Abstract
The transcription factor PU.1 is a key regulator of hematopoiesis and is essential for normal hematopoietic stem cell (HSC) function and myeloid and lymphoid differentiation. Knockout mice are embryonic or neonatal lethal and exhibit an early differentiation block at the transition from HSC to CMP and CLP. Mice with a deletion of a critical upstream regulatory element (URE) at −14kb has been deleted exhibited decreased PU.1 expression in HSC, progenitors and B cells to 20% of normal levels. In contrast, an increase of PU.1 expression was detected in early thymic progenitors. UREΔ/Δ animals exhibited a block in normal thymocyte differentiation and frequently progressed to fatal T cell lymphomas between 3 and 8 months of age. Tumors were clonal and transplantable into NOD-SCID recipients. Since aberrant PU.1 expression constituted the initiating mutation, we sought to identify cooperating mutations contributing to tumor development by surveying the genome for hypermethylated promoter regions using restriction landmark global scanning. Methylation patterns revealed a characteristic epigenetic footprint leading to selective tumor suppressor gene silencing in transformation of lymphoid but not myeloid UREΔ/Δ progenitors, since DNA methylation was significantly lower in the AML samples compared with lymphomas. One of the corresponding methylated genes that was silenced in all lymphomas analyzed but none of the AMLs was identified as Idb4 (encoding ID4, inhibitor of DNA binding 4). This observation is consistent with data describing ID4 as a strong tumor suppressor gene which is frequently silenced in neoplasms. The expression of Idb4 in 6 additional lymphomas was assessed by quantitative RT-PCR and showed a strong correlation between promoter hypermethylation and Idb4 downregulation. To test whether Idb4 silencing through promoter hypermethylation can be pharmacologically reversed, a UREΔ/Δ lymphoma cell line was established and treated with 5-aza-2′deoxycytidine. We found that Idb4 expression was restored to normal levels following increasing concentrations of this demethylating agent. Finally, we analyzed the functional relevance of the Idb4 downregulation by restoring Idb4 expression in the UREΔ/Δ tumor line. UREΔ/Δ lymphoma cells reexpressing Idb4 had a marked reduction in both the in vitro growth rate and the ability to cause tumors after transplantation into NOD/SCID recipient mice, indicating that Idb4 is a potent suppressor of UREΔ/Δ thymic tumorigenicity. Taken together, we provide evidence that epigenetic DNA modifications contribute significantly and selectively to lymphoid transformation in UREΔ/Δ mice, and suggest a role for Idb4 repression as a second event in PU.1-initiated transformation of lymphoid precursors.
Candidate Tumor-Suppressor Gene DLEC1 Is Frequently Downregulated by Promoter Hypermethylation and Histone Hypoacetylation in Human Epithelial Ovarian Cancer
