Search

SNCAIP duplication may promote Group 4 medulloblastoma via induction of PRDM6, a poorly characterized member of the PRDF1 and RIZ1 homology domain-containing (PRDM) family of transcription factors. Here, we investigated the function of PRDM6 in human hindbrain neuroepithelial stem cells and tested PRDM6 as a driver of Group 4 medulloblastoma. We report that human PRDM6 localizes predominantly to the nucleus, where it causes widespread repression of chromatin accessibility and complex alterations of gene expression patterns. Genome-wide mapping of PRDM6 binding reveals that PRDM6 binds to chromatin regions marked by histone H3 lysine 27 trimethylation that are located within, or proximal to, genes. Moreover, we show that PRDM6 expression in neuroepithelial stem cells promotes medulloblastoma. Surprisingly, medulloblastomas derived from PRDM6-expressing neuroepithelial stem cells match human Group 3, but not Group 4, medulloblastoma. We conclude that PRDM6 expression has oncogenic potential but is insufficient to drive Group 4 medulloblastoma from neuroepithelial stem cells. We propose that both PRDM6 and additional factors, such as specific cell-of-origin features, are required for Group 4 medulloblastoma. Given the lack of PRDM6 expression in normal tissues and its oncogenic potential shown here, we suggest that PRDM6 inhibition may have therapeutic value in PRDM6-expressing medulloblastomas.

BackgroundRigorous preclinical studies of chimeric antigen receptor (CAR) immunotherapy will require large quantities of consistent and high-quality CAR-transduced T (CART) cells that can be used in syngeneic mouse glioblastoma (GBM) models. To this end, we developed a novel transgenic (Tg) mouse strain with a fully murinized CAR targeting epidermal growth factor receptor variant III (EGFRvIII).MethodsWe first established the murinized version of EGFRvIII-CAR and validated its function using a retroviral vector (RV) in C57BL/6J mice bearing syngeneic SB28 GBM expressing EGFRvIII. Next, we created C57BL/6J-background Tg mice carrying the anti-EGFRvIII-CAR downstream of a Lox-Stop-Lox cassette in the Rosa26 locus. We bred these mice with CD4-Cre Tg mice to allow CAR expression on T cells and evaluated the function of the CART cells both in vitro and in vivo. To inhibit immunosuppressive myeloid cells within SB28 GBM, we also evaluated a combination approach of CART and an anti-EP4 compound (ONO-AE3-208).ResultsBoth RV- and Tg-CART cells demonstrated specific cytotoxic activities against SB28-EGFRvIII cells. A single intravenous infusion of EGFRvIII-CART cells prolonged the survival of glioma-bearing mice when preceded by a lymphodepletion regimen with recurrent tumors displaying profound EGFRvIII loss. The addition of ONO-AE3-208 resulted in long-term survival in a fraction of CART-treated mice and those survivors demonstrated delayed growth of subcutaneously re-challenged both EGFRvIII+ and parental EGFRvIII- SB28.ConclusionOur new syngeneic CAR Tg mouse model can serve as a useful tool to address clinically relevant questions and develop future immunotherapeutic strategies.

Recent studies revealed classes of recurrent DNA double-strand breaks (DSBs) in neural stem/progenitor cells, including transcription-associated, promoter-proximal breaks and recurrent DSB clusters in late-replicating, long neural genes that may give rise to somatic brain mosaicism. The mechanistic factors promoting these different classes of DSBs in neural stem/progenitor cells are not understood. Here, we elucidated the genome-wide landscape of RNA:DNA hybrid structures called "R-loops" in primary neural stem/progenitor cells undergoing aphidicolin-induced, mild replication stress to assess the potential contribution of R-loops to the different, recurrent classes of DNA break "hotspots". We find that R-loops in neural stem/progenitor cells undergoing mild replication stress are present primarily in early-replicating, transcribed regions and in genes with promoter GC skew that are associated with cell lineage-specific processes. Surprisingly, most long, neural genes that form recurrent DSB clusters do not show R-loop formation under conditions of mild replication stress. Our findings are consistent with a role of R-loop-associated processes in promoter-proximal DNA break formation in highly transcribed, early replicating regions but suggest that R-loops do not drive replication stress-induced, recurrent DSB cluster formation in most long, neural genes.

We provide a protocol for generating forebrain structures in vivo from mouse embryonic stem cells (ESCs) via neural blastocyst complementation (NBC). We developed this protocol for studies of development and function of specific forebrain regions, including the cerebral cortex and hippocampus. We describe a complete workflow, from methods for modifying a given genomic locus in ESCs via CRISPR-Cas9-mediated editing to the generation of mouse chimeras with ESC-reconstituted forebrain regions that can be directly analyzed. The procedure begins with genetic editing of mouse ESCs via CRISPR-Cas9, which can be accomplished in ~4-8 weeks. We provide protocols to achieve fluorescent labeling of ESCs in ~2-3 weeks, which allows tracing of the injected, ESC-derived donor cells in chimeras generated via NBC. Once modified ESCs are ready, NBC chimeras are generated in ~3 weeks via injection of ESCs into genetically programmed blastocysts that are subsequently transferred into pseudo-pregnant fosters. Our in vivo brain organogenesis platform is efficient, allowing functional and systematic analysis of genes and other genomic factors in as little as 3 months, in the context of a whole organism.

Early work from about two decades ago implicated DNA double-strand break (DSB) formation and repair in neuronal development. Findings emerging from recent studies of DSBs in proliferating neural progenitors and in mature, non-dividing neurons suggest important roles of DSBs in brain physiology, aging, cancer, psychiatric and neurodegenerative disorders. We provide an overview of some findings and speculate on what may lie ahead.

Genetically modified mice are commonly generated by the microinjection of pluripotent embryonic stem (ES) cells into wild-type host blastocysts1, producing chimeric progeny that require breeding for germline transmission and homozygosity of modified alleles. As an alternative approach and to facilitate studies of the immune system, we previously developed RAG2-deficient blastocyst complementation2. Because RAG2-deficient mice cannot undergo V(D)J recombination, they do not develop B or T lineage cells beyond the progenitor stage2: injecting RAG2-sufficient donor ES cells into RAG2-deficient blastocysts generates somatic chimaeras in which all mature lymphocytes derive from donor ES cells. This enables analysis, in mature lymphocytes, of the functions of genes that are required more generally for mouse development3. Blastocyst complementation has been extended to pancreas organogenesis4, and used to generate several other tissues or organs5-10, but an equivalent approach for brain organogenesis has not yet been achieved. Here we describe neural blastocyst complementation (NBC), which can be used to study the development and function of specific forebrain regions. NBC involves targeted ablation, mediated by diphtheria toxin subunit A, of host-derived dorsal telencephalic progenitors during development. This ablation creates a vacant forebrain niche in host embryos that results in agenesis of the cerebral cortex and hippocampus. Injection of donor ES cells into blastocysts with forebrain-specific targeting of diphtheria toxin subunit A enables donor-derived dorsal telencephalic progenitors to populate the vacant niche in the host embryos, giving rise to neocortices and hippocampi that are morphologically and neurologically normal with respect to learning and memory formation. Moreover, doublecortin-deficient ES cells-generated via a CRISPR-Cas9 approach-produced NBC chimaeras that faithfully recapitulated the phenotype of conventional, germline doublecortin-deficient mice. We conclude that NBC is a rapid and efficient approach to generate complex mouse models for studying forebrain functions; this approach could more broadly facilitate organogenesis based on blastocyst complementation.

Single-stranded DNA binding proteins (SSBs) regulate multiple DNA transactions, including replication, transcription, and repair. We recently identified SSB1 as a novel protein critical for the initiation of ATM signaling and DNA double-strand break repair by homologous recombination. Here we report that germline Ssb1(-/-) embryos die at birth from respiratory failure due to severe rib cage malformation and impaired alveolar development, coupled with additional skeletal defects. Unexpectedly, Ssb1(-/-) fibroblasts did not exhibit defects in Atm signaling or γ-H2ax focus kinetics in response to ionizing radiation (IR), and B-cell specific deletion of Ssb1 did not affect class-switch recombination in vitro. However, conditional deletion of Ssb1 in adult mice led to increased cancer susceptibility with broad tumour spectrum, impaired male fertility with testicular degeneration, and increased radiosensitivity and IR-induced chromosome breaks in vivo. Collectively, these results demonstrate essential roles of Ssb1 in embryogenesis, spermatogenesis, and genome stability in vivo.

Sirtuins are NAD(+)-dependent protein deacetylases. They mediate adaptive responses to a variety of stresses, including calorie restriction and metabolic stress. Sirtuin 3 (SIRT3) is localized in the mitochondrial matrix, where it regulates the acetylation levels of metabolic enzymes, including acetyl coenzyme A synthetase 2 (refs 1, 2). Mice lacking both Sirt3 alleles appear phenotypically normal under basal conditions, but show marked hyperacetylation of several mitochondrial proteins. Here we report that SIRT3 expression is upregulated during fasting in liver and brown adipose tissues. During fasting, livers from mice lacking SIRT3 had higher levels of fatty-acid oxidation intermediate products and triglycerides, associated with decreased levels of fatty-acid oxidation, compared to livers from wild-type mice. Mass spectrometry of mitochondrial proteins shows that long-chain acyl coenzyme A dehydrogenase (LCAD) is hyperacetylated at lysine 42 in the absence of SIRT3. LCAD is deacetylated in wild-type mice under fasted conditions and by SIRT3 in vitro and in vivo; and hyperacetylation of LCAD reduces its enzymatic activity. Mice lacking SIRT3 exhibit hallmarks of fatty-acid oxidation disorders during fasting, including reduced ATP levels and intolerance to cold exposure. These findings identify acetylation as a novel regulatory mechanism for mitochondrial fatty-acid oxidation and demonstrate that SIRT3 modulates mitochondrial intermediary metabolism and fatty-acid use during fasting.

DNA damage plays a causal role in numerous human pathologies including cancer, premature aging, and chronic inflammatory conditions. In response to genotoxic insults, the DNA damage response (DDR) orchestrates DNA damage checkpoint activation and facilitates the removal of DNA lesions. The DDR can also arouse the immune system by for example inducing the expression of antimicrobial peptides as well as ligands for receptors found on immune cells. The activation of immune signaling is triggered by different components of the DDR including DNA damage sensors, transducer kinases, and effectors. In this review, we describe recent advances on the understanding of the role of DDR in activating immune signaling. We highlight evidence gained into (i) which molecular and cellular pathways of DDR activate immune signaling, (ii) how DNA damage drives chronic inflammation, and (iii) how chronic inflammation causes DNA damage and pathology in humans. [ABSTRACT FROM AUTHOR]