Recurrent Chromosomal Intermingling Interactions at the BCL2 Locus in T(14;18) +Ve and −Ve Cell Lines.

Introduction Chromosomes locate non-randomly within the nucleus where they physically associate via intermingling. This physical association leads to co-ordinated regulation of gene expression but also makes these regions prone to translocations. The loci involved in many of the common translocations in lymphoma co-localise together and include IGH and BCL2 which are juxtaposed by t(14,18)(q32q21) in ~90% of Follicular Lymphoma (FL). This frequency suggests the BCL2 locus may be prone to intermingling, with the major breakpoint region (MBR) representing a potential interactive hotspot as it flanks a matrix attachment region (MAR) which tethers chromatin.

Methods-Results To investigate this possibility, intermingling associations with the BCL2 locus were determined using the Chromosome Conformation Capture technique (3C) in B-lympho-blastoid (HRC57 and NcNc), t(14;18) +ve (RL, SuDHL4, SuDHL6, DoHH2) and -ve (HT, SuDHL8, SuDHL5, RAJI) B-NHL cell lines. The 3C technique allows cross-linking of associating chromosomal segments, generating a library of DNADNA hybrid molecules. Intermingling profiles were established for the following three BCL2 fragments contained within this library:

• a 10kb section containing the BCL2 promoter region including exons 1 and 2;

• a 7kb segment within intron 2 containing a highly Conserved Non-coding Sequence (CNS) (associated with gene regulation) and overlapping MAR; and

• a 6kb section containing part of exon 3 and the MBR with flanking MAR.

This selection contained 81% of the BCL2 coding sequence. The intermingling partners of these BCL2 fragments were amplified using either BCL2- “unknown DNA walking primer” or BCL2-IGH enhancer primer combinations and their identity determined by direct sequencing. Nine intermingling partners were identified from the 3C libraries of at least two cell lines. Of these, IGH enhancer–BCL2 promoter associations were identified in RL, SuDHL4 and DoHH2 and appear restricted to t(14;18)+ve cell lines. MYCBP2 (chr13q22), which functions as a transcriptional activator of MYC, associated with the CNS+MAR sequence within intron 2 and this association was detected solely in malignant cell lines. In comparison, OPCML (chr11q25), a tumour-related gene, associated with the BCL2 MBR+MAR region in all cell lines examined.

Conclusions These preliminary studies suggest that the BCL2 locus has multiple recurring intermingling partners. These may be specific to BCL2, as demonstrated for OPCML which was a consistent partner in all lines investigated, or may be restricted to cells containing the translocated allele as shown for the IGH enhancer.