Tissue specific patterns of methylated cytosine residues vary with age, can be altered by environmental factors, and are often abnormal in human disease yet the cellular consequences of DNA methylation are incompletely understood. Although the bodies of highly expressed genes are often extensively methylated in plants, the relationship between intragenic methylation and expression is less clear in mammalian cells. We performed genome-wide analyses of DNA methylation and gene expression to determine how the pattern of intragenic methylation correlates with transcription and to assess the relationship between methylation of exonic and intronic portions of the gene body. We found that dense exonic methylation is far more common than previously recognized or expected statistically, yet first exons are relatively spared compared to more downstream exons and introns. Dense methylation surrounding the transcription start site (TSS) is uncoupled from methylation within more downstream regions suggesting that there are at least two classes of intragenic methylation. Whereas methylation surrounding the TSS is tightly linked to transcriptional silencing, methylation of more downstream regions is unassociated with the magnitude of gene expression. Notably, we found that DNA methylation downstream of the TSS, in the region of the first exon, is much more tightly linked to transcriptional silencing than is methylation in the upstream promoter region. These data provide direct evidence that DNA methylation is interpreted dissimilarly in different regions of the gene body and suggest that first exon methylation blocks transcript initiation, or vice versa. Our data also show that once initiated, downstream methylation is not a significant impediment to polymerase extension. Thus, the consequences of most intragenic DNA methylation must extend beyond the modulation of transcription magnitude.Sequencing data and gene expression microarray data have been submitted to the GEO online database (accession number SRA012081.1). Supporting information including expanded methods and ten additional figures in support of the manuscript is provided.

Inactivation of the von Hippel-Lindau tumor suppressor (VHL) is an archetypical tumor-initiating event in clear cell renal carcinoma (ccRCC), leading to the activation of hypoxia-inducible transcription factors (HIFs). However, VHL mutation status in ccRCC is not correlated with clinical outcome. Here we show that during ccRCC progression, cancer cells exploit diverse epigenetic alterations to empower a branch of the VHL-HIF pathway for metastasis, and the strength of this activation is associated with poor clinical outcome. By analyzing metastatic subpopulations of VHL-deficient ccRCC cells, we discovered an epigenetically altered VHL-HIF response specific to metastatic ccRCC. Focusing on the two most prominent pro-metastatic VHL-HIF target genes, we show that liberation from PRC2-dependent repressive histone methylation (H3K27me3) activates HIF-driven CXCR4 expression in support of chemotactic cell invasion, whereas loss of DNA methylation enables HIF-driven CYTIP expression to protect cancer cells from death cytokine signals. Thus, metastasis in ccRCC is based on an epigenetically expanded output of the tumor-initiating pathway.

No abstract

We conducted an open-label phase 1 study exploring the feasibility, safety, and biologic activity of epigenetic priming with decitabine before standard induction chemotherapy in patients with less-thanfavorable risk of acute myelogenous leukemia (AML). We directly compared the clinical and DNA-hypomethylating activity of decitabine delivered at 20 mg/m 2 by either a 1-hour infusion (Arm A) or a continuous infusion (Arm B) for 3, 5, or 7 days before a single, standard induction with infusional cytarabine (100 mg/m 2 for 7 days) and daunorubicin (60 mg/m 2 ؋ 3 doses). Toxicity was similar to that of standard induction chemotherapy alone. Although we did not identify a maximum tolerated dose, there was more gastrointestinal toxicity with 7 days of decitabine priming. Decitabine induced DNA hypomethylation at all dose levels and there was a trend toward greater hypomethylation in CD34 ؉ bone marrow cells when decitabine was delivered by a short pulse (Arm A). Twenty-seven subjects (90%) responded to therapy: 17 with complete remission (57%) and 10 with partial remission (33%). Of the patients with partial remission to protocol treatment, 8 achieved remission to their next therapy, bringing the overall complete remission rate to 83%. We conclude that epigenetic priming of intensive chemotherapy can be safely delivered in an attempt to improve response rates. This trial was registered at www.clinicaltrials. gov as NCT00538876. (Blood. 2011;118(6): 1472-1480) IntroductionComplete remission (CR) is required for the cure of patients with acute myelogenous leukemia (AML). Modern treatment has improved the survival of patients with AML and favorable molecular features, but the majority of patients do not have these favorable features. Standard therapy for younger patients with suboptimalrisk AML is associated with only 19%-37% long-term survival, and 17%-38% of these patients have persistent AML after standard induction chemotherapy. 1-3 Accordingly, new treatment strategies to improve induction success rates are needed to increase survival.Aberrant DNA methylation is a common means by which tumor suppressor genes (TSGs) are inactivated during carcinogenesis. 4,5 Unlike genetic mechanisms of inactivation such as gene deletion or mutation, the epigenetic silencing of TSGs by DNA methylation is potentially reversible. This has led to broad interest by cancer biologists and oncologists in the study of DNA methylation and provides the rationale for incorporating DNA-hypomethylating agents into cancer therapy.Acquired abnormalities of DNA methylation are frequently observed in AML and are particularly prevalent among patients with adverse risk features. 6,7 Decitabine has single-agent activity in patients with poor-risk, relapsed, and resistant AML. [8][9][10][11][12] This antileukemic activity is believed to result from the epigenetic induction of differentiation and cytotoxicity, 13,14 but a link between clinical outcomes and the decitabine-induced hypomethylation of specific genomic loci has not yet been established. 10,[15]...

We explored diverse alterations contributing to liposarcomagenesis by sequencing the genome, exome, transcriptome, and cytosine methylome of a primary and recurrent dedifferentiated liposarcoma (DLPS) from distinct chemotherapy/radiotherapy-naïve patients. The liposarcoma genomes had complex structural rearrangements, but in different patterns, and with varied effects on the structure and expression of affected genes. While the point mutation rate was modest, integrative analyses and additional screening identified somatic mutations in HDAC1 in 8.3% of DLPS. Liposarcoma methylomes revealed alterations in differentiation pathway genes, including CEBPA methylation in 24% of DLPS. Treatment with demethylating agents, which restored CEBPA expression in DLPS cells, was anti-proliferative and pro-apoptotic in vitro and reduced tumor growth in vivo. Both genetic and epigenetic abnormalities established a role for small RNAs in liposarcomagenesis, typified by methylation-induced silencing of microRNA-193b in DLPS but not its well-differentiated counterpart. These findings reveal an unanticipated role for epigenetic abnormalities in DLPS tumors and suggest demethylating agents as potential therapeutics.