Chromosomal translocations are the key inception point of many cancers and yet the molecular mechanism of translocations in humans is unclear. We have a unique database of 1,800 neoplastic chromosomal translocations from cancer patients for which the translocation junctions have been sequenced. We exploit these data from human B-cell lymphomas to define what is distinctive about the DNA regions where these patient translocations occurred. We have identified 7 well-defined DNA fragile zones that account for the majority of human B cell lymphomas. Although the translocations can potentially span 10-100 kb regions near oncogenes, the fragile zones we have identified are only 25-600 bp in length and are 100-1000-fold more susceptible to DNA breakage than nearby DNA. Determining the molecular and structural basis of these fragile zones is the major focus of this proposal. We have determined that all of the CG and WGCW sites within these fragile zones are sites of action of AID (activation-induced deaminase) which generates G:U base-pair mismatches by converting either C to U or methyl-C to T. The resulting base-pair mismatches can then be converted to double-strand DNA breaks (DSBs). None of the 7 human fragile zones we have identified are located in or near promoters, which is where translocations occur in murine models. Importantly, AID requires single-stranded DNA (ssDNA) as a substrate. Thus, one of our major goals is to determine what causes these 25-600 bp fragile zones to achieve a ssDNA state that leads to DSBs. All of the translocations studied in this proposal occurred during human pre-B cell differentiation. We have three sources of human pre-B cells for our analyses and two of these are primary cells.
Aim 1 uses four parallel approaches to define the ssDNA character of the 7 fragile zones relative to nearby DNA.
In Aim 1 A & B, we identify regions of ssDNA using bisulfite and permanganate chemical probing. We will determine how well the location, length and degree of ssDNA character is correlated among the 7 fragile zones and what sequences and features (DNA repeats, protein binding motifs, DNA structural motifs) are in common among the 7 fragile zones.
In Aim 1 C we map all noncoding RNAs in human pre-B cells to determine whether noncoding RNAs are initiated at the boundaries of the 7 fragile zones to create topological tension.
In Aim 1 D, we test for altered DNA structures called R-loops in the fragile zones (Yu & Lieber, 2003).
For Aim 2, we had already shown that the zones are fragile when moved to other nuclear locations, even in non-lymphoid human cells. New preliminary data in Aim 2 demonstrates that these zones are fragile in an extremely sensitive and quantitative genetic assay in S. cerevisiae. We show that the bcl-2 MBR is fragile only when transcription occurs through it and in a topologically-dependent manner.
Aim 2 A-C tests the other 6 fragile zones for transcription- and topologically-dependent fragility, and the effect of replication origin proximity.
Aim 2 D-E mutagenizes the bcl-2 MBR fragile zone to determine the minimal features and its sensitivity to activated Artemis. These studies are broadly relevant to fragile zones in all cells and tumors.
Chromosomal translocations are the key starting point for many human cancers and particularly for the large majority of lymphomas in humans, and we are one of the few laboratories in the world that studies the locations in the human genome where the DNA breaks occur during these chromosomal translocations in patients. We have a unique database containing the precise locations of over 1800 patient translocations, and based on these data, we have identified 7 fragile zones that account for the majority of the translocations in these patients and most human lymphomas. Our aim is to understand the mechanism of DNA breakage during these translocations so that, in the future, we may design and test therapeutic treatments to prevent the breakage and thereby reduce the occurrence of human lymphoma.
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