Publications by authors named "Mario R Capecchi"

120 Publications

The clear cell sarcoma functional genomic landscape.

J Clin Invest 2021 Aug;131(15)

Departments of Orthopaedics and Oncological Sciences, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, Utah, USA.

Clear cell sarcoma (CCS) is a deadly malignancy affecting adolescents and young adults. It is characterized by reciprocal translocations resulting in expression of the chimeric EWSR1-ATF1 or EWSR1-CREB1 fusion proteins, driving sarcomagenesis. Besides these characteristics, CCS has remained genomically uncharacterized. Copy number analysis of human CCSs showed frequent amplifications of the MITF locus and chromosomes 7 and 8. Few alterations were shared with Ewing sarcoma or desmoplastic, small round cell tumors, which are other EWSR1-rearranged tumors. Exome sequencing in mouse tumors generated by expression of EWSR1-ATF1 from the Rosa26 locus demonstrated no other repeated pathogenic variants. Additionally, we generated a new CCS mouse by Cre-loxP-induced chromosomal translocation between Ewsr1 and Atf1, resulting in copy number loss of chromosome 6 and chromosome 15 instability, including amplification of a portion syntenic to human chromosome 8, surrounding Myc. Additional experiments in the Rosa26 conditional model demonstrated that Mitf or Myc can contribute to sarcomagenesis. Copy number observations in human tumors and genetic experiments in mice rendered, for the first time to our knowledge, a functional landscape of the CCS genome. These data advance efforts to understand the biology of CCS using innovative models that will eventually allow us to validate preclinical therapies necessary to achieve longer and better survival for young patients with this disease.
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http://dx.doi.org/10.1172/JCI146301DOI Listing
August 2021

ETV4 and ETV5 drive synovial sarcoma through cell cycle and DUX4 embryonic pathway control.

J Clin Invest 2021 Jul;131(13)

Department of Medicine, Division of Medical Oncology.

Synovial sarcoma is an aggressive malignancy with no effective treatments for patients with metastasis. The synovial sarcoma fusion SS18-SSX, which recruits the SWI/SNF-BAF chromatin remodeling and polycomb repressive complexes, results in epigenetic activation of FGF receptor (FGFR) signaling. In genetic FGFR-knockout models, culture, and xenograft synovial sarcoma models treated with the FGFR inhibitor BGJ398, we show that FGFR1, FGFR2, and FGFR3 were crucial for tumor growth. Transcriptome analyses of BGJ398-treated cells and histological and expression analyses of mouse and human synovial sarcoma tumors revealed prevalent expression of two ETS factors and FGFR targets, ETV4 and ETV5. We further demonstrate that ETV4 and ETV5 acted as drivers of synovial sarcoma growth, most likely through control of the cell cycle. Upon ETV4 and ETV5 knockdown, we observed a striking upregulation of DUX4 and its transcriptional targets that activate the zygotic genome and drive the atrophy program in facioscapulohumeral dystrophy patients. In addition to demonstrating the importance of inhibiting all three FGFRs, the current findings reveal potential nodes of attack for the cancer with the discovery of ETV4 and ETV5 as appropriate biomarkers and molecular targets, and activation of the embryonic DUX4 pathway as a promising approach to block synovial sarcoma tumors.
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http://dx.doi.org/10.1172/JCI141908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8245179PMC
July 2021

Lrig1 expression prospectively identifies stem cells in the ventricular-subventricular zone that are neurogenic throughout adult life.

Neural Dev 2020 03 17;15(1). Epub 2020 Mar 17.

Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT, 84112-5331, USA.

Background: Leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1) regulates stem cell quiescence. As a marker, it identifies stem cells in multiple organs of the mouse. We had detected Lrig1 expression in cultured Id1 neural stem cells obtained from the lateral walls lining the lateral ventricles of the adult mouse brain. Thus, we investigated whether Lrig1 expression also identifies stem cells in that region in vivo.

Methods: Publicly available single cell RNA sequencing datasets were analyzed with Seurat and Monocle. The Lrig1+ cells were lineage traced in vivo with a novel non-disruptive co-translational Lrig1 reporter mouse line.

Results: Analysis of single cell RNA sequencing datasets suggested Lrig1 was highly expressed in the most primitive stem cells of the neurogenic lineage in the lateral wall of the adult mouse brain. In support of their neurogenic stem cell identity, cell cycle entry was only observed in two morphologically distinguishable Lrig1+ cells that could also be induced into activation by Ara-C infusion. The Lrig1+ neurogenic stem cells were observed throughout the lateral wall. Neuroblasts and neurons were lineage traced from Lrig1+ neurogenic stem cells at 1 year after labeling.

Conclusions: We identified Lrig1 as a marker of long-term neurogenic stem cells in the lateral wall of the mouse brain. Lrig1 expression revealed two morphotypes of the Lrig1+ cells that function as long-term neurogenic stem cells. The spatial distribution of the Lrig1+ neurogenic stem cells suggested all subtypes of the adult neurogenic stem cells were labeled.
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http://dx.doi.org/10.1186/s13064-020-00139-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077007PMC
March 2020

HDAC2 Regulates Site-Specific Acetylation of MDM2 and Its Ubiquitination Signaling in Tumor Suppression.

iScience 2019 Mar 15;13:43-54. Epub 2019 Feb 15.

HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806, USA. Electronic address:

Histone deacetylases (HDACs) are promising targets for cancer therapy, although their individual actions remain incompletely understood. Here, we identify a role for HDAC2 in the regulation of MDM2 acetylation at previously uncharacterized lysines. Upon inactivation of HDAC2, this acetylation creates a structural signal in the lysine-rich domain of MDM2 to prevent the recognition and degradation of its downstream substrate, MCL-1 ubiquitin ligase E3 (MULE). This mechanism further reveals a therapeutic connection between the MULE ubiquitin ligase function and tumor suppression. Specifically, we show that HDAC inhibitor treatment promotes the accumulation of MULE, which diminishes the t(X; 18) translocation-associated synovial sarcomagenesis by directly targeting the fusion product SS18-SSX for degradation. These results uncover a new HDAC2-dependent pathway that integrates reversible acetylation signaling to the anticancer ubiquitin response.
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http://dx.doi.org/10.1016/j.isci.2019.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393697PMC
March 2019

Silencing of retrotransposon-derived imprinted gene RTL1 is the main cause for postimplantational failures in mammalian cloning.

Proc Natl Acad Sci U S A 2018 11 31;115(47):E11071-E11080. Epub 2018 Oct 31.

State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, 100193 Beijing, China;

Substantial rates of fetal loss plague all in vitro procedures involving embryo manipulations, including human-assisted reproduction, and are especially problematic for mammalian cloning where over 90% of reconstructed nuclear transfer embryos are typically lost during pregnancy. However, the epigenetic mechanism of these pregnancy failures has not been well described. Here we performed methylome and transcriptome analyses of pig induced pluripotent stem cells and associated cloned embryos, and revealed that aberrant silencing of imprinted genes, in particular the retrotransposon-derived RTL1 gene, is the principal epigenetic cause of pregnancy failure. Remarkably, restoration of RTL1 expression in pig induced pluripotent stem cells rescued fetal loss. Furthermore, in other mammals, including humans, low RTL1 levels appear to be the main epigenetic cause of pregnancy failure.
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http://dx.doi.org/10.1073/pnas.1814514115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6255163PMC
November 2018

Two distinct ontogenies confer heterogeneity to mouse brain microglia.

Development 2018 07 4;145(13). Epub 2018 Jul 4.

Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT 84112, USA

mutant mice show compulsive behavior similar to trichotillomania, a human obsessive-compulsive-spectrum disorder. The only lineage-labeled cells in the brains of mice are microglia, suggesting that defective microglia caused the disorder. What is the source of the microglia? It has been posited that all microglia progenitors arise at embryonic day (E) 7.5 during yolk sac hematopoiesis, and colonize the brain at E9.5. In contrast, we show the presence of two microglia subpopulations: canonical, non- microglia and microglia. Unlike non- microglia, microglia progenitors appear to be generated during the second wave of yolk sac hematopoiesis, then detected in the aorto-gonad-mesonephros (AGM) and fetal liver, where they are greatly expanded, prior to infiltrating the E12.5 brain. Further, we demonstrate that hematopoietic progenitor cells taken from fetal liver are competent to give rise to microglia Although the two microglial subpopulations are very similar molecularly, and in their response to brain injury and participation in synaptic pruning, they show distinct brain distributions which might contribute to pathological specificity. Non- microglia significantly outnumber microglia, but they cannot compensate for the loss of function in microglia, suggesting further crucial differences between the two subpopulations.
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http://dx.doi.org/10.1242/dev.152306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6053660PMC
July 2018

The SS18-SSX Oncoprotein Hijacks KDM2B-PRC1.1 to Drive Synovial Sarcoma.

Cancer Cell 2018 03 1;33(3):527-541.e8. Epub 2018 Mar 1.

Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA; Howard Hughes Medical Institute, New York, NY 10065, USA. Electronic address:

Synovial sarcoma is an aggressive cancer invariably associated with a chromosomal translocation involving genes encoding the SWI-SNF complex component SS18 and an SSX (SSX1 or SSX2) transcriptional repressor. Using functional genomics, we identify KDM2B, a histone demethylase and component of a non-canonical polycomb repressive complex 1 (PRC1.1), as selectively required for sustaining synovial sarcoma cell transformation. SS18-SSX1 physically interacts with PRC1.1 and co-associates with SWI/SNF and KDM2B complexes on unmethylated CpG islands. Via KDM2B, SS18-SSX1 binds and aberrantly activates expression of developmentally regulated genes otherwise targets of polycomb-mediated repression, which is restored upon KDM2B depletion, leading to irreversible mesenchymal differentiation. Thus, SS18-SSX1 deregulates developmental programs to drive transformation by hijacking a transcriptional repressive complex to aberrantly activate gene expression.
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http://dx.doi.org/10.1016/j.ccell.2018.01.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5881394PMC
March 2018

Paracrine osteoprotegerin and β-catenin stabilization support synovial sarcomagenesis in periosteal cells.

J Clin Invest 2018 01 20;128(1):207-218. Epub 2017 Nov 20.

Departments of Orthopaedics and Oncological Sciences, and.

Synovial sarcoma (SS) is an aggressive soft-tissue sarcoma that is often discovered during adolescence and young adulthood. Despite the name, synovial sarcoma does not typically arise from a synoviocyte but instead arises in close proximity to bones. Previous work demonstrated that mice expressing the characteristic SS18-SSX fusion oncogene in myogenic factor 5-expressing (Myf5-expressing) cells develop fully penetrant sarcomagenesis, suggesting skeletal muscle progenitor cell origin. However, Myf5 is not restricted to committed myoblasts in embryos but is also expressed in multipotent mesenchymal progenitors. Here, we demonstrated that human SS and mouse tumors arising from SS18-SSX expression in the embryonic, but not postnatal, Myf5 lineage share an anatomic location that is frequently adjacent to bone. Additionally, we showed that SS can originate from periosteal cells expressing SS18-SSX alone and from preosteoblasts expressing the fusion oncogene accompanied by the added stabilization of β-catenin, which is a common secondary change in SS. Expression and secretion of the osteoclastogenesis inhibitory factor osteoprotegerin enabled early growth of SS18-SSX2-transformed cells, indicating a paracrine link between the bone and synovial sarcomagenesis. These findings explain the skeletal contact frequently observed in human SS and may provide alternate means of enabling SS18-SSX-driven oncogenesis in cells as differentiated as preosteoblasts.
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http://dx.doi.org/10.1172/JCI94955DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749548PMC
January 2018

The Influential Role of BCL2 Family Members in Synovial Sarcomagenesis.

Mol Cancer Res 2017 12 29;15(12):1733-1740. Epub 2017 Aug 29.

Department of Orthopaedics, University of Utah School of Medicine, Salt Lake City, Utah.

Synovial sarcomas are deadly soft tissue malignancies associated with t(X;18) balanced chromosomal translocations. Expression of the apoptotic regulator BCL2 is prominent in synovial sarcomas and has prompted the hypothesis that synovial sarcomagenesis may depend on it. Herein, it is demonstrated that overexpression enhances synovial sarcomagenesis in an animal model. Furthermore, we determined increased familial clustering of human synovial sarcoma patients with victims of other BCL2-associated malignancies in the Utah Population Database. Conditional genetic disruption of in mice also led to reduced sarcomagenesis. Pharmacologic inhibition specific to BCL2 had no demonstrable efficacy against human synovial sarcoma cell lines or mouse tumors. However, targeting BCLxL in human and mouse synovial sarcoma with the small molecule BH3 domain inhibitor, BXI-72, achieved significant cytoreduction and increased apoptotic signaling. Thus, the contributory role of BCL2 in synovial sarcomagenesis does not appear to render it as a therapeutic target, but mitochondrial antiapoptotic BCL2 family members may be. The association of BCL2 expression with synovial sarcoma is found to fit with a subtle, but significant, impact of its enhanced presence or absence during early tumorigenesis. However, specific pharmacologic inhibition of BCL2 does not demonstrate a persistent dependence in fully developed tumors. Conversely, inhibition of the BCL2 family member BCLxL resulted in nanomolar potency against human synovial sarcoma cell lines and 50% tumor reduction in a genetically engineered mouse model. .
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http://dx.doi.org/10.1158/1541-7786.MCR-17-0315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5816685PMC
December 2017

Mouse fitness measures reveal incomplete functional redundancy of Hox paralogous group 1 proteins.

PLoS One 2017 5;12(4):e0174975. Epub 2017 Apr 5.

Department of Biology, University of Utah, Salt Lake City, Utah, United States of America.

Here we assess the fitness consequences of the replacement of the Hoxa1 coding region with its paralog Hoxb1 in mice (Mus musculus) residing in semi-natural enclosures. Previously, this Hoxa1B1 swap was reported as resulting in no discernible embryonic or physiological phenotype (i.e., functionally redundant), despite the 51% amino acid sequence differences between these two Hox proteins. Within heterozygous breeding cages no differences in litter size nor deviations from Mendelian genotypic expectations were observed in the outbred progeny; however, within semi-natural population enclosures mice homozygous for the Hoxa1B1 swap were out-reproduced by controls resulting in the mutant allele being only 87.5% as frequent as the control in offspring born within enclosures. Specifically, Hoxa1B1 founders produced only 77.9% as many offspring relative to controls, as measured by homozygous pups, and a 22.1% deficiency of heterozygous offspring was also observed. These data suggest that Hoxa1 and Hoxb1 have diverged in function through either sub- or neo-functionalization and that the HoxA1 and HoxB1 proteins are not mutually interchangeable when expressed from the Hoxa1 locus. The fitness assays conducted under naturalistic conditions in this study have provided an ultimate-level assessment of the postulated equivalence of competing alleles. Characterization of these differences has provided greater understanding of the forces shaping the maintenance and diversifications of Hox genes as well as other paralogous genes. This fitness assay approach can be applied to any genetic manipulation and often provides the most sensitive way to detect functional differences.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0174975PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5381901PMC
September 2017

Derivation of Transgene-Free Rat Induced Pluripotent Stem Cells Approximating the Quality of Embryonic Stem Cells.

Stem Cells Transl Med 2017 02 13;6(2):340-351. Epub 2016 Sep 13.

State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China.

Although a variety of reprogramming strategies have been reported to create transgene-free induced pluripotent stem (iPS) cells from differentiated cell sources, a fundamental question still remains: Can we generate safe iPS cells that have the full spectrum of features of corresponding embryonic stem (ES) cells? Studies in transgene-free mouse iPS cells have indicated a positive answer to this question. However, the reality is that no other species have a derived transgene-free iPS cell line that can truly mimic ES cell quality. Specifically, critical data for chimera formation and germline transmission are generally lacking. To date, the rat is the only species, other than the mouse, that has commonly recognized authentic ES cells that can be used for direct comparison with measure features of iPS cells. To help find the underlying reasons of the current inability to derive germline-competent ES/iPS cells in nonrodent animals, we first used optimized culture conditions to isolate and establish rat ES cell lines and demonstrated they are fully competent for chimeric formation and germline transmission. We then used episomal vectors bearing eight reprogramming genes to improve rat iPS (riPS) cell generation from Sprague-Dawley rat embryonic fibroblasts. The obtained transgene-free riPS cells exhibit the typical characteristics of pluripotent stem cells; moreover, they are amenable to subsequent genetic modification by homologous recombination. Although they can contribute significantly to chimeric formation, no germline transmission has been achieved. Although this partial success in achieving competency is encouraging, it suggests that more efforts are still needed to derive ground-state riPS cells. Stem Cells Translational Medicine 2017;6:340-351.
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http://dx.doi.org/10.5966/sctm.2015-0390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5442795PMC
February 2017

piggyBac mediates efficient in vivo CRISPR library screening for tumorigenesis in mice.

Proc Natl Acad Sci U S A 2017 01 6;114(4):722-727. Epub 2017 Jan 6.

State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China;

CRISPR/Cas9 is becoming an increasingly important tool to functionally annotate genomes. However, because genome-wide CRISPR libraries are mostly constructed in lentiviral vectors, in vivo applications are severely limited as a result of difficulties in delivery. Here, we examined the piggyBac (PB) transposon as an alternative vehicle to deliver a guide RNA (gRNA) library for in vivo screening. Although tumor induction has previously been achieved in mice by targeting cancer genes with the CRISPR/Cas9 system, in vivo genome-scale screening has not been reported. With our PB-CRISPR libraries, we conducted an in vivo genome-wide screen in mice and identified genes mediating liver tumorigenesis, including known and unknown tumor suppressor genes (TSGs). Our results demonstrate that PB can be a simple and nonviral choice for efficient in vivo delivery of CRISPR libraries.
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http://dx.doi.org/10.1073/pnas.1615735114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5278437PMC
January 2017

Modeling synovial sarcoma metastasis in the mouse: PI3'-lipid signaling and inflammation.

J Exp Med 2016 12 9;213(13):2989-3005. Epub 2016 Nov 9.

Department of Orthopaedics, University of Utah, Salt Lake City, UT 84112

Solid tumor metastasis is a complex biology, impinged upon by a variety of dysregulated signaling pathways. PI3'-lipid signaling has been associated with metastasis and inflammation in many cancers, but the relationship between tumor cell-intrinsic PI3'-lipid signaling and inflammatory cell recruitment has remained enigmatic. Elevated PI3'-lipid signaling associates with progression of synovial sarcoma, a deadly soft tissue malignancy initiated by a t(X;18) chromosomal translocation that generates an SS18-SSX fusion oncoprotein. Here, we show in genetically engineered mouse models of locally induced expression of SS18-SSX1 or SS18-SSX2 that Pten silencing dramatically accelerated and enhanced sarcomagenesis without compromising synovial sarcoma characteristics. PTEN deficiency increased tumor angiogenesis, promoted inflammatory gene expression, and enabled highly penetrant spontaneous pulmonary metastasis. PTEN-deficient sarcomas revealed infiltrating myeloid-derived hematopoietic cells, particularly macrophages and neutrophils, recruited via PI3'-lipid-induced CSF1 expression in tumor cells. Moreover, in a large panel of human synovial sarcomas, enhanced PI3'-lipid signaling also correlated with increased inflammatory cell recruitment and CSF1R signal transduction in both macrophages and endothelial cells. Thus, both in the mouse model and in human synovial sarcomas, PI3'-lipid signaling drives CSF1 expression and associates with increased infiltration of the monocyte/macrophage lineage as well as neutrophils.
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http://dx.doi.org/10.1084/jem.20160817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154942PMC
December 2016

Genome-wide piggyBac transposon mediated screening reveals genes related to reprogramming.

Protein Cell 2017 02;8(2):134-139

State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China.

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http://dx.doi.org/10.1007/s13238-016-0332-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291772PMC
February 2017

Efficient generation of selection-gene-free rat knockout models by homologous recombination in ES cells.

FEBS Lett 2016 Oct 18;590(19):3416-3424. Epub 2016 Sep 18.

State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China.

Embryonic stem cell (ES cell)-based rat knockout technology, although successfully developed in 2010, has seen very limited usage to date due to low targeting efficiency and a lack of optimized procedures. In this study, we performed gene targeting in ES cells from the Sprague-Dawley (SD) and the Fischer 344 (F344) rat strains using an optimized procedure and the self-excising neomycin (neo)-positive selection cassette ACN to successfully generate Leptin and Trp53 knockout rats that did not carry the selection gene. These results demonstrate that our simplified targeting strategy using ACN provides an efficient approach to knock out many other rat genes.
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http://dx.doi.org/10.1002/1873-3468.12388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5129459PMC
October 2016

ASPM regulates symmetric stem cell division by tuning Cyclin E ubiquitination.

Nat Commun 2015 Nov 19;6:8763. Epub 2015 Nov 19.

Department of Human Genetics, University of Utah, Salt Lake City, Utah 84112, USA.

We generate a mouse model for the human microcephaly syndrome by mutating the ASPM locus, and demonstrate a premature exhaustion of the neuronal progenitor pool due to dysfunctional self-renewal processes. Earlier studies have linked ASPM mutant progenitor excessive cell cycle exit to a mitotic orientation defect. Here, we demonstrate a mitotic orientation-independent effect of ASPM on cell cycle duration. We pinpoint the cell fate-determining factor to the length of time spent in early G1 before traversing the restriction point. Characterization of the molecular mechanism reveals an interaction between ASPM and the Cdk2/Cyclin E complex, regulating the Cyclin activity by modulating its ubiquitination, phosphorylation and localization into the nucleus, before the cell is fated to transverse the restriction point. Thus, we reveal a novel function of ASPM in mediating the tightly coordinated Ubiquitin- Cyclin E- Retinoblastoma- E2F bistable-signalling pathway controlling restriction point progression and stem cell maintenance.
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http://dx.doi.org/10.1038/ncomms9763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5025044PMC
November 2015

Type I IFNs Act upon Hematopoietic Progenitors To Protect and Maintain Hematopoiesis during Pneumocystis Lung Infection in Mice.

J Immunol 2015 Dec 30;195(11):5347-57. Epub 2015 Oct 30.

Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59718; and

Although acquired bone marrow failure (BMF) is considered a T cell-mediated autoimmune disease, few studies have considered contributing roles of innate immune deviations following otherwise innocuous infections as a cause underlying the immune defects that lead to BMF. Type I IFN signaling plays an important role in protecting hematopoiesis during systemic stress responses to the opportunistic fungal pathogen Pneumocystis. During Pneumocystis lung infection, mice deficient in both lymphocytes and type I IFN receptor (IFrag(-/-)) develop rapidly progressing BMF associated with accelerated hematopoietic cell apoptosis. However, the communication pathway eliciting the induction of BMF in response to this strictly pulmonary infection has been unclear. We developed a conditional-null allele of Ifnar1 and used tissue-specific induction of the IFrag(-/-) state and found that, following Pneumocystis lung infection, type I IFNs act not only in the lung to prevent systemic immune deviations, but also within the progenitor compartment of the bone marrow to protect hematopoiesis. In addition, transfer of sterile-filtered serum from Pneumocystis-infected mice as well as i.p. injection of Pneumocystis into uninfected IFrag(-/-) mice induced BMF. Although specific cytokine deviations contribute to induction of BMF, immune-suppressive treatment of infected IFrag(-/-) mice ameliorated its progression but did not prevent loss of hematopoietic progenitor functions. This suggested that additional, noncytokine factors also target and impair progenitor functions; and interestingly, fungal β-glucans were also detected in serum. In conclusion, our data demonstrate that type 1 IFN signaling protects hematopoiesis within the bone marrow compartment from the damaging effects of proinflammatory cytokines elicited by Pneumocystis in the lung and possibly at extrapulmonary sites via circulating fungal components.
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http://dx.doi.org/10.4049/jimmunol.1501553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655130PMC
December 2015

Hoxc8 initiates an ectopic mammary program by regulating Fgf10 and Tbx3 expression and Wnt/β-catenin signaling.

Development 2015 Dec 12;142(23):4056-67. Epub 2015 Oct 12.

Department of Human Genetics and Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT 84112, USA.

The role of Hox genes in the formation of cutaneous accessory organs such as hair follicles and mammary glands has proved elusive, a likely consequence of overlapping function and expression among various homeobox factors. Lineage and immunohistochemical analysis of Hoxc8 in mice revealed that this midthoracic Hox gene has transient but strong regional expression in ventrolateral surface ectoderm at E10.5, much earlier than previously reported. Targeted mice were generated to conditionally misexpress Hoxc8 from the Rosa locus using select Cre drivers, which significantly expanded the domain of thoracic identity in mutant embryos. Accompanying this expansion was the induction of paired zones of ectopic mammary development in the cervical region, which generated between three and five pairs of mammary placodes anterior to the first wild-type mammary rudiment. These rudiments expressed the mammary placode markers Wnt10b and Tbx3 and were labeled by antibodies to the mammary mesenchyme markers ERα and androgen receptor. Somitic Fgf10 expression, which is required for normal mammary line formation, was upregulated in mutant cervical somites, and conditional ablation of ectodermal Tbx3 expression eliminated all normally positioned and ectopic mammary placodes. We present evidence that Hoxc8 participates in regulating the initiation stages of mammary placode morphogenesis, and suggest that this and other Hox genes are likely to have important roles during regional specification and initiation of these and other cutaneous accessory organs.
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http://dx.doi.org/10.1242/dev.128298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4712843PMC
December 2015

β-catenin stabilization enhances SS18-SSX2-driven synovial sarcomagenesis and blocks the mesenchymal to epithelial transition.

Oncotarget 2015 Sep;6(26):22758-66

Department of Orthopaedics and Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT 84112, USA.

β-catenin is a master regulator in the cellular biology of development and neoplasia. Its dysregulation is implicated as a driver of colorectal carcinogenesis and the epithelial-mesenchymal transition in other cancers. Nuclear β-catenin staining is a poor prognostic sign in synovial sarcoma, the most common soft-tissue sarcoma in adolescents and young adults. We show through genetic experiments in a mouse model that expression of a stabilized form of β-catenin greatly enhances synovial sarcomagenesis. Stabilization of β-catenin enables a stem-cell phenotype in synovial sarcoma cells, specifically blocking epithelial differentiation and driving invasion. β-catenin achieves its reprogramming in part by upregulating transcription of TCF/LEF target genes. Even though synovial sarcoma is primarily a mesenchymal neoplasm, its progression towards a more aggressive and invasive phenotype parallels the epithelial-mesenchymal transition observed in epithelial cancers, where β-catenin's transcriptional contribution includes blocking epithelial differentiation.
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http://dx.doi.org/10.18632/oncotarget.4283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4673197PMC
September 2015

Intracellular calcium dynamics in cortical microglia responding to focal laser injury in the PC::G5-tdT reporter mouse.

Front Mol Neurosci 2015 8;8:12. Epub 2015 May 8.

Department of Human Genetics, University of Utah Salt Lake City, UT, USA ; Howard Hughes Medical Institute Chevy Chase, MD, USA.

Microglia, the resident immune cells of the brain parenchyma, are highly responsive to tissue injury. Following cell damage, microglial processes redirect their motility from randomly scouting the extracellular space to specifically reaching toward the compromised tissue. While the cell morphology aspects of this defense mechanism have been characterized, the intracellular events underlying these responses remain largely unknown. Specifically, the role of intracellular Ca(2+) dynamics has not been systematically investigated in acutely activated microglia due to technical difficulty. Here we used live two-photon imaging of the mouse cortex ubiquitously expressing the genetically encoded Ca(2+) indicator GCaMP5G and fluorescent marker tdTomato in central nervous system microglia. We found that spontaneous Ca(2+) transients in microglial somas and processes were generally low (only 4% of all microglia showing transients within 20 min), but baseline activity increased about 8-fold when the animals were treated with LPS 12 h before imaging. When challenged with focal laser injury, an additional surge in Ca(2+) activity was observed in the somas and protruding processes. Notably, coherent and simultaneous Ca(2+) rises in multiple microglial cells were occasionally detected in LPS-treated animals. We show that Ca(2+) transients were pre-dominantly mediated via purinergic receptors. This work demonstrates the usefulness of genetically encoded Ca(2+) indicators for investigation of microglial physiology.
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http://dx.doi.org/10.3389/fnmol.2015.00012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4424843PMC
May 2015

Imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporation.

Front Mol Neurosci 2015 15;8:10. Epub 2015 Apr 15.

Neuronal Dynamics Laboratory, Department of Bioengineering, University of Utah Salt Lake City, UT, USA.

Complex interactions between networks of astrocytes and neurons are beginning to be appreciated, but remain poorly understood. Transgenic mice expressing fluorescent protein reporters of cellular activity, such as the GCaMP family of genetically encoded calcium indicators (GECIs), have been used to explore network behavior. However, in some cases, it may be desirable to use long-established rat models that closely mimic particular aspects of human conditions such as Parkinson's disease and the development of epilepsy following status epilepticus. Methods for expressing reporter proteins in the rat brain are relatively limited. Transgenic rat technologies exist but are fairly immature. Viral-mediated expression is robust but unstable, requires invasive injections, and only works well for fairly small genes (<5 kb). In utero electroporation (IUE) offers a valuable alternative. IUE is a proven method for transfecting populations of astrocytes and neurons in the rat brain without the strict limitations on transgene size. We built a toolset of IUE plasmids carrying GCaMP variants 3, 6s, or 6f driven by CAG and targeted to the cytosol or the plasma membrane. Because low baseline fluorescence of GCaMP can hinder identification of transfected cells, we included the option of co-expressing a cytosolic tdTomato protein. A binary system consisting of a plasmid carrying a piggyBac inverted terminal repeat (ITR)-flanked CAG-GCaMP-IRES-tdTomato cassette and a separate plasmid encoding for expression of piggyBac transposase was employed to stably express GCaMP and tdTomato. The plasmids were co-electroporated on embryonic days 13.5-14.5 and astrocytic and neuronal activity was subsequently imaged in acute or cultured brain slices prepared from the cortex or hippocampus. Large spontaneous transients were detected in slices obtained from rats of varying ages up to 127 days. In this report, we demonstrate the utility of this toolset for interrogating astrocytic and neuronal activity in the rat brain.
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http://dx.doi.org/10.3389/fnmol.2015.00010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4397926PMC
April 2015

Response: Contributions of the Myf5-independent lineage to myogenesis.

Dev Cell 2014 Dec;31(5):539-41

Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT 84112, USA; Department of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA. Electronic address:

In this issue, Comai et al. present evidence that previous support for Myf5-independent myogenic cell lineages was confounded by inefficiencies in lineage marking and ablation. Here, Haldar et al. discuss other possible explanations for the inconsistencies between different data sets and reiterate their views on Myf5-independent myogenesis.
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http://dx.doi.org/10.1016/j.devcel.2014.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5026122PMC
December 2014

Modeling alveolar soft part sarcomagenesis in the mouse: a role for lactate in the tumor microenvironment.

Cancer Cell 2014 Dec 26;26(6):851-862. Epub 2014 Nov 26.

Department of Orthopaedics, University of Utah, Salt Lake City, UT 84112, USA; Center for Children's Cancer Research at the Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA. Electronic address:

Alveolar soft part sarcoma (ASPS), a deadly soft tissue malignancy with a predilection for adolescents and young adults, associates consistently with t(X;17) translocations that generate the fusion gene ASPSCR1-TFE3. We proved the oncogenic capacity of this fusion gene by driving sarcomagenesis in mice from conditional ASPSCR1-TFE3 expression. The completely penetrant tumors were indistinguishable from human ASPS by histology and gene expression. They formed preferentially in the anatomic environment highest in lactate, the cranial vault, expressed high levels of lactate importers, harbored abundant mitochondria, metabolized lactate as a metabolic substrate, and responded to the administration of exogenous lactate with tumor cell proliferation and angiogenesis. These data demonstrate lactate's role as a driver of alveolar soft part sarcomagenesis.
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http://dx.doi.org/10.1016/j.ccell.2014.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4327935PMC
December 2014

Pro-proliferative and inflammatory signaling converge on FoxO1 transcription factor in pulmonary hypertension.

Nat Med 2014 Nov 26;20(11):1289-300. Epub 2014 Oct 26.

1] Max-Planck-Institute for Heart and Lung Research, Department of Lung Development and Remodeling, member of the German Center for Lung Research (DZL), Bad Nauheim, Germany. [2] Department of Internal Medicine, Universities of Giessen and Marburg Lung Center (UGMLC), member of the DZL, Justus-Liebig University, Giessen, Germany.

Pulmonary hypertension (PH) is characterized by increased proliferation and apoptosis resistance of pulmonary artery smooth muscle cells (PASMCs). Forkhead box O (FoxO) transcription factors are key regulators of cellular proliferation. Here we show that in pulmonary vessels and PASMCs of human and experimental PH lungs, FoxO1 expression is downregulated and FoxO1 is inactivated via phosphorylation and nuclear exclusion. These findings could be reproduced using ex vivo exposure of PASMCs to growth factors and inflammatory cytokines. Pharmacological inhibition and genetic ablation of FoxO1 in smooth muscle cells reproduced PH features in vitro and in vivo. Either pharmacological reconstitution of FoxO1 activity using intravenous or inhaled paclitaxel, or reconstitution of the transcriptional activity of FoxO1 by gene therapy, restored the physiologically quiescent PASMC phenotype in vitro, linked to changes in cell cycle control and bone morphogenic protein receptor type 2 (BMPR2) signaling, and reversed vascular remodeling and right-heart hypertrophy in vivo. Thus, PASMC FoxO1 is a critical integrator of multiple signaling pathways driving PH, and reconstitution of FoxO1 activity offers a potential therapeutic option for PH.
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http://dx.doi.org/10.1038/nm.3695DOI Listing
November 2014

Imaging activity in neurons and glia with a Polr2a-based and cre-dependent GCaMP5G-IRES-tdTomato reporter mouse.

Neuron 2014 Sep 21;83(5):1058-72. Epub 2014 Aug 21.

Department of Human Genetics, University of Utah, Salt Lake City, UT 84112. Electronic address:

Unlabelled: New strategies for introducing genetically encoded activity indicators into animal models facilitate the investigation of nervous system function. We have developed the PC::G5-tdT mouse line that expresses the GCaMP5G calcium indicator in a Cre-dependent fashion. Instead of targeting the ROSA26 locus, we inserted the reporter cassette nearby the ubiquitously expressed Polr2a gene without disrupting locus integrity. The indicator was tagged with IRES-tdTomato to aid detection of positive cells. This reporter system is effective in a wide range of developmental and cellular contexts. We recorded spontaneous cortical calcium waves in intact awake newborns and evaluated concentration-dependent responses to odorants in the adult olfactory bulb. Moreover, PC::G5-tdT effectively reports intracellular calcium dynamics in somas and fine processes of astrocytes and microglial cells. Through electrophysiological and behavioral analyses, we determined that GCaMP5G expression had no major impact on nervous system performance. PC::G5-tdT will be instrumental for a variety of brain mapping experiments.

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http://dx.doi.org/10.1016/j.neuron.2014.07.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4156920PMC
September 2014

Lineage of origin in rhabdomyosarcoma informs pharmacological response.

Genes Dev 2014 Jul;28(14):1578-91

Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Germans Trias i Pujol Health Sciences Research Institute (IGTP), 08916 Badalona, Barcelona, Spain;

Lineage or cell of origin of cancers is often unknown and thus is not a consideration in therapeutic approaches. Alveolar rhabdomyosarcoma (aRMS) is an aggressive childhood cancer for which the cell of origin remains debated. We used conditional genetic mouse models of aRMS to activate the pathognomonic Pax3:Foxo1 fusion oncogene and inactivate p53 in several stages of prenatal and postnatal muscle development. We reveal that lineage of origin significantly influences tumor histomorphology and sensitivity to targeted therapeutics. Furthermore, we uncovered differential transcriptional regulation of the Pax3:Foxo1 locus by tumor lineage of origin, which led us to identify the histone deacetylase inhibitor entinostat as a pharmacological agent for the potential conversion of Pax3:Foxo1-positive aRMS to a state akin to fusion-negative RMS through direct transcriptional suppression of Pax3:Foxo1.
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http://dx.doi.org/10.1101/gad.238733.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4102765PMC
July 2014

Efficient germ-line transmission obtained with transgene-free induced pluripotent stem cells.

Proc Natl Acad Sci U S A 2014 Jul 7;111(29):10678-83. Epub 2014 Jul 7.

Department of Human Genetics andHoward Hughes Medical Institute, University of Utah School of Medicine, Salt Lake City, UT 84112

Induced pluripotent stem (iPS) cells hold great promise for regenerative medicine. To overcome potential problems associated with transgene insertions, efforts have been directed over the past several years to generate transgene-free iPS cells by using non-viral-vector approaches. To date, however, cells generated through such procedures have had problems producing reproductively competent animals, suggesting that their quality needed further improvement. Here we report the use of optimized assemblies of reprogramming factors and selection markers incorporated into single plasmids as nonintegrating episomes to generate germ-line-competent iPS cells. In particular, the pMaster12 episome can produce transgene-free iPS cells that, when grown in 2i medium, recapitulate good mouse ES cells, in terms of their competency for generating germ-line chimeras.
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http://dx.doi.org/10.1073/pnas.1409933111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4115572PMC
July 2014

Fine-tuning of iPSC derivation by an inducible reprogramming system at the protein level.

Stem Cell Reports 2014 May 8;2(5):721-33. Epub 2014 May 8.

State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, PR China.

Induced pluripotent stem cells (iPSCs) generated from somatic cells by ectopic expression of reprogramming factors, e.g., POU5F1 (OCT4), KLF4, and SOX2, have great potential for regenerative medicine. However, before they can be used in a clinical setting, the mechanism of reprogramming needs to be better understood. Here, by engineering reprogramming factors to a destabilizing protein domain, we achieved inducible generation of mouse and pig iPSCs. Stability of the fusion protein was precisely regulated by the addition of the cell-permeable small molecule trimethoprim (TMP) in a dose-dependent manner. With these tools, we found that during the early and middle stages of reprogramming, exogenous OCT4 or KLF4 could be omitted, whereas exogenous SOX2 expression at early and middle stages was required for successful reprogramming. Our TMP reprogramming system is useful for defining the stoichiometry and temporal requirements of transcription factors for reprogramming.
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http://dx.doi.org/10.1016/j.stemcr.2014.03.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4050490PMC
May 2014
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