Publication Date:
2007-11-16
Description:
The t(11;17)(q23;q21) translocation involves the production of reciprocal fusion proteins PLZF-RARα and RARα-PLZF, which mediate malignant transformation by binding to and dysregulating RARα/RXR and PLZF target genes, respectively. In order to investigate the molecular basis for PLZF-RARα induced leukemogenesis, we used a gain of function model in which PLZF-RARα was ectopically expressed in U937 leukemia cells. After demonstrating in our system that PLZF-RARα is capable of inducing a G1 cell cycle arrest and inhibiting cell growth and myeloid differentiation, we sought to identify genes directly bound and transcriptionally regulated by PLZF-RARα. Chromatin from U937PLZF-RARα expressing cells (+10nM RA) was immunoprecipitated using PLZF antibodies, amplified by ligation-mediated PCR and biological triplicates were hybridized to NimbleGen 2.7kB promoter arrays, which represent 24,275 human promoters. We identified 1797 genes that are directly bound by PLZF-RARα in at least 2 out of 3 arrays, and the majority of these genes (89%) are also bound in the absence of exogenously added RA. Quantitative real time PCR using primary ChIP samples was used to validate ChIP-on-CHIP results and all genes tested to date (n=11) were confirmed as direct targets of PLZF-RARα. Ontological analyses of genes identified by ChIP-on-CHIP revealed enrichment for genes involved in myeloid cell functions including immune, inflammatory and defense responses, in addition to genes involved in apoptosis and signal transduction pathways. Furthermore, genes encoding nuclear proteins were also highly enriched and these included previously identified RARα/RXR target genes (ie. CEBPε, RARβ2, PRAM1, NFE-2), which are likely targeted by the PLZF-RARα oncoprotein, as well as novel PLZF-RARα targets, many of which have roles in blood cell development and have been implicated in leukemia (ie. RUNX1, MLL2, MCL1, PIM1, FANCB). Of these 1797 genes, a significant percentage (22%) are also transcriptionally regulated by PLZF-RARα (〉1.5 fold, p2 fold, p
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
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