Increased nicotine response in iPSC-derived human neurons carrying the CHRNA5 N398 allele

Genetic variation in nicotinic receptor alpha 5 (CHRNA5) has been associated with increased risk of addiction-associated phenotypes in humans yet little is known the underlying neural basis. Induced pluripotent stem cells (iPSCs) were derived from donors homozygous for either the major (D398) or the minor (N398) allele of the nonsynonymous single nucleotide polymorphism (SNP), rs16969968, in CHRNA5. To understand the impact of these nicotinic receptor variants in humans, we differentiated these iPSCs to dopamine (DA) or glutamatergic neurons and then tested their functional properties and response to nicotine. Results show that N398 variant human DA neurons differentially express genes associated with ligand receptor interaction and synaptic function. While both variants exhibited physiological properties consistent with mature neuronal function, the N398 neuronal population responded more actively with an increased excitatory postsynaptic current response upon the application of nicotine in both DA and glutamatergic neurons. Glutamatergic N398 neurons responded to lower nicotine doses (0.1 μM) with greater frequency and amplitude but they also exhibited rapid desensitization, consistent with previous analyses of N398-associated nicotinic receptor function. This study offers a proof-of-principle for utilizing human neurons to study gene variants contribution to addiction

Ago2 Immunoprecipitation Identifies Predicted MicroRNAs in Human Embryonic Stem Cells and Neural Precursors

MicroRNAs are required for maintenance of pluripotency as well as differentiation, but since more microRNAs have been computationally predicted in genome than have been found, there are likely to be undiscovered microRNAs expressed early in stem cell differentiation.SOLiD ultra-deep sequencing identified >10(7) unique small RNAs from human embryonic stem cells (hESC) and neural-restricted precursors that were fit to a model of microRNA biogenesis to computationally predict 818 new microRNA genes. These predicted genomic loci are associated with chromatin patterns of modified histones that are predictive of regulated gene expression. 146 of the predicted microRNAs were enriched in Ago2-containing complexes along with 609 known microRNAs, demonstrating association with a functional RISC complex. This Ago2 IP-selected subset was consistently expressed in four independent hESC lines and exhibited complex patterns of regulation over development similar to previously-known microRNAs, including pluripotency-specific expression in both hESC and iPS cells. More than 30% of the Ago2 IP-enriched predicted microRNAs are new members of existing families since they share seed sequences with known microRNAs.Extending the classic definition of microRNAs, this large number of new microRNA genes, the majority of which are less conserved than their canonical counterparts, likely represent evolutionarily recent regulators of early differentiation. The enrichment in Ago2 containing complexes, the presence of chromatin marks indicative of regulated gene expression, and differential expression over development all support the identification of 146 new microRNAs active during early hESC differentiation

Transcriptomic profiling of splenic B lymphomas spontaneously developed in B cell-specific TRAF3-deficient mice

AbstractTRAF3, a critical regulator of B cell survival, was recently recognized as a tumor suppressor gene in B lymphocytes. Specific deletion of TRAF3 from B lymphocytes leads to spontaneous development of marginal zone lymphomas (MZL) or B1 lymphomas in mice. To identify novel oncogenes and tumor suppressive genes involved in malignant transformation of TRAF3-deficient B cells, we performed a microarray analysis to identify genes differentially expressed in TRAF3−/− mouse splenic B lymphomas. We have identified 160 up-regulated genes and 244 down-regulated genes in TRAF3−/−B lymphomas as compared to littermate control splenocytes. Here we describe the samples, quality control assessment, as well as the data analysis methods in detail for the transcriptomic profiling study. Data are archived at NIH GEO with accession number GSE48818

A positive feedback mechanism that regulates expression of miR-9 during neurogenesis.

MiR-9, a neuron-specific miRNA, is an important regulator of neurogenesis. In this study we identify how miR-9 is regulated during early differentiation from a neural stem-like cell. We utilized two immortalized rat precursor clones, one committed to neurogenesis (L2.2) and another capable of producing both neurons and non-neuronal cells (L2.3), to reproducibly study early neurogenesis. Exogenous miR-9 is capable of increasing neurogenesis from L2.3 cells. Only one of three genomic loci capable of encoding miR-9 was regulated during neurogenesis and the promoter region of this locus contains sufficient functional elements to drive expression of a luciferase reporter in a developmentally regulated pattern. Furthermore, among a large number of potential regulatory sites encoded in this sequence, Mef2 stood out because of its known pro-neuronal role. Of four Mef2 paralogs, we found only Mef2C mRNA was regulated during neurogenesis. Removal of predicted Mef2 binding sites or knockdown of Mef2C expression reduced miR-9-2 promoter activity. Finally, the mRNA encoding the Mef2C binding partner HDAC4 was shown to be targeted by miR-9. Since HDAC4 protein could be co-immunoprecipitated with Mef2C protein or with genomic Mef2 binding sequences, we conclude that miR-9 regulation is mediated, at least in part, by Mef2C binding but that expressed miR-9 has the capacity to reduce inhibitory HDAC4, stabilizing its own expression in a positive feedback mechanism

Spontaneous ATM Gene Reversion in A-T iPSC to Produce an Isogenic Cell Line

A spontaneously reverted iPSC line was identified from an A-T subject with heterozygous ATM truncation mutations. The reverted iPSC line expressed ATM protein and was capable of radiation-induced phosphorylation of CHK2 and H2A.X. Genome-wide SNP analysis confirmed a match to source T cells and also to a distinct, non-reverted iPSC line from the same subject. Rearranged T cell receptor sequences predict that the iPSC culture originated as several independently reprogrammed cells that resolved into a single major clone, suggesting that gene correction likely occurred early in the reprogramming process. Gene expression analysis comparing ATM−/− iPSC lines to unrelated ATM+/− cells identifies a large number of differences, but comparing only the isogenic pair of A-T iPSC lines reveals that the primary pathway affected by loss of ATM is a diminished expression of p53-related mRNAs. Gene reversion in culture, although likely a rare event, provided a novel, reverted cell line for studying ATM function

Mef2C is regulated during neurogenesis, in turn contributing to miR-9 induction.

<p>(A) qRT-PCR analysis of each of the four Mef2 paralogs during differentiation of L2.2 cells. Results are normalized to FGF-treated L2.3 cultures (not shown). Comparisons were determined to be significantly different by Student's T-test with p<0.05. As anticipated, Mef2C is the only isoform that is significantly increased during neurogenesis in the L2.2 cells. Mef2A and Mef2D transcripts both decreased during differentiation of L2.3 cells. Mef2B transcripts were not detected; however primers were able to amplify a genomic DNA positive control (not shown). (B) Splice variants of the mammalian Mef2C transcript. (C) Deletion of Mef2 binding sites in the upstream regulatory region diminishes transcription activity of the miR-9-2 gene locus. A 5 kb upstream sequence of the rat miR-9-2 gene was cloned into the promoter-less pGL4.10 luciferase reporter vector (wild-type, WT). Two highly conserved Mef2 binding sites (Mef2.1 and Mef2.2 respectively; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094348#pone-0094348-g001" target="_blank">Fig. 1E</a>) were deleted in separate plasmids and a double-deletion plasmid was constructed as well. Plasmids were co-transfected with a Renilla transfection control reporter into undifferentiated L2.2 cultures. pGL4.10 and the full-length 5 Kb upstream sequence were transfected as controls. Ratios are expressed here as percentage of full-length miR-9-2 upstream sequence activity and differences from WT promoter were determined to be significant via Student's T-test with p<0.05. (D) Knockdown of Mef2C in neural precursors leads to a decrease in miR-9-2 promoter activity. FL/RL ratios were determined for replicate cultures of differentiated L2.2 cells with or without Mef2c shRNAs (Student's T-test, p<0.05). In this assay, a significant decrease in the abundance of luciferase was detected in the cultures electroporated with inhibitors for Mef2c. This reduction was abolished with a control hairpin with a specific mismatch for the Mef2c protein.</p

Model of Mef2c/HDAC4 regulation of miR-9 expression during neurogenesis.

<p>Cells becoming non-neuronal cells (top) would express both MEF2C and HDAC4, forming an inhibitory complex binding with sites upstream of miR-9-2 to suppress transcription. Cells becoming neurons (bottom) switch to a state where miR-9 inhibits production of HDAC4, allowing a pro-transcriptional binding of MEF2C upstream of miR-9-2.</p

miR-9 targets HDAC4 mRNA.

<p>(A) The wild-type version of the miR-9 response element 1 (RE1) of the HDAC4 3′UTR aligned with the miR-9 sequence and the mutated form (RE1mt). (B) Differentiation of L2.2 cells causes repression of a 1.2 kb fragment of the HDAC4 3′UTR containing miR-9 response element. Results show the FL/RL luciferase ratio expressed relative to the pMir control to compare multiple differentiation days. The reduced luciferase expression at Day 3 correlates with increased miR-9 expression upon FGF withdrawal-induced differentiation. (C) Exogenous expression of miR-9 reduces luciferase activity from a clone containing the 1.2 kb fragment of the HDAC4 3′UTR containing miR-9 response element. Levels of miR-9 were increased by transfecting HeLa cells with either a plasmid vector expressing rat genomic sequences surrounding miR-9 (pSI miR-9) or a synthetic miR-9 gene constructed in the Block-IT expression vector (Life Technologies). Both cases led to increased miR-9 levels as assayed by qPCR (not shown). (D) A single predicted miR-9 wild-type (RE1) or mutant (RE1mt) response element was cloned into the pMir luciferase construct. Mutating the miR-9 RE derepresses luciferase expression, with or without FGF (p = 0.029 for RE1 vs. RE1mt, ANOVA). pMir vector containing the miR-9 RE1 was transfected into L2.2 cells. Cells were assayed for luciferase one day later, either with or without FGF. (E) Inhibiting endogenous miR-9 levels in L2.2 cells using Anti-miR-9 increases luciferase expression. Anti-miR-9 or scrambled negative control RNA (Life Technologies) were nucleofected into L2.2 cells. Cells were collected for luciferase assays after one day. *p<0.05 by Student's t-test.</p

HDAC4 negatively regulates miR-9-2 promoter by binding Mef2C.

<p>(A) Infection of multipotential L2.3 cells with lentiviral-encoded shRNA specific for HDAC4 leads to increased miR-9 levels. Cultures were infected with lentiviruses and sampled 2 or 3 days later. MiR-9 was assessed using a TaqMan miRNA assay (Life Technologies). Relative Quantity (RQ) was calculated relative to RNU43 endogenous control. *p<0.05 by Student's t-test, compared with 3 days of no virus. (B) Co-immunoprecipitation of Mef2C and HDAC4. A GFP-HDAC4 fusion cDNA was expressed in N2a cells with or without a Mef2C expression clone and immunoprecipitated with antibodies against GFP or MEF2C as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094348#pone.0094348-Li3" target="_blank">[37]</a>. (C) Chromatin immunoprecipitation with anti-HDAC4 enriches Mef2 binding sites upstream of mouse miR-9-2. HDAC4 immunoprecipitated chromatin from homogenized mouse brain was PCR amplified for three predicted Mef2 binding sites (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094348#pone.0094348.s003" target="_blank">Fig. S3</a>). Primer sequences are found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094348#pone.0094348.s005" target="_blank">Table S1</a>. Results are expressed as fold increase relative to IgG-immunoprecipitated chromatin (*p<0.05 Student's t-test, compared with IgG).</p