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<p>Structural and functional plasticity of synapses are critical neuronal mechanisms underlying learning and memory. While activity-dependent regulation of synaptic strength has been extensively studied, much less is known about the transcriptional control of synapse maintenance and plasticity. Hippocampal mossy fiber (MF) synapses connect dentate granule cells to CA3 pyramidal neurons and are important for spatial memory formation and consolidation. The transcription factor Bcl11b/Ctip2 is expressed in dentate granule cells and required for postnatal hippocampal development. Ablation of Bcl11b/Ctip2 in the adult hippocampus results in impaired adult neurogenesis and spatial memory. The molecular mechanisms underlying the behavioral impairment remained unclear. Here we show that selective deletion of Bcl11b/Ctip2 in the adult mouse hippocampus leads to a rapid loss of excitatory synapses in CA3 as well as reduced ultrastructural complexity of remaining mossy fiber boutons (MFBs). Moreover, a dramatic decline of long-term potentiation (LTP) of the dentate gyrus-CA3 (DG-CA3) projection is caused by adult loss of Bcl11b/Ctip2. Differential transcriptomics revealed the deregulation of genes associated with synaptic transmission in mutants. Together, our data suggest Bcl11b/Ctip2 to regulate maintenance and function of MF synapses in the adult hippocampus.</p

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<p>Structural and functional plasticity of synapses are critical neuronal mechanisms underlying learning and memory. While activity-dependent regulation of synaptic strength has been extensively studied, much less is known about the transcriptional control of synapse maintenance and plasticity. Hippocampal mossy fiber (MF) synapses connect dentate granule cells to CA3 pyramidal neurons and are important for spatial memory formation and consolidation. The transcription factor Bcl11b/Ctip2 is expressed in dentate granule cells and required for postnatal hippocampal development. Ablation of Bcl11b/Ctip2 in the adult hippocampus results in impaired adult neurogenesis and spatial memory. The molecular mechanisms underlying the behavioral impairment remained unclear. Here we show that selective deletion of Bcl11b/Ctip2 in the adult mouse hippocampus leads to a rapid loss of excitatory synapses in CA3 as well as reduced ultrastructural complexity of remaining mossy fiber boutons (MFBs). Moreover, a dramatic decline of long-term potentiation (LTP) of the dentate gyrus-CA3 (DG-CA3) projection is caused by adult loss of Bcl11b/Ctip2. Differential transcriptomics revealed the deregulation of genes associated with synaptic transmission in mutants. Together, our data suggest Bcl11b/Ctip2 to regulate maintenance and function of MF synapses in the adult hippocampus.</p

Identification of <i>Rtl1</i>, a Retrotransposon-Derived Imprinted Gene, as a Novel Driver of Hepatocarcinogenesis

<div><p>We previously utilized a Sleeping Beauty (SB) transposon mutagenesis screen to discover novel drivers of HCC. This approach identified recurrent mutations within the <i>Dlk1-Dio3</i> imprinted domain, indicating that alteration of one or more elements within the domain provides a selective advantage to cells during the process of hepatocarcinogenesis. For the current study, we performed transcriptome and small RNA sequencing to profile gene expression in SB–induced HCCs in an attempt to clarify the genetic element(s) contributing to tumorigenesis. We identified strong induction of <i>Retrotransposon-like 1</i> (<i>Rtl1</i>) expression as the only consistent alteration detected in all SB–induced tumors with <i>Dlk1-Dio3</i> integrations, suggesting that <i>Rtl1</i> activation serves as a driver of HCC. While previous studies have identified correlations between disrupted expression of multiple <i>Dlk1-Dio3</i> domain members and HCC, we show here that direct modulation of a single domain member, <i>Rtl1</i>, can promote hepatocarcinogenesis <i>in vivo</i>. Overexpression of <i>Rtl1</i> in the livers of adult mice using a hydrodynamic gene delivery technique resulted in highly penetrant (86%) tumor formation. Additionally, we detected overexpression of <i>RTL1</i> in 30% of analyzed human HCC samples, indicating the potential relevance of this locus as a therapeutic target for patients. The <i>Rtl1</i> locus is evolutionarily derived from the domestication of a retrotransposon. In addition to identifying <i>Rtl1</i> as a novel driver of HCC, our study represents one of the first direct <i>in vivo</i> demonstrations of a role for such a co-opted genetic element in promoting carcinogenesis.</p> </div

Rtl1 promotes growth of cultured hepatocytes in extracellular matrix.

<p>Two weeks after plating cultured hepatocytes in a matrix of Matrigel, cells transfected with an empty vector construct (A) failed to grow significantly. Cells transfected with an <i>Rtl1</i> expression construct (B) grew to form several large colonies. (C–D) Rtl1 promotes growth of large cyst-like structures. Increased magnification reveals that cells lacking Rtl1 (C) form small, dense colonies, while those expressing Rtl1 (D) form large cyst-like colonies composed of several cells. (E) Quantification of colonies per well formed by each cell line in a 24-well plate. The results depicted are based on three experimental replicates per condition and are representative of experiments conducted on three separate days. Scale bars = 0.5 cm (A–B) and 100 µm (C–D).</p

Expression of <i>RTL1</i> in human HCC.

<p>(A) <i>RTL1</i> expression in human HCC and matched benign liver samples was analyzed by RT-PCR. Plotted values represent normalized band intensities from imaged gels. The threshold above which a sample was scored as positive for significant <i>RTL1</i> expression (dashed line) was set at three standard deviations above the average intensity value in benign samples lacking detectable expression. For the one patient with significant <i>RTL1</i> expression detected in benign tissue, the matched HCC sample also displayed expression (indicated with arrows). (B) Plot of <i>RTL1</i> expression in human HCC and normal liver samples based on RNASeq data available through TCGA. The threshold above which a sample was scored as positive for significant <i>RTL1</i> expression (dashed line) was set at one standard deviation above the average expression level in tumor-free liver. RSEM, RNASeq by Expectation Maximization.</p

<i>Dlk1-Dio3</i> domain transposon integration sites in SB–induced HCC and effects on domain expression.

<p>(A) The <i>Dlk1-Dio3</i> imprinted domain spans ∼800 kilobases at the distal end of mouse chromosome 12 (human chr14q32). Three protein-coding genes are expressed from the paternal allele (<i>Dlk1</i>, <i>Rtl1</i>, and <i>Dio3</i>). The maternal allele encodes four lncRNAs (<i>Meg3</i>, <i>Rtl1as</i>, <i>Rian</i>, and <i>Mirg</i>), as well as several miRNAs and snoRNAs. SB transposon and AAV integration sites found to be associated with HCC development in mice are depicted. (B) Intensity plot showing normalized expression levels of long transcripts within and surrounding the <i>Dlk1-Dio3</i> domain in SB-induced HCCs and normal livers. (C) Intensity plot showing normalized expression of <i>Dlk1-Dio3</i> domain miRNAs. miRNAs contained within lncRNAs are indicated. miRNAs with no detected expression across all samples were omitted.</p

<i>Rtl1</i>-expressing mouse HCCs resemble human S1 subclass.

<p>Expression levels for the gene sets defining human HCC subclasses S1, S2, and S3 were analyzed in SB-induced HCCs and normal livers. Gene Set Enrichment Analysis (GSEA) was conducted for each subclass independently to assess the significance of differential expression between tumor and normal samples. Heat maps generated by GSEA are shown. This analysis revealed a significant (p = 0.039) overexpression of the genes defining human subclass S1 in SB-induced HCCs, as compared to normal liver.</p

<i>In vivo</i> hepatic overexpression of <i>Rtl1</i> promotes tumorigenesis.

<p>(A–C) Macroscopic images of tumor-containing whole livers from mice injected with <i>Rtl1</i> overexpression constructs via hydrodynamic tail vein injection. Mice were euthanized and livers collected nine months post-injection. (D) A normal liver from a hydrodynamically injected mouse is shown for comparison. Scale bars = 1 cm.</p

Integrated transposons drive overexpression of <i>Rtl1</i>.

<p>(A) Strand-specific RT-PCR detected activation of <i>Rtl1</i> expression in SB-induced HCCs, with minimal activation of <i>Rtl1as</i> observed. No expression of either transcript was detected in normal liver. (B) Transposons integrated upstream of <i>Rtl1</i> drive its expression by generating fusion transcripts. Transcription initiated from the CAG promoter within the transposon splices into <i>Rtl1</i>, either directly or via inclusion of an upstream cryptic exon. PCR to detect fusion transcripts was performed on cDNA from SB-induced HCCs using the indicated primers. Transposon-driven expression of <i>Rtl1</i> was detected for all of the tumor samples harboring <i>Dlk1-Dio3</i> domain integrations. sd, splice donor.</p

Cardiac malformations and failure in Evi1^δex3/δex3 mice.

<p>(A) Transverse sections and (B) 3D reconstruction (left-ventral oblique view) of hearts from Evi1^δex3/δex3 or wild type littermate (+/+) E15.5 embryos analyzed by magnetic resonance imaging (MRI). The aorta (Ao), right ventricle (RV), left ventricle (LV), ventricular septum (VS), trachea (Tr), aortic arch (AoA) and ductus arteriosus (DA) are indicated. Ventricular septal defect (VSD), interrupted aortic arch (IAA) and common arterial trunk (CAT) were observed in Evi1^δex3/δex3 hearts. (C) List of the congenital heart defects identified in fifteen E15.5 embryos of various different genotypes by MRI and 3D reconstruction. (D) Hematoxylin and eosin staining of 5 µm sections of a sick Evi1^δex3/δex3 pup. Subcutaneous and other tissue edema (white spaces) was present, consistent with heart failure.</p