SUMMARY MYC contributes to the pathogenesis of a majority of human cancers, yet strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have targeted MYC transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative co-activator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain proteins as regulatory factors for c-Myc. BET inhibition by JQ1 downregulates MYC transcription, followed by genome-wide downregulation of Myc-dependent target genes. In experimental models of multiple myeloma, a Myc-dependent hematologic malignancy, JQ1 produces a potent antiproliferative effect associated with cell cycle arrest and cellular senescence. Efficacy of JQ1 in three murine models of multiple myeloma establishes the therapeutic rationale for BET bromodomain inhibition in this disease and other malignancies characterized by pathologic activation of c-Myc.

High throughput cDNA sequencing technologies have advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and because modifications are not retained. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies (ONT). Our study generated 9.9 million aligned sequence reads for the human cell line GM12878, using thirty MinION flow cells at six institutions. These native RNA reads had a median length of 771 bases, and a maximum aligned length of over 21,000 bases. Mitochondrial poly(A) reads provided an internal measure of read length quality. We combined these long nanopore reads with higher accuracy short-reads and annotated GM12878 promoter regions, to identify 33,984 plausible RNA isoforms. We describe strategies for assessing 3′ poly(A) tail length, base modifications, and transcript haplotypes.

Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current methods 1 - 4 are limited by the loss of native modifications, short read length, high input requirements, low yield, or long protocols. Here, we describe nanopore Cas9-targeted sequencing (nCATS), an enrichment strategy that uses targeted cleavage of chromosomal DNA with Cas9 to ligate adaptors for nanopore sequencing. We show that nCATS can simultaneously assess haplotype-resolved single-nucleotide variants (SNVs), structural variations (SVs) and CpG methylation. We apply nCATS to four cell lines, a cell-line-derived xenograft, and normal and paired tumor/normal primary human breast tissue. Median sequencing coverage was 675X using a minION flow cell and 34X using the smaller flongle flow cell. nCATS requires only ~3μg of genomic DNA and can target a large number of loci in a single reaction. The method will facilitate the use of long-read sequencing in research and in the clinic.

High throughput cDNA sequencing technologies have dramatically advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and because modifications are not carried forward in cDNA. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies (ONT). Our study focused on poly(A) RNA from the human cell line GM12878, generating 9.9 million aligned sequence reads. These native RNA reads had an aligned N50 length of 1294 bases, and a maximum aligned length of over 21,000 bases. A total of 78,199 high-confidence isoforms were identified by combining long nanopore reads with short higher accuracy Illumina reads. We describe strategies for assessing 3′ poly(A) tail length, base modifications and transcript haplotypes from nanopore RNA data. Together, these nanopore-based techniques are poised to deliver new insights into RNA biology.

Probing epigenetic features on DNA has tremendous potential to advance our understanding of the phased epigenome. In this study, we use nanopore sequencing to evaluate CpG methylation and chromatin accessibility simultaneously on long strands of DNA by applying GpC methyltransferase to exogenously label open chromatin. We performed nanopore sequencing of Nucleosome Occupancy and Methylome (nanoNOMe) on four human cell lines (GM12878, MCF-10A, MCF-7, MDA-MB-231). The single-molecule resolution allows footprinting of protein and nucleosome binding and determining the combinatorial promoter epigenetic signature on individual molecules. Long-read sequencing makes it possible to robustly assign reads to haplotypes, allowing us to generate the first fully phased human epigenome, consisting of chromosome-level allele-specific profiles of CpG methylation and chromatin accessibility. We further apply this to a breast cancer model to evaluate differential methylation and accessibility between cancerous and non-cancerous cells.