Publications by authors named "Dirk Hockemeyer"

50 Publications

Launching a saliva-based SARS-CoV-2 surveillance testing program on a university campus.

PLoS One 2021 26;16(5):e0251296. Epub 2021 May 26.

University of California, Berkeley, California, United States of America.

Regular surveillance testing of asymptomatic individuals for SARS-CoV-2 has been center to SARS-CoV-2 outbreak prevention on college and university campuses. Here we describe the voluntary saliva testing program instituted at the University of California, Berkeley during an early period of the SARS-CoV-2 pandemic in 2020. The program was administered as a research study ahead of clinical implementation, enabling us to launch surveillance testing while continuing to optimize the assay. Results of both the testing protocol itself and the study participants' experience show how the program succeeded in providing routine, robust testing capable of contributing to outbreak prevention within a campus community and offer strategies for encouraging participation and a sense of civic responsibility.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0251296PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8153421PMC
June 2021

Generation of a DAT-P2A-Flpo mouse line for intersectional genetic targeting of dopamine neuron subpopulations.

Cell Rep 2021 May;35(6):109123

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA. Electronic address:

Dopaminergic projections exert widespread influence over multiple brain regions and modulate various behaviors including movement, reward learning, and motivation. It is increasingly appreciated that dopamine neurons are heterogeneous in their gene expression, circuitry, physiology, and function. Current approaches to target dopamine neurons are largely based on single gene drivers, which either label all dopamine neurons or mark a subset but concurrently label non-dopaminergic neurons. Here, we establish a mouse line with Flpo recombinase expressed from the endogenous Slc6a3 (dopamine active transporter [DAT]) locus. DAT-P2A-Flpo mice can be used together with Cre-expressing mouse lines to efficiently and selectively label dopaminergic subpopulations using Cre/Flp-dependent intersectional strategies. We demonstrate the utility of this approach by generating DAT-P2A-Flpo;NEX-Cre mice that specifically label Neurod6-expressing dopamine neurons, which project to the nucleus accumbens medial shell. DAT-P2A-Flpo mice add to a growing toolbox of genetic resources that will help parse the diverse functions mediated by dopaminergic circuits.
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http://dx.doi.org/10.1016/j.celrep.2021.109123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8240967PMC
May 2021

Cancer-associated POT1 mutations lead to telomere elongation without induction of a DNA damage response.

EMBO J 2021 Jun 2;40(12):e107346. Epub 2021 May 2.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.

Mutations in the shelterin protein POT1 are associated with chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, angiosarcoma, melanoma, and other cancers. These cancer-associated POT1 (caPOT1) mutations are generally heterozygous, missense, or nonsense mutations occurring throughout the POT1 reading frame. Cancers with caPOT1 mutations have elongated telomeres and show increased genomic instability, but which of the two phenotypes promotes tumorigenesis is unclear. We tested the effects of CAS9-engineered caPOT1 mutations in human embryonic and hematopoietic stem cells (hESCs and HSCs, respectively). HSCs with caPOT1 mutations did not show overt telomere damage. In vitro and in vivo competition experiments showed the caPOT1 mutations did not confer a selective disadvantage. Since DNA damage signaling is known to affect the fitness of HSCs, the data argue that caPOT1 mutations do not cause significant telomere damage. Furthermore, hESC lines with caPOT1 mutations showed no detectable telomere damage response while showing consistent telomere elongation. Thus, caPOT1 mutations are likely selected for during cancer progression because of their ability to elongate telomeres and extend the proliferative capacity of the incipient cancer cells.
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http://dx.doi.org/10.15252/embj.2020107346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8204863PMC
June 2021

is a haploinsufficient tumor suppressor that limits telomere length.

Elife 2020 12 1;9. Epub 2020 Dec 1.

Laboratory for Cell Biology and Genetics, Rockefeller University, New York, United States.

Telomere shortening is a presumed tumor suppressor pathway that imposes a proliferative barrier (the Hayflick limit) during tumorigenesis. This model predicts that excessively long somatic telomeres predispose to cancer. Here, we describe cancer-prone families with two unique mutations that truncate TIN2, a shelterin subunit that controls telomere length. Patient lymphocyte telomeres were unusually long. We show that the truncated TIN2 proteins do not localize to telomeres, suggesting that the mutations create loss-of-function alleles. Heterozygous knock-in of the mutations or deletion of one copy of resulted in excessive telomere elongation in clonal lines, indicating that is haploinsufficient for telomere length control. In contrast, telomere protection and genome stability were maintained in all heterozygous clones. The data establish that the truncations predispose to a tumor syndrome. We conclude that acts as a haploinsufficient tumor suppressor that limits telomere length to ensure a timely Hayflick limit.
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http://dx.doi.org/10.7554/eLife.61235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7707837PMC
December 2020

Telomere length set point regulation in human pluripotent stem cells critically depends on the shelterin protein TPP1.

Mol Biol Cell 2020 11 9;31(23):2583-2596. Epub 2020 Sep 9.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720.

Telomere maintenance is essential for the long-term proliferation of human pluripotent stem cells, while their telomere length set point determines the proliferative capacity of their differentiated progeny. The shelterin protein TPP1 is required for telomere stability and elongation, but its role in establishing a telomere length set point remains elusive. Here, we characterize the contribution of the shorter isoform of TPP1 (TPP1S) and the amino acid L104 outside the TEL patch, TPP1's telomerase interaction domain, to telomere length control. We demonstrate that cells deficient for TPP1S (TPP1S knockout [KO]), as well as the complete TPP1 KO cell lines, undergo telomere shortening. However, TPP1S KO cells are able to stabilize short telomeres, while TPP1 KO cells die. We compare these phenotypes with those of TPP1 mutant cells, which have short and stable telomeres similar to the TPP1S KO. In contrast to TPP1S KO cells, TPP1 cells respond to increased telomerase levels and maintain protected telomeres. However, TPP1 shows altered sensitivity to expression changes of shelterin proteins suggesting the mutation causes a defect in telomere length feedback regulation. Together this highlights TPP1 as the first shelterin mutant engineered at the endogenous locus of human stem cells with an altered telomere length set point.
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http://dx.doi.org/10.1091/mbc.E19-08-0447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851873PMC
November 2020

Controlled Cycling and Quiescence Enables Efficient HDR in Engraftment-Enriched Adult Hematopoietic Stem and Progenitor Cells.

Cell Rep 2020 09;32(9):108093

Innovative Genomics Institute, University of California, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA; Department of Biology, ETH Zürich, 8093 Zürich, Switzerland. Electronic address:

Genome editing often takes the form of either error-prone sequence disruption by non-homologous end joining (NHEJ) or sequence replacement by homology-directed repair (HDR). Although NHEJ is generally effective, HDR is often difficult in primary cells. Here, we use a combination of immunophenotyping, next-generation sequencing, and single-cell RNA sequencing to investigate and reprogram genome editing outcomes in subpopulations of adult hematopoietic stem and progenitor cells. We find that although quiescent stem-enriched cells mostly use NHEJ, non-quiescent cells with the same immunophenotype use both NHEJ and HDR. Inducing quiescence before editing results in a loss of HDR in all cell subtypes. We develop a strategy of controlled cycling and quiescence that yields a 6-fold increase in the HDR/NHEJ ratio in quiescent stem cells ex vivo and in vivo. Our results highlight the tension between editing and cellular physiology and suggest strategies to manipulate quiescent cells for research and therapeutic genome editing.
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http://dx.doi.org/10.1016/j.celrep.2020.108093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7487781PMC
September 2020

Analysis of muntjac deer genome and chromatin architecture reveals rapid karyotype evolution.

Commun Biol 2020 09 1;3(1):480. Epub 2020 Sep 1.

Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.

Closely related muntjac deer show striking karyotype differences. Here we describe chromosome-scale genome assemblies for Chinese and Indian muntjacs, Muntiacus reevesi (2n = 46) and Muntiacus muntjak vaginalis (2n = 6/7), and analyze their evolution and architecture. The genomes show extensive collinearity with each other and with other deer and cattle. We identified numerous fusion events unique to and shared by muntjacs relative to the cervid ancestor, confirming many cytogenetic observations with genome sequence. One of these M. muntjak fusions reversed an earlier fission in the cervid lineage. Comparative Hi-C analysis showed that the chromosome fusions on the M. muntjak lineage altered long-range, three-dimensional chromosome organization relative to M. reevesi in interphase nuclei including A/B compartment structure. This reshaping of multi-megabase contacts occurred without notable change in local chromatin compaction, even near fusion sites. A few genes involved in chromosome maintenance show evidence for rapid evolution, possibly associated with the dramatic changes in karyotype.
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http://dx.doi.org/10.1038/s42003-020-1096-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463020PMC
September 2020

TERT promoter mutations and telomeres during tumorigenesis.

Curr Opin Genet Dev 2020 02 9;60:56-62. Epub 2020 Mar 9.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address:

Telomerase regulation and telomere shortening act as a strong tumor suppressor mechanism in human somatic cells. Point mutations in the promoter of telomerase reverse transcriptase (TERT) are the most frequent non-coding mutation in cancer. These TERT promoter mutations (TPMs) create de novo ETS factor binding sites upstream of the start codon of the gene, which can be bound by different ETS factors. TPMs can occur early during tumorigenesis and are thought to be among the first mutations in melanoma, glioblastoma and hepatocellular carcinoma. Despite their association with increased TERT levels, TPMs do not prohibit telomere shortening and TPM-harboring cancers present with short telomeres. Their short telomere length combined with their high prevalence and specificity for cancer makes TPMs an attractive target for future therapeutic exploitation of telomerase inhibition and telomere deprotection-induced cell death.
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http://dx.doi.org/10.1016/j.gde.2020.02.001DOI Listing
February 2020

Transient activation of the UPR is an essential step in the acquisition of pluripotency during reprogramming.

Sci Adv 2019 04 10;5(4):eaaw0025. Epub 2019 Apr 10.

Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA.

Somatic cells can be reprogrammed into pluripotent stem cells using the Yamanaka transcription factors. Reprogramming requires both epigenetic landscape reshaping and global remodeling of cell identity, structure, basic metabolic processes, and organelle form and function. We hypothesize that variable regulation of the proteostasis network and its influence upon the protein-folding environment within cells and their organelles is responsible for the low efficiency and stochasticity of reprogramming. We find that the unfolded protein response of the endoplasmic reticulum (UPR), the mitochondrial UPR, and the heat shock response, which ensure proteome quality during stress, are activated during reprogramming. The UPR is particularly crucial, and its ectopic, transient activation, genetically or pharmacologically, enhances reprogramming. Last, stochastic activation of the UPR predicts reprogramming efficiency in naïve cells. Thus, the low efficiency and stochasticity of cellular reprogramming are due partly to the inability to properly initiate the UPR to remodel the ER and its proteome.
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http://dx.doi.org/10.1126/sciadv.aaw0025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6457941PMC
April 2019

4D cell biology: big data image analytics and lattice light-sheet imaging reveal dynamics of clathrin-mediated endocytosis in stem cell-derived intestinal organoids.

Mol Biol Cell 2018 11 6;29(24):2959-2968. Epub 2018 Sep 6.

Department of Molecular and Cell Biology, Berkeley, Berkeley, CA 94720.

New methods in stem cell 3D organoid tissue culture, advanced imaging, and big data image analytics now allow tissue-scale 4D cell biology, but currently available analytical pipelines are inadequate for handing and analyzing the resulting gigabytes and terabytes of high-content imaging data. We expressed fluorescent protein fusions of clathrin and dynamin2 at endogenous levels in genome-edited human embryonic stem cells, which were differentiated into hESC-derived intestinal epithelial organoids. Lattice light-sheet imaging with adaptive optics (AO-LLSM) allowed us to image large volumes of these organoids (70 × 60 × 40 µm xyz) at 5.7 s/frame. We developed an open-source data analysis package termed pyLattice to process the resulting large (∼60 Gb) movie data sets and to track clathrin-mediated endocytosis (CME) events. CME tracks could be recorded from ∼35 cells at a time, resulting in ∼4000 processed tracks per movie. On the basis of their localization in the organoid, we classified CME tracks into apical, lateral, and basal events and found that CME dynamics is similar for all three classes, despite reported differences in membrane tension. pyLattice coupled with AO-LLSM makes possible quantitative high temporal and spatial resolution analysis of subcellular events within tissues.
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http://dx.doi.org/10.1091/mbc.E18-06-0375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6329908PMC
November 2018

Genetically engineered human cortical spheroid models of tuberous sclerosis.

Nat Med 2018 10 20;24(10):1568-1578. Epub 2018 Aug 20.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.

Tuberous sclerosis complex (TSC) is a multisystem developmental disorder caused by mutations in the TSC1 or TSC2 genes, whose protein products are negative regulators of mechanistic target of rapamycin complex 1 signaling. Hallmark pathologies of TSC are cortical tubers-regions of dysmorphic, disorganized neurons and glia in the cortex that are linked to epileptogenesis. To determine the developmental origin of tuber cells, we established human cellular models of TSC by CRISPR-Cas9-mediated gene editing of TSC1 or TSC2 in human pluripotent stem cells (hPSCs). Using heterozygous TSC2 hPSCs with a conditional mutation in the functional allele, we show that mosaic biallelic inactivation during neural progenitor expansion is necessary for the formation of dysplastic cells and increased glia production in three-dimensional cortical spheroids. Our findings provide support for the second-hit model of cortical tuber formation and suggest that variable developmental timing of somatic mutations could contribute to the heterogeneity in the neurological presentation of TSC.
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http://dx.doi.org/10.1038/s41591-018-0139-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6261470PMC
October 2018

Genome-edited human stem cells expressing fluorescently labeled endocytic markers allow quantitative analysis of clathrin-mediated endocytosis during differentiation.

J Cell Biol 2018 09 6;217(9):3301-3311. Epub 2018 Jul 6.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA

We developed a general approach for investigation of how cellular processes become adapted for specific cell types during differentiation. Previous studies reported substantial differences in the morphology and dynamics of clathrin-mediated endocytosis (CME) sites. However, associating specific CME properties with distinct differentiated cell types and determining how these properties are developmentally specified during differentiation have been elusive. Using genome-edited human embryonic stem cells, and isogenic fibroblasts and neuronal progenitor cells derived from them, we established by live-cell imaging and platinum replica transmission electron microscopy that CME site dynamics and ultrastructure on the plasma membrane are precisely reprogrammed during differentiation. Expression levels for the endocytic adaptor protein AP2μ2 were found to underlie dramatic changes in CME dynamics and structure. Additionally, CME dependency on actin assembly and phosphoinositide-3 kinase activity are distinct for each cell type. Collectively, our results demonstrate that key CME properties are reprogrammed during differentiation at least in part through AP2μ2 expression regulation.
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http://dx.doi.org/10.1083/jcb.201710084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123002PMC
September 2018

Single Molecule Imaging in Live Embryos Using Lattice Light-Sheet Microscopy.

Methods Mol Biol 2018 ;1814:541-559

Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.

In the past decade, live-cell single molecule imaging studies have provided unique insights on how DNA-binding molecules such as transcription factors explore the nuclear environment to search for and bind to their targets. However, due to technological limitations, single molecule experiments in living specimens have largely been limited to monolayer cell cultures. Lattice light-sheet microscopy overcomes these limitations and has now enabled single molecule imaging within thicker specimens such as embryos. Here we describe a general procedure to perform single molecule imaging in living Drosophila melanogaster embryos using lattice light-sheet microscopy. This protocol allows direct observation of both transcription factor diffusion and binding dynamics. Finally, we illustrate how this Drosophila protocol can be extended to other thick samples using single molecule imaging in live mouse embryos as an example.
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http://dx.doi.org/10.1007/978-1-4939-8591-3_32DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6225527PMC
March 2019

Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms.

Science 2018 04;360(6386)

Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA.

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes that we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution, without inducing undue stress on either. We combined lattice light-sheet microscopy with adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.
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http://dx.doi.org/10.1126/science.aaq1392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6040645PMC
April 2018

Widespread Translational Remodeling during Human Neuronal Differentiation.

Cell Rep 2017 Nov;21(7):2005-2016

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA 94143, USA; Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address:

Faithful cellular differentiation requires temporally precise activation of gene expression programs, which are coordinated at the transcriptional and translational levels. Neurons express the most complex set of mRNAs of any human tissue, but translational changes during neuronal differentiation remain incompletely understood. Here, we induced forebrain neuronal differentiation of human embryonic stem cells (hESCs) and measured genome-wide RNA and translation levels with transcript-isoform resolution. We found that thousands of genes change translation status during differentiation without a corresponding change in RNA level. Specifically, we identified mTOR signaling as a key driver for elevated translation of translation-related genes in hESCs. In contrast, translational repression in active neurons is mediated by regulatory sequences in 3' UTRs. Together, our findings identify extensive translational control changes during human neuronal differentiation and a crucial role of 3' UTRs in driving cell-type-specific translation.
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http://dx.doi.org/10.1016/j.celrep.2017.10.095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5759054PMC
November 2017

Mutations in the promoter of the telomerase gene contribute to tumorigenesis by a two-step mechanism.

Science 2017 09 17;357(6358):1416-1420. Epub 2017 Aug 17.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.

promoter mutations (TPMs) are the most common noncoding mutations in cancer. The timing and consequences of TPMs have not been fully established. Here, we show that TPMs acquired at the transition from benign nevus to malignant melanoma do not support telomere maintenance. In vitro experiments revealed that TPMs do not prevent telomere attrition, resulting in cells with critically short and unprotected telomeres. Immortalization by TPMs requires a gradual up-regulation of telomerase, coinciding with telomere fusions. These data suggest that TPMs contribute to tumorigenesis by promoting immortalization and genomic instability in two phases. In an initial phase, TPMs do not prevent bulk telomere shortening but extend cellular life span by healing the shortest telomeres. In the second phase, the critically short telomeres lead to genome instability and telomerase is further up-regulated to sustain cell proliferation.
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http://dx.doi.org/10.1126/science.aao0535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5942222PMC
September 2017

Defined and Scalable Differentiation of Human Oligodendrocyte Precursors from Pluripotent Stem Cells in a 3D Culture System.

Stem Cell Reports 2017 06 25;8(6):1770-1783. Epub 2017 May 25.

Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720-1762, USA; Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA 94720-1462, USA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3370, USA; The Helen Wills Neuroscience Institute, University of California, Berkeley, CA 94720-3370, USA. Electronic address:

Oligodendrocyte precursor cells (OPCs) offer considerable potential for the treatment of demyelinating diseases and injuries of the CNS. However, generating large quantities of high-quality OPCs remains a substantial challenge that impedes their therapeutic application. Here, we show that OPCs can be generated from human pluripotent stem cells (hPSCs) in a three-dimensional (3D), scalable, and fully defined thermoresponsive biomaterial system. We used CRISPR/Cas9 to create a NKX2.2-EGFP human embryonic stem cell reporter line that enabled fine-tuning of early OPC specification and identification of conditions that markedly increased the number of OLIG2 and NKX2.2 cells generated from hPSCs. Transplantation of 50-day-old OPCs into the brains of NOD/SCID mice revealed that progenitors generated in 3D without cell selection or purification subsequently engrafted, migrated, and matured into myelinating oligodendrocytes in vivo. These results demonstrate the potential of harnessing lineage reporter lines to develop 3D platforms for rapid and large-scale production of OPCs.
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http://dx.doi.org/10.1016/j.stemcr.2017.04.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5470111PMC
June 2017

Endogenous Telomerase Reverse Transcriptase N-Terminal Tagging Affects Human Telomerase Function at Telomeres In Vivo.

Mol Cell Biol 2017 02 19;37(3). Epub 2017 Jan 19.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA

Telomerase action at telomeres is essential for the immortal phenotype of stem cells and the aberrant proliferative potential of cancer cells. Insufficient telomere maintenance can cause stem cell and tissue failure syndromes, while increased telomerase levels are associated with tumorigenesis. Both pathologies can arise from only small perturbation of telomerase function. To analyze telomerase at its low endogenous expression level, we genetically engineered human pluripotent stem cells (hPSCs) to express various N-terminal fusion proteins of the telomerase reverse transcriptase from its endogenous locus. Using this approach, we found that these modifications can perturb telomerase function in hPSCs and cancer cells, resulting in telomere length defects. Biochemical analysis suggests that this defect is multileveled, including changes in expression and activity. These findings highlight the unknown complexity of telomerase structural requirements for expression and function in vivo.
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http://dx.doi.org/10.1128/MCB.00541-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5247607PMC
February 2017

Minimized human telomerase maintains telomeres and resolves endogenous roles of H/ACA proteins, TCAB1, and Cajal bodies.

Elife 2016 08 15;5. Epub 2016 Aug 15.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

We dissected the importance of human telomerase biogenesis and trafficking pathways for telomere maintenance. Biological stability of human telomerase RNA (hTR) relies on H/ACA proteins, but other eukaryotes use other RNP assembly pathways. To investigate additional rationale for human telomerase assembly as H/ACA RNP, we developed a minimized cellular hTR. Remarkably, with only binding sites for telomerase reverse transcriptase (TERT), minimized hTR assembled biologically active enzyme. TERT overexpression was required for cellular interaction with minimized hTR, indicating that H/ACA RNP assembly enhances endogenous hTR-TERT interaction. Telomere maintenance by minimized telomerase was unaffected by the elimination of the telomerase holoenzyme Cajal body chaperone TCAB1 or the Cajal body scaffold protein Coilin. Surprisingly, wild-type hTR also maintained and elongated telomeres in TCAB1 or Coilin knockout cells, with distinct changes in telomerase action. Overall, we elucidate trafficking requirements for telomerase biogenesis and function and expand mechanisms by which altered telomere maintenance engenders human disease.
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http://dx.doi.org/10.7554/eLife.18221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5005035PMC
August 2016

Induced Pluripotent Stem Cells Meet Genome Editing.

Cell Stem Cell 2016 05;18(5):573-86

The Whitehead Institute for Biomedical Research and Department of Biology, MIT, Cambridge, MA 02142, USA.

It is extremely rare for a single experiment to be so impactful and timely that it shapes and forecasts the experiments of the next decade. Here, we review how two such experiments-the generation of human induced pluripotent stem cells (iPSCs) and the development of CRISPR/Cas9 technology-have fundamentally reshaped our approach to biomedical research, stem cell biology, and human genetics. We will also highlight the previous knowledge that iPSC and CRISPR/Cas9 technologies were built on as this groundwork demonstrated the need for solutions and the benefits that these technologies provided and set the stage for their success.
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http://dx.doi.org/10.1016/j.stem.2016.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4871596PMC
May 2016

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation.

J Vis Exp 2016 Feb 2(108):e53583. Epub 2016 Feb 2.

Department of Molecular and Cell Biology, University of California, Berkeley;

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell's endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent stem cells.
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http://dx.doi.org/10.3791/53583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4781717PMC
February 2016

Control of telomerase action at human telomeres.

Nat Struct Mol Biol 2015 Nov;22(11):848-52

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.

Recent progress has greatly increased the understanding of telomere-bound shelterin proteins and the telomerase holoenzyme, predominantly as separate complexes. Pioneering studies have begun to investigate the requirements for shelterin-telomerase interaction. From this vantage point, focusing on human cells, we review and discuss models for how telomerase and shelterin subunits coordinate to achieve balanced telomere-length homeostasis.
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http://dx.doi.org/10.1038/nsmb.3083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4765361PMC
November 2015

Human stem cell-based disease modeling: prospects and challenges.

Curr Opin Cell Biol 2015 Dec 11;37:84-90. Epub 2015 Nov 11.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address:

Human stem cell-based disease models have great promise to advance our understanding of human disease. These models can be derived from patients with genetic disorders and manipulated with genome editing and myriad differentiation protocols to model pathologies in vitro. However, several challenges have impeded the full potential of stem cell-based in vitro disease modeling. Many genetically predisposed diseases take time to manifest and occur in specific tissue microenvironments, and these parameters are often not adequately modeled using conventional shorter-term monolayer cultures. These challenges must be overcome especially for cases where animal models also incompletely recapitulate the complex pathologies found in humans. As prominent ways to tackle these challenges we discuss here how advanced genome editing tools in human stem cells and human organoid cultures, specifically the example of intestinal organoids, contribute genetically defined models that recapitulate phenotypes of disease.
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http://dx.doi.org/10.1016/j.ceb.2015.10.007DOI Listing
December 2015

Cancer-associated TERT promoter mutations abrogate telomerase silencing.

Elife 2015 Jul 21;4. Epub 2015 Jul 21.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

Mutations in the human telomerase reverse transcriptase (TERT) promoter are the most frequent non-coding mutations in cancer, but their molecular mechanism in tumorigenesis has not been established. We used genome editing of human pluripotent stem cells with physiological telomerase expression to elucidate the mechanism by which these mutations contribute to human disease. Surprisingly, telomerase-expressing embryonic stem cells engineered to carry any of the three most frequent TERT promoter mutations showed only a modest increase in TERT transcription with no impact on telomerase activity. However, upon differentiation into somatic cells, which normally silence telomerase, cells with TERT promoter mutations failed to silence TERT expression, resulting in increased telomerase activity and aberrantly long telomeres. Thus, TERT promoter mutations are sufficient to overcome the proliferative barrier imposed by telomere shortening without additional tumor-selected mutations. These data establish that TERT promoter mutations can promote immortalization and tumorigenesis of incipient cancer cells.
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http://dx.doi.org/10.7554/eLife.07918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4507476PMC
July 2015

TALEN gene knockouts reveal no requirement for the conserved human shelterin protein Rap1 in telomere protection and length regulation.

Cell Rep 2014 Nov 6;9(4):1273-80. Epub 2014 Nov 6.

Laboratory for Cell Biology and Genetics, The Rockefeller University, New York, NY 10065, USA. Electronic address:

The conserved protein Rap1 functions at telomeres in fungi, protozoa, and vertebrates. Like yeast Rap1, human Rap1 has been implicated in telomere length regulation and repression of nonhomologous end-joining (NHEJ) at telomeres. However, mouse telomeres lacking Rap1 do not succumb to NHEJ. To determine the functions of human Rap1, we generated several transcription activator-like effector nuclease (TALEN)-mediated human cell lines lacking Rap1. Loss of Rap1 did not affect the other components of shelterin, the modification of telomeric histones, the subnuclear position of telomeres, or the 3' telomeric overhang. Telomeres lacking Rap1 did not show a DNA damage response, NHEJ, or consistent changes in their length, indicating that Rap1 does not have an important function in protection or length regulation of human telomeres. As human Rap1, like its mouse and unicellular orthologs, affects gene expression, we propose that the conservation of Rap1 reflects its role in transcriptional regulation rather than a function at telomeres.
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http://dx.doi.org/10.1016/j.celrep.2014.10.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4254571PMC
November 2014

Genome editing in human pluripotent stem cells using site-specific nucleases.

Methods Mol Biol 2015 ;1239:267-80

Department of Molecular and Cell Biology, University of California, Berkeley, 400B Li Ka Shing Center, Berkeley, CA, 94720-3370, USA.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) (Thomson, Science 282:1145-1147, 1998; Takahashi et al. Cell 131:861-872, 2007), collectively referred to as pluripotent stem cells (hPSCs), are currently used in disease modeling to address questions specific to humans and to complement our insight gained from model organisms (Soldner et al. Cell 146:318-331, 2011; Soldner and Jaenisch, Science 338:1155-1156, 2012). Recently, genetic engineering using site-specific nucleases has been established in hPSCs (Hockemeyer et al. Nat Biotechnol 27:851-857, 2009; Hockemeyer et al., Nat Biotechnol 29:731-734, 2011; Zou et al., Cell Stem Cell 5:97-110, 2011; Yusa et al., Nature 478:391-394, 2011; DeKelver et al., Genome Res 20:1133-1142, 2010), allowing a level of genetic control previously limited to model systems. Thus, we can now perform targeted gene knockouts, generate tissue-specific cell lineage reporters, overexpress genes from a defined locus, and introduce and repair single point mutations in hPSCs. This ability to genetically engineer pluripotent stem cells will significantly facilitate the study of human disease in a defined genetic context. Here we outline protocols for efficient gene targeting in hPSCs.
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http://dx.doi.org/10.1007/978-1-4939-1862-1_15DOI Listing
July 2015

Genetic and molecular identification of three human TPP1 functions in telomerase action: recruitment, activation, and homeostasis set point regulation.

Genes Dev 2014 Sep 15;28(17):1885-99. Epub 2014 Aug 15.

Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA;

Telomere length homeostasis is essential for the long-term survival of stem cells, and its set point determines the proliferative capacity of differentiated cell lineages by restricting the reservoir of telomeric repeats. Knockdown and overexpression studies in human tumor cells showed that the shelterin subunit TPP1 recruits telomerase to telomeres through a region termed the TEL patch. However, these studies do not resolve whether the TPP1 TEL patch is the only mechanism for telomerase recruitment and whether telomerase regulation studied in tumor cells is representative of nontransformed cells such as stem cells. Using genome engineering of human embryonic stem cells, which have physiological telomere length homeostasis, we establish that the TPP1 TEL patch is genetically essential for telomere elongation and thus long-term cell viability. Furthermore, genetic bypass, protein fusion, and intragenic complementation assays define two distinct additional mechanisms of TPP1 involvement in telomerase action at telomeres. We demonstrate that TPP1 provides an essential step of telomerase activation as well as feedback regulation of telomerase by telomere length, which is necessary to determine the appropriate telomere length set point in human embryonic stem cells. These studies reveal and resolve multiple TPP1 roles in telomere elongation and stem cell telomere length homeostasis.
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http://dx.doi.org/10.1101/gad.246819.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4197946PMC
September 2014

Human intestinal tissue with adult stem cell properties derived from pluripotent stem cells.

Stem Cell Reports 2014 Jun 3;2(6):838-52. Epub 2014 Jun 3.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3370, USA.

Genetically engineered human pluripotent stem cells (hPSCs) have been proposed as a source for transplantation therapies and are rapidly becoming valuable tools for human disease modeling. However, many applications are limited due to the lack of robust differentiation paradigms that allow for the isolation of defined functional tissues. Here, using an endogenous LGR5-GFP reporter, we derived adult stem cells from hPSCs that gave rise to functional human intestinal tissue comprising all major cell types of the intestine. Histological and functional analyses revealed that such human organoid cultures could be derived with high purity and with a composition and morphology similar to those of cultures obtained from human biopsies. Importantly, hPSC-derived organoids responded to the canonical signaling pathways that control self-renewal and differentiation in the adult human intestinal stem cell compartment. This adult stem cell system provides a platform for studying human intestinal disease in vitro using genetically engineered hPSCs.
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http://dx.doi.org/10.1016/j.stemcr.2014.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4050346PMC
June 2014

Genome editing 101: let's go digital.

Nat Methods 2014 Mar;11(3):248-9

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.

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http://dx.doi.org/10.1038/nmeth.2859DOI Listing
March 2014

Global transcriptional and translational repression in human-embryonic-stem-cell-derived Rett syndrome neurons.

Cell Stem Cell 2013 Oct;13(4):446-58

The Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.

Rett syndrome (RTT) is caused by mutations of MECP2, a methyl CpG binding protein thought to act as a global transcriptional repressor. Here we show, using an isogenic human embryonic stem cell model of RTT, that MECP2 mutant neurons display key molecular and cellular features of this disorder. Unbiased global gene expression analyses demonstrate that MECP2 functions as a global activator in neurons but not in neural precursors. Decreased transcription in neurons was coupled with a significant reduction in nascent protein synthesis and lack of MECP2 was manifested as a severe defect in the activity of the AKT/mTOR pathway. Lack of MECP2 also leads to impaired mitochondrial function in mutant neurons. Activation of AKT/mTOR signaling by exogenous growth factors or by depletion of PTEN boosted protein synthesis and ameliorated disease phenotypes in mutant neurons. Our findings indicate a vital function for MECP2 in maintaining active gene transcription in human neuronal cells.
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http://dx.doi.org/10.1016/j.stem.2013.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4053296PMC
October 2013
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