Summary DNA methylation was described almost a century ago. However, the rules governing its establishment and maintenance remain elusive. Here, we present data demonstrating that active transcription regulates levels of genomic methylation. We identified a novel RNA arising from the CEBPA gene locus critical in regulating the local DNA methylation profile. This RNA binds to DNMT1 and prevents CEBPA gene locus methylation. Deep sequencing of transcripts associated with DNMT1 combined with genome-scale methylation and expression profiling extended the generality of this finding to numerous gene loci. Collectively, these results delineate the nature of DNMT1-RNA interactions and suggest strategies for gene selective demethylation of therapeutic targets in disease.

Quantification of circulating DNA by real-time PCR at diagnosis can identify patients with elevated levels that are associated with disease characteristics indicating aggressive disease and poor prognosis.

The transcription factor C/EBP␣ (CCAAT/ enhancer binding protein ␣) is critical for granulopoiesis. Gene disruption in mice blocks early granulocyte differentiation and disruption of C/EBP␣ function has been implicated in human acute myeloid leukemia (AML), but no systematic structure-function analysis has been undertaken to identify the mechanisms involved in C/EBP␣-mediated granulocyte differentiation. Here we demonstrate that loss of either of 2 key regions results in disruption of C/EBP␣ granulocytic development: the amino terminus and specific residues residing on the non-DNA binding face of the basic region. Mutation of either results in loss of C/EBP␣ inhibition of E2F and down-regulation of c-Myc, but only mutation of the basic region results in loss of physical interaction with E2F. In contrast, while the amino terminal mutant retains the ability to interact with E2F, this mutant fails to bind a C/EBP␣ site efficiently, fails to activate C/EBP␣ target genes, and is also defective in inhibition of E2F activity. These results further emphasize the importance of inhibition of proliferative pathways in granulopoiesis and demonstrate that several regions of the C/EBP␣ protein are involved in this mechanism. ( IntroductionRecent studies have emphasized the importance of transcription factors in cell differentiation. One such factor is CCAAT/enhancer binding protein ␣ (C/EBP␣). C/EBP␣ is expressed in a number of different tissues, but in the hematopoietic system it is expressed in stem and myeloid progenitor cells and up-regulated with granulocytic differentiation and is not expressed in other blood lineages. [1][2][3] Consistent with this specific pattern of expression is its critical role in granulopoiesis supported by studies demonstrating that disruption of the C/EBP␣ gene in mice results in a specific loss of granulocyte development and the accumulation of early myeloid blasts in the fetal liver. 4 In addition, expression of C/EBP␣ in bipotential myeloid cells promotes granulocytic and blocks monocytic differentiation. 2,5 Finally, consistent with its role in promoting granulopoiesis, a number of studies have implicated mutation and/or loss of C/EBP␣ expression as contributing to the pathogenesis of acute myeloid leukemia (AML), a disease characterized by an early block in granulopoiesis. [6][7][8][9] A number of studies have investigated the function of the domains of C/EBP␣ in an effort to understand its mechanism of action. C/EBP␣ was the first transcription factor described to have a basic-leucine zipper (bZip) domain, a characteristic shared by several families of transcription factors. 10 The carboxyl-terminal leucine zipper forms a domain mediating homo-and heterodimerization with other members of the C/EBP family. Adjacent to the leucine zipper, in the direction of the amino terminus, is a basic region essential for DNA binding activity. Early studies also defined a number of transactivation domains, largely based on transactivation studies using nonmyeloid cells (Figure 1). 11,12 In addition, a ...

Purpose: The Epstein-Barr virus (EBV) is present in the malignant Hodgkin/Reed-Sternberg (HRS) cells of 20% to 40% cases of Hodgkin lymphoma (HL) in Western countries. We were interested in the detection and quantification of cell-free plasma EBV-DNA as an indicator of biological and clinical characteristics in EBV-associated HL.Experimental Design: EBV was detected in peripheral blood compartments (whole blood, plasma, and mononuclear cells) at diagnosis by real-time PCR for the EBNA (EB nuclear antigen) region (n ¼ 93) and in HRS cells by in situ hybridization for EBV-encoded small RNAs (EBER; n ¼ 63). These data were correlated to histological and clinical characteristics, EBV serology, circulating cell-free DNA, and interleukin (IL)-6 levels.Results: Detection of EBV-DNA in plasma had a high specificity (90%), but a relatively low sensitivity (65%) to predict for EBV association. The viral load was higher in patients with advanced stage disease, older age in the presence of B-symptoms, and international prognostic score more than 2. The presence of EBV in HRS cells and higher plasma EBV-DNA copy numbers correlated to an increased frequency of tumor-infiltrating CD68þ macrophages in lymph node biopsies. Plasma EBV-DNA load correlated to circulating cell-free DNA and IL-6 levels, and inversely correlated to lymphocyte counts and EBNA1 antibody titers.Conclusion: Although the presence of EBV-DNA in peripheral blood cannot be regarded as a surrogate marker for EBER, the plasma EBV-DNA load at HL diagnosis is an indicator of disease activity and biological characteristics associated with negative prognosis. Moreover, the inverse correlation to EBNA1 antibody titers and lymphocyte counts may indicate a reduction in immunosurveillance, favoring the expansion of EBV-HRS cells in HL. Clin Cancer Res; 17(9); 2885-92. Ó2011 AACR.