Cell lines were not tested for mycoplasma contamination. Commonly misidentified lines (See ICLAC register) No commonly misidentified cell lines were used.

The PVT1 locus is identified as a cluster of T(2;8) and T(8;22) "variant" MYC-activating chromosomal translocation breakpoints extending 400 kb downstream of MYC in a subset (approximately 20%) of Burkitt's lymphoma (vBL). Recent reports that microRNAs (miRNA) may be associated with fragile sites and cancer-associated genomic regions prompted us to investigate whether the PVT1 region on chromosome 8q24 may contain miRNAs. Computational analysis of the genomic sequence covering the PVT1 locus and experimental verification identified seven miRNAs. One miRNA, hsa-miR-1204, resides within a previously described PVT1 exon (1b) that is often fused to the immunoglobulin light chain constant region in vBLs and is present in high copy number in MYC/PVT1-amplified tumors. Like its human counterpart, mouse mmu-miR-1204 represents the closest miRNA to Myc (~50 kb) and is found only 1 to 2 kb downstream of a cluster of retroviral integration sites. Another miRNA, mmu-miR-1206, is close to a cluster of variant translocation breakpoints associated with mouse plasmacytoma and exon 1 of mouse Pvt1. Virtually all the miRNA precursor transcripts are expressed at higher levels in late-stage B cells (including plasmacytoma and vBL cell lines) compared with immature B cells, suggesting possible roles in lymphoid development and/or lymphoma. In addition, lentiviral vector-mediated overexpression of the miR-1204 precursor (human and mouse) in a mouse pre-B-cell line increased expression of Myc. High levels of expression of the hsa-miR-1204 precursor is also seen in several epithelial cancer cell lines with MYC/PVT1 coamplification, suggesting a potentially broad role for these miRNAs in tumorigenesis.

The mangrove killifish (Kryptolebias marmoratus) is the only vertebrate known to be capable of self-fertilization. Its gonad is typically an ovotestis that simultaneously produces eggs and sperm, and fertilization is internal. Although most populations of this species consist primarily or exclusively of hermaphroditic individuals, gonochoristic males occur at Ϸ20% frequency in a natural population at Twin Cays, Belize. Here we use a battery of 36 microsatellite loci to document a striking genetic pattern (high intraspecimen heterozygosities and low within-population linkage disequilibria) that differs qualitatively from the highly homozygous (or ''clonal'') genetic architecture characteristic of killifish populations previously studied in Florida, where males are much rarer. These findings document that outcrossing (probably between gonochoristic males and hermaphrodites) is common at the Belize site, and, more importantly, they demonstrate the dramatic impact that functional androdioecy can have on the population genetic architecture of this reproductively unique vertebrate species.A ndrodioecy is a rare reproductive system in which a natural population consists of functional males and hermaphrodites but no true female gonochorists. Previously known only in a few plants (1-10) and invertebrate animals (11-15), androdioecy has also evolved independently in a vertebrate species: the mangrove killifish, Kryptolebias (formerly Rivulus) marmoratus. Most surveyed populations of K. marmoratus consist primarily or exclusively of hermaphroditic individuals, but gonochoristic males are observed occasionally, and recent genetic evidence suggests that such individuals may mediate infrequent outcross events in this otherwise self-fertilizing species (16-18). The net result, documented most clearly for Florida locales (16), is a mixed-mating population genetic architecture consisting mostly of highly homozygous inbred strains (traditionally referred to as ''clones'') plus low percentages of highly heterozygous specimens stemming from recent outcross events. Thus, the genetic variety generated by outcrossing (16), and subsequently converted into new arrays of recombinant inbred lines upon resumptions of selfing, can significantly augment mutation and interlocality gene flow that formerly were thought to be the sole sources of ''clonal'' diversity in K. marmoratus (19)(20)(21).Gonochoristic males, which are phenotypically recognizable by coloration and histology, seem to be extremely rare in Florida (in our experience, Ͻ1% frequency among Ͼ1,000 individuals examined). However, in a collection from Twin Cays, Belize, made during 1988 and 1989, 53 males (18.8%) were present among 282 specimens surveyed (22). In laboratory-reared progeny from another collection (in 1991) of hermaphrodites from this Belize locale, segregating genetic variation was detected, a result interpreted to imply that natural outcrossing had occurred between males and hermaphrodites (17, 18).In this study, we use a battery of 36 microsatellite loci to exami...

Chromatin immunoprecipitation followed by next-generation DNA sequencing (ChIP-seq) is a widely used technique for identifying transcription factor (TF) binding events throughout an entire genome. However, ChIP-seq is limited by the availability of suitable ChIP-seq grade antibodies, and the vast majority of commercially available antibodies fail to generate usable data sets. To ameliorate these technical obstacles, we present a robust methodological approach for performing ChIPseq through epitope tagging of endogenous TFs. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing technology to develop CRISPR epitope tagging ChIP-seq (CETCh-seq) of DNA-binding proteins. We assessed the feasibility of CETCh-seq by tagging several DNA-binding proteins spanning a wide range of endogenous expression levels in the hepatocellular carcinoma cell line HepG2. Our data exhibit strong correlations between both replicate types as well as with standard ChIP-seq approaches that use TF antibodies. Notably, we also observed minimal changes to the cellular transcriptome and to the expression of the tagged TF. To examine the robustness of our technique, we further performed CETCh-seq in the breast adenocarcinoma cell line MCF7 as well as mouse embryonic stem cells and observed similarly high correlations. Collectively, these data highlight the applicability of CETCh-seq to accurately define the genome-wide binding profiles of DNA-binding proteins, allowing for a straightforward methodology to potentially assay the complete repertoire of TFs, including the large fraction for which ChIP-quality antibodies are not available.

Transcription factors are DNA-binding proteins that have key roles in gene regulation 1,2. Genome-wide occupancy maps of transcriptional regulators are important for understanding gene regulation and its effects on diverse biological processes 3-6. However, only a minority of the more than 1,600 transcription factors encoded in the human genome has been assayed. Here we present, as part of the ENCODE (Encyclopedia of DNA Elements) project, data and analyses from chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) experiments using the human HepG2 cell line for 208 chromatinassociated proteins (CAPs). These comprise 171 transcription factors and 37 transcriptional cofactors and chromatin regulator proteins, and represent nearly one-quarter of CAPs expressed in HepG2 cells. The binding profiles of these CAPs form major groups associated predominantly with promoters or enhancers, or with both. We confirm and expand the current catalogue of DNA sequence motifs for transcription factors, and describe motifs that correspond to other transcription factors that are co-enriched with the primary ChIP target. For example, FOX family motifs are enriched in ChIP-seq peaks of 37 other CAPs. We show that motif content and occupancy patterns can distinguish between promoters and enhancers. This catalogue reveals high-occupancy target regions at which many CAPs associate, although each contains motifs for only a minority of the numerous associated transcription factors. These analyses provide a more complete overview of the gene regulatory networks that define this cell type, and demonstrate the usefulness of the large-scale production efforts of the ENCODE Consortium. There are an estimated 1,639 transcription factors (TFs) in the human genome 2 , and up to 2,500 CAPs when we include transcriptional cofactors, RNA polymerase-associated proteins, histone-binding regulators, and chromatin-modifying enzymes 1,7. A typical TF binds to a short DNA sequence motif, and, in vivo, some TFs exhibit additional chromosomal occupancy mediated by their interactions with other CAPs 8-10. CAPs are vital for orchestrating cell type-and cell state-specific gene regulation, including the temporal coordination of gene expression in developmental processes, environmental responses, and disease states 3-6,11-13. Identifying genomic regions with which a TF is physically associated, referred to as TF binding sites (TFBSs), is an important step towards understanding its biological roles. The most common genome-wide assay for identifying TFBSs is ChIP-seq 14-16. In addition to highlighting potentially active regulatory DNA elements by direct measurement, ChIP-seq data can define DNA sequence motifs that can be used, often in conjunction with expression data and chromatin accessibility maps, to infer likely binding events in other cellular contexts without performing direct assays. Although motifs identified by ChIP-seq are often representative of direct binding, this is not always the case, as co-occurrence of other TFs could ...