Research on the Initiative Scientific Research and Innovation Behavior of Postgraduates: Based on the Theory of Planned Behavior.

To clarify the generative mechanism and influencing factors of graduate students' willingness and behavior of initiative scientific research and innovation, this research builds a quantitative model consisting of six variables: academic interest, regulatory pressure, conditions for improving scientific research and innovation capabilities, willingness to take initiative in scientific research and innovation, the promotion of achievements in scientific research, and initiative scientific research and innovation behavior. In total, 684 valid questionnaires were distributed and collected through WeChat Moments. Descriptive statistical analysis, reliability and validity analysis, factor analysis, path analysis, and conditioning analysis were conducted using the SPSS and structural equation model (SEM). The results showed that: (1) academic interest, regulatory pressure, and conditions for improving scientific research and innovation capabilities have a positive impact on the intention of initiative scientific research and innovation, but the impact of regulatory pressure is extremely insignificant. (2) The willingness to take initiative in scientific research and innovation has a positive and significant impact on initiative scientific research and innovation behavior, and the promotion of achievements in scientific research plays a negative moderating effect. Finally, this manuscript puts forward the suggestions on how to promote the initiative scientific research and innovation behavior of postgraduate students based on the research results.

Lin28A Binds Active Promoters and Recruits Tet1 to Regulate Gene Expression.

Lin28, a well-known RNA-binding protein, regulates diverse cellular properties. All physiological functions of Lin28A characterized so far have been attributed to its repression of let-7 miRNA biogenesis or modulation of mRNA translational efficiency. Here we show that Lin28A directly binds to a consensus DNA sequence in vitro and in mouse embryonic stem cells in vivo. ChIP-seq and RNA-seq reveal enrichment of Lin28A binding around transcription start sites and a positive correlation between its genomic occupancy and expression of many associated genes. Mechanistically, Lin28A recruits 5-methylcytosine-dioxygenase Tet1 to genomic binding sites to orchestrate 5-methylcytosine and 5-hydroxymethylcytosine dynamics. Either Lin28A or Tet1 knockdown leads to dysregulated DNA methylation and expression of common target genes. These results reveal a surprising role for Lin28A in transcriptional regulation via epigenetic DNA modifications and have implications for understanding mechanisms underlying versatile functions of Lin28A in mammalian systems.

DNA methylation presents distinct binding sites for human transcription factors.

DNA methylation, especially CpG methylation at promoter regions, has been generally considered as a potent epigenetic modification that prohibits transcription factor (TF) recruitment, resulting in transcription suppression. Here, we used a protein microarray-based approach to systematically survey the entire human TF family and found numerous purified TFs with methylated CpG (mCpG)-dependent DNA-binding activities. Interestingly, some TFs exhibit specific binding activity to methylated and unmethylated DNA motifs of distinct sequences. To elucidate the underlying mechanism, we focused on Kruppel-like factor 4 (KLF4), and decoupled its mCpG- and CpG-binding activities via site-directed mutagenesis. Furthermore, KLF4 binds specific methylated or unmethylated motifs in human embryonic stem cells in vivo. Our study suggests that mCpG-dependent TF binding activity is a widespread phenomenon and provides a new framework to understand the role and mechanism of TFs in epigenetic regulation of gene transcription. DOI:http://dx.doi.org/10.7554/eLife.00726.001.

Quantitative assessment of Tet-induced oxidation products of 5-methylcytosine in cellular and tissue DNA.

Recent studies showed that Ten-eleven translocation (Tet) family dioxygenases can oxidize 5-methyl-2'-deoxycytidine (5-mdC) in DNA to yield the 5-hydroxymethyl, 5-formyl and 5-carboxyl derivatives of 2'-deoxycytidine (5-HmdC, 5-FodC and 5-CadC). 5-HmdC in DNA may be enzymatically deaminated to yield 5-hydroxymethyl-2'-deoxyuridine (5-HmdU). After their formation at CpG dinucleotide sites, these oxidized pyrimidine nucleosides, particularly 5-FodC, 5-CadC, and 5-HmdU, may be cleaved from DNA by thymine DNA glycosylase, and subsequent action of base-excision repair machinery restores unmethylated cytosine. These processes are proposed to be important in active DNA cytosine demethylation in mammals. Here we used a reversed-phase HPLC coupled with tandem mass spectrometry (LC-MS/MS/MS) method, along with the use of stable isotope-labeled standards, for accurate measurements of 5-HmdC, 5-FodC, 5-CadC and 5-HmdU in genomic DNA of cultured human cells and multiple mammalian tissues. We found that overexpression of the catalytic domain of human Tet1 led to marked increases in the levels of 5-HmdC, 5-FodC and 5-CadC, but only a modest increase in 5-HmdU, in genomic DNA of HEK293T cells. Moreover, 5-HmdC is present at a level that is approximately 2-3 and 3-4 orders of magnitude greater than 5-FodC and 5-CadC, respectively, and 35-400 times greater than 5-HmdU in the mouse brain and skin, and human brain. The robust analytical method built a solid foundation for dissecting the molecular mechanisms of active cytosine demethylation, for measuring these 5-mdC derivatives and assessing their involvement in epigenetic regulation in other organisms and for examining whether these 5-mdC derivatives can be used as biomarkers for human diseases.

p53 binds to and is required for the repression of Arf tumor suppressor by HDAC and polycomb.

The expression of tumor suppressor Arf is tightly repressed during normal cell growth at a young age and is activated by oncogenic insults, and during aging, results in p53 activation and cell-cycle arrest to prevent hyperproliferation. The mechanisms of both transcriptional repression and activation of Arf are not understood. We show that p53 binds to and represses Arf expression and that this repression requires the function of both histone deacetylases (HDAC) and polycomb group (PcG) proteins. Inactivation of p53 leads to increased Arf transcription in both mouse embryonic fibroblasts (MEF) cultured in vitro and in tissues and organs of p53 null mice. Activation of endogenous p53 enhances Arf repression, and reintroduction of p53 back into p53 null MEFs restores Arf repression. Both DNA binding and transactivation activities of p53 are required for Arf repression. We show that p53 is required for both HDAC and PcG to repress Arf expression. Bindings of both HDAC and PcG to Arf are disrupted by inactivation of p53 and can be restored in p53 null MEFs by the reintroduction of wild-type, but not mutant, p53. These results indicate that p53 recruits both HDAC and PcG to Arf locus to repress its expression, and this repression constitutes a second feedback loop in p53 regulation.

[Screen in Saccharomyces cerevisiae for transposon insertion sites able to rescue phenotype of MTM1 deletion mutant using mTn-lacZ/LEU2 transposon library].

OBJECTIVE: MTM1 gene is essential for superoxide dismutase 2 activity and normal mitochondrial functions. MTM1 deletion results in decreased superoxide dismutase 2 activity, impaired mitochondrial functions and growth defect on nonfermentable carbon source. To promote understanding of MTM1 gene, we started a genetic screen for transposon insertions which are able to rescue the growth defect resulting from MTM1 deletion. METHODS: Routine screening strategy didnt work because of the irreversible damage caused by MTM1 deletion. So we adopted the following screening strategy: we transformed a plasmid overexpressing MTM1 into wild type before deleting MTM1 in chromosome and got the resulting strain, designated YES2MTM1. Then we transformed mTn-lacZ/LEU2 transposon library into the YES2MTM1 strain. Transformants lost the plasmid overexpressing MTM1 after 5-Fluoroorotic acid treatment. We picked up the yeast strains able to grow on nonfermentable carbon source with MTM1 deletion and some transposon insertion and identified the insertion sites. RESULTS: We found transposon insertions in two genes were able to rescue the growth defect resulting from MTM1 deletion on nonfermentable carbon source. CONCLUSION: Our study will provide reference for thorough understanding of MTM1 gene function.

Regulation of cellular metabolism by protein lysine acetylation.

Protein lysine acetylation has emerged as a key posttranslational modification in cellular regulation, in particular through the modification of histones and nuclear transcription regulators. We show that lysine acetylation is a prevalent modification in enzymes that catalyze intermediate metabolism. Virtually every enzyme in glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, the urea cycle, fatty acid metabolism, and glycogen metabolism was found to be acetylated in human liver tissue. The concentration of metabolic fuels, such as glucose, amino acids, and fatty acids, influenced the acetylation status of metabolic enzymes. Acetylation activated enoyl-coenzyme A hydratase/3-hydroxyacyl-coenzyme A dehydrogenase in fatty acid oxidation and malate dehydrogenase in the TCA cycle, inhibited argininosuccinate lyase in the urea cycle, and destabilized phosphoenolpyruvate carboxykinase in gluconeogenesis. Our study reveals that acetylation plays a major role in metabolic regulation.

DDB1-CUL4 and MLL1 mediate oncogene-induced p16INK4a activation.

The induction of cellular senescence by oncogenic signals acts as a barrier to cellular transformation and is attained, in part, by the elevation of the p16(INK4a) tumor suppressor gene. p16 expression is repressed epigenetically by Polycomb, but how p16 is induced is not known. We report here that the p16 locus is H3K4-methylated in highly expressing cells. H3K4 methyltransferase MLL1 directly binds to and is required, along with its core component RbBP5, for the induction of p16 by oncogenic Ras. We further show that damaged DNA binding protein DDB1 and CUL4, which assemble distinct E3 ubiquitin ligases by recruiting various WD40 proteins, act upstream of MLL1-mediated H3K4 methylation. We showed that CUL4A directly binds to p16 and that silencing DDB1 blocks Ras-induced p16 activation. Ras expression dissociates BMI1 from the p16 locus, whereas both CUL4 and MLL1 bind to the p16 locus similarly in both normal and oncogenic stimulated cells. These results suggest that DDB1-CUL4 and MLL1 complexes constitute a novel pathway that mediates p16 activation during oncogenic checkpoint response and is repressed by the polycomb repression complexes during normal growth of young cells.

Control of MAPK specificity by feedback phosphorylation of shared adaptor protein Ste50.

Many different signaling pathways share common components but nevertheless invoke distinct physiological responses. In yeast, the adaptor protein Ste50 functions in multiple mitogen-activated protein (MAP) kinase pathways, each with unique dynamical and developmental properties. Although Kss1 activity is sustained and promotes invasive growth, Hog1 activity is transient and promotes cell adaptation to osmotic stress. Here we show that osmotic stress activates Kss1 as well as Hog1. We show further that Hog1 phosphorylates Ste50 and that phosphorylation of Ste50 limits the duration of Kss1 activation and prevents invasive growth under high osmolarity growth conditions. Thus feedback regulation of a shared component can restrict the activity of a competing MAP kinase to ensure signal fidelity.

DDB1 functions as a linker to recruit receptor WD40 proteins to CUL4-ROC1 ubiquitin ligases.

Cullins assemble the largest family of ubiquitin ligases by binding with ROC1 and various substrate receptors. CUL4 function is linked with many cellular processes, but its substrate-recruiting mechanism remains elusive. We identified a protein motif, the DWD box (DDB1-binding WD40 protein), and demonstrated the binding of 15 DWD proteins with DDB1-CUL4A. We provide evidence supporting the critical function of the DWD box and DDB1's role as the linker mediating DWD protein association with CUL4A. A database search predicts that about one-third of WD40 proteins, 90 in humans, contain the DWD box, suggesting a potentially large number of DWD-DDB1-CUL4-ROC1 E3 ligases.