur within the same time window as those on chromosome 14.

BCL6 breaks occur at different AID sequence motifs in Ig–BCL6 and non-Ig–BCL6 rearrangements

BCL6 translocations are common in B-cell lymphomas and frequently have chromosomal breaks in immunoglobulin heavy chain (IgH) switch regions, suggesting that they occur during class-switch recombination. We analyze 120 BCL6 translocation breakpoints clustered in a 2156-bp segment of BCL6 intron 1, including 62 breakpoints (52%) joined to IgH, 12 (10%) joined to Ig light chains, and 46 (38%) joined to non-Ig partners. The BCL6 breaks in Ig-BCL6 translocations prefer known activation-induced cytosine deaminase (AID) hotspots such as WGCW and WRC (W = A/T, R = A/G), whereas BCL6 breaks in non-Ig rearrangements occur at CpG/CGC sites in addition to WGCW. Unlike previously identified CpG breaks in pro-B/pre-B-cell translocations, the BCL6 breaks do not show evidence of recombination activating gene or terminal deoxynucleotidyl transferase activity. Both WGCW/WRC and CpG/CGC breaks at BCL6 are most likely initiated by AID in germinal center B-cells, and their differential use suggests subtle mechanistic differences between Ig-BCL6 and non-Ig-BCL6 rearrangements.