A therapeutically targetable positive feedback loop between lnc-HLX-2-7, HLX, and MYC that promotes group 3 medulloblastoma.

Recent studies suggest that long non-coding RNAs (lncRNAs) contribute to medulloblastoma (MB) formation and progression. We have identified an lncRNA, lnc-HLX-2-7, as a potential therapeutic target in group 3 (G3) MBs. lnc-HLX-2-7 RNA specifically accumulates in the promoter region of HLX, a sense-overlapping gene of lnc-HLX-2-7, which activates HLX expression by recruiting multiple factors, including enhancer elements. RNA sequencing and chromatin immunoprecipitation reveal that HLX binds to and activates the promoters of several oncogenes, including TBX2, LIN9, HOXM1, and MYC. Intravenous treatment with cerium-oxide-nanoparticle-coated antisense oligonucleotides targeting lnc-HLX-2-7 (CNP-lnc-HLX-2-7) inhibits tumor growth by 40%-50% in an intracranial MB xenograft mouse model. Combining CNP-lnc-HLX-2-7 with standard-of-care cisplatin further inhibits tumor growth and significantly prolongs mouse survival compared with CNP-lnc-HLX-2-7 monotherapy. Thus, the lnc-HLX-2-7-HLX-MYC axis is important for regulating G3 MB progression, providing a strong rationale for using lnc-HLX-2-7 as a therapeutic target for G3 MBs.

Novel circular RNAs of the apoptosis-related BAX and BCL2L12 genes identified in a chronic lymphocytic leukemia cell line using nanopore sequencing.

Circular RNAs (circRNAs), a novel RNA type generated by back-splicing, are key regulators of gene expression, with deregulated expression and established involvement in leukemia. The products of BCL2 and its homologs, including BAX and BCL2L12, are implicated in chronic lymphocytic leukemia (CLL). However, to the best of our knowledge, nothing is known about circRNAs produced by these two genes and their role in CLL. We sought to further elucidate the contribution of BAX and BCL2L12 in CLL by unraveling the identity, localization, and potential role of their circRNAs. Therefore, total RNA from the EHEB cell line and peripheral blood mononuclear cells (PBMCs) of CLL patients and non-leukemic blood donors was extracted and reverse-transcribed using random hexamers. Next, nested PCRs with divergent primers were performed and the purified PCR products were subjected to 3rd generation nanopore sequencing. Nested PCRs were also applied to first-strand cDNAs synthesized from total RNA extracts of PBMCs from CLL patients and non-leukemic blood donors. Lastly, a single-molecule resolution fluorescent in situ hybridization method called circFISH was used to visualize the circRNA distribution in EHEB cells. We discovered several novel circRNAs produced by BAX and BCL2L12, which were characterized by great exon structure diversity. In addition, intriguing findings regarding their formation emerged. Interestingly, visualization of the most abundant circRNAs showed distinct intracellular localization. Moreover, a complex BAX and BCL2L12 circRNA expression pattern was revealed in CLL patients and non-leukemic blood donors. Our data suggest a multifaceted role of BAX and BCL2L12 circRNAs in B-cell CLL.

circCsnk1g3- and circAnkib1-regulated interferon responses in sarcoma promote tumorigenesis by shaping the immune microenvironment.

Exonic circular RNAs (circRNAs) produce predominantly non-coding RNA species that have been recently profiled in many tumors. However, their functional contribution to cancer progression is still poorly understood. Here, we identify the circRNAs expressed in soft tissue sarcoma cells and explore how the circRNAs regulate sarcoma growth in vivo. We show that circCsnk1g3 and circAnkib1 promote tumor growth by shaping a pro-tumorigenic microenvironment, possibly due to their capabilities to regulate tumor-promoting elements extrinsic to the tumor cells. Accordingly, circCsnk1g3 and circAnkib1 can control the expression of interferon-related genes and pro-inflammatory factors in the sarcoma cells, thus directing immune cell recruitment into the tumor mass, and hence their activation. Mechanistically, circRNAs may repress pro-inflammatory elements by buffering activation of the pathways mediated by RIG-I, the cytosolic viral RNA sensor. The current findings suggest that the targeting of specific circRNAs could augment the efficacy of tumor and immune response to mainstay therapies.

Exon skipping induced by CRISPR-directed gene editing regulates the response to chemotherapy in non-small cell lung carcinoma cells.

We have been developing CRISPR-directed gene editing as an augmentative therapy for the treatment of non-small cell lung carcinoma (NSCLC) by genetic disruption of Nuclear Factor Erythroid 2-Related Factor 2 (NRF2). NRF2 promotes tumor cell survival in response to therapeutic intervention and thus its disablement should restore or enhance effective drug action. Here, we report how NRF2 disruption leads to collateral damage in the form of CRISPR-mediated exon skipping. Heterogeneous populations of transcripts and truncated proteins produce a variable response to chemotherapy, dependent on which functional domain is missing. We identify and characterize predicted and unpredicted transcript populations and discover that several types of transcripts arise through exon skipping; wherein one or two NRF2 exons are missing. In one specific case, the presence or absence of a single nucleotide determines whether an exon is skipped or not by reorganizing Exonic Splicing Enhancers (ESEs). We isolate and characterize the diversity of clones induced by CRISPR activity in a NSCLC tumor cell population, a critical and often overlooked genetic byproduct of this exciting technology. Finally, gRNAs must be designed with care to avoid altering gene expression patterns that can account for variable responses to solid tumor therapy.

CircFISH: A Novel Method for the Simultaneous Imaging of Linear and Circular RNAs.

Circular RNAs (circRNAs) are regulatory RNAs which have recently been shown to have clinical significance in several diseases, including, but not limited to, various cancers, neurological diseases and cardiovascular diseases. The function of such regulatory RNAs is largely dependent on their subcellular localization. Several circRNAs have been shown to conduct antagonistic roles compared to the products of the linear isoforms, and thus need to be characterized distinctly from the linear RNAs. However, conventional fluorescent in situ hybridization (FISH) techniques cannot be employed directly to distinguish the signals from linear and circular isoforms because most circRNAs share the same sequence with the linear RNAs. In order to address this unmet need, we adapted the well-established method of single-molecule FISH by designing two sets of probes to differentiate the linear and circular RNA isoforms by virtue of signal colocalization. We call this method 'circular fluorescent in situ hybridization' (circFISH). Linear and circular RNAs were successfully visualized and quantified at a single-molecule resolution in fixed cells. RNase R treatment during the circFISH reduced the levels of linear RNAs while the circRNA levels remain unaltered. Furthermore, cells with shRNAs specific to circRNA showed the loss of circRNA levels, whereas the linear RNA levels were unaffected. The optimization of the in-situ RNase R treatment allowed the multiplexing of circFISH to combine it with organelle staining. CircFISH was found to be compatible with multiple sample types, including cultured cells and fresh-frozen and formalin-fixed tissue sections. Thus, we present circFISH as a versatile method for the simultaneous visualization and quantification of the distribution and localization of linear and circular RNA in fixed cells and tissue samples.

Apiaceous vegetables protect against acrolein-induced pulmonary injuries through modulating hepatic detoxification and inflammation in C57BL/6 male mice.

Acrolein (Acr) is a reactive aldehyde in the environment. Acr causes oxidative stress and a cascade of catalytic events and has, thereby, been associated with increased risk of pulmonary diseases. Whether apiaceous vegetables (API) consumption can prevent Acr-induced pulmonary toxicity has not yet been explored hence, we investigated the effects of API on Acr-induced pulmonary damages in C57BL/6J mice. The mice were assigned into either negative control [NEG group; American Institute of Nutrition (AIN)-93G diet only], positive control (POS group; AIN-93G+Acr) or API intervention group (API group; AIN-93G+21% API+Acr). After 1 week of dietary intervention, the POS and API mice were exposed to Acr (10 µmol/kg body weight/day) for 5 days. During the exposure period, assigned diets remained the same. Prominent indicators lung of toxicity of POS mice were found, including mucus accumulation, macrophage infiltration, and hemorrhage, all of which were ameliorated by the API. Serum and lung inflammation markers, such as a tumor necrosis factor alpha were also increased by Acr while reduced by API. In the liver, API upregulated expression of glutathione S-transferases, which enhanced the metabolism of Acr into water-soluble 3-hydroxypropyl mercapturic acid for excretion. This is consistent with observed reductions in serum Acr-protein adducts. Taken together, our results suggest that API may provide protection against Acr-induced pulmonary damages and inflammation via enhancement of the hepatic detoxification of Acr.

Structural basis of cyclic oligoadenylate binding to the transcription factor Csa3 outlines cross talk between type III and type I CRISPR systems.

RNA interference by type III CRISPR systems results in the synthesis of cyclic oligoadenylate (cOA) second messengers, which are known to bind and regulate various CARF domain-containing nuclease receptors. The CARF domain-containing Csa3 family of transcriptional factors associated with the DNA-targeting type I CRISPR systems regulate expression of various CRISPR and DNA repair genes in many prokaryotes. In this study, we extend the known receptor repertoire of cOA messengers to include transcriptional factors by demonstrating specific binding of cyclic tetra-adenylate (cA4) to Saccharolobus solfataricus Csa3 (Csa3Sso). Our 2.0-Å resolution X-ray crystal structure of cA4-bound full-length Csa3Sso reveals the binding of its CARF domain to an elongated conformation of cA4. Using cA4 binding affinity analyses of Csa3Sso mutants targeting the observed Csa3Sso•cA4 structural interface, we identified a Csa3-specific cA4 binding motif distinct from a more widely conserved cOA-binding CARF motif. Using a rational surface engineering approach, we increased the cA4 binding affinity of Csa3Sso up to ∼145-fold over the wildtype, which has potential applications for future second messenger-driven CRISPR gene expression and editing systems. Our in-solution Csa3Sso structural analysis identified cA4-induced allosteric and asymmetric conformational rearrangement of its C-terminal winged helix-turn-helix effector domains, which could potentially be incompatible to DNA binding. However, specific in vitro binding of the purified Csa3Sso to its putative promoter (PCas4a) was found to be cA4 independent, suggesting a complex mode of Csa3Sso regulation. Overall, our results support cA4-and Csa3-mediated cross talk between type III and type I CRISPR systems.

The Landscape of Regulatory Noncoding RNAs in Ewing's Sarcoma.

Ewing's sarcoma (ES) is a pediatric sarcoma caused by a chromosomal translocation. Unlike in most cancers, the genomes of ES patients are very stable. The translocation product of the EWS-FLI1 fusion is most often the predominant genetic driver of oncogenesis, and it is pertinent to explore the role of epigenetic alterations in the onset and progression of ES. Several types of noncoding RNAs, primarily microRNAs and long noncoding RNAs, are key epigenetic regulators that have been shown to play critical roles in various cancers. The functions of these epigenetic regulators are just beginning to be appreciated in ES. Here, we performed a comprehensive literature review to identify these noncoding RNAs. We identified clinically relevant tumor suppressor microRNAs, tumor promoter microRNAs and long noncoding RNAs. We then explored the known interplay between different classes of noncoding RNAs and described the currently unmet need for expanding the noncoding RNA repertoire of ES. We concluded the review with a discussion of epigenetic regulation of ES via regulatory noncoding RNAs. These noncoding RNAs provide new avenues of exploration to develop better therapeutics and identify novel biomarkers.

Identification of a New Transcriptional Co-Regulator of STEAP1 in Ewing's Sarcoma.

Ewing's sarcoma (ES) is caused by a chromosomal translocation leading to the formation of the fused EWSFLI1 gene, which codes for an aberrant transcription factor EWSFLI1. The transcriptional targets of EWSFLI1 have been viewed as promising and novel drug targets in the treatment of ES. One such target is six transmembrane epithelial antigen of the prostate 1 (STEAP1), a transmembrane protein that is upregulated by EWSFLI1 in ES. STEAP1 is a hallmark of tumor invasiveness and an indicator of tumor responsiveness to therapy. EWSFLI1 binds to the STEAP1 promoter region, but the mechanism of action by which it upregulates STEAP1 expression in ES is not entirely understood. Upon analysis of the STEAP1 promoter, we predicted two binding sites for NKX2.2, another crucial transcription factor involved in ES pathogenesis. We confirmed the interaction of NKX2.2 with the STEAP1 promoter using chromatin immunoprecipitation (ChIP) analysis. We used single-molecule RNA imaging, biochemical, and genetic studies to identify the novel role of NKX2.2 in regulating STEAP1 expression in ES. Our results show that NKX2.2 is a co-regulator of STEAP1 expression and functions by interacting with the STEAP1 promoter at sites proximal to the reported EWSFLI1 sites. The co-operative interaction of NKX2.2 with EWSFLI1 in regulating STEAP1 holds potential as a new target for therapeutic interventions for ES.

Structural basis of KdpD histidine kinase binding to the second messenger c-di-AMP.

The KdpDE two-component system regulates potassium homeostasis and virulence in various bacterial species. The KdpD histidine kinases (HK) of this system contain a universal stress protein (USP) domain which binds to the second messenger cyclic-di-adenosine monophosphate (c-di-AMP) for regulating transcriptional output from this two-component system in Firmicutes such as Staphylococcus aureus. However, the structural basis of c-di-AMP specificity within the KdpD-USP domain is not well understood. Here, we resolved a 2.3 Å crystal structure of the S. aureus KdpD-USP domain (USPSa) complexed with c-di-AMP. Binding affinity analyses of USPSa mutants targeting the observed USPSa:c-di-AMP structural interface enabled the identification of the sequence residues that are required for c-di-AMP specificity. Based on the conservation of these residues in other Firmicutes, we identified the binding motif, (A/G/C)XSXSX2N(Y/F), which allowed us to predict c-di-AMP binding in other KdpD HKs. Furthermore, we found that the USPSa domain contains structural features distinct from the canonical standalone USPs that bind ATP as a preferred ligand. These features include inward-facing conformations of its ß1-α1 and ß4-α4 loops, a short α2 helix, the absence of a triphosphate-binding Walker A motif, and a unique dual phospho-ligand binding mode. It is therefore likely that USPSa-like domains in KdpD HKs represent a novel subfamily of the USPs.

Author Correction: Intragenic antagonistic roles of protein and circRNA in tumorigenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

EWS-FLI1 modulated alternative splicing of ARID1A reveals novel oncogenic function through the BAF complex.

Connections between epigenetic reprogramming and transcription or splicing create novel mechanistic networks that can be targeted with tailored therapies. Multiple subunits of the chromatin remodeling BAF complex, including ARID1A, play a role in oncogenesis, either as tumor suppressors or oncogenes. Recent work demonstrated that EWS-FLI1, the oncogenic driver of Ewing sarcoma (ES), plays a role in chromatin regulation through interactions with the BAF complex. However, the specific BAF subunits that interact with EWS-FLI1 and the precise role of the BAF complex in ES oncogenesis remain unknown. In addition to regulating transcription, EWS-FLI1 also alters the splicing of many mRNA isoforms, but the role of splicing modulation in ES oncogenesis is not well understood. We have identified a direct connection between the EWS-FLI1 protein and ARID1A isoform protein variant ARID1A-L. We demonstrate here that ARID1A-L is critical for ES maintenance and supports oncogenic transformation. We further report a novel feed-forward cycle in which EWS-FLI1 leads to preferential splicing of ARID1A-L, promoting ES growth, and ARID1A-L reciprocally promotes EWS-FLI1 protein stability. Dissecting this interaction may lead to improved cancer-specific drug targeting.

Intragenic antagonistic roles of protein and circRNA in tumorigenesis.

circRNAs arise from back splicing events during mRNA processing, and when deregulated can play an active role in cancer. Here we characterize a new circRNA (circPOK) encoded by the Zbtb7a gene (also kown as POKEMON, LRF) in the context of mesenchymal tumor progression. circPOK functions as a non-coding proto-oncogenic RNA independently and antithetically to its linear transcript counterpart, which acts as a tumor suppressor by encoding the Pokemon transcription factor. We find that circPOK regulates pro-proliferative and pro-angiogenic factors by co-activation of the ILF2/3 complex. Importantly, the expression of Pokemon protein and circRNA is aberrantly uncoupled in cancer through differential post-transcriptional regulation. Thus, we identify a novel type of genetic unit, the iRegulon, that yields biochemically distinct RNA products, circular and linear, with diverse and antithetical functions. Our findings further expand the cellular repertoire towards the control of normal biological outputs, while aberrant expression of such components may underlie disease pathogenesis including cancer.

Analysis of the androgen receptor-regulated lncRNA landscape identifies a role for ARLNC1 in prostate cancer progression.

The androgen receptor (AR) plays a critical role in the development of the normal prostate as well as prostate cancer. Using an integrative transcriptomic analysis of prostate cancer cell lines and tissues, we identified ARLNC1 (AR-regulated long noncoding RNA 1) as an important long noncoding RNA that is strongly associated with AR signaling in prostate cancer progression. Not only was ARLNC1 induced by the AR protein, but ARLNC1 stabilized the AR transcript via RNA-RNA interaction. ARLNC1 knockdown suppressed AR expression, global AR signaling and prostate cancer growth in vitro and in vivo. Taken together, these data support a role for ARLNC1 in maintaining a positive feedback loop that potentiates AR signaling during prostate cancer progression and identify ARLNC1 as a novel therapeutic target.

The long noncoding RNA SPRIGHTLY acts as an intranuclear organizing hub for pre-mRNA molecules.

Molecular mechanisms by which long noncoding RNA (lncRNA) molecules may influence cancerous condition are poorly understood. The aberrant expression of SPRIGHTLY lncRNA, encoded within the drosophila gene homolog Sprouty-4 intron, is correlated with a variety of cancers, including human melanomas. We demonstrate by SHAPE-seq and dChIRP that SPRIGHTLY RNA secondary structure has a core pseudoknotted domain. This lncRNA interacts with the intronic regions of six pre-mRNAs: SOX5, SMYD3, SND1, MEOX2, DCTN6, and RASAL2, all of which have cancer-related functions. Hemizygous knockout of SPRIGHTLY by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 in melanoma cells significantly decreases SPRIGHTLY lncRNA levels, simultaneously decreases the levels of its interacting pre-mRNA molecules, and decreases anchorage-independent growth rate of cells and the rate of in vivo tumor growth in mouse xenografts. These results provide the first demonstration of an lncRNA's three-dimensional coordinating role in facilitating cancer-related gene expression in human melanomas.

Astrocyte-produced leukemia inhibitory factor expands the neural stem/progenitor pool following perinatal hypoxia-ischemia.

Brain injuries, such as cerebral hypoxia-ischemia (H-I), induce a regenerative response from the neural stem/progenitors (NSPs) of the subventricular zone (SVZ); however, the mechanisms that regulate this expansion have not yet been fully elucidated. The Notch- Delta-Serrate-Lag2 (DSL) signaling pathway is considered essential for the maintenance of neural stem cells, but it is not known if it is necessary for the expansion of the NSPs subsequent to perinatal H-I injury. Therefore, the aim of this study was to investigate whether this pathway contributes to NSP expansion in the SVZ after H-I and, if so, to establish whether this pathway is directly induced by H-I or regulated by paracrine factors. Here we report that Notch1 receptor induction and one of its ligands, Delta-like 1, precedes NSP expansion after perinatal H-I in P6 rat pups and that this increase occurs specifically in the most medial cell layers of the SVZ where the stem cells reside. Pharmacologically inhibiting Notch signaling in vivo diminished NSP expansion. With an in vitro model of H-I, Notch1 was not induced directly by hypoxia, but was stimulated by soluble factors, specifically leukemia inhibitory factor, produced by astrocytes within the SVZ. These data confirm the importance both of the Notch-DSL signaling pathway in the expansion of NSPs after H-I and in the role of the support cells in their niche. They further support the body of evidence that indicates that leukemia inhibitory factor is a key injury-induced cytokine that is stimulating the regenerative response of the NSPs. © 2016 Wiley Periodicals, Inc.

Profiling stem cell states in three-dimensional biomaterial niches using high content image informatics.

A predictive framework for the evolution of stem cell biology in 3-D is currently lacking. In this study we propose deep image informatics of the nuclear biology of stem cells to elucidate how 3-D biomaterials steer stem cell lineage phenotypes. The approach is based on high content imaging informatics to capture minute variations in the 3-D spatial organization of splicing factor SC-35 in the nucleoplasm as a marker to classify emergent cell phenotypes of human mesenchymal stem cells (hMSCs). The cells were cultured in varied 3-D culture systems including hydrogels, electrospun mats and salt leached scaffolds. The approach encompasses high resolution 3-D imaging of SC-35 domains and high content image analysis (HCIA) to compute quantitative 3-D nuclear metrics for SC-35 organization in single cells in concert with machine learning approaches to construct a predictive cell-state classification model. Our findings indicate that hMSCs cultured in collagen hydrogels and induced to differentiate into osteogenic or adipogenic lineages could be classified into the three lineages (stem, adipogenic, osteogenic) with ⩾80% precision and sensitivity, within 72h. Using this framework, the augmentation of osteogenesis by scaffold design exerted by porogen leached scaffolds was also profiled within 72h with ∼80% high sensitivity. Furthermore, by employing 3-D SC-35 organizational metrics, differential osteogenesis induced by novel electrospun fibrous polymer mats incorporating decellularized matrix could also be elucidated and predictably modeled at just 3days with high precision. We demonstrate that 3-D SC-35 organizational metrics can be applied to model the stem cell state in 3-D scaffolds. We propose that this methodology can robustly discern minute changes in stem cell states within complex 3-D architectures and map single cell biological readouts that are critical to assessing population level cell heterogeneity. STATEMENT OF SIGNIFICANCE: The sustained development and validation of bioactive materials relies on technologies that can sensitively discern cell response dynamics to biomaterials, while capturing cell-to-cell heterogeneity and preserving cellular native phenotypes. In this study, we illustrate the application of a novel high content image informatics platform to classify emergent human mesenchymal stem cell (hMSC) phenotypes in a diverse range of 3-D biomaterial scaffolds with high sensitivity and precision, and track cell responses to varied external stimuli. A major in silico innovation is the proposed image profiling technology based on unique three dimensional textural signatures of a mechanoreporter protein within the nuclei of stem cells cultured in 3-D scaffolds. This technology will accelerate the pace of high-fidelity biomaterial screening.

Role of mir-15a/16-1 in early B cell development in a mouse model of chronic lymphocytic leukemia.

In both human chronic lymphocytic leukemia (CLL) and the New Zealand Black (NZB) murine model of CLL, decreased levels of microRNAs miR-15a/16 play an important role in the disease. Here we investigate the effects of this microRNA on early steps of B cell development and the capacity of miR-15a-deficient hematopoietic stem cells (HSC) and B1 progenitor cells (B1P) to reproduce CLL-like phenotype both in vitro and in vivo. Our results demonstrate that both miR-15a deficient HSC and B1P cells are capable of repopulating irradiated recipients and produce higher numbers of B1 cells than sources with normal miR-15a/16 levels. Furthermore, induced pluripotent stem (iPS) cells derived for the first time from NZB mice, provided insights into the B cell differentiation roadblock inherent in this strain. In addition, exogenously delivered miR-15a into the NZB derived B cell line provided valuable clues into novel targets such as Mmp10 and Mt2. Our data supports the hypothesis that miR-15a/16 deficient stem cells and B1Ps experience a maturation blockage, which contributes to B1 cells bias in development. This work will help understand the role of miR-15a in early events of CLL and points to B1P cells as potential cells of origin for this incurable disease.

Alterations in the mir-15a/16-1 Loci Impairs Its Processing and Augments B-1 Expansion in De Novo Mouse Model of Chronic Lymphocytic Leukemia (CLL).

New Zealand Black (NZB) mice, a de novo model of CLL, share multiple characteristics with CLL patients, including decreased expression of miR-15a/16-1. We previously discovered a point mutation and deletion in the 3' flanking region of mir-16-1 of NZB and a similar mutation has been found in a small number of CLL patients. However, it was unknown whether the mutation is the cause for the reduced miR-15a/16-1 expression and CLL development. Using PCR and in vitro microRNA processing assays, we found that the NZB sequence alterations in the mir-15a/16-1 loci result in deficient processing of the precursor forms of miR-15a/16-1, in particular, we observe impaired conversion of pri-miR-15a/16-1 to pre-miR-15a/16-1. The in vitro data was further supported by derivation of congenic strains with replaced mir-15a/16-1 loci at one or both alleles: NZB congenic mice (NmiR+/-) and DBA congenic mice (DmiR-/-). The level of miR-15a/16-1 reflected the configuration of the mir-15a/16-1 loci with DBA congenic mice (DmiR-/-) showing reduced miR-15a levels compared to homozygous wild-type allele, while the NZB congenic mice (NmiR+/-) showed an increase in miR-15a levels relative to homozygous mutant allele. Similar to Monoclonal B-cell Lymphocytosis (MBL), the precursor stage of the human disease, an overall expansion of the B-1 population was observed in DBA congenic mice (DmiR-/-) relative to wild-type (DmiR+/+). These studies support our hypothesis that the mutations in the mir-15a/16-1 loci are responsible for decreased expression of this regulatory microRNA leading to B-1 expansion and CLL development.

Fusion FISH imaging: single-molecule detection of gene fusion transcripts in situ.

Double-stranded DNA breaks occur on a regular basis in the human genome as a consequence of genotoxic stress and errors during replication. Usually these breaks are rapidly and faithfully repaired, but occasionally different chromosomes, or different regions of the same chromosome, are fused to each other. Some of these aberrant chromosomal translocations yield functional recombinant genes, which have been implicated as the cause of a number of lymphomas, leukemias, sarcomas, and solid tumors. Reliable methods are needed for the in situ detection of the transcripts encoded by these recombinant genes. We have developed just such a method, utilizing single-molecule fluorescence in situ hybridization (sm-FISH), in which approximately 50 short fluorescent probes bind to adjacent sites on the same mRNA molecule, rendering each target mRNA molecule visible as a diffraction-limited spot in a fluorescence microscope. Utilizing this method, gene fusion transcripts are detected with two differently colored probe sets, each specific for one of the two recombinant segments of a target mRNA; enabling the fusion transcripts to be seen in the microscope as distinct spots that fluoresce in both colors. We demonstrate this method by detecting the BCR-ABL fusion transcripts that occur in chronic myeloid leukemia cells, and by detecting the EWSR1-FLI1 fusion transcripts that occur in Ewing's sarcoma cells. This technology should pave the way for accurate in situ typing of many cancers that are associated with, or caused by, fusion transcripts.