Environmental stresses are universally encountered by microbes, plants and animals. Yet systematic studies of stress-responsive transcription factor (TF) networks in multi-cellular organisms have been limited. The phytohormone abscisic acid (ABA) influences the expression of thousands of genes, allowing us to characterize complex stress-responsive regulatory networks. Using chromatin immunoprecipitation sequencing, we identified genome-wide targets of 21 ABA-related TFs to construct a comprehensive regulatory network in Arabidopsis thaliana. Determinants of dynamic TF binding and a hierarchy among TFs were defined, illuminating the relationship between differential gene expression patterns and ABA pathway feedback regulation. By extrapolating regulatory characteristics of observed canonical ABA pathway components, we identified a new family of transcriptional regulators modulating ABA and salt responsiveness and demonstrated their utility to modulate plant resilience to osmotic stress.
3,7,3',4'-Tetrahydroxyflavone (fisetin) is a flavonoid found in vegetables and fruits having broad biological activities. Here the effects of fisetin on adipogenesis and its regulatory mechanism in mouse adipocytic 3T3-L1 cells are studied. Fisetin inhibited the accumulation of intracellular lipids and lowered the expression of adipogenic genes such as peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein (C/EBP) α and fatty acid-binding protein 4 (aP2) during adipogenesis. Moreover, the mRNA levels of genes such as acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase involved in the fatty acid biosynthesis (lipogenesis) were reduced by the treatment with fisetin. The expression level of the glucose transporter 4 (GLUT4) gene was also decreased by fisetin, resulting in down-regulation of glucose uptake. Furthermore, fisetin inhibited the phosphorylation of the mammalian target of rapamycin (mTOR) and that of p70 ribosomal S6 kinase, a target of the mTOR complex, the inhibition of which was followed by a decreased mRNA level of the C/EBPα gene. The results obtained from a chromatin immunoprecipitation assay demonstrated that the ability of C/EBPα to bind to the GLUT4 gene promoter was reduced by the treatment with fisetin, which agreed well with those obtained when 3T3-L1 cells were allowed to differentiate into adipocytes in medium in the presence of rapamycin, an inhibitor for mTOR. These results indicate that fisetin suppressed the accumulation of intracellular lipids by inhibiting GLUT4-mediated glucose uptake through inhibition of the mTOR-C/EBPα signaling in 3T3-L1 cells.
Many studies have looked at how dogs respond to human communicative information. Here, we examined which human communicative factors were important in influencing dogs' responses. Eleven healthy pet dogs with no apparent aggressive behaviour toward people were recruited. Five sensory conditions (all cues presented; either a visual, an auditory, or an olfactory cue presented; no cues presented) were provided three times randomly to each dog during the tests. All tests were video recorded, and both the dogs' behaviour and time taken to reach the person when she presented each of the sensory cue conditions were observed. Total rates of reaching the person were as follows: 97.0% (all cues), 87.9% (auditory cues), 84.4% (visual cues), 84.4% (olfactory cues), and 69.7% (no cues). The time taken for the dog to notice the person in the box and then obtain a reward from her differed among the five conditions: all cues (6.00 ± 0.32 s) and visual cues (6.02 ± 0.91 s) were significantly faster than auditory cues (18.56 ± 9.57 s) and no cues (26.55 ± 11.72 s). Thus the type of information input was important in recognition of the person by the dogs and influenced the dogs' response times; visual cues appeared advantageous in confirming the person's presence.
Vacuolar-type H + -ATPase (v-ATPase), a multi-subunit protein complex, has two distinct functions on lysosomes: acidifying the lysosomal lumen and controlling mTOR-S6K (mTORC1) signaling, both of which are crucial for several biological processes. However, little is known about how both functions of v-ATPase are coordinated and whether lysosomes are also involved in mTOR-AKT (mTORC2) signaling. We found that knocking down (KD) of a subunit of v-ATPase impairs cell proliferation of undifferentiated induced pluripotent stem cells (iPSCs) although all cells do not die. As expected, lysosomal pH increased and mTORC1 signaling was attenuated in the KD cells. Unexpectedly, mTORC2 signaling was also impaired. Treatment of iPSCs with bafilomycin A1, a specific inhibitor of v-ATPase proton pump, increased lysosomal pH, and impaired both mTORC1 and mTORC2 signaling pathways. When treating Hek293, a cancer cell line, with the inhibitor, attenuation of mTORC2 activity was observed. Therefore, in addition to mTORC1, v-ATPase regulates the mTORC2 activity. We are now investigating how the proton pump affects the mTOR signaling using deletion mutants of the subunit and some chemicals that affect pH in lysosomes. We will discuss our results in this meeting.
Recent developments of induced pluripotent stem cell (iPSC) and genome editing technologies enable us to create countless new opportunities in clinical researches for previously incurable diseases. With these new technologies, we aim to gain deeper understandings of a group of related untreatable disorders, Cockayne Syndrome, Xeroderma Pigmentosa and the ultraviolet (UV)-Sensitive Syndrome. The joint possession of these diseases is their hereditary and also insufficiency in repair systems for DNA damages. The impairment of DNA repair systems causes successive accumulation of genomic mutations that frequently, but not always, elevates the risk of cancer, UV sensitivity of skins and neuronal damages tremendously. However, despite dysfunction of the same DNA repair machinery in the diseases, there are differences in symptoms among them, and molecular mechanisms that underpin the different manifestations are poorly understood.Here, to elucidate the mechanisms, we used the Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) technology. The CRISPRi utilizes catalytically dead (d)Cas9 fused with KRAB, a transcription repressor domain, to transcriptionally repress genes of interest. Mechanistically, the repression is achieved by recruiting dCas9-KRAB to a promoter region of the gene in a single guide (sg)RNA dependent manner. By employing a disease-free CRISPRi human (h)iPSC line in which dCas9-KRAB can be induced by doxycycline (Dox) treatment, we first established CRISPRi hiPSC lines where each disease-causative gene can be repressed. Then, we examined if suppressing the causative gene by Dox treatment can recapitulate disease phenotypes. We observed that, for instance, when we suppressed ERCC6, a gene responsible for Cockayne syndrome, cell proliferation of iPSCs was impaired. Additionally, production of reactive oxygen species, which involves DNA damages, was elevated in the hiPSCs regardless of UV irradiation. The similar phenotypes were observed using hiPSCs generated from a Cockayne Syndrome patient, suggesting the CRISPRi can mirror the disease phenotypes. In this meeting, I will discuss differences in phenotypes at molecular and cellular levels by suppressing the genes that are responsible for the related disorders not only in undifferentiated iPSCs but also in differentiated hiPSCs relevant to the diseases.
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