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Zhang W.,CAS Institute of Biophysics | Li J.,Peking University | Suzuki K.,Salk Institute for Biological Studies | Qu J.,CAS Institute of Zoology | And 37 more authors.
Science | Year: 2015

Werner syndrome (WS) is a premature aging disorder caused by WRN protein deficiency. Here, we report on the generation of a human WS model in human embryonic stem cells (ESCs). Differentiation of WRN-null ESCs to mesenchymal stem cells (MSCs) recapitulates features of premature cellular aging, a global loss of H3K9me3, and changes in heterochromatin architecture. We show that WRN associates with heterochromatin proteins SUV39H1 and HP1a and nuclear lamina-heterochromatin anchoring protein LAP2β. Targeted knock-in of catalytically inactive SUV39H1 in wild-type MSCs recapitulates accelerated cellular senescence, resembling WRN-deficient MSCs. Moreover decrease in WRN and heterochromatin marks are detected in MSCs from older individuals Our observations uncover a role for WRN in maintaining heterochromatin stability and highlight heterochromatin disorganization as a potential determinant of human aging.


Duan S.,CAS Institute of Biophysics | Yuan G.,CAS Institute of Biophysics | Liu X.,Peking University | Ren R.,CAS Institute of Biophysics | And 31 more authors.
Nature Communications | Year: 2015

PTEN is a tumour suppressor frequently mutated in many types of cancers. Here we show that targeted disruption of PTEN leads to neoplastic transformation of human neural stem cells (NSCs), but not mesenchymal stem cells. PTEN-deficient NSCs display neoplasm-associated metabolic and gene expression profiles and generate intracranial tumours in immunodeficient mice. PTEN is localized to the nucleus in NSCs, binds to the PAX7 promoter through association with cAMP responsive element binding protein 1 (CREB)/CREB binding protein (CBP) and inhibits PAX7 transcription. PTEN deficiency leads to the upregulation of PAX7, which in turn promotes oncogenic transformation of NSCs and instates 'aggressiveness' in human glioblastoma stem cells. In a large clinical database, we find increased PAX7 levels in PTEN-deficient glioblastoma. Furthermore, we identify that mitomycin C selectively triggers apoptosis in NSCs with PTEN deficiency. Together, we uncover a potential mechanism of how PTEN safeguards NSCs, and establish a cellular platform to identify factors involved in NSC transformation, potentially permitting personalized treatment of glioblastoma.


Pan H.,CAS Institute of Biophysics | Pan H.,University of Chinese Academy of Sciences | Guan D.,CAS Institute of Biophysics | Liu X.,Peking University | And 32 more authors.
Cell Research | Year: 2016

SIRT6 belongs to the mammalian homologs of Sir2 histone NAD + -dependent deacylase family. In rodents, SIRT6 deficiency leads to aging-associated degeneration of mesodermal tissues. It remains unknown whether human SIRT6 has a direct role in maintaining the homeostasis of mesodermal tissues. To this end, we generated SIRT6 knockout human mesenchymal stem cells (hMSCs) by targeted gene editing. SIRT6-deficient hMSCs exhibited accelerated functional decay, a feature distinct from typical premature cellular senescence. Rather than compromised chromosomal stability, SIRT6-null hMSCs were predominately characterized by dysregulated redox metabolism and increased sensitivity to the oxidative stress. In addition, we found SIRT6 in a protein complex with both nuclear factor erythroid 2-related factor 2 (NRF2) and RNA polymerase II, which was required for the transactivation of NRF2-regulated antioxidant genes, including heme oxygenase 1 (HO-1). Overexpression of HO-1 in SIRT6-null hMSCs rescued premature cellular attrition. Our study uncovers a novel function of SIRT6 in maintaining hMSC homeostasis by serving as a NRF2 coactivator, which represents a new layer of regulation of oxidative stress-associated stem cell decay. © 2016 IBCB, SIBS, CAS.


Li X.,Peking University | Wang W.,Chinese Academy of Sciences | Zhu P.,Peking University | Zhu P.,Tsinghua University | And 13 more authors.
Nature | Year: 2016

Haematopoietic stem cells (HSCs) are derived early from embryonic precursors, such as haemogenic endothelial cells and pre-haematopoietic stem cells (pre-HSCs), the molecular identity of which still remains elusive. Here we use potent surface markers to capture the nascent pre-HSCs at high purity, as rigorously validated by single-cell-initiated serial transplantation. Then we apply single-cell RNA sequencing to analyse endothelial cells, CD45 â' and CD45 + pre-HSCs in the aorta-gonad-mesonephros region, and HSCs in fetal liver. Pre-HSCs show unique features in transcriptional machinery, arterial signature, metabolism state, signalling pathway, and transcription factor network. Functionally, activation of mechanistic targets of rapamycin (mTOR) is shown to be indispensable for the emergence of HSCs but not haematopoietic progenitors. Transcriptome data-based functional analysis reveals remarkable heterogeneity in cell-cycle status of pre-HSCs. Finally, the core molecular signature of pre-HSCs is identified. Collectively, our work paves the way for dissection of complex molecular mechanisms regulating stepwise generation of HSCs in vivo, informing future efforts to engineer HSCs for clinical applications. © 2016 Macmillan Publishers Limited. All rights reserved.


Hou Y.,Peking University | Guo H.,Capital Medical University | Guo H.,Peking University | Cao C.,Peking University | And 14 more authors.
Cell Research | Year: 2016

Single-cell genome, DNA methylome, and transcriptome sequencing methods have been separately developed. However, to accurately analyze the mechanism by which transcriptome, genome and DNA methylome regulate each other, these omic methods need to be performed in the same single cell. Here we demonstrate a single-cell triple omics sequencing technique, scTrio-seq, that can be used to simultaneously analyze the genomic copy-number variations (CNVs), DNA methylome, and transcriptome of an individual mammalian cell. We show that large-scale CNVs cause proportional changes in RNA expression of genes within the gained or lost genomic regions, whereas these CNVs generally do not affect DNA methylation in these regions. Furthermore, we applied scTrio-seq to 25 single cancer cells derived from a human hepatocellular carcinoma tissue sample. We identified two subpopulations within these cells based on CNVs, DNA methylome, or transcriptome of individual cells. Our work offers a new avenue of dissecting the complex contribution of genomic and epigenomic heterogeneities to the transcriptomic heterogeneity within a population of cells.


Reddy P.,Salk Institute for Biological Studies | Ocampo A.,Salk Institute for Biological Studies | Suzuki K.,Salk Institute for Biological Studies | Luo J.,Salk Institute for Biological Studies | And 23 more authors.
Cell | Year: 2015

Mitochondrial diseases include a group of maternally inherited genetic disorders caused by mutations in mtDNA. In most of these patients, mutated mtDNA coexists with wild-type mtDNA, a situation known as mtDNA heteroplasmy. Here, we report on a strategy toward preventing germline transmission of mitochondrial diseases by inducing mtDNA heteroplasmy shift through the selective elimination of mutated mtDNA. As a proof of concept, we took advantage of NZB/BALB heteroplasmic mice, which contain two mtDNA haplotypes, BALB and NZB, and selectively prevented their germline transmission using either mitochondria-targeted restriction endonucleases or TALENs. In addition, we successfully reduced human mutated mtDNA levels responsible for Leber's hereditary optic neuropathy (LHOND), and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP), in mammalian oocytes using mitochondria-targeted TALEN (mito-TALENs). Our approaches represent a potential therapeutic avenue for preventing the transgenerational transmission of human mitochondrial diseases caused by mutations in mtDNA. PaperClip © 2015 Elsevier Inc.


News Article | February 10, 2017
Site: www.biosciencetechnology.com

A team of biomedical scientists has identified a molecule that targets a gene known to play a critical role in the rapid progression of amyotrophic lateral sclerosis (ALS), sometimes known as Lou Gehrig's disease, the neurodegenerative disease that affects motor neurons - nerve cells in the brain and spinal cord that link the nervous system to the voluntary muscles of the body. The research, done on a mouse model of ALS, aims at blocking the gene, thus providing an important stepping stone for the development of novel treatments to delay the progression of ALS and, potentially, other human diseases. Specifically, the team, led by Maurizio Pellecchia, a professor of biomedical sciences in the School of Medicine at the University of California, Riverside, reports in the journal Cell Chemical Biology on the design of 123C4, a molecule the lab developed that targets the EphA4 receptor, a gene in animal models and in humans that is efficacious in delaying the progression of ALS. Importantly, the expression of EphA4 is associated not only with the progression of motor neuron disease, but also with other conditions including abnormal blood clotting, spinal cord and brain injury, Alzheimer's disease, as well as gastric and pancreatic cancers. "Research in assessing the therapeutic value of EphA4 for these diseases has been hampered, however, by the lack of suitable pharmacological EphA4-inhibitors," said Pellecchia, who holds the Daniel Hays Endowed Chair in Cancer Research and is the director of the Center for Molecular and Translational Medicine. "While the exact mechanism responsible for the therapeutic efficacy of our agent, 123C4, is still to be fully understood, we are confident that 123C4 - or its derivatives - will find wide application in preclinical studies as well as human clinical trials for the treatment of ALS and potentially other human disorders." Pellecchia said that only recently genetic studies on humans affected by the disease, as well as animal models of ALS, clearly indicated that the EphA4 receptor could be a suitable drug target to delay the progression of motor neuron death. "Prior to this current work, no bona fide EphA4 targeting agent with demonstrated efficacy in animal models of ALS had been reported," he said. "It has been a long and difficult journey to derive 123C4." To derive the molecule, Pellecchia's lab used an approach it developed recently that merges combinatorial chemistry and biophysical methods based on Nuclear Magnetic Resonance Spectroscopy, and tested more than 100,000 possible candidates. The research entailed a combination also of a wide variety of other sophisticated techniques and approaches, ranging from medicinal chemistry, to cell biology and imaging, to in vivo pharmacology, and efficacy studies using transgenic mice models of ALS. "My lab has had a long-standing interest in developing approaches to target protein-protein interactions and to apply these to relevant drug targets," Pellecchia said. "Targeting EphA4 has been particularly challenging, though. But its association with the progression of ALS inspired us to increase our efforts in this field in the past years." He noted that most studies suggest that decreasing EphA4 levels genetically in transgenic animal models of ALS result in prolonged survival. Intuitively, it can be imagined, therefore, that blocking EphA4 with drugs would have the same effect. "Indeed 123C4 increases survival in mice models of ALS, but acts as an EphA4 agonist and not antagonist," Pellecchia said, going on to explain that an agonist is a substance that stimulates chemical action, while an antagonist blocks such action. "We show that 123C4 interacting with EphA4 causes the receptor to be internalized by a process known as endocytosis - a process initiated only by an agonist. We hypothesize that by inducing receptor internalization, 123C4 effectively removes EphA4 from the surface of motor neurons." The transgenic mouse model Pellecchia and his colleagues used for the study has been widely adopted as a standard to select drug candidates as potential ALS therapeutics. But challenges lie ahead for Pellecchia's team to bring 123C4 to the clinic to confirm that the laboratory studies effectively translate to patients affected by ALS. "As in any preclinical study, we must acknowledge that several obstacles are still in the way of translating agents like 123C4 into viable therapeutics," Pellecchia said. "But Iron Horse Therapeutics, a biotech company in San Diego, is taking steps to progress this class of agents into the clinic." Pellecchia was joined in the study by Surya K. De, Anna Kulinich, Ahmed F. Salem, Jordan Joeppen, Elisa Barile and Iryna Ethell, a professor of biomedical sciences, at UC Riverside; and Bainan Wu (the first author of the paper), Rengang Wang, Si Wang, and Dongxiang Zhang at Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, Calif. "The collaboration with Dr. Ethell and her laboratory, which specializes in cell biology and the imaging of primary neurons, was particularly fruitful in our endeavors," Pellecchia said. "I consider it a perfect example of the power of collaborative research at UCR" Next, the researchers plan to develop additional agents based on 123C4 with either antagonist activity or enhanced agonist activity, and to test these in motor neurons and in animal models of ALS. "In collaboration with Iron Horse Therapeutics, we hope these additional studies will further facilitate the translation of these agents into novel treatments," Pellecchia said.


Deng L.,CAS Institute of Biophysics | Ren R.,CAS Institute of Biophysics | Wu J.,Salk Institute for Biological Studies | Suzuki K.,Salk Institute for Biological Studies | And 4 more authors.
Protein and Cell | Year: 2015

Nuclease-based genome editing has proven to be a powerful and promising tool for disease modeling and gene therapy. Recent advances in CRISPR/Cas and TALE indicate that they could also be used as a targeted regulator of gene expression, as well as being utilized for illuminating specific chromosomal structures or genomic regions. © 2015, The Author(s).


Wang L.,CAS Institute of Biophysics | Wu J.,Salk Institute for Biological Studies | Fang W.,Beijing Hospital of the Ministry of Health | Liu G.-H.,CAS Institute of Biophysics | And 3 more authors.
Cell Research | Year: 2015

The CRISPR/Cas system has proven to be a powerful gene editing tool both in vitro and in vivo. A recent flurry of studies of in vivo gene editing using the CRISPR/Cas system bring bright prospects in creating animal models and targeted gene therapy of human genetic diseases. © 2015 IBCB, SIBS, CAS All rights reserved.

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