We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.

Previously, identification of promoters regulated by mammalian transcription factors has relied upon overexpression studies. Here we present the identification of a large set of promoters that are bound by E2F in physiological conditions. Probing a human CpG microarray with chromatin immunoprecipitated using an antibody to E2F4, we have identified 68 unique target loci; 15% are bidirectional promoters and 25% recruit E2F via a mechanism distinct from the defined consensus site. Interestingly, although E2F has been shown previously to regulate genes involved in cell cycle progression, many of the new E2F target genes encode proteins involved in DNA repair or recombination. We suggest that human CpG microarrays, in combination with chromatin immunoprecipitation, will allow rapid identification of target promoters for many mammalian transcription factors.

Using ChIP-chip assays (employing ENCODE arrays and core promoter arrays), we examined the binding patterns of three members of the E2F family in five cell types. We determined that most E2F1, E2F4, and E2F6 binding sites are located within 2 kb of a transcription start site, in both normal and tumor cells. In fact, the majority of promoters that are active (as defined by TAF1 or POLR2A binding) in GM06990 B lymphocytes and Ntera2 carcinoma cells were also bound by an E2F. This very close relationship between E2F binding sites and binding sites for general transcription factors in both normal and tumor cells suggests that a chromatin-bound E2F may be a signpost for active transcription initiation complexes. In general, we found that several E2Fs bind to a given promoter and that there is only modest cell type specificity of the E2F family. Thus, it is difficult to assess the role of any particular E2F in transcriptional regulation, due to extreme redundancy of target promoters. However, Ntera2 carcinoma cells were exceptional in that a large set of promoters were bound by E2F6, but not by E2F1 or E2F4. It has been proposed that E2F6 contributes to gene silencing by recruiting enzymes involved in methylating histone H3. To test this hypothesis, we created Ntera2 cell lines harboring shRNAs to E2F6. We found that reduction of E2F6 only induced minimal alteration of the transcriptome of Ntera2 transcriptome. Our results support the concept of functional redundancy in the E2F family and suggest that E2F6 is not critical for histone methylation.

Treatment with dose-adjusted EPOCH (etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone) chemotherapy and rituximab (DA-EPOCH-R) has become the standard of care for primary mediastinal B-cell lymphoma (PMBCL) at many institutions despite limited data in the multi-centre setting. We report a large, multi-centre retrospective analysis of children and adults with PMBCL treated with DA-EPOCH-R to characterize outcomes and evaluate prognostic factors. We assessed 156 patients with PMBCL treated with DA-EPOCH-R across 24 academic centres, including 38 children and 118 adults. All patients received at least one cycle of DA-EPOCH-R. Radiation therapy was administered in 14·9% of patients. With median follow-up of 22·6 months, the estimated 3-year event-free survival (EFS) was 85·9% [95% confidence interval (CI) 80·3-91·5] and overall survival was 95·4% (95% CI 91·8-99·0). Outcomes were not statistically different between paediatric and adult patients. Thrombotic complications were reported in 28·2% of patients and were more common in paediatric patients (45·9% vs. 22·9%, P = 0·011). Seventy-five per cent of patients had a negative fluorodeoxyglucose positron emission tomography (FDG-PET) scan at the completion of DA-EPOCH-R, defined as Deauville score 1-3. Negative FDG-PET at end-of-therapy was associated with improved EFS (95·4% vs. 54·9%, P < 0·001). Our data support the use of DA-EPOCH-R for the treatment of PMBCL in children and adults. Patients with a positive end-of-therapy FDG-PET scan have an inferior outcome.

Obesity is associated with poorer outcome for many cancers. Previously, we observed that adipocytes protect acute lymphoblastic leukemia (ALL) cells from the anthracycline, daunorubicin (DNR). In the present study, it is determined whether adipocytes clear DNR from the tumor microenvironment (TME). Intracellular DNR concentrations were evaluated using fluorescence. DNR and its largely inactive metabolite, daunorubicinol, were analytically measured in media, cells, and tissues using liquid chromatography/mass spectrometry (LC/MS). Expression of DNR-metabolizing enzymes: aldo-keto reductases (AKR1A1, AKR1B1, AKR1C1, AKR1C2, AKR1C3, and AKR7A2) and carbonyl reductases (CBR1, CBR3) in human adipose tissue were queried using public databases, and directly measured by quantitative PCR (qPCR) and immunoblot. Adipose tissue AKR activity was measured by colorimetric assay. Adipocytes absorbed and efficiently metabolized DNR to daunorubicinol reducing its anti-leukemia effect in the local microenvironment. Murine studies confirmed adipose tissue conversion of DNR to daunorubicinol in vivo. Adipocytes expressed high levels of AKR and CBR isoenzymes that deactivate anthracyclines. Indeed, adipocyte protein levels of AKR1C1, AKR1C2, and AKR1C3 are higher than all other human non-cancerous cell types. To our knowledge, this is the first demonstration that adipocytes metabolize and inactivate a therapeutic drug. Adipocyte-mediated DNR metabolism reduces active drug concentration in the TME. These results could be clinically important for adipocyte-rich cancer microenvironments such as omentum, breast, and marrow. Since AKR and CBR enzymes metabolize several drugs, and can be expressed at higher levels in obese individuals, this proof-of-principle finding has important implications across many diseases.

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E2F6 contains a DNA binding domain that is very similar to that of the other members of the E2F family of transcriptional regulators. However, E2F6 cannot bind to all promoters that contain consensus E2F-binding sites. Therefore, we used a combination of chromatin immunoprecipitation and genomic microarrays to identify promoters bound by E2F6 in human cells. Although most of the identified promoters were bound by multiple E2F family members, one promoter was bound only by E2F6. To determine which of the newly identified promoters were regulated by E2F6, we reduced the level of E2F6 by using RNA interference technology. We found that mRNA transcribed from promoters bound by E2F6 was increased after reduction of the amount of E2F6 protein in the cell. Interestingly, many of the E2F6-regulated genes encoded functions involved in tumor suppression and the maintenance of chromatin structure. Specifically, our results suggest that E2F6 represses transcription of the brca1, ctip, art27, hp1␣, and the rbap48 genes. E2F6 has been postulated to mediate transcriptional repression by recruiting a histone H3 methyltransferase to the DNA. However, we found that the E2F6-regulated promoters did not contain histone H3 methylated at lysine 9. To determine the mechanism by which E2F6 regulates transcription, we performed chromatin immunoprecipitation before and after the introduction of small inhibitory ribonucleic acids specific to E2F6. We found that depletion of E2F6 resulted in the recruitment of E2F1 to the target promoters. In summary, we have identified 48 endogenous target genes of E2F6 and have shown that E2F6 can repress target promoters in a manner that does not require histone H3 methylation at lysine 9.The E2F family consists of six members, E2Fs 1-6, and two obligate heterodimeric partners, DP1 and DP2, which are required for binding to DNA (1). All of the E2Fs contain a conserved DNA binding and dimerization domain, and the different E2F-DP heterodimers can bind to the same consensus sequence. E2Fs 1-5 each contain a C-terminal transactivation domain that can interact with a variety of transcriptional coactivators such as CREB 1 -binding protein and TFIIH (2, 3).The C-terminal domain also contains sequences required for binding to the pocket protein family of transcriptional repressors (retinoblastoma, p107, and p130). E2Fs 1-3 interact preferentially with retinoblastoma, whereas E2F4 and -5 mainly associate with p107 or p130 (4). Depending upon exactly which proteins associate with the C-terminal domain, E2Fs1-5 can therefore mediate either activation or repression. E2F6, the most recently identified E2F family member, lacks the C-terminal transactivation domain found in the other E2Fs. Therefore, E2F6 likely cannot serve as a transcriptional activator or bind to the pocket protein family. However, E2F6 has been shown to be a potent transcriptional repressor (5-8). Because E2F6 lacks the pocket protein interaction domain, transcriptional repression may be mediated via interaction with other proteins. Yeast two-hyb...

Pediatric large B-cell lymphomas (LBCLs) share morphological and phenotypic features with adult types but have better prognosis. The higher frequency of some subtypes such as LBCL with IRF4 rearrangement (LBCL-IRF4) in children suggests that some age-related biological differences may exist. To characterize the genetic and molecular heterogeneity of these tumors, we studied 31 diffuse LBCLs (DLBCLs), not otherwise specified (NOS); 20 LBCL-IRF4 cases; and 12 cases of high-grade B-cell lymphoma (HGBCL), NOS in patients ≤25 years using an integrated approach, including targeted gene sequencing, copy-number arrays, and gene expression profiling. Each subgroup displayed different molecular profiles. LBCL-IRF4 had frequent mutations in IRF4 and NF-κB pathway genes (CARD11, CD79B, and MYD88), losses of 17p13 and gains of chromosome 7, 11q12.3-q25, whereas DLBCL, NOS was predominantly of germinal center B-cell (GCB) subtype and carried gene mutations similar to the adult counterpart (eg, SOCS1 and KMT2D), gains of 2p16/REL, and losses of 19p13/CD70. A subset of HGBCL, NOS displayed recurrent alterations of Burkitt lymphoma–related genes such as MYC, ID3, and DDX3X and homozygous deletions of 9p21/CDKN2A, whereas other cases were genetically closer to GCB DLBCL. Factors related to unfavorable outcome were age &gt;18 years; activated B-cell (ABC) DLBCL profile, HGBCL, NOS, high genetic complexity, 1q21-q44 gains, 2p16/REL gains/amplifications, 19p13/CD70 homozygous deletions, and TP53 and MYC mutations. In conclusion, these findings further unravel the molecular heterogeneity of pediatric and young adult LBCL, improve the classification of this group of tumors, and provide new parameters for risk stratification.