Abstract
Multiple myeloma (MM) is a malignancy of plasma cells characterized by frequent chromosomal translocations of the immunoglobulin heavy chain (IgH) locus. The multiple myeloma SET domain (MMSET) gene is a recurrent chromosomal partner in the t(4;14) translocation, and MMSET levels are elevated in these patients relative to other myeloma cases and normal cells. Previously, we showed that MMSET is a histone methyltransferase with specific activity for lysine 20 on histone H4 and acts as a transcriptional repressor when tethered to a model target gene. To reveal the function of MMSET in t(4;14) MM in vivo, we identified MMSET target genes in the KMS11 t(4;14) MM cell line. Chromatin from these cells was subjected to immunoprecipitation with a polyclonal anti-MMSET antibody in biological replicate, amplified by ligation-mediated PCR and hybridized to NimbleGen 2.7kB promoter arrays, which represent 24,659 human promoters. Data analysis using the MaxFour algorithm ranked putative binding sites based upon intensities of 4 consecutive probes. The top 2000 promoters identified from each experiment were combined to yield a list of 1,412 putative MMSET target genes. This list was analyzed using the DAVID program (david.abcc.ncifcrf.gov/). Genes bound by MMSET includes those implicated in antigen processing and presentation (p<8.7×10-4), cell cycle (p<2.2×10-3), the p53 signaling pathway (p<0.03) apoptosis (p<1.6×10-6) and DNA repair (p<3.3×10-4) Among genes bound by MMSET were XBP1, IRF2, and BCL6, all important transcription factors regulating B cell development. Real-time quantitative PCR validated MMSET binding in 6/6 promoters tested so far. To investigate the role of MMSET in transcriptional regulation, we profiled gene expression in KMS11 cells using Illumina arrays to determine the expression of MMSET bound genes. Nearly 50% of genes bound by MMSET had very low levels of expression (≤100, Range on arrays 10–18,000) while only 13% of genes bound by MMSET were expressed at high levels (>1000). This supports the notion that MMSET represses target genes in vivo. Functional annotation of genes bound by MMSET and expressed at low levels showed over-representation of genes implicated in toll-like receptor signaling pathway (p<2.8×10-3), cytokine-signaling (p<3.3×10-3) and JAK2/STAT signaling (p<0.08), transmembrane receptor function (p<4×10-8), and apoptosis (p<0.01), while those bound yet expressed at high levels were implicated in oxidative phosphorylation (p<3.9×10-4) and protein synthesis (p<4.1×10-6). The effects of MMSET on gene expression were further investigated using KMS11KO cells in which the rearranged MMSET allele was ablated by homologous recombination. RNA from KMS11 and KMS11KO cells was profiled by Illumina arrays and genes showing a significant change in gene expression were determined by significance analysis of microarray (SAM) testing with 1% of false discovery rate. Among the 720 genes bound by MMSET and expressed at a level of >100 in the wild-type KMS11 cells, 35 genes were up-regulated and 20 genes were down-regulated (>1.5 fold) in the KMS11KO cell line. Among the 692 genes bound by MMSET and expressed at a level of ≤100, 9 genes were up-regulated in KMS11KO cells. The up-regulated genes (presumably bound and repressed by MMSET) were categorized in cytokine receptor (p<0.02) and JAK2/STAT signaling pathway (p<0.05), nucleosome assembly (p<6×10-4), apoptosis (p<0.01), and cell differentiation (p<0.05). Collectively these data suggest that MMSET may interfere with signal transduction, chromatin modulation and apoptosis pathways involved in the terminal differentiation of the plasma cell. Intriguingly MMSET also bound and was associated with repression of RB1 and RBL2 suggesting a role of MMSET in cell cycle control. Chromatin immunoprecipitation analysis of a MMSET bound gene (ARHGAP25) revealed that MMSET binding was correlated with increased tri-methylation of H4K20, a repression-associated chromatin mark. MMSET binding of this promoter was decreased but still detectable in the KMS11KO cells. Collectively these data suggest that MMSET binds and represses many target genes in vivo. However MMSET could still bind to genes expressed at a high level and MMSET ablation was associated with activation of some MMSET target genes, suggesting that its role in gene regulation may be complex and potentially gene-specific.
The HiNF-P/p220NPAT Cell Cycle Signaling Pathway Controls Nonhistone Target Genes
