Epigenetic Editing of Ascl1 Gene in Neural Stem Cells by Optogenetics

Enzymes involved in epigenetic processes such as methyltransferases or demethylases are becoming highly utilized for their persistent DNA or histone modifying efficacy. Herein, we have developed an optogenetic toolbox fused to the catalytic domain (CD) of DNA-methyltransferase3A (DNMT3A-CD) or Ten-Eleven Dioxygenase-1 (TET1-CD) for loci-specific alteration of the methylation state at the promoter of Ascl1 (Mash1), a candidate proneuron gene. Optogenetical protein pairs, CRY2 linked to DNMT3A-CD or TET1-CD and CIB1 fused to a Transcription Activator-Like Element (TALE) locating an Ascl1 promoter region, were designed for site specific epigenetic editing. A differentially methylated region at the Ascl1 promoter, isolated from murine dorsal root ganglion (hypermethylated) and striated cells (hypomethylated), was targeted with these optogenetic-epigenetic constructs. Optimized blue-light illumination triggered the co-localization of TALE constructs with DNMT3A-CD or TET1-CD fusion proteins at the targeted site of the Ascl1 promoter. We found that this spatiotemporal association of the fusion proteins selectively alters the methylation state and also regulates gene activity. This proof of concept developed herein holds immense promise for the ability to regulate gene activity via epigenetic modulation with spatiotemporal precision

Cell-Wide DNA De-Methylation and Re-Methylation of Purkinje Neurons in the Developing Cerebellum

Global DNA de-methylation is thought to occur only during pre-implantation and gametogenesis in mammals. Scalable, cell-wide de-methylation has not been demonstrated beyond totipotent stages. Here, we observed a large scale de-methylation and subsequent re-methylation (CDR) (including 5-methylcytosine (5mC) and 5-hydroxylmethylcytosine (5hmC)) in post-mitotic cerebellar Purkinje cells (PC) through the course of normal development. Through single cell immuno-identification and cell-specific quantitative methylation assays, we demonstrate that the CDR event is an intrinsically scheduled program, occurring in nearly every PC. Meanwhile, cerebellar granule cells and basket interneurons adopt their own DNA methylation program, independent of PCs. DNA de-methylation was further demonstrated at the gene level, on genes pertinent to PC development. The PC, being one of the largest neurons in the brain, may showcase an amplified epigenetic cycle which may mediate stage transformation including cell cycle arrest, vast axonal-dendritic growth, and synaptogenesis at the onset of neuronal specificity. This discovery is a key step toward better understanding the breadth and role of DNA methylation and de-methylation during neural ontology

Cis‐acting allele specific expression (ASE) differences induced by alcohol and impacted by sex as well as parental genotype of origin

Background Alcohol use disorders (AUDs) are influenced by complex interactions between the genetics of the individual and their environment. We have previously identified hundreds of polygenic genetic variants between the selectively bred high and low alcohol drinking (HAD and LAD) rat lines. Here we report allele specific expression (ASE) differences, between the HAD2 and LAD2 rat lines. Methods The HAD2 and LAD2 rats which have been sequenced were reciprocally crossed to generate 10 litters of F1 progeny. For 5 of these litters, the sire was HAD2; and, for the other 5 litters, the sire was a LAD2. From these 10 litters, two males and two females were picked from each F1 litter (N = 40 total). The F1‐pups were divided, with balancing for sex and direction of cross, into an alcohol (15%) vs a water control group. Alcohol‐drinking started in the middle of adolescence (~PND 35) and lasted 9 weeks. At the end of these treatments, rats were euthanized, the nucleus accumbens was dissected, and RNA was processed for RNA‐sequencing and ASE analyses. Results Analyses revealed that adolescent ethanol drinking, individual ethanol drinking levels, parentage, and sex‐of‐animal affected ASEs of about 300 genes. The identified genes included those associated with ethanol metabolism (e.g., Aldh2); neuromodulatory function [e.g., Cckbr, Slc6a7, and Slc1a1]; ion channel activity (e.g., Kcnc3); as well as other synaptic and epigenetic function. Conclusion These data indicate that ethanol drinking differentially amplified paternal vs maternal allelic contribution to the transcriptome. We hypothesize that this was due, at least in part, to ethanol‐induced changes in cis‐regulation of polymorphisms previously identified between the HAD2 and LAD2 rat lines. This report highlights the complexity of gene‐by‐environment interactions mediating a genetic predisposition for, and/or the active development of, alcohol use disorders

Optogenetic regulation of site-specific subtelomeric DNA methylation

Telomere length homeostasis, critical for chromosomal integrity and genome stability, is controlled by intricate molecular regulatory machinery that includes epigenetic modifications. Here, we examine site-specific and spatiotemporal alteration of the subtelomeric methylation of CpG islands using optogenetic tools to understand the epigenetic regulatory mechanisms of telomere length maintenance. Human DNA methyltransferase3A (DNMT3A) were assembled selectively at chromosome ends by fusion to cryptochrome 2 protein (CRY2) and its interacting complement, the basic helix loop helix protein-1 (CIB1). CIB1 was fused to the telomere-associated protein telomere repeat binding factor-1 (TRF1), which localized the protein complex DNMT3A-CRY2 at telomeric regions upon excitation by blue-light monitored by single-molecule fluorescence analyses. Increased methylation was achieved selectively at subtelomeric CpG sites on the six examined chromosome ends specifically after blue-light activation, which resulted in progressive increase in telomere length over three generations of HeLa cell replications. The modular design of the fusion constructs presented here allows for the selective substitution of other chromatin modifying enzymes and for loci-specific targeting to regulate the epigenetic pathways at telomeres and other selected genomic regions of interest

Expert-Novice Differences in SMR Activity during Dart Throwing

Cheng M-Y, Hung C-L, Huang C-J, et al. Expert-Novice Differences in SMR Activity during Dart Throwing. Biological Psychology. 2015;110:212-218.Previous evidence suggests that augmented sensorimotor rhythm (SMR) activity is related to the superior regulation of processing cognitive-motor information in motor performance. However, no published studies have examined the relationship between SMR and performance in precision sports; thus, this study examined the relationship between SMR activity and the level of skilled performance in tasks requiring high levels of attention (e.g., dart throwing). We hypothesized that skilled performance would be associated with higher SMR activity. Fourteen dart-throwing experts and eleven novices were recruited. Participants were requested to perform 60 dart throws while EEG was recorded. The 2 (Group: Expert, Novice) x 2 (Time window: –2000 ms to –1000 ms, –1000 ms to 0 ms) ANOVA showed that the dart-throwing experts maintained a relatively higher SMR power than the novices before dart release. These results suggest that SMR might reflect the adaptive regulation of cognitive-motor processing during the preparatory period

High Resolution Genomic Scans Reveal Genetic Architecture Controlling Alcohol Preference in Bidirectionally Selected Rat Model

Investigations on the influence of nature vs. nurture on Alcoholism (Alcohol Use Disorder) in human have yet to provide a clear view on potential genomic etiologies. To address this issue, we sequenced a replicated animal model system bidirectionally-selected for alcohol preference (AP). This model is uniquely suited to map genetic effects with high reproducibility, and resolution. The origin of the rat lines (an 8-way cross) resulted in small haplotype blocks (HB) with a corresponding high level of resolution. We sequenced DNAs from 40 samples (10 per line of each replicate) to determine allele frequencies and HB. We achieved ~46X coverage per line and replicate. Excessive differentiation in the genomic architecture between lines, across replicates, termed signatures of selection (SS), were classified according to gene and region. We identified SS in 930 genes associated with AP. The majority (50%) of the SS were confined to single gene regions, the greatest numbers of which were in promoters (284) and intronic regions (169) with the least in exon\u27s (4), suggesting that differences in AP were primarily due to alterations in regulatory regions. We confirmed previously identified genes and found many new genes associated with AP. Of those newly identified genes, several demonstrated neuronal function involved in synaptic memory and reward behavior, e.g. ion channels (Kcnf1, Kcnn3, Scn5a), excitatory receptors (Grin2a, Gria3, Grip1), neurotransmitters (Pomc), and synapses (Snap29). This study not only reveals the polygenic architecture of AP, but also emphasizes the importance of regulatory elements, consistent with other complex traits

Notchless interacts with multiple signaling pathways during mouse peri-implantation development

During peri-implantation, the embryo transitions from a suspension environment in the fallopian tubes to an adherent system within the uterus. Successful transition requires maternal and fetal signaling cascades that establish maternal-fetal boundaries. Failure is common, as ~ 25% of all human pregnancies terminate during these steps. A large-scale mutation study in mice produced two mutants (l11Jus1 and l11Jus4) that are excellent models of this transition. l11Jus1 and l11Jus4 contain missense mutations in the Notchless homolog 1 ( Drosophila) (Nle1) gene. NLE1 is thought to signal via the canonical NOTCH pathway in vertebrates. Although in invertebrates and lower vertebrates, NOTCH signaling directs cell fate prior to gastrulation, it is dispensable for gastrulation in mice. Moreover, in yeast and plants, which lack NOTCH signaling, Nle1 is crucial for ribosome biogenesis. These seemingly contradictory data led me to hypothesize that mutation of Nle1 causes a lethal trauma to the embryo that disrupts multiple signaling pathways during peri-implantation development. I present data that: 1. Refute the presumption that Nle1 functions as a negative regulator of NOTCH during pre-implantation development; 2. Demonstrate that mutations in Nle1 lead to mis-expression of several members of the Wnt pathway; and 3. Show that mutant embryos enter cell cycle arrest; when that fails, they undergo p53-mediated programmed cell death. To understand the trauma(s) that precipitated these lethal cascades, I discovered that Nle1 mutants display delays in ribosomal RNA processing and nucleogenesis. These results uncover novel functions for NLE1 in the ribosomal biogenesis, TRP53 and WNT pathways during mammalian embryonic development

Study on the Impact Factors of using Residential Photovoltaic System via the Technology Acceptance Model (TAM)

[[abstract]]To this day, human beings have globally faced so many serious problems, such as oil crisis, environmental pollution, global warming, energy security and so on. The oil crisis brings the fears of energy supply competition among the countries. The environments are also impacted seriously by all kinds of pollutions and the trend of global warming. Recently, the energy security has become a threatened issue for human society such as the tragedy of Fukushima Accident in 2011. Therefore, many countries worldwide devote funding to develop Renewable energy resources. It is important to understand the development and changes in this field further. This study focuses on exploring the buildings employed with a residential photovoltaic system in Taiwan through the Technology Acceptance Model (TAM) to understanding the customer perception influenced by attitude and intention of buying a residential photovoltaic system as well as other possible factors. Besides, we import External Variables and Environmental Attitude to explore this issue. This study is based on questionnaire survey. In total of 280 questionnaires were issued and 278 valid questionnaires were collected. The data was analyzed by descriptive statistics, reliability analysis, validity analysis, etc. Partial Least Squares is applied to test and verify the research results. According to the results, four conclusions can be made below: 1.The acceptability and attitude of customer perception are positive impacts on usage intention toward using a residential photovoltaic system. 2.External Variables is a positive impact on customers’ perceived usefulness toward using a residential photovoltaic system. 3.Environmental Attitude is a positive impact on customers’ attitude toward using a residential photovoltaic system. 4.Overall, the impact factors of using a residential photovoltaic system not only bases on the frame work of the Technology Acceptance Model itself but also include Environmental Attitude and External Variables such as oil price, commodity price, generating efficiency, and government subsidy policy. All of these factors are related to the customers’ decision of using a residential photovoltaic system

[[alternative]]The influences of gender stereotype in prior knowledge and teaching intervention on children's classification

[[abstract]]　　鑑於國內探討幼兒分類概念行為的相關研究十分有限，幼兒分類概念的發展與分類能力的培養，一直深為研究者所關切。本研究即在探討大班幼兒先備知識裡的性別刻板印象與多重概念相衝突時的分類行為，及經由教師教學活動後幼兒的分類反應，以進行二階段實驗以獲取資料。第一階段是透過實驗設計，瞭解受試幼兒在同時面對具有性別刻板印象（先備知識）及腳本關係（多重概念系統）的圖片時，如何抉擇進行分類任務。第二階段先將受試幼兒分為實驗組與對照組，針對實驗組進行為期兩個月，每週兩次的性別教育故事教學活動，之後再重複進行分類任務，以瞭解故事教學活動的介入是否會改變幼兒分類行為。實驗結果顯示：大班幼兒在前測上，不分組別，男女生的分類方式並無顯著的差異，且分類方式與月齡並無相關，而再觀看幼兒父母的社經地位背景，發現性別刻板化的程度與父親學歷與職業有顯著的差異，但與母親學歷與職業卻未有顯著的不同。實驗組在教學活動過後，發現故事教學活動能影響大班幼兒的性別刻板分類方式，其中，男生前測與後測的分類方式並沒有顯著的不同，但女生卻有明顯的差異

Taking a Different Pathway: Nle1 Plays with Wnt During Embryogenesis

<p>We discovered that mutations in <em>Nle1</em>, a gene that is implicated in the Notch pathway in <em>Drosophila</em>, leads to misregulation of several <em>Wnt</em> family members, but not <em>Notch</em> target genes, in mouse embryos.</p