Gene Transcriptional Activity Analysis in the 6q Region in Waldenstrom Macroglobulinemia (WM) Using ChIP-on-Chip Technology

Background. Multi-level genetic characterization of Waldenstrom’s Macroglobulinemia (WM) is required to improve our understanding of the underlying molecular changes that lead to the initiation and progression of this rare disease. Cytogenetic and molecular studies using gene expression analysis at the mRNA level have demonstrated minimal changes in WM cells. The most commonly identified abnormality in WM is the deletion of the 6q arm. Karyotype studies showed this abnormality in 16% of WM patients, but recent studies have shown that this deletion is present in >50% of patients using cIgMFISH. We therefore sought to investigate molecular changes that occur at the level of the “transcriptome”, indicating genes that have been actively transcribed by PolIymerase II (Poll II) enzyme using ChIP on chip technology. This technique offers the advantage of discovery of genes deregulated in WM with greater confidence than when cDNA (from RNA) is hybridized to tiling arrays.

Methods. Experiments were performed using genomewide location analysis with ChIP-on-Chip TranscriptionPath technology by Genpathway (San Diego, CA). TranscriptionPath is a novel assay that identifies and quantifies genomic DNA sequences undergoing active transcription within cells. We studied CD19+ selected WM samples (N=7) and CD19+ control cells obtained from normal bone marrows (N=2). The chromatin was immunoprecipitated using an antibody specific for pol II. DNA was labeled using the Affymetrix GeneChip^® WT Double-Stranded DNA Terminal Labeling Kit and hybridized to the human promoter tiling arrays. Raw data from the scans were analyzed using Tiling array analysis algorithm MAT (Johnson et al., 2006) to determine the difference in Pol II binding sites/activities using a bandwidth of 300bp and p value cut off of . RefSeq mapping were performed using Galaxy (Giardine et al., 2005). Gene ontology analysis was conducted using DAVID at http://david.adcc.ncifcrf.gov/. Results: Analysis showed that most samples with WM demonstrated a higher level of transcribed genes compared to normal control. Overall, there were 9304 regions corresponding to 4975 known genes (7405 unique Genebank accession) in the WM samples that had enriched Pol II binding activities compared to normal control. We then focused on the long arm of chromosome 6 to determine changes that occur at the “transcriptome”. We did not analyze the samples for 6q deletion to avoid bias in the analysis. At p-value of level, 193 regions were identified with higher Pol II binding activities in WM patients compared with normal donor. With at least 100 bp overlap, the regions hit 144 genes (144 unique Gene Bank accession), which contained 106 unique genes. Of these, proteasome subunit beta type 1, ubiqutin-conjugating enzyme E2, mitogen activated protein kinase, cyclin C, transcription factor B1, BCL2-associated transcription factor 1, ribosomal protein 12, wilms tumor 1 associated protein (WT1), Forkhead Box O3A (FOXO), CBP/P300-interacting transactivator, Interferon gamma receptor and chemokine receptor CCR6 were among the most overtranscripted genes observed in WM compared to control. In addition, poly(a)- specific ribonuclease (parn)-like domain containing 1 (PNLDC1) showed decreased Pol II binding activity in WM compared to control.

Conclusion: TranscriptionPath ChIP on Chip/ Tiling provides the first assay for comprehensive identification of sequences undergoing active transcription across the genome in WM with high sensitivity and specificity. These included both known genes such as regulators of the proteosome/ubiqutin pathways, MAPK pathways, and Bcl-2, and previously unknown genes such as FOXO and WT1. Translocation of FOXO with the MLL gene is associated with secondary acute leukemia. Similarly, WT1 has been associated with leukemias. Interestingly, we did not identify a significant number of genes underexpressed in WM samples compared to control in the 6q. Further confirmation of the role of these genes in the regulation of tumor progression in WM is underway.