Epigenomic profiling of neuroblastoma cell lines.

Understanding the aberrant transcriptional landscape of neuroblastoma is necessary to provide insight to the underlying influences of the initiation, progression and persistence of this developmental cancer. Here, we present chromatin immunoprecipitation sequencing (ChIP-Seq) data for the oncogenic transcription factors, MYCN and MYC, as well as regulatory histone marks H3K4me1, H3K4me3, H3K27Ac, and H3K27me3 in ten commonly used human neuroblastoma-derived cell line models. In addition, for all of the profiled cell lines we provide ATAC-Seq as a measure of open chromatin. We validate specificity of global MYCN occupancy in MYCN amplified cell lines and functional redundancy of MYC occupancy in MYCN non-amplified cell lines. Finally, we show with H3K27Ac ChIP-Seq that these cell lines retain expression of key neuroblastoma super-enhancers (SE). We anticipate this dataset, coupled with available transcriptomic profiling on the same cell lines, will enable the discovery of novel gene regulatory mechanisms in neuroblastoma

The epigenetic modifier Usp22 regulates the transition of embryonic stem cells from self-renewal to differentiation via transcriptional repression of SOX2

Pluripotent embryonic stem cells (ESCs) undergo self-renewal until stimulated to differentiate down specific lineage pathways. Many of the transcriptional networks that drive the movement from a self-renewing ESC to a differentiating cell have been identified. However, fundamental questions remain unanswered about the epigenetic programs that control these changes in gene expression. Here we report that the histone ubiquitin hydrolase, Usp22, is a critical epigenetic modifier that controls this transition from self-renewal to differentiation. This study reveals that Usp22 is induced as ESCs differentiate. More importantly, both human and mouse ESCs require USP22 for proper differentiation. Conversely, ectopic expression of USP22 is sufficient to trigger the spontaneous differentiation of ESCs, even in the absence of other differentiation signals. Gene expression profiling in ESCs demonstrated broad defects in the transcription of genes linked to all three germ layers when USP22 levels were depleted. Remarkably, this analysis revealed that USP22 is also required for the repression of a limited number of pluripotency genes, including SOX2. These findings suggested that the loss of USP22 might result in both defective differentiation and lineage-specific gene expression due to the failure to repress pluripotency factors. As the core pluripotency factor most affected by USP22 depletion, SOX2\u27s role in this phenotype was explored. Epistasis experiments suggest the possibility that SOX2 de-repression may be responsible for the effects of USP22 depletion, as blocking the increase in SOX2 reversed the USP22 phenotype. Mechanistically, USP22 was found to directly occupy the SOX2 locus where it controls the relative level of histone H2B ubiquitylation. USP22-mediated changes in H2B ubiquitylation at SOX2 likely explain its effects on SOX2 transcription and pluripotency, since our studies show that RNF20, the E3 ligase responsible for H2B ubiquitylation, is essential for SOX2 expression in ESCs. Our study reveals an epigenetic mechanism that represses the core pluripotency transcriptional network in ESCs, allowing ESCs to transition from a state of self-renewal into lineage-specific differentiation programs.

Validation of a Next-Generation Sequencing Assay Targeting RNA for the Multiplexed Detection of Fusion Transcripts and Oncogenic Isoforms

Context.-Next-generation sequencing is a high-throughput method for detecting genetic abnormalities and providing prognostic and therapeutic information for patients with cancer. Oncogenic fusion transcripts are among the various classifications of genetic abnormalities present in tumors and are typically detected clinically with fluorescence in situ hybridization (FISH). However, FISH probes only exist for a limited number of targets, do not provide any information about fusion partners, cannot be multiplex, and have been shown to be limited in specificity for common targets such as ALK.Objective.-To validate an anchored multiplex polymerase chain reaction-based panel for the detection of fusion transcripts in a university hospital-based clinical molecular diagnostics laboratory.Design.-We used 109 unique clinical specimens to validate a custom panel targeting 104 exon boundaries from 17 genes involved in fusions in solid tumors. The panel can accept as little as 100 ng of total nucleic acid from PreservCyt-fixed tissue, and formalin-fixed, paraffinembedded specimens with as little as 10% tumor nuclei.Results.-Using FISH as the gold standard, this assay has a sensitivity of 88.46% and a specificity of 95.83% for the detection of fusion transcripts involving ALK, RET, and ROS1 in lung adenocarcinomas. Using a validated next-generation sequencing assay as the orthogonal gold standard for the detection of EGFR variant Ill (EGFRvIII) in glioblastomas, the assay is 92.31% sensitive and 100% specific.Conclusions.-This multiplexed assay is tumor and fusion partner agnostic and will provide clinical utility in therapy selection for patients with solid tumors

A delayed h3k27me3 accumulation after DNA replication of embryonic stem cells opens chromatin for lineage specific transcription factors to bind and initiate differentiation

Introduction Pluripotent stem cells (PSCs) have been useful to generate differentiated progenies for cell replacement therapy, and disease models. The Parkinson’s Disease (PD) field was arguably one of the first to have embraced the promise of stem cells. However, regardless of the differentiation protocols used, cultures and grafts continue to contain multiple cell types with midbrain dopamine (mDA) neural progenitors (NPs) and neurons representing only a fraction of total cells in the dish or graft. During cell differentiation, recruitment of transcription factors (TFs) to repressed genes in euchromatin is essential to activate new transcriptional programs, which is impeded by condensed H3K27me3-containing chromatin. Here, using single-cell and gene-specific analyses, we tested the hypothesis that during the first hours of induction of differentiation of human embryonic stem cells (ESCs), accumulation of the repressive histone mark H3K27me3 is delayed after DNA replication, indicative of decondensed chromatin structure, potentially providing a critical ‘window of opportunity’ for recruitment of lineage-specific TFs to DNA. If true, it may be possible to direct the differentiation of ESCs or iPSCs into homogeneous populations of any desired cell type needed to study, model and potentially treat different diseases including PD. Poster presented at: ISSCR 2017 in Boston MA, United States