Publications by authors named "T Michael Underhill"

121 Publications

Murine Limb Bud Organ Cultures for Studying Musculoskeletal Development.

Methods Mol Biol 2021 ;2230:115-137

Biomedical Research Centre, University of British Columbia, Vancouver, BC, Canada.

The biological signals that coordinate the three-dimensional outgrowth and patterning of the vertebrate limb bud have been well delineated. These include a number of vital embryonic signaling pathways, including the fibroblast growth factor, WNT, transforming growth factor, and hedgehog. Collectively these signals converge on multiple progenitor populations to drive the formation of a variety of tissues that make up the limb musculoskeletal system, such as muscle, tendon, cartilage, stroma, and bone. The basic mechanisms regulating the commitment and differentiation of diverse limb progenitor populations has been successfully modeled in vitro using high density primary limb mesenchymal or micromass cultures. However, this approach is limited in its ability to more faithfully recapitulate the assembly of progenitors into organized tissues that span the entire musculoskeletal system. Other biological systems have benefitted from the development and availability of three-dimensional organoid cultures which have transformed our understanding of tissue development, homeostasis and regeneration. Such a system does not exist that effectively models the complexity of limb development. However, limb bud organ cultures while still necessitating the use of collected embryonic tissue have proved to be a powerful model system to elucidate the molecular underpinning of musculoskeletal development. In this methods article, the derivation and use of limb bud organ cultures from murine limb buds will be described, along with strategies to manipulate signaling pathways, examine gene expression and for longitudinal lineage tracking.
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http://dx.doi.org/10.1007/978-1-0716-1028-2_8DOI Listing
March 2021

Abortive γδTCR rearrangements suggest ILC2s are derived from T-cell precursors.

Blood Adv 2020 11;4(21):5362-5372

The Biomedical Research Centre, University of British Columbia, Vancouver, BC, Canada; and.

Innate lymphoid cells (ILCs) are a recently identified subset of leukocytes that play a central role in pathogen surveillance and resistance, modulation of immune response, and tissue repair. They are remarkably similar to CD4+ T-helper subsets in terms of function and transcription factors required for their development but are distinguished by their lack of antigen-specific receptors. Despite their similarities, the absence of a surface T-cell receptor (TCR) and presence of ILCs and precursors in adult bone marrow has led to speculation that ILCs and T cells develop separately from lineages that branch at the point of precursors within the bone marrow. Considering the common lineage markers and effector cytokine profiles shared between ILCs and T cells, it is surprising that the status of the TCR loci in ILCs was not fully explored at the time of their discovery. Here, we demonstrate that a high proportion of peripheral tissue ILC2s have TCRγ chain gene rearrangements and TCRδ locus deletions. Detailed analyses of these loci show abundant frameshifts and premature stop codons that would encode nonfunctional TCR proteins. Collectively, these data argue that ILC2 can develop from T cells that fail to appropriately rearrange TCR genes, potentially within the thymus.
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http://dx.doi.org/10.1182/bloodadvances.2020002758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7656916PMC
November 2020

Distinct Regulatory Programs Control the Latent Regenerative Potential of Dermal Fibroblasts during Wound Healing.

Cell Stem Cell 2020 09 4;27(3):396-412.e6. Epub 2020 Aug 4.

Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada; Alberta Children's Hospital Research Institute, University of Calgary, Calgary, AB T2N 4N1, Canada; Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada. Electronic address:

Dermal fibroblasts exhibit considerable heterogeneity during homeostasis and in response to injury. Defining lineage origins of reparative fibroblasts and regulatory programs that drive fibrosis or, conversely, promote regeneration will be essential for improving healing outcomes. Using complementary fate-mapping approaches, we show that hair follicle mesenchymal progenitors make limited contributions to wound repair. In contrast, extrafollicular progenitors marked by the quiescence-associated factor Hic1 generated the bulk of reparative fibroblasts and exhibited functional divergence, mediating regeneration in the center of the wound neodermis and scar formation in the periphery. Single-cell RNA-seq revealed unique transcriptional, regulatory, and epithelial-mesenchymal crosstalk signatures that enabled mesenchymal competence for regeneration. Integration with scATAC-seq highlighted changes in chromatin accessibility within regeneration-associated loci. Finally, pharmacological modulation of RUNX1 and retinoic acid signaling or genetic deletion of Hic1 within wound-activated fibroblasts was sufficient to modulate healing outcomes, suggesting that reparative fibroblasts have latent but modifiable regenerative capacity.
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http://dx.doi.org/10.1016/j.stem.2020.07.008DOI Listing
September 2020

Development and function of smooth muscle cells is modulated by in mouse testis.

Development 2020 07 13;147(13). Epub 2020 Jul 13.

Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada

In mammalian testis, contractile peritubular myoid cells (PMCs) regulate the transport of sperm and luminal fluid, while secreting growth factors and extracellular matrix proteins to support the spermatogonial stem cell niche. However, little is known about the role of testicular smooth muscle cells during postnatal testicular development. Here we report age-dependent expression of hypermethylated in cancer 1 (; also known as ) in testicular smooth muscle cells, including PMCs and vascular smooth muscle cells, in the mouse. Postnatal deletion of in smooth muscle cells led to their increased proliferation and resulted in dilatation of seminiferous tubules, with increased numbers of PMCs. These seminiferous tubules contained fewer Sertoli cells and more spermatogonia, and fibronectin was not detected in their basement membrane. The expression levels of genes encoding smooth muscle contractile proteins, and , were downregulated in the smooth muscle cells lacking , and the seminiferous tubules appeared to have reduced contractility. These data imply a role for in determining the size of seminiferous tubules by regulating postnatal smooth muscle cell proliferation, subsequently affecting spermatogenesis in adulthood.
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http://dx.doi.org/10.1242/dev.185884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375483PMC
July 2020

Loss of Thymine DNA Glycosylase Causes Dysregulation of Bile Acid Homeostasis and Hepatocellular Carcinoma.

Cell Rep 2020 04;31(1):107475

Department of Biochemistry, Western University, London, ON, Canada; Department of Oncology, The London Regional Cancer Program, London, ON, Canada; Lawson Health Research Institute, London Health Science Centre, London, ON, Canada. Electronic address:

Thymine DNA glycosylase (TDG) is a nuclear receptor coactivator that plays an essential role in the maintenance of epigenetic stability in cells. Here, we demonstrate that the conditional deletion of TDG in adult mice results in a male-predominant onset of hepatocellular carcinoma (HCC). TDG loss leads to a prediabetic state, as well as bile acid (BA) accumulation in the liver and serum of male mice. Consistent with these data, TDG deletion led to dysregulation of the farnesoid X receptor (FXR) and small heterodimer partner (SHP) regulatory cascade in the liver. FXR and SHP are tumor suppressors of HCC and play an essential role in BA and glucose homeostasis. These results indicate that TDG functions as a tumor suppressor of HCC by regulating a transcriptional program that protects against the development of glucose intolerance and BA accumulation in the liver.
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http://dx.doi.org/10.1016/j.celrep.2020.03.039DOI Listing
April 2020

Pathogenic Potential of Hic1-Expressing Cardiac Stromal Progenitors.

Cell Stem Cell 2020 02 23;26(2):205-220.e8. Epub 2020 Jan 23.

Biomedical Research Centre, University of British Columbia, 2222 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada. Electronic address:

The cardiac stroma contains multipotent mesenchymal progenitors. However, lineage relationships within cardiac stromal cells are poorly defined. Here, we identified heart-resident PDGFRa SCA-1 cells as cardiac fibro/adipogenic progenitors (cFAPs) and show that they respond to ischemic damage by generating fibrogenic cells. Pharmacological blockade of this differentiation step with an anti-fibrotic tyrosine kinase inhibitor decreases post-myocardial infarction (post-MI) remodeling and leads to improvement in cardiac function. In the undamaged heart, activation of cFAPs through lineage-specific deletion of the gene encoding the quiescence-associated factor HIC1 reveals additional pathogenic potential, causing fibrofatty infiltration within the myocardium and driving major pathological features pathognomonic in arrhythmogenic cardiomyopathy (AC). In this regard, cFAPs contribute to multiple pathogenic cell types within cardiac tissue and therapeutic strategies aimed at modifying their activity are expected to have tremendous benefit for the treatment of diverse cardiac diseases.
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http://dx.doi.org/10.1016/j.stem.2019.12.008DOI Listing
February 2020

Hic1 Defines Quiescent Mesenchymal Progenitor Subpopulations with Distinct Functions and Fates in Skeletal Muscle Regeneration.

Cell Stem Cell 2019 Dec;25(6):797-813.e9

Department of Cellular and Physiological Sciences, 2222 Health Sciences Mall, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada; School of Biomedical Engineering and the Biomedical Research Centre, 2222 Health Sciences Mall, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada. Electronic address:

Many adult tissues contain resident stem cells, such as the Pax7 satellite cells within skeletal muscle, that regenerate parenchymal elements following damage. Tissue-resident mesenchymal progenitors (MPs) also participate in regeneration, although their function and fate in this process are unclear. Here, we identify Hypermethylated in cancer 1 (Hic1) as a marker of MPs in skeletal muscle and further show that Hic1 deletion leads to MP hyperplasia. Single-cell RNA-seq and ATAC-seq analysis of Hic1 MPs in skeletal muscle shows multiple subpopulations, which we further show have distinct functions and lineage potential. Hic1 MPs orchestrate multiple aspects of skeletal muscle regeneration by providing stage-specific immunomodulation and trophic and mechanical support. During muscle regeneration, Hic1 derivatives directly contribute to several mesenchymal compartments including Col22a1-expressing cells within the myotendinous junction. Collectively, these findings demonstrate that HIC1 regulates MP quiescence and identifies MP subpopulations with transient and enduring roles in muscle regeneration.
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http://dx.doi.org/10.1016/j.stem.2019.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941576PMC
December 2019

Clonal Decomposition and DNA Replication States Defined by Scaled Single-Cell Genome Sequencing.

Cell 2019 Nov;179(5):1207-1221.e22

Department of Molecular Oncology, BC Cancer Research Centre, 675 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC V6T 2B5, Canada. Electronic address:

Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.
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http://dx.doi.org/10.1016/j.cell.2019.10.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912164PMC
November 2019

The Transcription Factor RORα Preserves ILC3 Lineage Identity and Function during Chronic Intestinal Infection.

J Immunol 2019 12 1;203(12):3209-3215. Epub 2019 Nov 1.

The Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada; and

Innate lymphoid cells (ILCs) are critical for host defense and tissue repair but can also contribute to chronic inflammatory diseases. The transcription factor RORα is required for ILC2 development but is also highly expressed by other ILC subsets where its function remains poorly defined. We previously reported that bone marrow chimeric mice (C57BL/6J) were protected from -induced intestinal fibrosis due to defective ILC3 responses. In this study, single-cell RNA analysis of ILCs isolated from inflamed tissues indicates that RORα perturbation led to a reduction in ILC3 lineages. Furthermore, residual ILC3s have decreased expression of key signature genes, including and activating cytokine receptors. Collectively, our data suggest that RORα plays a key role in preserving functional ILC3s by modulating their ability to integrate environmental cues to efficiently produce cytokines.
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http://dx.doi.org/10.4049/jimmunol.1900781DOI Listing
December 2019

Mapping Retinoic Acid-Dependant 5mC Derivatives in Mouse Embryonic Fibroblasts.

Methods Mol Biol 2019 ;2019:129-141

Department of Biochemistry, Western University, London, ON, Canada.

Methylase-assisted bisulfite sequencing (MAB-seq) is a derivatization technique to evaluate the presence of 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) at base-pair resolution. Although MAB-seq was originally designed to study these metabolites under steady-state conditions, we have developed an alternative protocol to evaluate the dynamics of 5-fC/5-caC accumulation in response to agonists, such as all-trans retinoic acid (ATRA). In addition, this protocol utilizes a lower quantity of the M.SssI enzyme without compromising methylation efficiency and requires less bench time. Herein, we describe the use of MAB-seq assay to evaluate the generation of 5-fC/5-caC in response to ATRA in mouse embryonic fibroblasts, using the hypermethylated in cancer 1 (Hic1) locus as a model system.
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http://dx.doi.org/10.1007/978-1-4939-9585-1_10DOI Listing
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

Podocalyxin is required for maintaining blood-brain barrier function during acute inflammation.

Proc Natl Acad Sci U S A 2019 03 20;116(10):4518-4527. Epub 2019 Feb 20.

Biomedical Research Centre, University of British Columbia, Vancouver, BC, Canada V6T 1Z3;

Podocalyxin (Podxl) is broadly expressed on the luminal face of most blood vessels in adult vertebrates, yet its function on these cells is poorly defined. In the present study, we identified specific functions for Podxl in maintaining endothelial barrier function. Using electrical cell substrate impedance sensing and live imaging, we found that, in the absence of Podxl, human umbilical vein endothelial cells fail to form an efficient barrier when plated on several extracellular matrix substrates. In addition, these monolayers lack adherens junctions and focal adhesions and display a disorganized cortical actin cytoskeleton. Thus, Podxl has a key role in promoting the appropriate endothelial morphogenesis required to form functional barriers. This conclusion is further supported by analyses of mutant mice in which we conditionally deleted a floxed allele of in vascular endothelial cells (vECs) using Tie2Cre mice (). Although we did not detect substantially altered permeability in naïve mice, systemic priming with lipopolysaccharide (LPS) selectively disrupted the blood-brain barrier (BBB) in mice. To study the potential consequence of this BBB breach, we used a selective agonist (TFLLR-NH) of the protease-activated receptor-1 (PAR-1), a thrombin receptor expressed by vECs, neuronal cells, and glial cells. In response to systemic administration of TFLLR-NH, LPS-primed mice become completely immobilized for a 5-min period, coinciding with severely dampened neuroelectric activity. We conclude that Podxl expression by CNS tissue vECs is essential for BBB maintenance under inflammatory conditions.
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http://dx.doi.org/10.1073/pnas.1814766116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6410846PMC
March 2019

Histone Deacetylase Inhibitors Synergize with Catalytic Inhibitors of EZH2 to Exhibit Antitumor Activity in Small Cell Carcinoma of the Ovary, Hypercalcemic Type.

Mol Cancer Ther 2018 12 19;17(12):2767-2779. Epub 2018 Sep 19.

Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare but extremely lethal malignancy that mainly impacts young women. SCCOHT is characterized by a diploid genome with loss of SMARCA4 and lack of SMARCA2 expression, two mutually exclusive ATPases of the SWI/SNF chromatin-remodeling complex. We and others have identified the histone methyltransferase EZH2 as a promising therapeutic target for SCCOHT, suggesting that SCCOHT cells depend on the alternation of epigenetic pathways for survival. In this study, we found that SCCOHT cells were more sensitive to pan-HDAC inhibitors compared with other ovarian cancer lines or immortalized cell lines tested. Pan-HDAC inhibitors, such as quisinostat, reversed the expression of a group of proteins that were deregulated in SCCOHT cells due to SMARCA4 loss, leading to growth arrest, apoptosis, and differentiation and suppressed tumor growth of xenografted tumors of SCCOHT cells. Moreover, combined treatment of HDAC inhibitors and EZH2 inhibitors at sublethal doses synergistically induced histone H3K27 acetylation and target gene expression, leading to rapid induction of apoptosis and growth suppression of SCCOHT cells and xenografted tumors. Therefore, our preclinical study highlighted the therapeutic potential of combined treatment of HDAC inhibitors with EZH2 catalytic inhibitors to treat SCCOHT.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-0348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279577PMC
December 2018

HIC1 links retinoic acid signalling to group 3 innate lymphoid cell-dependent regulation of intestinal immunity and homeostasis.

PLoS Pathog 2018 02 22;14(2):e1006869. Epub 2018 Feb 22.

The Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia, Canada.

The intestinal immune system must be able to respond to a wide variety of infectious organisms while maintaining tolerance to non-pathogenic microbes and food antigens. The Vitamin A metabolite all-trans-retinoic acid (atRA) has been implicated in the regulation of this balance, partially by regulating innate lymphoid cell (ILC) responses in the intestine. However, the molecular mechanisms of atRA-dependent intestinal immunity and homeostasis remain elusive. Here we define a role for the transcriptional repressor Hypermethylated in cancer 1 (HIC1, ZBTB29) in the regulation of ILC responses in the intestine. Intestinal ILCs express HIC1 in a vitamin A-dependent manner. In the absence of HIC1, group 3 ILCs (ILC3s) that produce IL-22 are lost, resulting in increased susceptibility to infection with the bacterial pathogen Citrobacter rodentium. Thus, atRA-dependent expression of HIC1 in ILC3s regulates intestinal homeostasis and protective immunity.
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http://dx.doi.org/10.1371/journal.ppat.1006869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823476PMC
February 2018

Death by HDAC Inhibition in Synovial Sarcoma Cells.

Mol Cancer Ther 2017 Dec 6;16(12):2656-2667. Epub 2017 Sep 6.

Faculty of Medicine, Vancouver Coastal Health Research Institute, University of British Columbia, Vancouver, British Columbia, Canada.

Conventional cytotoxic therapies for synovial sarcoma provide limited benefit, and no drugs specifically targeting the causative SS18-SSX fusion oncoprotein are currently available. Histone deacetylase (HDAC) inhibition has been shown in previous studies to disrupt the synovial sarcoma oncoprotein complex, resulting in apoptosis. To understand the molecular effects of HDAC inhibition, RNA-seq transcriptome analysis was undertaken in six human synovial sarcoma cell lines. HDAC inhibition induced pathways of cell-cycle arrest, neuronal differentiation, and response to oxygen-containing species, effects also observed in other cancers treated with this class of drugs. More specific to synovial sarcoma, polycomb group targets were reactivated, including tumor suppressor , and proapoptotic transcriptional patterns were induced. Functional analyses revealed that ROS-mediated FOXO activation and proapoptotic factors BIK, BIM, and BMF were important to apoptosis induction following HDAC inhibition in synovial sarcoma. HDAC inhibitor pathway activation results in apoptosis and decreased tumor burden following a 7-day quisinostat treatment in the mouse model of synovial sarcoma. This study provides mechanistic support for a particular susceptibility of synovial sarcoma to HDAC inhibition as a means of clinical treatment. .
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http://dx.doi.org/10.1158/1535-7163.MCT-17-0397DOI Listing
December 2017

Methyltransferase G9A Regulates Osteogenesis via Twist Gene Repression.

J Dent Res 2017 Sep 23;96(10):1136-1144. Epub 2017 Jun 23.

1 Department of Oral Health Sciences, Life Sciences Institute, Faculty of Dentistry, The Biomedical Research Centre, University of British Columbia, Vancouver, Canada.

Here we investigate the role of epigenetic factors in controlling the timing of cranial neural crest cell differentiation. The gene coding for histone H3 lysine 9 methyltransferase G9A was conditionally deleted in neural crest cells with Wnt1-Cre. The majority of homozygous-null animals survived to birth but thereafter failed to thrive. Phenotypic analysis of postnatal animals revealed that the mutants displayed incomplete ossification and 20% shorter jaws as compared to their wild-type littermates. At E13.5, patterns of expression of the osteogenic transcription factor RUNX2 and the mesenchymal transcription factor TWIST are similar in controls and mutants; both overlap in areas of future intramembranous bone formation. At E14.5, the nonosteogenic mesenchyme expressed TWIST, whereas the ossification center had strong RUNX2 and osteopontin expression. In the mutants, TWIST protein was present in the osteogenic mesenchyme, while osteopontin was not expressed until E15.5. In addition, in mutants, small regions of TWIST-positive osteogenic mesenchyme were visible until E15.5. The delay in ossification and reduction in size of the ossification centers were correlated with an earlier decrease in proliferation. We used micromass cultures of the face to investigate the direct effects of G9A inhibition on skeletal differentiation. Addition of a small molecule inhibitor for G9A, BIX-01294, to wild-type cells upregulated Twist genes similar to what was observed in vivo. The inhibitor also caused decreases in several osteogenic markers. Chromatin immunoprecipitation analysis of primary osteogenic mesenchyme from calvaria revealed that Twist1 and Twist2 regulatory regions contain the repressive H3K9me2 marks catalyzed by G9A, which are removed when BIX-01294 is added. Our results establish a role for G9A and H3K9me2 in the regulation of Twist genes and provide novel insights into the significance of epigenetic mechanisms in controlling temporal and tissue-specific gene expression during development.
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http://dx.doi.org/10.1177/0022034517716438DOI Listing
September 2017

Regulation of Active DNA Demethylation through RAR-Mediated Recruitment of a TET/TDG Complex.

Cell Rep 2017 05;19(8):1685-1697

Department of Biochemistry, Western University, London, ON N6A 5C1, Canada; Department of Oncology, The London Regional Cancer Program and the Lawson Health Research Institute, London, ON N6A 4L6, Canada. Electronic address:

Retinoic acid (RA) plays important roles in development, growth, and homeostasis through regulation of the nuclear receptors for RA (RARs). Herein, we identify Hypermethylated in Cancer 1 (Hic1) as an RA-inducible gene. HIC1 encodes a tumor suppressor, which is often silenced by promoter hypermethylation in cancer. Treatment of cells with an RAR agonist causes a rapid recruitment of an RAR/RXR complex consisting of TDG, the lysine acetyltransferase CBP, and TET 1/2 to the Hic1 promoter. Complex binding coincides with a transient accumulation of 5fC/5caC and concomitant upregulation of Hic1 expression, both of which are TDG dependent. Furthermore, conditional deletion of Tdg in vivo is associated with Hic1 silencing and DNA hypermethylation of the Hic1 promoter. These findings suggest that the catalytic and scaffolding activities of TDG are essential for RA-dependent gene expression and provide important insights into the mechanisms underlying targeting of TET-TDG complexes.
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http://dx.doi.org/10.1016/j.celrep.2017.05.007DOI Listing
May 2017

The transcriptional repressor HIC1 regulates intestinal immune homeostasis.

Mucosal Immunol 2017 11 22;10(6):1518-1528. Epub 2017 Mar 22.

The Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia, Canada.

The intestine is a unique immune environment that must respond to infectious organisms but remain tolerant to commensal microbes and food antigens. However, the molecular mechanisms that regulate immune cell function in the intestine remain unclear. Here we identify the POK/ZBTB family transcription factor hypermethylated in cancer 1 (HIC1, ZBTB29) as a central component of immunity and inflammation in the intestine. HIC1 is specifically expressed in immune cells in the intestinal lamina propria (LP) in the steady state and mice with a T-cell-specific deletion of HIC1 have reduced numbers of T cells in the LP. HIC1 expression is regulated by the Vitamin A metabolite retinoic acid, as mice raised on a Vitamin A-deficient diet lack HIC1-positive cells in the intestine. HIC1-deficient T cells overproduce IL-17A in vitro and in vivo, and fail to induce intestinal inflammation, identifying a critical role for HIC1 in the regulation of T-cell function in the intestinal microenvironment under both homeostatic and inflammatory conditions.
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http://dx.doi.org/10.1038/mi.2017.17DOI Listing
November 2017

Tumor-homing cytotoxic human induced neural stem cells for cancer therapy.

Sci Transl Med 2017 02;9(375)

Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Engineered neural stem cells (NSCs) are a promising approach to treating glioblastoma (GBM). The ideal NSC drug carrier for clinical use should be easily isolated and autologous to avoid immune rejection. We transdifferentiated (TD) human fibroblasts into tumor-homing early-stage induced NSCs (h-iNSC), engineered them to express optical reporters and different therapeutic gene products, and assessed the tumor-homing migration and therapeutic efficacy of cytotoxic h-iNSC in patient-derived GBM models of surgical and nonsurgical disease. Molecular and functional analysis revealed that our single-factor SOX2 TD strategy converted human skin fibroblasts into h-iNSC that were nestin and expressed pathways associated with tumor-homing migration in 4 days. Time-lapse motion analysis showed that h-iNSC rapidly migrated to human GBM cells and penetrated human GBM spheroids, a process inhibited by blockade of CXCR4. Serial imaging showed that h-iNSC delivery of the proapoptotic agent tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) reduced the size of solid human GBM xenografts 250-fold in 3 weeks and prolonged median survival from 22 to 49 days. Additionally, h-iNSC thymidine kinase/ganciclovir enzyme/prodrug therapy (h-iNSC-TK) reduced the size of patient-derived GBM xenografts 20-fold and extended survival from 32 to 62 days. Mimicking clinical NSC therapy, h-iNSC-TK therapy delivered into the postoperative surgical resection cavity delayed the regrowth of residual GBMs threefold and prolonged survival from 46 to 60 days. These results suggest that TD of human skin into h-iNSC is a platform for creating tumor-homing cytotoxic cell therapies for cancer, where the potential to avoid carrier rejection could maximize treatment durability in human trials.
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http://dx.doi.org/10.1126/scitranslmed.aah6510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719790PMC
February 2017

HDAC and Proteasome Inhibitors Synergize to Activate Pro-Apoptotic Factors in Synovial Sarcoma.

PLoS One 2017 5;12(1):e0169407. Epub 2017 Jan 5.

Faculty of Medicine, Vancouver Coastal Health Research Institute, University of British Columbia, Vancouver, British Columbia, Canada.

Conventional cytotoxic therapies for synovial sarcoma provide limited benefit, and no drugs specifically targeting its driving SS18-SSX fusion oncoprotein are currently available. Patients remain at high risk for early and late metastasis. A high-throughput drug screen consisting of over 900 tool compounds and epigenetic modifiers, representing over 100 drug classes, was undertaken in a panel of synovial sarcoma cell lines to uncover novel sensitizing agents and targetable pathways. Top scoring drug categories were found to be HDAC inhibitors and proteasomal targeting agents. We find that the HDAC inhibitor quisinostat disrupts the SS18-SSX driving protein complex, thereby reestablishing expression of EGR1 and CDKN2A tumor suppressors. In combination with proteasome inhibition, HDAC inhibitors synergize to decrease cell viability and elicit apoptosis. Quisinostat inhibits aggresome formation in response to proteasome inhibition, and combination treatment leads to elevated endoplasmic reticulum stress, activation of pro-apoptotic effector proteins BIM and BIK, phosphorylation of BCL-2, increased levels of reactive oxygen species, and suppression of tumor growth in a murine model of synovial sarcoma. This study identifies and provides mechanistic support for a particular susceptibility of synovial sarcoma to the combination of quisinostat and proteasome inhibition.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0169407PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5215898PMC
August 2017

Methods and Strategies for Lineage Tracing of Mesenchymal Progenitor Cells.

Methods Mol Biol 2016 ;1416:171-203

Department of Cellular and Physiological Sciences and Biomedical Research Centre, University of British Columbia, 2222 Health Sciences Mall, Vancouver, BC, V6T 1Z3, USA.

Mesenchymal progenitors (MP) are found to varying extents in most tissues and organs. Their relationship to bone marrow-derived mesenchymal stem cells (MSCs) remains unclear, however, both populations appear to share a number of properties as defined by functional assays, clonogenic activity, and genetic and cell surface markers. MSCs were originally defined by their in vitro colony forming unit-fibroblast (CFU-F) activity and their ability to contribute to various mesenchymal lineages (i.e. cartilage, bone, and fat). MSCs also appear to exhibit some unique properties, in that expanded clones in the absence of bone-inducing factors generate bone spicules/organs in vivo. Subsequent analysis of these elements has demonstrated that the transplanted cells directly contribute to multiple mesenchymal lineages. Our ability to study MP and/or MSC behavior and lineage potential in vivo has been hampered by a lack of suitable Cre lines in which to effectively genetically mark and follow the fate and activity of these cells in development, growth, homeostasis and following injury or in disease. The emergence of several new genetic lines is enabling us to now address critical questions regarding MP/MSC location, behavior, function, and fate. The use of these lines and others in conjunction with suitable reporter lines will be described for MP/MSC cell fate analysis.
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http://dx.doi.org/10.1007/978-1-4939-3584-0_10DOI Listing
December 2017

Impact of a CXCL12/CXCR4 Antagonist in Bleomycin (BLM) Induced Pulmonary Fibrosis and Carbon Tetrachloride (CCl4) Induced Hepatic Fibrosis in Mice.

PLoS One 2016 21;11(3):e0151765. Epub 2016 Mar 21.

The Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia, Canada.

Modulation of chemokine CXCL12 and its receptor CXCR4 has been implicated in attenuation of bleomycin (BLM)-induced pulmonary fibrosis and carbon tetrachloride (CCl4)-induced hepatic injury. In pulmonary fibrosis, published reports suggest that collagen production in the injured lung is derived from fibrocytes recruited from the circulation in response to release of pulmonary CXCL12. Conversely, in hepatic fibrosis, resident hepatic stellate cells (HSC), the key cell type in progression of fibrosis, upregulate CXCR4 expression in response to activation. Further, CXCL12 induces HSC proliferation and subsequent production of collagen I. In the current study, we evaluated AMD070, an orally bioavailable inhibitor of CXCL12/CXCR4 in alleviating BLM-induced pulmonary and CCl4-induced hepatic fibrosis in mice. Similar to other CXCR4 antagonists, treatment with AMD070 significantly increased leukocyte mobilization. However, in these two models of fibrosis, AMD070 had a negligible impact on extracellular matrix deposition. Interestingly, our results indicated that CXCL12/CXCR4 signaling has a role in improving mortality associated with BLM induced pulmonary injury, likely through dampening an early inflammatory response and/or vascular leakage. Together, these findings indicate that the CXCL12-CXCR4 signaling axis is not an effective target for reducing fibrosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0151765PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4801399PMC
August 2016

Retinoid regulation of antiviral innate immunity in hepatocytes.

Hepatology 2016 06 21;63(6):1783-95. Epub 2016 Jan 21.

Department of Medicine, USC Research Center for Liver Diseases, Division of Gastrointestinal and Liver Diseases, University of Southern California, Los Angeles, CA.

Unlabelled: Persistent infection of hepatitis C virus (HCV) is one of the leading causes of end-stage liver disease (ESLD), such as decompensated cirrhosis and liver cancer. Of particular note, nearly half of HCV-infected people in the United States are reported to be heavy drinkers. This particular group of patients is known to rapidly progress to the ESLD. Although accelerated disease progression among alcohol abusers infected with HCV is clinically well recognized, the molecular pathophysiology behind this manifestation has not been well elucidated. Hepatocytes metabolize ethanol (EtOH) primarily through two steps of oxidative catabolism in which alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) play central roles. The ADH-ALDH pathway also governs the metabolism of retinol (vitamin A) to its transcriptionally active metabolite, retinoic acid (RA). In this study, we defined that the ADH-ALDH pathway serves as a potent antiviral host factor in hepatocytes, which regulates the expression of interferon (IFN)-stimulated genes (ISGs) by biogenesis of RA. ISGs constitute over 300 antiviral effectors, which cooperatively govern intracellular antiviral innate immunity. Our study revealed that intracellular RA levels greatly influence ISG expression under basal conditions. Moreover, RA augments ISG induction in response to viral infection or exposure to IFN in a gene-specific manner. Lastly, our results demonstrated that EtOH attenuates the antiviral function of the ADH-ALDH pathway, which suggests the possibility that EtOH-retinol metabolic competition is one of the molecular mechanisms for the synergism between HCV and alcohol abuse in liver disease progression.

Conclusions: RA plays a critical role in the regulation of intracellular antiviral innate immunity in hepatocytes. (Hepatology 2016;63:1783-1795).
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http://dx.doi.org/10.1002/hep.28380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4874888PMC
June 2016

SOX9 modulates the expression of key transcription factors required for heart valve development.

Development 2015 Dec 2;142(24):4340-50. Epub 2015 Nov 2.

Terry Fox Laboratory, BC Cancer Agency, Vancouver, Canada V5Z 1L3 Program in Cell and Developmental Biology, University of British Columbia, Vancouver, Canada V6T 1Z4 Department of Medical Genetics, University of British Columbia, Vancouver, Canada V6T 1Z4

Heart valve formation initiates when endothelial cells of the heart transform into mesenchyme and populate the cardiac cushions. The transcription factor SOX9 is highly expressed in the cardiac cushion mesenchyme, and is essential for heart valve development. Loss of Sox9 in mouse cardiac cushion mesenchyme alters cell proliferation, embryonic survival, and valve formation. Despite this important role, little is known about how SOX9 regulates heart valve formation or its transcriptional targets. Therefore, we mapped putative SOX9 binding sites by ChIP-Seq in E12.5 heart valves, a stage at which the valve mesenchyme is actively proliferating and initiating differentiation. Embryonic heart valves have been shown to express a high number of genes that are associated with chondrogenesis, including several extracellular matrix proteins and transcription factors that regulate chondrogenesis. Therefore, we compared regions of putative SOX9 DNA binding between E12.5 heart valves and E12.5 limb buds. We identified context-dependent and context-independent SOX9-interacting regions throughout the genome. Analysis of context-independent SOX9 binding suggests an extensive role for SOX9 across tissues in regulating proliferation-associated genes including key components of the AP-1 complex. Integrative analysis of tissue-specific SOX9-interacting regions and gene expression profiles on Sox9-deficient heart valves demonstrated that SOX9 controls the expression of several transcription factors with previously identified roles in heart valve development, including Twist1, Sox4, Mecom and Pitx2. Together, our data identify SOX9-coordinated transcriptional hierarchies that control cell proliferation and differentiation during valve formation.
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http://dx.doi.org/10.1242/dev.125252DOI Listing
December 2015

Two novel disease-causing variants in BMPR1B are associated with brachydactyly type A1.

Eur J Hum Genet 2015 Dec 11;23(12):1640-5. Epub 2015 Mar 11.

Faculty of Medicine, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada.

Brachydactyly type A1 is an autosomal dominant disorder primarily characterized by hypoplasia/aplasia of the middle phalanges of digits 2-5. Human and mouse genetic perturbations in the BMP-SMAD signaling pathway have been associated with many brachymesophalangies, including BDA1, as causative mutations in IHH and GDF5 have been previously identified. GDF5 interacts directly as the preferred ligand for the BMP type-1 receptor BMPR1B and is important for both chondrogenesis and digit formation. We report pathogenic variants in BMPR1B that are associated with complex BDA1. A c.975A>C (p.(Lys325Asn)) was identified in the first patient displaying absent middle phalanges and shortened distal phalanges of the toes in addition to the significant shortening of middle phalanges in digits 2, 3 and 5 of the hands. The second patient displayed a combination of brachydactyly and arachnodactyly. The sequencing of BMPR1B in this individual revealed a novel c.447-1G>A at a canonical acceptor splice site of exon 8, which is predicted to create a novel acceptor site, thus leading to a translational reading frameshift. Both mutations are most likely to act in a dominant-negative manner, similar to the effects observed in BMPR1B mutations that cause BDA2. These findings demonstrate that BMPR1B is another gene involved with the pathogenesis of BDA1 and illustrates the continuum of phenotypes between BDA1 and BDA2.
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http://dx.doi.org/10.1038/ejhg.2015.38DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4795202PMC
December 2015

The methyltransferase G9a regulates HoxA9-dependent transcription in AML.

Genes Dev 2014 Feb;28(4):317-27

University of British Columbia, Biomedical Research Centre, Vancouver, British Columbia V6T 1Z3, Canada;

Chromatin modulators are emerging as attractive drug targets, given their widespread implication in human cancers and susceptibility to pharmacological inhibition. Here we establish the histone methyltransferase G9a/EHMT2 as a selective regulator of fast proliferating myeloid progenitors with no discernible function in hematopoietic stem cells (HSCs). In mouse models of acute myeloid leukemia (AML), loss of G9a significantly delays disease progression and reduces leukemia stem cell (LSC) frequency. We connect this function of G9a to its methyltransferase activity and its interaction with the leukemogenic transcription factor HoxA9 and provide evidence that primary human AML cells are sensitive to G9A inhibition. Our results highlight a clinical potential of G9A inhibition as a means to counteract the proliferation and self-renewal of AML cells by attenuating HoxA9-dependent transcription.
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http://dx.doi.org/10.1101/gad.236794.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3937511PMC
February 2014

Analysis of chondrogenesis using micromass cultures of limb mesenchyme.

Methods Mol Biol 2014 ;1130:251-265

University of British Columbia, Vancouver, BC, Canada.

High-density micromass cultures of embryonic mesenchymal cells have proved to be an invaluable model for studying the entire chondrogenic program, from precartilaginous condensations through to chondrocyte hypertrophy. This culture model also provides a powerful system in which to explore the function of various factors in the commitment and differentiation of mesenchymal cells to the chondrogenic lineage. In this regard, micromass cultures provide a consistent and robust model for investigating the effects of genetic manipulations on skeletal phenotypes and for delineating their molecular basis. In this methods chapter, the derivation and use of micromass cultures from murine limb buds are described, but these techniques are also applicable to other organisms and mesenchymal cell sources.
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http://dx.doi.org/10.1007/978-1-62703-989-5_19DOI Listing
September 2014

The retinoic acid-metabolizing enzyme Cyp26b1 regulates CD4 T cell differentiation and function.

PLoS One 2013 22;8(8):e72308. Epub 2013 Aug 22.

The Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia, Canada.

The vitamin A metabolite retinoic acid (RA) has potent immunomodulatory properties that affect T cell differentiation, migration and function. However, the precise role of RA metabolism in T cells remains unclear. Catabolism of RA is mediated by the Cyp26 family of cytochrome P450 oxidases. We examined the role of Cyp26b1, the T cell-specific family member, in CD4(+) T cells. Mice with a conditional knockout of Cyp26b1 in T cells (Cyp26b1 (-/-) mice) displayed normal lymphoid development but showed an increased sensitivity to serum retinoids, which led to increased differentiation under both inducible regulatory T (iTreg) cell- and TH17 cell-polarizing conditions in vitro. Further, Cyp26b1 expression was differentially regulated in iTreg and TH17 cells. Transfer of naïve Cyp26b1 (-/-) CD4(+) T cells into Rag1 (-/-) mice resulted in significantly reduced disease in a model of T cell-dependent colitis. Our results show that T cell-specific expression of Cyp26b1 is required for the development of T cell-mediated colitis and may be applicable to the development of therapeutics that target Cyp26b1 for the treatment of inflammatory bowel disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0072308PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3750006PMC
June 2014