Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

Background: Transgenic zebrafish lines with the expression of a fluorescent reporter under the control of a cell-type specific promoter, enable transcriptome analysis of FACS sorted cell populations. RNA quality and yield are key determinant factors for accurate expression profiling. Limited cell number and FACS induced cellular stress make RNA isolation of sorted zebrafish cells a delicate process. We aimed to optimize a workflow to extract sufficient amounts of high-quality RNA from a limited number of FACS sorted cells from Tg(fli1a:GFP) zebrafish embryos, which can be used for accurate gene expression analysis. Results: We evaluated two suitable RNA isolation kits (theRNAqueous micro and the RNeasy plus micro kit) and determined that sorting cells directly into lysis buffer is a critical step for success. For low cell numbers, this ensures direct cell lysis, protects RNA from degradation and results in a higher RNA quality and yield. We showed that this works well up to 0.5x dilution of the lysis buffer with sorted cells. In our sort settings, this corresponded to 30,000 and 75,000 cells for the RNAqueous micro kit and RNeasy plus micro kit respectively. Sorting more cells dilutes the lysis buffer too much and requires the use of a collection buffer. We also demonstrated that an additional genomic DNA removal step after RNA isolation is required to completely clear the RNA from any contaminating genomic DNA. For cDNA synthesis and library preparation, we combined SmartSeq v4 full length cDNA library amplification, Nextera XT tagmentation and sample barcoding. Using this workflow, we were able to generate highly reproducible RNA sequencing results. Conclusions: The presented optimized workflow enables to generate high quality RNA and allows accurate transcriptome profiling of small populations of sorted zebrafish cells

In silico discovery of a FOXM1 driven embryonal signaling pathway in therapy resistant neuroblastoma tumors

Chemotherapy resistance is responsible for high mortality rates in neuroblastoma. MYCN, an oncogenic driver in neuroblastoma, controls pluripotency genes including LIN28B. We hypothesized that enhanced embryonic stem cell (ESC) gene regulatory programs could mark tumors with high pluripotency capacity and subsequently increased risk for therapy failure. An ESC miRNA signature was established based on publicly available data. In addition, an ESC mRNA signature was generated including the 500 protein coding genes with the highest positive expression correlation with the ESC miRNA signature score in 200 neuroblastomas. High ESC m(i)RNA expression signature scores were significantly correlated with poor neuroblastoma patient outcome specifically in the subgroup of MYCN amplified tumors and stage 4 nonamplified tumors. Further data-mining identified FOXM1, as the major predicted driver of this ESC signature, controlling a large set of genes implicated in cell cycle control and DNA damage response. Of further interest, re-analysis of published data showed that MYCN transcriptionally activates FOXM1 in neuroblastoma cells. In conclusion, a novel ESC m(i)RNA signature stratifies neuroblastomas with poor prognosis, enabling the identification of therapy-resistant tumors. The finding that this signature is strongly FOXM1 driven, warrants for drug design targeted at FOXM1 or key components controlling this pathway

PHF6 expression levels impact human hematopoietic stem cell differentiation

Transcriptional control of hematopoiesis involves complex regulatory networks and functional perturbations in one of these components often results in malignancies. Loss-of-function mutations in PHF6, encoding a presumed epigenetic regulator, have been primarily described in T cell acute lymphoblastic leukemia (T-ALL) and the first insights into its function in normal hematopoiesis only recently emerged from mouse modeling experiments. Here, we investigated the role of PHF6 in human blood cell development by performing knockdown studies in cord blood and thymus-derived hematopoietic precursors to evaluate the impact on lineage differentiation in well-established in vitro models. Our findings reveal that PHF6 levels differentially impact the differentiation of human hematopoietic progenitor cells into various blood cell lineages, with prominent effects on lymphoid and erythroid differentiation. We show that loss of PHF6 results in accelerated human T cell development through reduced expression of NOTCH1 and its downstream target genes. This functional interaction in developing thymocytes was confirmed in vivo using a phf6-deficient zebrafish model that also displayed accelerated developmental kinetics upon reduced phf6 or notch1 activation. In summary, our work reveals that appropriate control of PHF6 expression is important for normal human hematopoiesis and provides clues towards the role of PHF6 in T-ALL development

Expressed repetitive elements are broadly applicable reference targets for normalization of reverse transcription-qPCR data in mice

Reverse transcription quantitative PCR (RT-qPCR) is the gold standard method for gene expression analysis on mRNA level. To remove experimental variation, expression levels of the gene of interest are typically normalized to the expression level of stably expressed endogenous reference genes. Identifying suitable reference genes and determining the optimal number of reference genes should precede each quantification study. Popular reference genes are not necessarily stably expressed in the examined conditions, possibly leading to inaccurate results. Stably and universally expressed repetitive elements (ERE) have previously been shown to be an excellent alternative for normalization using classic reference genes in human and zebrafish samples. Here, we confirm that in mouse tissues, EREs are broadly applicable reference targets for RT-qPCR normalization, provided that the RNA samples undergo a thorough DNase treatment. We identified Orr1a0, Rltr2aiap, and Rltr13a3 as the most stably expressed mouse EREs across six different experimental conditions. Therefore, we propose this set of ERE reference targets as good candidates for normalization of RT-qPCR data in a plethora of conditions. The identification of widely applicable stable mouse RT-qPCR reference targets for normalization has great potential to facilitate future murine gene expression studies and improve the validity of RT-qPCR data

TBX2 is a neuroblastoma core regulatory circuitry component enhancing MYCN/FOXM1 reactivation of DREAM targets

Chromosome 17q gains are almost invariably present in high-risk neuroblastoma cases. Here, we perform an integrative epigenomics search for dosage-sensitive transcription factors on 17q marked by H3K27ac defined super-enhancers and identify TBX2 as top candidate gene. We show that TBX2 is a constituent of the recently established core regulatory circuitry in neuroblastoma with features of a cell identity transcription factor, driving proliferation through activation of p21-DREAM repressed FOXM1 target genes. Combined MYCN/TBX2 knockdown enforces cell growth arrest suggesting that TBX2 enhances MYCN sustained activation of FOXM1 targets. Targeting transcriptional addiction by combined CDK7 and BET bromodomain inhibition shows synergistic effects on cell viability with strong repressive effects on CRC gene expression and p53 pathway response as well as several genes implicated in transcriptional regulation. In conclusion, we provide insight into the role of the TBX2 CRC gene in transcriptional dependency of neuroblastoma cells warranting clinical trials using BET and CDK7 inhibitors

Cellular senescence in neuroblastoma

Neuroblastoma is a tumour that arises from the sympathoadrenal lineage occurring predominantly in children younger than five years. About half of the patients are diagnosed with high-risk tumours and undergo intensive multi-modal therapy. The success rate of current treatments for high-risk neuroblastoma is disappointingly low and survivors suffer from multiple therapy-related long-term side effects. Most chemotherapeutics drive cancer cells towards cell death or senescence. Senescence has long been considered to represent a terminal non-proliferative state and therefore an effective barrier against tumorigenesis. This dogma, however, has been challenged by recent observations that infer a much more dynamic and reversible nature for this process, which may have implications for the efficacy of therapy-induced senescence-oriented treatment strategies. Neuroblastoma cells in a dormant, senescent-like state may escape therapy, whilst their senescence-associated secretome may promote inflammation and invasiveness, potentially fostering relapse. Conversely, due to its distinct molecular identity, senescence may also represent an opportunity for the development of novel (combination) therapies. However, the limited knowledge on the molecular dynamics and diversity of senescence signatures demands appropriate models to study this process in detail. This review summarises the molecular knowledge about cellular senescence in neuroblastoma and investigates current and future options towards therapeutic exploration

Cellular senescence in neuroblastoma

Neuroblastoma is a tumour that arises from the sympathoadrenal lineage occurring predominantly in children younger than five years. About half of the patients are diagnosed with high-risk tumours and undergo intensive multi-modal therapy. The success rate of current treatments for high-risk neuroblastoma is disappointingly low and survivors suffer from multiple therapy-related long-term side effects. Most chemotherapeutics drive cancer cells towards cell death or senescence. Senescence has long been considered to represent a terminal non-proliferative state and therefore an effective barrier against tumorigenesis. This dogma, however, has been challenged by recent observations that infer a much more dynamic and reversible nature for this process, which may have implications for the efficacy of therapy-induced senescence-oriented treatment strategies. Neuroblastoma cells in a dormant, senescent-like state may escape therapy, whilst their senescence-associated secretome may promote inflammation and invasiveness, potentially fostering relapse. Conversely, due to its distinct molecular identity, senescence may also represent an opportunity for the development of novel (combination) therapies. However, the limited knowledge on the molecular dynamics and diversity of senescence signatures demands appropriate models to study this process in detail. This review summarises the molecular knowledge about cellular senescence in neuroblastoma and investigates current and future options towards therapeutic exploration